Compounds and methods for modulating angiotensinogen expression

Abstract

To provide composition and compounds comprising modified oligonucleotides to modulate AGT and to modulate a RAAS pathway-related disease, disorder and / or condition in an individual in need thereof.SOLUTION: Provided herein is a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of a specific nucleobase sequence, where the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to the specific nucleobase sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing This application is filed with an electronic sequence listing, which is submitted as a file entitled BIOL0270WOSEQ_ST25.txt, created on October 3, 2016, and 456 kb in size. The information in this electronic sequence listing is incorporated herein by reference in its entirety.

[0002] The present invention provides compounds, compositions and methods for modulating the expression of angiotensinogen (AGT) for the purpose of modulating a RAAS pathway-related disease, disorder or condition in an animal. The present invention also provides compounds, compositions and methods for reducing hypertension and organ damage by administering an AGT inhibitor to an animal. [Background technology]

[0003] Angiotensinogen (AGT), also known as SERPINA8 or ANHU, is a member of the serpin family and a component of the renin-angiotensin-aldosterone system (RAAS). AGT is primarily produced in the liver and then released into the circulation where it is converted by renin to angiotensin I. Angiotensin I is then converted by angiotensin-converting enzyme (ACE) to angiotensin II. Angiotensin II is a peptide hormone that causes vasoconstriction, which can increase blood pressure. Angiotensin II also stimulates the secretion of the hormone aldosterone from the adrenal cortex. Aldosterone acts on the kidneys to increase sodium and water reabsorption, which can increase the amount of fluid in the body and result in increased blood pressure. Excessive stimulation or activity of the RAAS pathway can lead to high blood pressure. Chronically high blood pressure is known as hypertension. In subjects with high blood pressure, this high blood pressure requires the heart to work harder to circulate blood through the blood vessels.

[0004] The World Health Organization (WHO) has identified hypertension as the leading cause of cardiovascular disease. Hypertension is a major risk factor for a variety of diseases, disorders and conditions, such as shortened life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, vascular aneurysms (e.g., aortic aneurysms), peripheral arterial disease, cardiac disorders (e.g., cardiac enlargement or hypertrophy), and other cardiovascular-related diseases, disorders and / or conditions.

[0005] The prevalence of treatment-resistant hypertension (RHTN), i.e., high blood pressure that is resistant to drug treatment, is steadily increasing in number due to the aging of the population and the ever-increasing incidence of obesity. The current projection of approximately 10 million RHTN adults in the United States is expected to increase.

[0006] Antihypertensive drugs, renal denervation, baroreceptor activation therapy, dietary modification, and lifestyle modification may reduce hypertension and reduce hypertension-related diseases, disorders, and / or pathologies (Paulis et al., Nat Rev Cardiol, 2012, 9:276-285). However, currently approved therapies for the treatment of hypertension are limited because a significant portion of all hypertensive patients do not achieve adequate blood pressure control. For example, drugs such as ACE inhibitors and angiotensin receptor blockers (ARBs), which target part of the renin-angiotensin system (RAS) pathway, have limited ability to inhibit the RAAS pathway (Nobakht et al., Nat Rev Nephrol, 2011, 7:356-359). Furthermore, certain antihypertensive drugs, such as ACE inhibitors, are contraindicated in hypertensive patients with renal disease because they may weaken the patient's renal function.

[0007] Therefore, there is a need to find alternative therapies to inhibit the RAAS pathway and treat hypertension. Antisense technology has been attracting attention as an effective means to reduce the expression of certain gene products. However, early antisense oligonucleotides targeting AGT have only provided limited benefit (WO1997 / 33623) or target non-human AGT (WO2014 / 018930). The compounds and compositions herein provide novel highly potent and tolerable compounds that inhibit human AGT, which are also suitable for use in human subjects. Furthermore, the compounds disclosed herein are expected to reduce the tolerability issues of conventional RAS antagonists in patients at risk of hyperkalemia and / or renal disease by using conjugation methods to deliver antisense compounds to the liver and limit their intrarenal distribution and activity.

[0008] All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and papers, are expressly incorporated by reference in their entirety, including those documents that are discussed herein. Summary of the Invention

[0009] Provided herein are compositions, compounds and methods for reducing AGT mRNA and / or protein levels in an animal.

[0010] Certain embodiments disclosed herein provide compounds comprising modified oligonucleotides that target a nucleic acid sequence encoding AGT. In certain embodiments, the compounds target the AGT sequence shown in any of the nucleobase sequences of SEQ ID NOs: 1-6.

[0011] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 2250 to 2337 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0012] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 2281-2300 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0013] Certain embodiments disclosed herein comprise 12-30 linked nucleosides and are selected from the group consisting of SEQ ID NOs: 46, 53-54, 61, 68, 76, 83, 85, 93, 96-97, 109, 127, 129-130, 132, 134-15, 137-39, 142, 163-172, 180-184, 186, 189, 234, 236, 238~239, 267, 313, 411, 452, 463~470, 475~478, 480, 500~503, 512, 517~518, 524~526, 654, 689, 702, 725~726, 728, 738, 779, 786~787, 800, 808, 810~811, 825, 865, 868, 889, 894, 903, 905, 909, 954, 966, 1011, 1015, 1021, 1024, 1080, 1085, 1258~1259, 1261~1262, 1293~1294, 1299, 1325, 1470, 1472~1473, 1522, 1542, 1604, 1623~1624, 1667, 1670, 1682~1683, 1687, 1700, 1703~1704, 1708, 1714, 1716, 1719~1720, 1724~1726, 1729~1730, 1827, 1936, 1843~1844, 1846, 1886, 1893~1894, 1914, Compounds are provided, comprising modified oligonucleotides having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences: 1923, 1925, 1932, 1979, 1986, 1988, 1990, 2003, 2015, 2018, 2020, 2027-2028, 2035, 2037, 2039, and 2044.

[0014] Certain embodiments disclosed herein include compounds having the formula: mCes Aes mCes Aes Provided is a compound comprising a modified oligonucleotide according to Aes Ads mCds Ads Ads Gds mCds Tds Gds Gds Tds mCes Ges Ges Tes Te (SEQ ID NO: 1914), where A is adenine, mC is 5-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, and s is a phosphorothioate internucleoside linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0015] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide having the formula:

[0016] [ka] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] 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. As used herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including" as well as other forms such as "includes" and "included" is not limiting. Additionally, terms such as "element" or "component" include both elements and components that include one unit and elements and components that include more than one subunit, unless specifically stated otherwise.

[0018] The section headings used herein are for organizational purposes only and are not intended to limit 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 papers, are expressly incorporated herein by reference in their entirety, as well as any portions of documents discussed herein.

[0019] definition Unless specific definitions are provided, the terms used in and procedures and techniques relating to analytical chemistry, synthetic organic chemistry, and medicinal pharmaceutical chemistry described herein are understood to be within the skill of the art. Standard techniques may be used for chemical synthesis and chemical analysis. Where permitted, all patents, applications, published applications, and other publications referenced throughout this disclosure, GENBANK accession numbers and associated sequence information available through databases such as the National Center for Biotechnology Information (NCBI), and other data, are hereby incorporated by reference in their entirety, as well as for the portions of the documents discussed herein.

[0020] Unless otherwise indicated, the following terms have the following meanings.

[0021] "2'-O-Methoxyethyl" (also 2'-MOE and 2'-O(CH2)2-OCH3) refers to an O-methoxy-ethyl modification at the 2' position of the furosyl ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0022] "2'-O-methoxyethyl nucleotide" means a nucleotide containing a 2'-O-methoxyethyl modified sugar moiety.

[0023] "5-methylcytosine" refers to a modified cytosine having a methyl group attached to the 5' position. 5-Methylcytosine is a modified nucleobase.

[0024] "About" means within ±10% of a value. For example, if it is stated that "a marker may be increased by about 50%," it means that the marker may be increased by 45% to 55%.

[0025] "ACE escape", also known as angiotensin II reactivation, refers to the failure of currently available ACE inhibitor treatments to reliably suppress plasma angiotensin II levels. The increase in plasma angiotensin II levels during ACE inhibition is caused by other enzymes that convert angiotensin I to angiotensin. This incomplete blockade of angiotensin II levels prevents effective treatment of some hypertensive subjects with ACE inhibitors. Angiotensin receptor blockers (ARBs) can similarly be affected by ACE escape, as they have other receptors besides the AT1 receptor that bind to angiotensin metabolites.

[0026] "Active agent" or "agent" refers to one or more substances in a pharmaceutical composition that provide a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to AGT is an active agent.

[0027] By "active target region" or "target region" is meant a region to which one or more active antisense compounds are targeted.

[0028] By "active antisense compound" is meant an antisense compound that reduces target nucleic acid levels or protein levels.

[0029] "Administered simultaneously" refers to the co-administration of two agents in any manner that results in the pharmacological effects of both agents being manifested in a patient at the same time. Simultaneous 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 do not have to be manifested at the same time. The effects need only overlap over a period of time, and do not have to have the same time span.

[0030] "Administering" means providing a medication to an individual, and includes, but is not limited to, administration by a medical professional and self-administration.

[0031] "Aldosterone escape" or "aldosterone breakthrough" refers to the failure of currently available ACE inhibitors, angiotensin receptor blockers (ARBs) and / or direct renin inhibitors (DRIs) to reliably suppress aldosterone release in some treated subjects. This incomplete blockade of aldosterone prevents effective treatment of some hypertensive subjects with ACE inhibitors, DRIs and ARBs.

[0032] "Agent" refers to an active substance that can provide a therapeutic benefit when administered to an animal. "First agent" refers to a therapeutic compound provided herein. For example, the first agent is an antisense oligonucleotide that targets AGT. "Second agent" refers to a second therapeutic compound described herein. For example, the second agent can be a second antisense oligonucleotide that targets AGT or a target other than AGT. Alternatively, the second agent can be a compound other than an antisense oligonucleotide.

[0033] "Amelioration" or "ameliorating" refers to a reduction in at least one indicator, marker, sign or symptom of the associated disease, disorder and / or condition. In certain embodiments, amelioration includes a delay or slowing of the progression of one or more indicators of the condition, disorder and / or disease. The severity of the indicator can be determined by subjective or objective measures known to those of skill in the art.

[0034] "Angiotensinogen" and "AGT" are used interchangeably herein. Angiotensinogen is also known as SERPINA8 and ANHU.

[0035] "Angiotensinogen nucleic acid" or "AGT nucleic acid" refers to any nucleic acid encoding AGT. For example, in certain embodiments, AGT nucleic acid includes DNA sequences encoding AGT, RNA sequences transcribed from DNA encoding AGT (including genomic DNA containing introns and exons), and mRNA sequences encoding AGT. "AGT mRNA" refers to the mRNA encoding the AGT protein.

[0036] "AGT specific inhibitor" refers to any agent capable of specifically inhibiting the expression of AGT mRNA and / or AGT protein. For example, AGT specific inhibitors include nucleic acids (including antisense compounds such as RNasH, siRNA and blockmir antisense compounds), peptides, antibodies, small molecules and other agents capable of specifically inhibiting the expression of AGT mRNA and / or AGT protein. In certain embodiments, AGT specific inhibitors can affect components of the renin-angiotensin-aldosterone system (RAAS) pathway by specifically regulating AGT mRNA levels and / or AGT protein expression. In certain embodiments, AGT specific inhibitors can affect RAAS pathway-related diseases, disorders and / or pathologies, such as blood pressure, by specifically regulating AGT mRNA levels and / or AGT protein expression. Similarly, in certain embodiments, AGT specific inhibitors can affect other molecular processes in animals.

[0037] "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).

[0038] "Antihypertensive drug" refers to a drug that can lower blood pressure. Examples of such drugs include, but are not limited to, RAAS inhibitors, diuretics, calcium channel antagonists, sympathetic receptor blockers, sympathomimetics, and vasodilators. As an example, the antihypertensive drug captopril can be used in combination with the AGT compounds described herein to treat animals having or at risk for a RAAS pathway-related disease, disorder, and / or condition. can be done.

[0039] "Antihypertensive treatment" refers to a medical treatment administered to a subject to reduce high blood pressure. Examples of such treatments include renal denervation and baroreceptor activation therapy.

[0040] "Antibody" refers to a molecule characterized by specifically reacting in some way with an antigen, where antibody and antigen are defined in terms of each other. Antibody refers to an intact antibody molecule or any fragment or region thereof, such as the heavy chain, light chain, Fab region, and Fc region.

[0041] "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 reduction in the amount or expression of a target nucleic acid or a protein encoded by the target nucleic acid.

[0042] "Antisense compound" means an oligomeric compound capable of hybridizing to a target nucleic acid through hydrogen bonding.

[0043] "Antisense inhibition" means a reduction in the level of a target nucleic acid or target protein in the presence of an antisense compound complementary to a target nucleic acid compared to the level of the target nucleic acid or target protein in the absence of the antisense compound.

[0044] By "antisense oligonucleotide" is meant a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

[0045] "Bicyclic sugar" means a furosyl ring modified by the bridging of two non-geminal ring atoms. A bicyclic sugar is a modified sugar.

[0046] "Bicyclic nucleic acid" or "BNA" refers to a nucleoside or nucleotide in which the furanose portion of the nucleoside or nucleotide contains a bridge connecting two carbon atoms on the furanose ring, thereby forming a bicyclic system.

[0047] "Blood pressure" refers to the pressure of blood against the walls of blood vessels in the circulatory system. Blood pressure is primarily due to the beating of an animal's heart. During each heartbeat, blood pressure fluctuates between a maximum (systolic) blood pressure (SBP) and a minimum (diastolic) blood pressure (DBP). Mean arterial pressure (MAP) is the average of the arterial pressure during the cardiac cycle. Blood pressure can be measured by a blood pressure measuring device (i.e., a sphygmomanometer). Normal blood pressure at rest is in the range of 100-140 mmHg systolic and 60-90 mmHg diastolic, and is commonly expressed as systolic pressure (highest reading) / diastolic pressure (lowest reading) mmHg.

[0048] By "cap structure" or "terminal cap moiety" is meant a chemical modification incorporated at either end of an antisense compound.

[0049] "cEt" or "constrained ethyl" means a bicyclic sugar moiety containing a bridge connecting the 4'-carbon and the 2'-carbon, said bridge having the formula: 4'-CH(CH3)-O-2'.

[0050] "Constrained ethyl nucleoside" (also cEt nucleoside) means a nucleoside that includes a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge.

[0051] "Chemically distinct region" refers to a region of an antisense compound that is in some way chemically distinct from another region of the same antisense compound. For example, a region having 2'-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides that do not contain 2'-O-methoxyethyl modifications.

[0052] By "chimeric antisense compound" is meant an antisense compound that has at least two chemically distinct regions.

[0053] "Co-administration" refers to the administration of two or more agents to an individual. The two or more agents may be in a single pharmaceutical composition or in separate pharmaceutical compositions. Each of the two or more agents may be administered by the same or different routes of administration. Co-administration includes simultaneous, parallel, or sequential administration.

[0054] "Complementarity" refers to the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid. In certain embodiments, the first nucleic acid is an antisense compound and the second nucleic acid is a target nucleic acid.

[0055] "Contiguous nucleobases" means nucleobases immediately adjacent to each other.

[0056] "Deoxyribonucleotide" means a nucleotide having a hydrogen at the 2' position of the sugar moiety of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.

[0057] "Diluent" refers to an ingredient in a composition that is not pharmacologically active, but is pharma- ceutically necessary or desirable. For example, a diluent in an injectable composition can be a liquid, such as phosphate buffered saline (PBS) or water.

[0058] "Formulation unit" refers to the form in which the drug is provided, such as a pill, tablet, or other formulation unit known in the art. In certain embodiments, the formulation unit is a vial containing lyophilized antisense oligonucleotide. In certain embodiments, the formulation unit is a vial containing reconstituted antisense oligonucleotide.

[0059] "Dose" refers to a specified amount of drug provided in one administration or within a specified period of time. In certain embodiments, a dose can 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 that is not easily accommodated by a single injection, and therefore, two or more injections can be used to achieve the desired dose. In certain embodiments, the drug is administered by infusion or continuous infusion over an extended period of time. The dose can be expressed as the amount of drug per hour, day, week or month.

[0060] "Effective amount" or "therapeutically effective amount" refers to an amount of active agent sufficient to produce a desired physiological result in an individual who needs the active agent. Effective amount may vary between individuals, depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, the evaluation of the individual's medical condition, and other relevant factors. As an example, an effective amount of AGT antisense oligonucleotide reduces blood pressure and / or improves organ damage caused by hypertension.

[0061] "Fully complementary" or "100% complementary" means that each nucleobase of a nucleobase sequence of a first nucleic acid has a complementary nucleobase in a second nucleobase sequence of a second nucleic acid. In certain embodiments, the first nucleic acid is an antisense compound and the second nucleic acid is a target nucleic acid.

[0062] "Gapmer" refers to a chimeric antisense compound in which an inner region having multiple nucleosides that support RNase H cleavage is positioned between an outer region having one or more nucleosides, where the nucleosides included in the inner region are chemically distinct from the one or more nucleosides included in the outer region. The inner region can be referred to as the "gap segment" and the outer region can be referred to as the "wing segment."

[0063] "Gap widened" refers to a chimeric antisense compound having a gap segment of 12 or more contiguous 2'-deoxynucleosides positioned between and immediately adjacent to 5' and 3' wing segments having from 1 to 6 nucleosides.

[0064] "Hybridization" refers to the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include antisense compounds and nucleic acid targets.

[0065] "Hypertension" or "HTN" refers to a chronic medical condition in which an animal has elevated blood pressure. Elevated blood pressure requires the heart to work harder to circulate blood through the blood vessels. Blood pressure that is persistently above 140 / 90 mmHg is said to be hypertension. Hypertension is classified as primary (essential) or secondary. Primary hypertension has no clear cause and is thought to be related to genetics, diet, lack of exercise, and obesity. Secondary hypertension is caused by another medical condition. Hypertension is a major risk factor for shortened life span, chronic kidney disease, stroke, myocardial infarction, heart failure, vascular aneurysms (e.g., aortic aneurysms), peripheral arterial disease, organ damage (e.g., cardiac enlargement or hypertrophy), and other cardiovascular diseases, disorders, and / or conditions or symptoms thereof. Antihypertensive drugs, dietary modifications, and lifestyle modifications may reduce hypertension and reduce hypertension-related diseases, disorders, and / or conditions. Hypertension may be amenable to drug intervention (ie, controllable by treatment with over-the-counter drugs) or resistant to drug intervention.

[0066] "Identifying an animal having or at risk for a RAAS-related disease, disorder and / or condition" means identifying an animal that has been diagnosed with a RAAS-related disease, disorder and / or condition or that is predisposed to developing a RAAS-related disease, disorder and / or condition. Individuals predisposed to developing a RAAS-related disease, disorder and / or condition include, for example, individuals with a family history of a RAAS-related disease, such as hypertension. Such identification can be accomplished by any method, including evaluation of the individual's medical history and standard laboratory tests or clinical evaluations.

[0067] By "immediately adjacent" it is meant that there are no intervening elements between the immediately adjacent elements.

[0068] By "individual" or "subject" or "animal" is meant a human or non-human animal selected for treatment or therapy.

[0069] "Inhibiting expression or activity" refers to reducing or blocking the expression or activity of an RNA or protein, and does not necessarily indicate a complete elimination of expression or activity.

[0070] "Internucleoside linkage" refers to the chemical bond between nucleosides.

[0071] "Intravenous administration" means administration into a vein.

[0072] "Linked nucleosides" means adjacent nucleosides that are linked to one another.

[0073] A "marker" or "biomarker" is any measurable and quantifiable biological parameter that is an indicator of an assessment of health or physiological function. For example, an increase in blood pressure or a reduction in organ damage (e.g., fibrosis) can be considered a marker of a RAAS-related disease, disorder, and / or condition.

[0074] A "mismatch" or "non-complementary nucleobase" or "MM" refers to an instance where a nucleobase of a first nucleic acid is not able to pair with the corresponding nucleobase of a second, or target, nucleic acid.

[0075] A "modified internucleoside linkage" refers to a substitution or any alteration from a naturally occurring internucleoside linkage (ie, a phosphodiester internucleoside linkage).

[0076] "Modified nucleobase" refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. For example, a modified nucleobase can be 5-methylcytosine. "Unmodified nucleobase" refers to the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).

[0077] "Modified nucleoside" means a nucleoside having, independently, a modified sugar moiety and / or a modified nucleobase.

[0078] "Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, and / or modified nucleobase.

[0079] "Modified oligonucleotide" means an oligonucleotide containing modified internucleoside linkages, modified sugars, and / or modified nucleobases.

[0080] "Modified sugar" refers to a substitution or alteration from a natural sugar. For example, the modified sugar can be 2'-MOE.

[0081] "Modulating" refers to changing or adjusting a certain characteristic of a cell, tissue, organ or organism. For example, modulating AGT mRNA can mean increasing or decreasing the level of AGT mRNA and / or AGT protein in a cell, tissue, organ or organism. Modulation of AGT mRNA and / or protein can result in an increase or decrease in a RAAS-related disease, disorder and / or pathology in a cell, tissue, organ or organism. A "modulator" produces a change in a cell, tissue, organ or organism. For example, an AGT antisense compound can be a modulator that increases or decreases the amount of AGT mRNA and / or AGT protein in a cell, tissue, organ or organism.

[0082] "Monomer" refers to one unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

[0083] By "motif" is meant a pattern of chemically distinct regions of an antisense compound.

[0084] "Naturally occurring internucleoside linkage" means a 3'-5' phosphodiester linkage. do.

[0085] By "natural sugar moiety" is meant a sugar found in DNA (2'-H) or RNA (2'-OH).

[0086] "Non-resistant hypertension", "non-controllable hypertension" or "controlled hypertension" is defined as hypertension that has responded to treatment with up to three concurrent antihypertensive agents, resulting in, for example, an SBP < 140 mmHg or DBP < 90 mmHg.

[0087] "Nucleic acid" refers to a molecule composed of nucleotide monomers. Nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid, double-stranded nucleic acid, small interfering ribonucleic acid (siRNA) and microRNA (miRNA).

[0088] "Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

[0089] By "nucleobase sequence" is meant the order of contiguous nucleobases regardless of any sugar, linkage or nucleobase modifications.

[0090] "Nucleoside" means a nucleobase linked to a sugar.

[0091] "Nucleoside mimetics" include structures used to replace the sugar or sugar and base, and, although not necessarily, the linkage, at one or more positions of an oligomeric compound, e.g., morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., nucleoside mimetics having sugar units other than furanose.

[0092] "Nucleotide" means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

[0093] "Nucleotide mimetics" include structures used to replace nucleosides and linkages at one or more positions in an oligomeric compound, such as, for example, peptide nucleic acids or morpholinos (morpholinos linked by -N(H)-C(=O)-O- or other non-phosphodiester linkages).

[0094] "Organ damage" or "end organ damage" refers to damage that occurs to major organs that are nourished by the circulatory system, such as the heart (e.g., myocardial hypertrophy, cardiac decline and / or heart failure), kidney (e.g., albuminuria, proteinuria, renal decline and / or renal failure), eye (e.g., hypertensive retinopathy), brain (e.g., stroke), etc. Organs can be damaged by hypertension in an animal. In certain embodiments, the cardiac damage is hypertrophy of cardiac cells and / or myocardium leading to fibrosis, cardiac enlargement.

[0095] "Oligomeric compound" or "oligomer" refers to a polymeric structure that contains two or more substructures (monomers) and can hybridize to a region of a nucleic acid molecule. In certain embodiments, an oligomeric compound is an oligonucleoside. In certain embodiments, an oligomeric compound is an oligonucleotide. In certain embodiments, an oligomeric compound is an antisense compound. In certain embodiments, an oligomeric compound is an antisense oligonucleotide. In certain embodiments, an oligomeric compound is a chimeric oligonucleotide.

[0096] "Oligonucleotide" refers to a polymer of linked nucleosides. Each of may be modified or unmodified, independently of the other.

[0097] "Parenteral administration" means administration by injection or infusion. Parenteral administration includes subcutaneous, intravenous, intramuscular, intraarterial, intraperitoneal or intracranial administration, e.g., intrathecal or intraventricular. Administration can be continuous, or chronic, or short-term, or intermittent.

[0098] "Peptide" refers to a molecule formed by joining at least two amino acids by amide bonds. Peptide refers to polypeptides and proteins.

[0099] "Pharmaceutical composition" means a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition can include one or more active agents and a sterile aqueous solution.

[0100] "Pharmaceutically acceptable carrier" means a medium or diluent that does not interfere with the structure of oligonucleotide. Such a specific carrier allows the formulation of pharmaceutical compositions for oral ingestion by subjects, such as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and lozenges. For example, pharmaceutically acceptable carrier can be sterile water or sterile aqueous solution such as PBS.

[0101] "Pharmaceutically acceptable derivatives" includes pharma- ceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein.

[0102] "Pharmaceutically acceptable salts" means physiologically and pharma- ceutically 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.

[0103] "Phosphorothioate bond" means a bond between nucleosides in which the phosphodiester bond has been modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate bond is a modified internucleoside bond.

[0104] "Portion" means a predetermined number of contiguous (i.e. linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a predetermined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a predetermined number of contiguous nucleobases of an antisense compound.

[0105] "Preventing" refers to delaying or forestalling the onset, development or progression of a disease, disorder or condition for a period of time ranging from minutes to indefinitely. Preventing can also mean reducing the risk of developing a disease, disorder or condition.

[0106] "Prodrug" means a therapeutic agent that is prepared in an inactive form and that is converted into an active form within the body or cells thereof by the action of endogenous enzymes or other chemicals or conditions.

[0107] "Renin-angiotensin-aldosterone system", "renin-angiotensin-aldosterone system pathway", "RAAS pathway" or "RAAS" refers to a multi-component enzymatic pathway in which a precursor component (angiotensinogen) is converted to downstream components such as angiotensin I and angiotensin II by various enzymes, such as renin and the enzyme angiotensin-converting enzyme (ACE). Angiotensin I stimulates the secretion of the steroid aldosterone along the way. The RAAS pathway controls blood pressure and fluid balance in the body.

[0108] "Renin-angiotensin system" or "RAS" or "RAS pathway" refers to a part of the RAAS pathway. Various components of this pathway are targeted by agonists or blockers that prevent the production of each component. For example, renin inhibitors, ACE inhibitors, angiotensin receptor blockers (ARBs), etc. have been developed to inhibit or block the RAS pathway. However, commercially available therapeutic agents that target various RAS pathway elements are not effective in completely inhibiting or blocking the RAS pathway due to diverse mechanisms (Nobakht et al., Nat Rev Nephrol, 2011, 7:356-359).

[0109] "RAAS-related disease, disorder and / or condition" or "RAAS pathway-related disease, disorder and / or condition" refers to any disease, disorder or condition associated with RAAS in an animal. Examples of RAAS-related disease, disorder and / or condition include shortened life span, hypertension (e.g., non-resistant hypertension, treatment-resistant hypertension), kidney disease (e.g., chronic kidney disease, polycystic kidney disease), stroke, heart disease (e.g., myocardial infarction, heart failure, valvular heart disease), vascular aneurysm (e.g., aortic aneurysm), peripheral arterial disease, organ damage (e.g., heart damage or cardiac hypertrophy), tissue fibrosis and other cardiovascular diseases, disorders and / or conditions or symptoms thereof. In certain embodiments, the RAAS-related disease, disorder and / or condition is not associated with hypertension.

[0110] "Resistant hypertension" or "RHTN" is defined as (1) blood pressure of SBP ≥ 140 mmHg or DBP ≥ 90 mmHg, regardless of the concomitant use of three antihypertensive agents from different drug classes, or (2) the use of four or more antihypertensive agents, regardless of blood pressure.

[0111] "Side effects" refers to physiological disorders and / or conditions that are not the desired effects believed to result from a treatment. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, myopathy, and disorders. For example, an increase in serum aminotransferase levels may indicate hepatotoxicity or hepatic abnormalities. For example, an increase in bilirubin may indicate hepatotoxicity or hepatic abnormalities.

[0112] By "single-stranded oligonucleotide" is meant an oligonucleotide that is not hybridized to a complementary strand.

[0113] "Specifically hybridizable" refers to an antisense compound that has a sufficient degree of complementarity between antisense oligonucleotide and target nucleic acid under conditions where specific binding is desired, such as physiological conditions in the case of in vivo assays and therapeutic treatments, to induce a desired effect, but has minimal or no effect on non-target nucleic acid.For example, an antisense compound can specifically hybridize to a target if the binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid, causing loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions where specific binding is desired.

[0114] "Subcutaneous administration" means administration just below the skin.

[0115] "Targeting" or "targeted" refers to the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

[0116] "Target nucleic acid," "target RNA," and "target RNA transcript" all refer to a nucleic acid capable of being targeted by antisense compounds.

[0117] "Target segment" means the sequence of nucleotides in a target nucleic acid to which an antisense compound is targeted. "5' target site" means the 5'-most nucleotide of a target segment. "3' target site" refers to the 3'-most nucleotide of a target segment.

[0118] By "therapeutically effective amount" is meant an amount of a drug that confers a therapeutic benefit on an animal.

[0119] "Treating" refers to administering a pharmaceutical composition to an animal to effect an alteration or amelioration of a disease, disorder, or condition in the animal. In certain embodiments, one or more pharmaceutical compositions can be administered to the animal.

[0120] "Unmodified nucleotide" refers to a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e., β-D-ribonucleotide) or a DNA nucleotide (i.e., β-D-deoxyribonucleotide).

[0121] Specific Embodiments Certain embodiments provide compounds that specifically regulate AGT. In certain embodiments, the AGT specific regulator is an AGT specific inhibitor used to treat, prevent or ameliorate RAAS-related diseases, disorders and / or pathologies. In certain embodiments, the AGT specific inhibitor is a nucleic acid compound that can inhibit the expression of AGT mRNA and / or AGT protein. In certain embodiments, the nucleic acid compound is an oligomeric compound. In certain embodiments, the oligomeric compound is an antisense oligonucleotide. In certain embodiments, the antisense oligonucleotide is a modified antisense oligonucleotide. In certain embodiments, the modified antisense oligonucleotide is a chimeric antisense oligonucleotide.

[0122] In certain embodiments, the compound targets the AGT nucleic acid. In certain embodiments, the AGT nucleic acid is any of the human sequences set forth in GENBANK Accession No. NM_000029.3 (incorporated herein as SEQ ID NO: 1), the complement of nucleotides 24354000-24370100 of GENBANK Accession No. NT_167186.1 (incorporated herein as SEQ ID NO: 2), GENBANK Accession No. AK307978.1 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. AK303755.1 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No. AK293507.1 (incorporated herein as SEQ ID NO: 5), and GENBANK Accession No. CR606672.1 (incorporated herein as SEQ ID NO: 6).

[0123] Certain embodiments disclosed herein provide compounds comprising modified oligonucleotides that target a nucleic acid sequence encoding AGT. In certain embodiments, the compounds target the AGT sequence shown in any of the nucleobase sequences of SEQ ID NOs: 1-6.

[0124] Certain embodiments disclosed herein provide compounds comprising modified oligonucleotides consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases complementary to equal length portions of SEQ ID NOs: 1-6.

[0125] In certain embodiments, the nucleobase sequence of the modified oligonucleotide is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150 ... In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence that is 100% complementary to an equal length portion of any of SEQ ID NOs: 1-6.

[0126] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 2027 to 2068 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0127] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 contiguous nucleobases complementary to an equal length portion of nucleobases 2027-2068 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0128] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 2250 to 2337 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0129] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 contiguous nucleobases complementary to an equal length portion of nucleobases 2250 to 2337 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0130] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 2266 to 2337 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0131] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 contiguous nucleobases complementary to an equal length portion of nucleobases 2266-2337 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0132] Certain embodiments disclosed herein include modified oligonucleotides consisting of 12 to 30 linked nucleosides having a nucleobase sequence that includes at least an 8 contiguous nucleobase portion complementary to an equal portion of nucleobases 2281 to 2300 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 8 times longer than SEQ ID NO:1. 0% complementary to the target gene.

[0133] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 contiguous nucleobases complementary to an equal length portion of nucleobases 2281-2300 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0134] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 2324 to 2346 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0135] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a nucleobase portion of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 contiguous nucleobases complementary to an equal length portion of nucleobases 2324 to 2346 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:1.

[0136] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence that includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 14-2051.

[0137] Certain embodiments disclosed herein include SEQ ID NOs: 40, 42, 46, 47, 49, 53-55, 61, 62, 68, 71, 76, 82, 84, 85, 89, 93, 96-98, 102, 109, 114, 119, 127, 129, 130-135, 137-140, 142, 143, 160, 162-207, 209, 210, 223, 225-227, 230-243, 252-254, 257, 258, 262-264, 264-266, 264-268, 264-269, 270-271, 272-273, 274-275, 276-277, 278-279, 279-280, 279-281, 279-282, 283-284, 285-286, 287-288, 289-290, 290-300, 300-301, 301-302, 302-303, 303-304, 304-305, 305-306, 306-307, 307-308, 308-309, 310-311, 311-312, 312-313, 313-314, 314-315, 315-316, 316-317, 317- 73, 276, 278, 279, 281, 284, 452, 463, 464, 466, 467, 470, 477, 480, 500, 502, 512, 517, 525, 526, 726, 728, 868, 905, 906, 954, 961, 962, 963, 965, 966, 971, 973, 986, 987, 989, 990, 991, 994, 997, 998, 1000, 1001, 1011, 1015, 1021, 10 24, 1035, 1080, 1085, 1150, 1258, 1259-1262, 1293, 1294, 1299, 1325, 1326, 1354, 1355-1357, 1370, 1384, 1391, 1393-1395, 1406-1408, 1431, 1467, 1468, 1470, 1472-1474, 1476, 1488, 1489, 1500, 1503, 1504, 1522, 1524, 1526, 15 28, 1535, 1536, 1539, 1542, 1543, 1545, 1585, 1592, 1594, 1595, 1599, 1604, 1610-1612, 1615, 1618, 1619-1624, 1626, 1628, 1629, 1631, 1632, 1635-1637, 1640, 1658, 1662, 1665-1671, 1673, 1676-1679, 1681-1683, 1686, 1687, 16 99~1710, 1712, 1714~1721, 1724~1726, 1728~1731, 1735, 1736, 1739~1741, 1751, 1755, 1771, 1778, 1781~1783, 1827, 1834, 1836, 1843~1846, 1872, 1874, 1875~188 8, 1890~1895, 1897, 1898, 1900, 1904~1927, 1931~1933, 1937, 1939, 1940, 1943, 1950, 1951, 1953, 1955~1959, 1962, 1964~1967, 1969~1971, 1973, 1977~1981, 1984 The present invention provides a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence that includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases of any of the nucleobase sequences of 1991, 1993 to 1996, 2000 to 2005, 2007 to 2012, 2014 to 2025, 2027, 2028, 2030, 2032 to 2037, 2039 to 2045, 2047, 2051.

[0138] Certain embodiments disclosed herein include SEQ ID NOs: 46, 53, 54, 68, 76, 85, 96, 97, 114, 127, 129-132, 134, 135, 137-139, 142, 162-207, 225, 226, 230-243, 252, 264, 266-270, 284, 464, 467, 962, 963, 965, 966, 973, 990, 991, 997, 1000, 1001, 1011, 1261, 1299, 1355, 1356, 1470, 1472, 1473, 1503 , 1504, 1522, 1526, 1535, 1536, 1542, 1543, 1545, 1595, 1599, 1604, 1620, 1623, 1624, 1626, 1640, 1662, 1666, 1667, 1669, 1670, 1673, 1682, 1683, 1687, 1699~1706, 1708, 1712, 1714~1716, 1719~1721, 1724~1726, 1729, 1730, 1736, 1778, 1783, 1836, 1843, 1875~1888, 1893~1895, 1897, 1900, 1904~1908, 1911, 1914~1918, 1920, 1922, 1923, 1925, 1926, 1931~1933, 1937, 1939, 1955, 1958, 1959, 1962, 1966, 1967, 1970, 1971, 1973, 1977, 1978~1981, 1985, 1986, 1987, 1988, 1990, 1991, 1994, 1996, 2000, 2002~2005, 2010, 2011, 2014~2 In one embodiment, the compound comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence that includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases of any of the nucleobase sequences listed in Table 1.

[0139] Certain embodiments disclosed herein are selected from the group consisting of SEQ ID NOs: 96, 127, 129-132, 139, 162-169, 171-189, 191-193, 195, 196, 198-206, 234, 236, 238-240, 267-270, 966, 1000, 1522, 1542, 1623, 1624, 1667, 1682, 1683, 1700, 1703, 1704, 1708, 1714, 1719, 1720, 1724-1726, 1729, 1875, 1876, 1878, 1884 At least 8, at least 9 of any of the nucleic acid base sequences of 1886, 1893, 1894, 1906, 1908, 1914, 1917, 1918, 1922, 1923, 1925, 1926, 1932, 1933, 1967, 1970, 1978-1981, 1985, 1986, 1988, 1990, 1991, 2003, 2010, 2015, 2016, 2018, 2020, 2021, 2024, 2025, 2027, 2028, 2035, 2037, 2039, 2044, Compounds are provided that include modified oligonucleotides consisting of 12 to 30 linked nucleosides having a nucleobase sequence that includes at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases.

[0140] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 129, 130, 132, 163-168, 171, 172, 175-186, 188, 189, 192, 193, 195, 198-206, 238, 239, 966, 1703, 1720, 1726, 1923, 1925, 2003, 2015.

[0141] Certain embodiments disclosed herein include SEQ ID NOs: 46, 53-54, 61, 68, 76, 83, 85, 93, 96-97, 109, 127, 129-130, 132, 134-15, 137-39, 142, 163-172, 180-184, 186, 189, 234, 236, 238-239, 267, 313, 411, 452, 463-470, 475-478, 480, 500-503, 512, 517-518, 524-526, 65 4, 689, 702, 725-726, 728, 738, 779, 786-787, 800, 808, 810-811, 825, 865, 868, 889, 894, 903, 905, 909, 954, 966, 1011, 1015, 1021, 1024, 1080, 1085, 1258-1259, 1261-1262, 1293-1294, 1299, 1325, 1470, 1472-1473, 1522, 1542, 1604, 1623-1 624, 1667, 1670, 1682~1683, 1687, 1700, 1703~1704, 1708, 1714, 1716, 1719~1720, 1724~1726, 1729~1730, 1827, 1936, 1843~1844, 1846, 1886, 1893~1894, 1914, 1923, 1925, 1932, 1979, 1986, 1988, 1990, 2003, 2015, 2018, 2020, 2027~2028, 2 In one embodiment, the compound comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence that includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13,Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence that includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 238, 1714, 1719, 1893-1894, 1914, 1923, 1925, 2003.

[0142] In certain embodiments, the compound comprises a modified oligonucleotide consisting of 8-80, 20-80, 10-50, 20-35, 10-30, 12-30, 15-30, 16-30, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 15-25, 16-25, 15-24, 16-24, 17-24, 18-24, 19-24, 19-22, 16-21, 18-21, or 16-20 linked nucleobases. In certain embodiments, the compound comprises a modified oligonucleotide consisting of 16 linked nucleosides. In certain embodiments, the compound comprises a modified oligonucleotide consisting of 20 linked nucleosides. Includes.

[0143] In certain embodiments, a compound comprises a modified oligonucleotide consisting of 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 nucleobases in length, or a range defined by any two of the aforesaid values.

[0144] In certain embodiments, the modified oligonucleotide is single-stranded.

[0145] In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside bond. In certain embodiments, the modified internucleoside bond is a phosphorothioate internucleoside bond. In certain embodiments, at least one modified internucleoside bond is a phosphorothioate internucleoside bond. In certain embodiments, each modified internucleoside bond is a phosphorothioate internucleoside bond.

[0146] In certain embodiments, the modified oligonucleotide comprises at least one nucleoside comprising a modified sugar. In certain embodiments, at least one modified sugar comprises a bicyclic sugar. In certain embodiments, at least one modified sugar comprises 2'-O-methoxyethyl, constrained ethyl, 3'-fluoro-HNA or 4'-(CH2) n Contains an -O-2' bridge, where n is 1 or 2.

[0147] In certain embodiments, a modified oligonucleotide comprises at least one nucleoside comprising a modified nucleobase. In certain embodiments, the modified nucleobase is 5-methylcytosine.

[0148] In certain embodiments, the modified oligonucleotide comprises a conjugate group. In certain embodiments, the conjugate is a carbohydrate moiety. In certain embodiments, the conjugate is a GalNAc moiety. In certain embodiments, the GalNAc is 5'-trishexylamino-(THA)-C6GalNAc3. In certain embodiments, the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate has the formula:

[0149] [ka]

[0150] In certain embodiments, the modified oligonucleotide is 5'- Attached to a trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the cleavable moiety is a phosphate group.

[0151] In certain embodiments, the compound comprises a modified oligonucleotide consisting of 12-30 linked nucleosides and targeted to or complementary to an equal length portion of the region 2250-2337 of SEQ ID NO:1, the modified oligonucleotide comprising (a) a gap segment consisting of a plurality of linked deoxynucleosides, (b) a 5' wing segment consisting of a plurality of linked nucleosides, and (c) a 3' wing segment consisting of a plurality of linked nucleosides, wherein the gap segment is disposed immediately adjacent between the 5' wing segment and the 3' wing segment, and each nucleoside of each wing segment comprises a modified sugar. In certain embodiments, the modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0152] In certain embodiments, the compound comprises a modified oligonucleotide consisting of 12-30 linked nucleosides and targeted to or complementary to an equal length portion of the region 2266-2337 of SEQ ID NO:1, the modified oligonucleotide comprising (a) a gap segment consisting of a plurality of linked deoxynucleosides, (b) a 5' wing segment consisting of a plurality of linked nucleosides, and (c) a 3' wing segment consisting of a plurality of linked nucleosides, wherein the gap segment is disposed immediately adjacent between the 5' wing segment and the 3' wing segment, and each nucleoside of each wing segment comprises a modified sugar. In certain embodiments, the modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0153] In certain embodiments, the compound comprises a modified oligonucleotide consisting of 12-30 linked nucleosides and targeted to or complementary to an equal length portion of the region 2281-2300 of SEQ ID NO:1, the modified oligonucleotide comprising (a) a gap segment consisting of a plurality of linked deoxynucleosides, (b) a 5' wing segment consisting of a plurality of linked nucleosides, and (c) a 3' wing segment consisting of a plurality of linked nucleosides, wherein the gap segment is disposed immediately adjacent between the 5' wing segment and the 3' wing segment, and each nucleoside of each wing segment comprises a modified sugar. In certain embodiments, the modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0154] In certain embodiments, the compound comprises a modified oligonucleotide consisting of 20 linked nucleosides and targeted to or complementary to an equal length portion of the region 2027-2068 of SEQ ID NO:1, the modified oligonucleotide comprising (a) a gap segment consisting of a plurality of linked deoxynucleosides, (b) a 5' wing segment consisting of a plurality of linked nucleosides, and (c) a 3' wing segment consisting of a plurality of linked nucleosides, wherein the gap segment is disposed immediately adjacent and between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, at least one internucleoside linkage is a phosphorothioate linkage, and each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0155] In certain embodiments, the compound comprises a modified oligonucleotide having a nucleobase sequence consisting of 16-20 linked nucleosides and comprising at least 8 contiguous nucleobases of SEQ ID NOs: 14-2051, the modified oligonucleotide comprising (a) a gap segment consisting of a plurality of linked deoxynucleosides, (b) a 5' wing segment consisting of a plurality of linked nucleosides, and (c) a 3' wing segment consisting of a plurality of linked nucleosides, wherein the gap segment is disposed immediately adjacent and between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a modified sugar, at least one internucleoside linkage is a phosphorothioate linkage, and each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.In certain embodiments, the compound consists of 16-20 linked nucleosides and is selected from the group consisting of SEQ ID NOs: 40, 42, 46, 47, 49, 53-55, 61, 62, 68, 71, 76, 82, 84, 85, 89, 93, 96-98, 102, 109, 114, 119, 127, 129, 130-135, 1 37~140, 142, 143, 160, 162~207, 209, 210, 223, 225~227, 230~243, 252~254, 257, 258, 262~273, 276, 278, 279, 281, 284, 452, 463, 464, 466, 467, 470, 477, 480, 500, 50 2, 512, 517, 525, 526, 726, 728, 868, 905, 906, 954, 961, 962, 963, 965, 966, 971, 973, 986, 987, 989, 990, 991, 994, 997, 998, 1000, 1001, 1011, 1015, 1021, 1024, 1035, 1080, 1085, 1150, 1258, 1259~1262, 1293, 1294, 1299, 1325, 1326, 1354, 1355~1357, 1370, 1384, 1391, 1393~1395, 1406~1408, 1431, 1467, 1468, 1470, 1472~1474, 1. 476, 1488, 1489, 1500, 1503, 1504, 1522, 1524, 1526, 1528, 1535, 1536, 1539, 1542, 1543, 1545, 1585, 1592, 1594, 1595, 1599, 1604, 1610-1612, 1615, 1618, 1619-1624, 1626, 1628, 1629, 1631, 1632, 1635-1637, 1640, 1658, 1662, 1665-1671, 1673, 1676-1679, 1681-1683, 16 86, 1687, 1699~1710, 1712, 1714~1721, 1724~1726, 1728~1731, 1735, 1736, 1739~1741, 1751, 1755, 1771, 1778, 1781~1783, 1827, 1834, 1836, 1843~1846, 1872, 1874, 1875~1888, 1890~1895, 1897, 1898, 1900, 1904~1927, 1931~1933, 1937, 1939, 1940, 1943, 1950, 1951, 195 3, 1955-1959, 1962, 1964-1967, 1969-1971, 1973, 1977-1981, 1984-1991, 1993-1996, 2000-2005, 2007-2012, 2014-2025, 2027, 2028, 2030, 2032-2037, 2039-2045, 2047, and 2051, wherein the modified oligonucleotide comprises (a) a plurality of linked deoxyribonucleic acid residues. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. ...In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0156] In certain embodiments, the compound consists of 16-20 linked nucleosides and is selected from the group consisting of SEQ ID NOs: 46, 53, 54, 68, 76, 85, 96, 97, 114, 127, 129-132, 134, 135, 137-139, 142, 162-207, 225, 226, 230-243, 252, 264, 266-270, 284, 464, 467, 962, 963, 965, 966, 973, 990, 991, 997, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, , 1011, 1261, 1299, 1355, 1356, 1470, 1472, 1473, 1503, 1504, 1522, 1526, 1535, 1536, 1542, 1543, 1545, 1595, 1599, 1604, 1620, 1623, 1624, 1626, 1640, 1662, 1666, 1667, 1669, 1670, 1673, 1682, 1683, 1687, 1699~1706, 1708, 1712, 1714~1716 , 1719~1721, 1724~1726, 1729, 1730, 1736, 1778, 1783, 1836, 1843, 1875~1888, 1893~1895, 1897, 1900, 1904~1908, 1911, 1914~1918, 1920, 1922, 1923, 1925, 1926, 1931~1933, 1937, 1939, 1955, 1958, 1959, 1962, 1966, 1967, 1970, 1971, 1973 , 1977, 1978-1981, 1985, 1986, 1987, 1988, 1990, 1991, 1994, 1996, 2000, 2002-2005, 2010, 2011, 2014-2025, 2027, 2028, 2035-2037, 2039, and 2041-2045, wherein the modified oligonucleotide comprises (a) a plurality of linked The modified oligonucleotide comprises: (a) a gap segment consisting of a deoxynucleoside; (b) a 5' wing segment consisting of a plurality of linked nucleosides; and (c) a 3' wing segment consisting of a plurality of linked nucleosides, wherein the gap segment is disposed immediately adjacent between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a modified sugar, at least one internucleoside linkage is a phosphorothioate linkage, and each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0157] In certain embodiments, the compound consists of 16-20 linked nucleosides and is selected from the group consisting of SEQ ID NOs: 96, 127, 129-132, 139, 162-169, 171-189, 191-193, 195, 196, 198-206, 234, 236, 238-240, 267-270, 966, 1000, 1522, 1542, 1623, 1624, 1667, 1682, 1683, 1700, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1800, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 19 704, 1708, 1714, 1719, 1720, 1724~1726, 1729, 1875, 1876, 1878, 1884~1886, 1893, 1894, 1906, 1908, 1914, 1917, 1918, 1922, 1923, 1925, 1926, 1932, 1933, 1967, 1970, 1978~1981, 1985, 1986, 1988, 1990, 1991, 2003, 2010, 2 and (c) a 3' wing segment consisting of a plurality of linked nucleosides, wherein the gap segment is disposed immediately adjacent and between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a modified sugar, at least one internucleoside linkage is a phosphorothioate linkage, and each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0158] In certain embodiments, the compound is a modified oligonucleoside having a nucleobase sequence consisting of 16-20 linked nucleosides and comprising at least 8 consecutive nucleobases of SEQ ID NOs: 129, 130, 132, 163-168, 171, 172, 175-186, 188, 189, 192, 193, 195, 198-206, 238, 239, 966, 1703, 1720, 1726, 1923, 1925, 2003, 2015. and (c) a 3' wing segment consisting of a plurality of linked nucleosides, wherein the gap segment is disposed immediately adjacent to and between the 5' wing segment and the 3' wing segment, and each nucleoside in each wing segment is a nucleotide. , a modified sugar, at least one internucleoside linkage is a phosphorothioate linkage, and each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0159] In certain embodiments, the compound consists of 16-20 linked nucleosides and is selected from the group consisting of SEQ ID NOs: 46, 53-54, 61, 68, 76, 83, 85, 93, 96-97, 109, 127, 129-130, 132, 134-15, 137-39, 142, 163-172, 180-184, 186, 189, 234, 236, 238-239, 267, 313, 411, 452, 463-470, 475-478, 480, 500-503, 512, 517-518, 524-526, 654, 689, 702, 725-730, 740-745, 750-751, 760-762, 770-775, 780-785, 790-791, 800-801, 810-811, 820-822, 830-831, 840-842, 850-851, 860-861, 870-871, 880-891, 892-901, 901-912, 913-923, 924-930, 930-941, 940-951, 951-962, 960-971, 970-982, 982-991, 990-1001, 1000-100 726, 728, 738, 779, 786-787, 800, 808, 810-811, 825, 865, 868, 889, 894, 903, 905, 909, 954, 966, 1011, 1015, 1021, 1024, 1080, 1085, 1258-1259, 1261-1262, 1293-1294, 1299, 1325, 1470, 1472-1473, 1522, 1542, 1604, 1623-1624, 1667, 1670, 1682-1683, 1687, 1700, 1703-1704, 1708, 17 14, 1716, 1719-1720, 1724-1726, 1729-1730, 1827, 1936, 1843-1844, 1846, 1886, 1893-1894, 1914, 1923, 1925, 1932, 1979, 1986, 1988, 1990, 2003, 2015, 2018, 2020, 2027-2028, 2035, 2037, 2039, or 2044, wherein the modified oligonucleotide comprises (a) a plurality of (b) a gap segment consisting of linked deoxynucleosides, (b) a 5' wing segment consisting of a plurality of linked nucleosides, and (c) a 3' wing segment consisting of a plurality of linked nucleosides, where the gap segment is disposed immediately adjacent and between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a modified sugar, at least one internucleoside linkage is a phosphorothioate linkage, and each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage.In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0160] In certain embodiments, compounds comprise a modified oligonucleotide comprising 16-20 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of SEQ ID NOs: 238, 1714, 1719, 1893-1894, 1914, 1923, 1925, 2003, the modified oligonucleotide comprising (a) a gap segment comprising a plurality of linked deoxynucleosides; (b) a 5' wing segment comprising a plurality of linked nucleosides; and (c) a 3' wing segment comprising a plurality of linked nucleosides, wherein the gap segment is disposed immediately adjacent and between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a modified sugar; and at least one internucleoside linkage is In certain embodiments, the internucleoside linkage is a phosphorothioate linkage, and each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide further comprises a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate, and each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage ... the modified oligonucleotide further comprises a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate, and each cytosine residue is a 5-methylcytosine. is attached to a GalNAc3 conjugate. In certain embodiments, the cleavable moiety is a phosphate group.

[0161] In certain embodiments, the compound comprises a modified oligonucleotide having a nucleobase sequence consisting of 20 linked nucleosides and comprising at least 8 consecutive nucleobases of SEQ ID NO: 1914, the modified oligonucleotide comprising (a) a gap segment consisting of 10 linked deoxynucleosides, (b) a 5' wing segment consisting of 5 linked nucleosides, and (c) a 3' wing segment consisting of 5 linked nucleosides, wherein the gap segment is disposed immediately adjacent between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, at least one internucleoside linkage is a phosphorothioate linkage, and each cytosine residue is a 5-methylcytosine. In certain embodiments, each internucleoside linkage is a phosphorothioate linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0162] Certain embodiments disclosed herein include compounds having the formula: mCes Aes mCes Aes Provided is a compound comprising a modified oligonucleotide according to Aes Ads mCds Ads Ads Gds mCds Tds Gds Gds Tds mCes Ges Ges Tes Te (SEQ ID NO: 1914), where A is adenine, mC is 5-methylcytosine, G is guanine, T is thymine, e is a 2'-O-methoxyethyl modified nucleoside, d is a 2'-deoxynucleoside, and s is a phosphorothioate internucleoside linkage. In certain embodiments, the modified oligonucleotide further comprises a GalNAc conjugate. In certain embodiments, the conjugate is a 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate. In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0163] Certain embodiments disclosed herein provide compounds comprising a modified oligonucleotide having the formula:

[0164] [ka]

[0165] In certain embodiments, a compound or composition disclosed herein comprises a salt of a modified oligonucleotide.

[0166] In certain embodiments, a compound or composition disclosed herein further comprises a pharma- ceutically acceptable carrier or diluent.

[0167] In certain embodiments, the animal is a human.

[0168] Certain embodiments provide compositions or compounds comprising modified oligonucleotides as described herein, wherein the viscosity is less than 40 cP. In certain embodiments, the composition has a viscosity of less than 15 cP. In certain embodiments, the composition has a viscosity of less than 12 cP. In certain embodiments, the composition has a viscosity of less than 10 cP.

[0169] Embodiments disclosed herein provide compounds and compositions comprising modified oligonucleotides targeting AGT for use in reducing AGT in a cell, tissue, organ, or animal. In certain embodiments, reduction of AGT results in the treatment, prevention, slowing of progression, slowing of onset, and / or alleviation of a RAAS pathway-related disease, disorder, and / or condition, or a symptom thereof. In certain embodiments, reduction of AGT reduces hypertension. In certain embodiments, the reduction of AGT reduces or prevents fibrosis. In certain embodiments, the reduction of AGT modulates the symptoms or markers of RAAS pathway-related diseases, disorders and / or conditions. In certain embodiments, the markers can be selected from one or more of shortened life span, hypertension, chronic kidney disease, stroke, myocardial infarction, heart failure, valvular heart disease, vascular aneurysm, peripheral artery disease, organ damage and other cardiovascular diseases, disorders and / or conditions or symptoms thereof.

[0170] In certain embodiments, compounds and compositions comprising modified oligonucleotides targeting AGT are provided for use in therapy, hi certain embodiments, compounds and compositions comprising modified oligonucleotides targeting AGT are administered to an animal in a therapeutically effective amount.

[0171] In certain embodiments, compounds and compositions are provided that include modified oligonucleotides targeting AGT for use in the preparation of a medicament, in certain embodiments, the medicament is used for treating, preventing, delaying progression, delaying onset and / or alleviating a RAAS pathway-related disease, disorder and / or condition or a symptom thereof.

[0172] In certain embodiments, a kit for treating, preventing or ameliorating a RAAS pathway-related disease and / or condition, disease, disorder or condition is provided, the kit comprising (i) an AGT specific inhibitor as described herein; and optionally (ii) an additional agent or therapy as described herein. The kit of the invention may further comprise instructions for using the kit to treat, prevent or ameliorate a RAAS pathway-related disease, disorder or condition as described herein.

[0173] In certain embodiments, the RAAS pathway-related disease, disorder or condition is shortened life span, hypertension, kidney disease (e.g., chronic kidney disease), stroke, heart disease (e.g., myocardial infarction, heart failure, valvular heart disease), vascular aneurysm, peripheral arterial disease, organ damage, and other RAAS-related disease, disorder and / or condition or symptoms thereof. In certain embodiments, the hypertension is non-treatment-resistant hypertension or treatment-resistant hypertension. In certain embodiments, the vascular aneurysm is an aortic aneurysm. In certain embodiments, the organ damage is myocardial hypertrophy or fibrosis of an organ or tissue. In certain embodiments, the organ is the heart, liver or kidney, and the tissue is derived from the heart, liver or kidney.

[0174] The compounds can be used in combination therapy with one or more additional agents or therapies described herein. The agents or therapies can be administered to the animal simultaneously or sequentially. In certain embodiments, the compositions or compounds comprising modified oligonucleotides targeting AGT are co-administered with one or more second agent(s). In certain embodiments, the second agents include treatments to reduce hypertension, dietary modifications, lifestyle modifications, antifibrotic and antihypertensive agents, such as RAS or RAAS inhibitors, diuretics, calcium channel antagonists, sympathetic receptor blockers, sympathomimetics, and vasodilators. In certain embodiments, the second agent is a second antisense compound. In further embodiments, the second antisense compound targets AGT. In other embodiments, the second antisense compound targets a compound other than AGT.

[0175] Antisense Compounds Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. Oligomeric compounds can be "antisense" to a target nucleic acid, meaning that the oligomeric compound can undergo hybridization to a target nucleic acid via hydrogen bonding.

[0176] 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 the target nucleic acid to which the antisense compound 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 the target nucleic acid to which the antisense compound is targeted.

[0177] In certain embodiments, antisense compounds targeted to AGT nucleic acids are 10-30 nucleotides in length. In other words, the antisense compounds are 10-30 linked nucleobases. In other embodiments, the antisense compounds comprise modified oligonucleotides consisting of 8-80, 10-80, 12-50, 15-30, 18-24, 19-22, or 20 linked nucleobases. 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, 60, 70, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 121, 131, 142, 143, 150, 161, 171 , 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 nucleobases in length, or a range defined by any two of the foregoing values. In some embodiments, the antisense compound is an antisense oligonucleotide.

[0178] In certain embodiments, the antisense compound comprises a shortened or truncated modified oligonucleotide. The shortened or truncated modified oligonucleotide may have one nucleoside deleted from the 5' end (5' truncation), the middle portion or the 3' end (3' truncation). The shortened or truncated oligonucleotide may have two or more nucleosides deleted from the 5' end, may have two or more nucleosides deleted from the middle portion, or may have two or more nucleosides deleted from the 3' end. Alternatively, the deleted nucleosides may be distributed throughout the modified oligonucleotide, such as, for example, an antisense compound with one or more nucleosides deleted from the 5' end, one or more nucleosides deleted from the middle portion, and / or one or more nucleosides deleted from the 3' end.

[0179] When there is one additional nucleoside in the extended oligonucleotide, the additional nucleoside can be located at the 5'-end, 3'-end or middle part of the oligonucleotide.When there is two or more additional nucleosides, the additional nucleosides can be adjacent to each other, such as an oligonucleotide with two additional nucleosides at the 5'-end (5'-addition), 3'-end (3'-addition) or middle part of the oligonucleotide.Alternatively, the additional nucleosides can be distributed throughout the antisense compound, such as an oligonucleotide with one or more additional nucleosides at the 5'-end, one or more additional nucleosides at the 3'-end and / or one or more additional nucleosides at the middle part.

[0180] 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, Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992) tested a series of antisense oligonucleotides, 13-25 nucleobases in length, for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length containing 8 or 11 mismatch bases near the ends of the antisense oligonucleotide were able to specifically cleave the target mRNA, although to a lesser extent than antisense oligonucleotides that contained no mismatches. Similarly, target-specific cleavage was also achieved using 13 nucleobase antisense oligonucleotides, including those containing one or three mismatches.

[0181] Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated that an oligonucleotide with 100% complementarity to bcl-2 mRNA and three mismatches to bcl-xL mRNA could reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide showed potent antitumor activity in vivo.

[0182] Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of 14 nucleobase tandem antisense oligonucleotides and 28 and 42 nucleobase antisense oligonucleotides consisting of two or three of the tandem antisense oligonucleotide sequences, respectively, for their ability to block translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides was able to inhibit translation alone, although to a lesser extent than the 28 or 42 nucleobase antisense oligonucleotides.

[0183] Specific antisense compound motifs and mechanisms In certain embodiments, antisense compounds have chemically modified subunits arranged in patterns or motifs to confer upon the antisense compounds properties such as improved inhibitory activity, increased binding affinity for target nucleic acids, or resistance to degradation by nucleases in vivo.

[0184] Chimeric antisense compounds typically contain at least one region modified 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 the chimeric antisense compound can confer another desired property, such as serving as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

[0185] Antisense activity may result from any mechanism involving hybridization of an antisense compound (e.g., an oligonucleotide) with a target nucleic acid, which ultimately results in a biological effect. In certain embodiments, the amount and / or activity of the target nucleic acid is modulated. In certain embodiments, the amount and / or activity of the target nucleic acid is decreased. In certain embodiments, hybridization of an antisense compound to a target nucleic acid ultimately results in degradation of the target nucleic acid. In certain embodiments, hybridization of an antisense compound to a target nucleic acid does not result in degradation of the target nucleic acid. In certain such embodiments, the presence (occupancy) of an antisense compound hybridized to a target nucleic acid results in modulation of antisense activity. In certain embodiments, antisense compounds with specific chemical motifs or patterns of chemical modifications are particularly suited to exploit one or more mechanisms. In certain embodiments, antisense compounds function via more than one mechanism and / or function via a mechanism that has not yet been elucidated. Thus, the antisense compounds described herein are not limited to a specific mechanism.

[0186] Antisense mechanisms include, but are not limited to, RNase H-mediated antisense; RNAi mechanisms that utilize the RISC pathway and include, but are not limited to, siRNA, ssRNA and microRNA mechanisms; and occupancy-based mechanisms. A particular antisense compound may act through more than one such mechanism and / or through additional mechanisms.

[0187] RNase H-mediated antisense In certain embodiments, antisense activity results, at least in part, from degradation of the target RNA by RNase H. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds that are "DNA-like" induce RNase H activity in mammalian cells. Thus, antisense compounds that include at least a portion of DNA or DNA-like nucleosides can activate RNase H, resulting in cleavage of the target nucleic acid. In certain embodiments, antisense compounds that utilize RNase H include one or more modified nucleosides. In certain embodiments, such antisense compounds have at least one block of 1-8 modified nucleosides. In certain such embodiments, the modified nucleosides do not support RNase H activity. In certain such embodiments, such antisense compounds are gapmers as described herein. In certain such embodiments, the gap of the gapmer includes DNA nucleosides. In certain such embodiments, the gap of the gapmer includes DNA-like nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.

[0188] Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer, an inner region having a number of nucleotides that support RNase H cleavage is disposed between an outer region having a number of nucleotides that are chemically distinct from the nucleosides of the inner region. In antisense oligonucleotides having a gapmer motif, the gap segment typically serves as a substrate for endonuclease cleavage, while the wing segments contain modified nucleosides. In certain embodiments, the regions of the gapmer are differentiated by the type of sugar moiety that each distinct region comprises. The types of sugar moieties used to differentiate the regions of the gapmer can include, in some embodiments, β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides can include, among others, 2'-MOE and 2'-O-CH3), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides can include those with a constrained ethyl, etc.). In certain embodiments, the nucleosides of the wings may comprise several modified sugar moieties, including, for example, 2'-MOE and bicyclic sugar moieties such as constrained ethyl (cEt) or LNA. In certain embodiments, the wings may comprise several modified and unmodified sugar moieties. In certain embodiments, the wings may comprise various combinations of 2'-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2'-deoxynucleosides.

[0189] Each distinct region may contain uniform sugar moieties, variants, or alternating sugar moieties. A wing-gap-wing motif is often described as "XYZ," where "X" represents the length of the 5'-wing, "Y" represents the length of the gap, and "Z" represents the length of the 3'-wing. "X" and "Z" may contain uniform sugar moieties, variants, or alternating sugar moieties. In certain embodiments, "X" and "Y" may contain one or more 2'-deoxynucleosides. "Y" may contain 2'-deoxynucleosides. As used herein, a gapmer described as "XYZ" has a configuration in which a gap is positioned immediately adjacent to each of the 5'-wing and 3'-wing. Thus, there are no intervening nucleotides between the 5'-wing and the gap, or between the gap and the 3'-wing. Any of the antisense compounds described herein may have a gapmer motif. In certain embodiments, "X" and "Z" are the same, and in other embodiments, "X" and "Z" are different. In certain embodiments, "Y" is 8 to 15 nucleosides. X, Y or Z can be 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.

[0190] In certain embodiments, antisense compounds targeted to an AGT nucleic acid have a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 linked nucleosides.

[0191] In certain embodiments, the antisense oligonucleotides have the sugar motif shown in Formula A below. (J) m -(B) n -(J) p -(B) r -(A) t -(D) g -(A) v -(B) w -(J) x -(B)y -(J) z During the ceremony, each A is independently a 2'-substituted nucleoside; each B is independently a bicyclic nucleoside; each J is independently either a 2'-substituted nucleoside or a 2'-deoxynucleoside; each D is a 2'-deoxynucleoside; m is 0-4, n is 0-2, p is 0-2, r is 0-2, t is 0-2, v is 0-2, w is 0-4, x is 0-2, y is 0-2, z is 0-4, and g is 6-14; however, At least one of m, n, and r is other than 0; At least one of w and y is other than 0; the sum of m, n, p, r, and t is 2 to 5; The sum of v, w, x, y, and z is 2 to 5.

[0192] RNAi compounds In certain embodiments, the antisense compounds are interfering RNA compounds (RNAi), including double-stranded RNA compounds (also called short interfering RNA or siRNA) and single-stranded RNAi compounds (i.e., ssRNA). Such compounds act at least in part through the RISC pathway to degrade and / or sequester target nucleic acids (and thus include microRNA / microRNA mimetic compounds). In certain embodiments, the antisense compounds contain modifications that make them particularly suitable for such mechanisms.

[0193] i.ssRNA compounds In certain embodiments, antisense compounds, including those that are particularly suitable for use as single-stranded RNAi compounds (ssRNA), comprise modified 5'-ends. In certain such embodiments, 5'-ends comprise modified phosphate moieties. In certain such embodiments, such modified phosphates are stabilized (e.g., resistant to degradation / cleavage compared to unmodified 5'-phosphates). In certain such embodiments, such 5'-end nucleosides stabilize 5'-phosphorus moieties. Certain modified 5'-end nucleosides can be found in the art, for example, in WO2011 / 139702.

[0194] In certain embodiments, the 5'-nucleoside of the ssRNA compound has the formula IIc:

[0195] [ka]

[0196] During the ceremony, T1 is an optionally protected phosphorus moiety, T2 is an internucleoside linking group that connects the compound of formula IIc to the oligomeric compound, and A has one of the following formulae:

[0197] [ka]

[0198] Q1 and Q2 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, or N(R3)(R4); Q3 is O, S, N(R5) or C(R6)(R7); each R3, R4, R5, R6 and R7 is independently H, C1-C6 alkyl, substituted C1-C6 alkyl or C1-C6 alkoxy; M3 is O, S, NR 14, C(R 15 )(R 16 ), C(R 15 )(R 16 )C(R 17 )(R 18 ), C(R 15 )=C(R 17 ), O.C.(R 15 )(R 16 ) or OC(R 15 )(Bx2), R 14 is H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; R 15 , R 16 , R 17 and R 18 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; Bx1 is a heterocyclic base moiety, Alternatively, when Bx2 is present, Bx2 is a heterocyclic base moiety and Bx1 is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; J4, J5, J6 and J7 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; Alternatively, J4 forms a bridge with one of J5 or J7, where the bridge is O, S, NR 19 , C(R 20 )(R 21 ), C(R 20 )=C(R 21 ), C[=C(R20 )(R 21 )] and C(=O), and the other two of J5, J6 and J7 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, , C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; Each R 19 , R 20 and R 21 are independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; G is H, OH, halogen or O-[C(R8)(R9)] n -[(C=O) m -X1] j -Z, each R8 and R9 is independently H, halogen, C1-C6 alkyl or substituted C1-C6 alkyl; X1 is O, S or N(E1); Z is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3); E1, E2, and E3 are each independently H, C1-C6 alkyl, or substituted C1-C6 alkyl; n is from 1 to about 6; m is 0 or 1; j is 0 or 1, each substituent comprises one or more optionally protected substituents independently selected from halogen, OJ1, N(J1)(J2), =NJ1, SJ1, N3, CN, OC(=X2)J1, OC(=X2)N(J1)(J2) and C(=X2)N(J1)(J2); X2 is O, S or NJ3; Each J1, J2, and J3 is independently H or C1-C6 alkyl; When j is 1, Z is other than halogen or N(E2)(E3); The oligomeric compounds contain from 8 to 40 monomeric subunits and are capable of hybridizing to at least a portion of a target nucleic acid.

[0199] In certain embodiments, M3 is O, CH=CH, OCH2 or OC(H)(Bx2). In certain embodiments, M3 is O.

[0200] In certain embodiments, J4, J5, J6 and J7 are each H. In certain embodiments, J4 forms a bridge with one of J5 or J7.

[0201] In certain embodiments, A has one of the following formulae:

[0202] [ka]

[0203] During the ceremony, Q1 and Q2 are each independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, or substituted C1-C6 alkoxy. In certain embodiments, Q1 and Q2 are each independently H. In certain embodiments, Q1 and Q2 are each independently H or halogen. In certain embodiments, Q1 and Q2 are H, and one of Q1 and Q2 is F, CH3, or OCH3.

[0204] In certain embodiments, T1 has the formula:

[0205] [ka]

[0206] During the ceremony, Ra and R c are each independently a protected hydroxyl, a protected thiol, a C1-C6 alkyl, a substituted C1-C6 alkyl, a C1-C6 alkoxy, a substituted C1-C6 alkoxy, a protected amino, or a substituted amino; R b is O or S. In certain embodiments, R b is O and R a and R c are each independently OCH3, OCH2CH3, or CH(CH3)2.

[0207] In certain embodiments, G is halogen, OCH, OCHF, OCHF, OCF, OCHCH, O(CH)F, OCHCHF, OCHCF, OCH-CH=CH, O(CH)-OCH, O(CH)-SCH, O(CH)-OCF, O(CH)-N(R 10 )(R 11 ), O(CH2)2-ON(R 10 )(R 11 ), O(CH2)2-O(CH2)2-N(R 10 )(R 11 ), OCH2C(=O)-N(R 10 )(R 11 ), OCH2C(=O)-N(R 12 )-(CH2)2-N(R 10 )(R 11 ) or O(CH2)2-N(R 12 )-C(=NR 13 )[N(R 10 )(R 11 )](where R 10 , R 11 , R 12 and R 13are each independently H or C1-C6 alkyl). In certain embodiments, G is halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2-CH=CH2, O(CH2)2-OCH3, O(CH2)2-O(CH2)2-N(CH3)2, OCH2C(=O)-N(H)CH3, OCH2C(=O)-N(H)-(CH2)2-N(CH3)2, or OCH2-N(H)-C(=NH)NH2. In certain embodiments, G is F, OCH3, or O(CH2)2-OCH3. In certain embodiments, G is O(CH2)2-OCH3.

[0208] In certain embodiments, the 5'-terminal nucleoside has the formula IIe.

[0209] [ka]

[0210] In certain embodiments, antisense compounds, including those particularly suited for ssRNA, contain one or more modified sugar moieties and / or naturally occurring sugar moieties arranged in a predetermined pattern or in a predetermined sugar modification motif along the oligonucleotide or region thereof, which may include any of the sugar modifications discussed herein and / or other well-known sugar modifications.

[0211] In certain embodiments, the oligonucleotide comprises or consists of a region having uniform sugar modifications. In certain such embodiments, each nucleoside in the region is , containing the same RNA-like sugar modification. In certain embodiments, each nucleoside in the region is a 2'-F nucleoside. In certain embodiments, each nucleoside in the region is a 2'-OMe nucleoside. In certain embodiments, each nucleoside in the region is a 2'-MOE nucleoside. In certain embodiments, each nucleoside in the region is a cEt nucleoside. In certain embodiments, each nucleoside in the region is an LNA nucleoside. In certain embodiments, the uniform region comprises all of the oligonucleotide or comprises essentially all of the oligonucleotide. In certain embodiments, the region comprises the entire oligonucleotide except for the terminal nucleosides 1-4.

[0212] In certain embodiments, the oligonucleotide comprises one or more regions of alternating sugar modifications, in which nucleosides alternate between nucleotides having a first type of sugar modification and nucleotides having a second type of sugar modification. In certain embodiments, both types of nucleosides are RNA-like nucleosides. In certain embodiments, the alternating nucleosides are selected from 2'-OMe, 2'-F, 2'-MOE, LNA, and cEt. In certain embodiments, the alternating modifications are 2'-F and 2'-OMe. Such regions may be contiguous or interrupted by differently modified nucleosides or conjugated nucleosides.

[0213] In certain embodiments, the alternating regions of alternating modification each consist of one nucleoside (i.e., the pattern is (AB) x A y (wherein A is a nucleoside having a first type of sugar modification, B is a nucleoside having a second type of sugar modification, x is 1-20, and y is 0 or 1). In certain embodiments, one or more alternating regions in an alternating motif contain two or more nucleosides of a certain type. For example, an oligonucleotide may contain one or more regions of any of the following nucleoside motifs: AABBAA; ABBABB; AABAAB; ABBABAABB; ABABAA; AABABAB; ABABAA; ABBAABBABABAA; BABBAABBABABAA; or ABABBABBABABAA; where A is a first type of nucleoside and B is a second type of nucleoside.

[0214] In certain embodiments, A and B are each selected from 2'-F, 2'-OMe, BNA, and MOE.

[0215] In certain embodiments, oligonucleotides having such alternating motifs also include a modified 5' terminal nucleoside, such as that of Formula IIc or IIe.

[0216] In certain embodiments, the oligonucleotide comprises a region having a 2-2-3 motif. Such a region includes the following motif: -(A)2-(B) x -(A)2-(C) y -(A)3- wherein A is a first type of modified nucleoside; B and C are nucleosides having a different modification from A, but B and C may have the same modification as each other or different modifications from each other; x and y are 1 to 15.

[0217] In certain embodiments, A is a 2'-OMe modified nucleoside. In certain embodiments, B and C are both 2'-F modified nucleosides. In certain embodiments, A is a 2'-OMe modified nucleoside and B and C are both 2'-F modified nucleosides.

[0218] In certain embodiments, the oligonucleosides have the following sugar motifs: 5'-(Q)-(AB)x A y -(D) z During the ceremony, Q is a nucleoside that includes a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having formula IIc or IIe: A is a first type of modified nucleoside, B is a second type of modified nucleoside; D is a modified nucleoside that contains a different modification than the adjacent nucleosides. Thus, when y is 0, D must be a different modification than B, and when y is 1, D must be a different modification than A. In certain embodiments, D is different from both A and B. X is 5 to 15; Y is 0 or 1; Z is 0 to 4.

[0219] In certain embodiments, the oligonucleosides have the following sugar motifs: 5'-(Q)-(A) x -(D) z During the ceremony, Q is a nucleoside that includes a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having formula IIc or IIe: A is a first type of modified nucleoside, D is a modified nucleoside that contains a different modification than A. X is between 11 and 30; Z is 0 to 4.

[0220] In certain embodiments, A, B, C and D in the motif are selected from 2'-OMe, 2'-F, 2'-MOE, LNA and cEt. In certain embodiments, D represents a terminal nucleoside. In certain embodiments, such terminal nucleosides are designed not to hybridize to the target nucleic acid (although one or more may hybridize by chance). In certain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments, the nucleobase of each D nucleoside is thymine.

[0221] In certain embodiments, antisense compounds, including those particularly suitable for use as ssRNA, comprise modified internucleoside linkages arranged in a predetermined pattern or in a predetermined modified internucleoside linkage motif along an oligonucleotide or region thereof. In certain embodiments, an oligonucleotide comprises a region having an alternating internucleoside linkage motif. In certain embodiments, an oligonucleotide comprises a region of uniformly modified internucleoside linkages. In certain such embodiments, an oligonucleotide comprises a region uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, an oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of an oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of an oligonucleotide is selected from phosphodiester and phosphorothioate, and at least one internucleoside linkage is phosphorothioate. do.

[0222] In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide.

[0223] Oligonucleotides having any of the various sugar motifs described herein can have any linkage motif, including but not limited to those listed above, can have a linkage motif selected from the non-limiting table below.

[0224] [Table 1]

[0225] ii.siRNA compounds In certain embodiments, antisense compounds are double-stranded RNAi compounds (siRNA).In such embodiments, one or both strands can contain any of the modified motifs described above for ssRNA.In certain embodiments, ssRNA compounds can be unmodified RNA.In certain embodiments, siRNA compounds contain unmodified RNA nucleosides but can contain modified internucleoside linkages.

[0226] Some embodiments relate to double-stranded compositions, each strand comprises a motif defined by the position of one or more modified or unmodified nucleosides.In certain embodiments, a composition is provided, comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a double-stranded region, and further comprising a region that is complementary to and hybridizes with a nucleic acid target.Such a composition preferably comprises a first oligomeric compound that is an antisense strand that has full or partial complementarity to a nucleic acid target, and a second oligomeric compound that is a sense strand that has one or more regions that are complementary to the first oligomeric compound and forms at least one double-stranded region with the first oligomeric compound.

[0227] The composition of some embodiments regulates gene expression by hybridizing to nucleic acid target, resulting in the loss of its normal function.In some embodiments, the target nucleic acid is AGT.In certain embodiments, the degradation of target AGT is promoted by the activated RISC complex formed together with the composition of the present invention.

[0228] Some embodiments relate to double-stranded compositions, one of which is useful for influencing the preferential loading of the paired strand into the RISC (or cleavage) complex. The compositions are useful for targeting selected nucleic acid molecules and regulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of the target RNA, thereby causing the loss of the normal function of the target RNA.

[0229] Certain embodiments relate to double-stranded compositions, in which both strands contain hemimer motifs, fully modified motifs, positionally modified motifs, or alternating motifs.Each strand of the composition of the present invention can be modified to play a specific role, for example, in the siRNA pathway.By using different motifs in each strand, or by using the same motif with different chemical modifications in each strand, the antisense strand can be targeted to the RISC complex while inhibiting the uptake of the sense strand.In this model, each strand can be independently modified to enhance its specific role.The antisense strand can be modified at the 5' end to enhance its role in one region of RISC, while the 3' end can be modified differently to enhance its role in a different region of RISC.

[0230] A double-stranded oligonucleotide molecule can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, where the antisense region comprises a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region has a nucleotide sequence corresponding to a target nucleic acid sequence or a portion thereof. A double-stranded oligonucleotide molecule can be assembled from two separate oligonucleotides, one strand being the sense strand and the other strand being the antisense strand, where the antisense and sense strands are self-complementary (i.e., each strand comprises a nucleotide sequence complementary to a nucleotide sequence in the other strand; e.g., the antisense and sense strands form a duplex or double-stranded structure, e.g., the double-stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs long. Yes; the antisense strand comprises a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense strand comprises a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double-stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the double-stranded oligonucleotide is assembled from a single oligonucleotide, and the self-complementary sense and antisense regions of the siRNA are linked by a nucleic acid-based or non-nucleic acid-based linker(s).

[0231] The double-stranded oligonucleotide can be a double-stranded, asymmetric double-stranded, hairpin or asymmetric hairpin secondary structure polynucleotide with self-complementary sense and antisense regions, where antisense region comprises nucleotide sequence complementary to nucleotide sequence in separate target nucleic acid molecule or part thereof, and sense region has nucleotide sequence corresponding to target nucleic acid sequence or part thereof.The double-stranded oligonucleotide can be a circular single-stranded polynucleotide with a stem comprising two or more loop structures and self-complementary sense and antisense regions, where antisense region comprises nucleotide sequence complementary to nucleotide sequence in target nucleic acid molecule or part thereof, and sense region has nucleotide sequence corresponding to target nucleic acid sequence or part thereof, and the circular polynucleotide can be processed in vivo or in vitro to generate active siRNA molecule that can mediate RNAi.

[0232] In certain embodiments, the double-stranded oligonucleotide comprises separate sense and antisense sequences or regions, where the sense and antisense regions are covalently linked by a nucleotide or non-nucleotide linker molecule known in the art. Alternatively, the double-stranded oligonucleotide is non-covalently bound to the target gene by ionic interactions, hydrogen bonds, van der Waals interactions, hydrophobic interactions, and / or stacking interactions. In certain embodiments, the double-stranded oligonucleotide comprises a nucleotide sequence that is complementary to the nucleotide sequence of the target gene. In another embodiment, the double-stranded oligonucleotide interacts with the nucleotide sequence of the target gene in a manner that causes inhibition of expression of the target gene.

[0233] As used herein, double-stranded oligonucleotide is not limited to molecules that contain only RNA, but also includes chemically modified nucleotides and non-nucleotides. In certain embodiments, small interfering nucleic acid molecules do not contain 2'-hydroxy (2'-OH)-containing nucleotides. In certain embodiments, small interfering nucleic acid molecules do not contain any ribonucleotides (e.g., nucleotides with 2'-OH groups) if desired. However, such double-stranded oligonucleotides that do not require the presence of ribonucleotides in the molecule to support RNAi can have one or more linking linkers or other linked or associated groups, moieties or strands that contain one or more nucleotides that contain 2'-OH groups. Optionally, double-stranded oligonucleotides can contain ribonucleotides at about 5, 10, 20, 30, 40 or 50% of nucleotide positions. As used herein, the term siRNA is intended to be equivalent to other terms used to describe nucleic acid molecules that can mediate sequence-specific RNAi, such as short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), small hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA) and others.Furthermore, as used herein, the term RNAi is intended to be equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translation inhibition or epigenetics.For example, double-stranded oligonucleotides can be used to perform epigenetic gene silencing at both post-transcriptional and pre-transcriptional levels. As non-limiting examples, epigenetic control of gene expression by the siRNA molecules of the present invention may result from siRNA-mediated modulation of chromatin structure or methylation patterns, which alter gene expression (e.g., Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2003, Science, 297, 1818-1819). et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al. al., 2002, Science, 297, 2232-2237).

[0234] Some embodiments of the compounds and compositions provided herein are believed to be capable of targeting AGT by a dsRNA-mediated gene silencing or RNAi mechanism, including, for example, "hairpin" or stem-loop double-stranded RNA effector molecules in which a single RNA strand containing a self-complementary sequence can adopt a double-stranded structure, or double-stranded dsRNA effector molecules containing two different RNA strands. In various embodiments, the dsRNA is composed entirely of ribonucleotides or a mixture of ribonucleotides and deoxynucleotides, such as the RNA / DNA hybrids disclosed in WO00 / 63364, filed April 19, 2000, or U.S. Patent Application No. 60 / 130,377, filed April 21, 1999. The dsRNA or dsRNA effector molecule may be a single molecule that contains a self-complementary region such that nucleotides in one segment of the molecule base-pair with nucleotides in another segment of the molecule. In various embodiments, a single molecule of dsRNA can be entirely composed of ribonucleotides or can contain a ribonucleotide region that is complementary to a deoxyribonucleotide region. Alternatively, the dsRNA can contain two different strands that have a complementary region to each other.

[0235] In various embodiments, both strands are entirely composed of ribonucleotides, or one strand is entirely composed of ribonucleotides and the other strand is entirely composed of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides. In certain embodiments, the complementary regions are at least 70, 80, 90, 95, 98 or 100% complementary to each other and to the target nucleic acid sequence. In certain embodiments, the region of the dsRNA present in the double-stranded structure comprises at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides, or all of the nucleotides in the cDNA or other target nucleic acid sequence present in the dsRNA. In some embodiments, the dsRNA does not contain any single-stranded region, such as a single-stranded end, or the dsRNA is a hairpin. In other embodiments, dsRNA has one or more single-stranded regions or overhangs.In certain embodiments, RNA / DNA hybrid comprises a DNA strand or region that is antisense strand or region (e.g., has at least 70, 80, 90, 95, 98 or 100% complementarity with target nucleic acid) and an RNA strand or region that is sense strand or region (e.g., has at least 70, 80, 90, 95, 98 or 100% identity with target nucleic acid), or vice versa.

[0236] In various embodiments, the RNA / DNA hybrid is made in vitro using enzymatic or chemical synthesis methods, such as those described herein or those described in WO 00 / 63364, filed April 19, 2000, or U.S. Patent Application No. 60 / 130,377, filed April 21, 1999. In other embodiments, the DNA strand synthesized in vitro is complexed with the RNA strand made in vivo or in vitro before, after, or simultaneously with the transformation of the DNA strand into the cell. In still other embodiments, the dsRNA is a single circular nucleic acid containing a sense region and an antisense region, or the dsRNA comprises a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, e.g., WO 00 / 63364, filed April 19, 2000, or U.S. Patent Application No. 60 / 130,377, filed April 21, 1999). Exemplary circular nucleic acids include a lariat structure in which the free 5' phosphoryl group of a nucleotide is joined to the 2' hydroxyl group of another nucleotide in a fold-back manner.

[0237] In other embodiments, the dsRNA contains one or more modified nucleotides that contain a halogen (such as a fluorine group) or an alkoxy group (such as a methoxy group) at the 2' position of the sugar. This increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA whose corresponding 2' position contains a hydrogen or hydroxyl group. In yet other embodiments, the dsRNA contains one or more linkages between adjacent nucleotides other than one or more naturally occurring phosphodiester linkages. Examples of such linkages include phosphoramide linkages, phosphorothioate linkages, and phosphorodithioate linkages. The dsRNA may also be a chemically modified nucleic acid molecule as taught in U.S. Patent No. 6,673,661. In other embodiments, the dsRNA contains one or two cap strands, as disclosed, for example, by WO 00 / 63364, filed April 19, 2000, or U.S. Patent Application No. 60 / 130,377, filed April 21, 1999.

[0238] In other embodiments, the dsRNA may be, at least in part, any of the dsRNA molecules disclosed in WO00 / 63364, as well as any of the dsRNA molecules described in U.S. Provisional Patent Application Nos. 60 / 399,998 and 60 / 419,532 and PCT / US2003 / 033466, the teachings of which are incorporated herein by reference. Any of the dsRNA may be synthesized in vitro or in vivo using standard methods, such as those described herein or in WO00 / 63364. or in vivo.

[0239] occupancy In certain embodiments, the antisense compounds are not intended to cause cleavage or removal of the target nucleic acid by RNase H or by the RISC pathway. In certain such embodiments, the antisense activity may result from occupancy where the presence of the hybridized antisense compound blocks the activity of the target nucleic acid. In certain such embodiments, the antisense compounds may be uniformly modified or may contain combinations of modifications and / or modified and unmodified nucleosides.

[0240] Target Nucleic Acids, Target Regions and Nucleotide Sequences Nucleotide sequences encoding AGT include, but are not limited to, the following: GENBANK Accession No. NM_000029.3 (incorporated herein as SEQ ID NO:1), the complement of nucleotides 24354000-24370100 of GENBANK Accession No. NT_167186.1 (incorporated herein as SEQ ID NO:2), GENBANK Accession No. AK307978.1 (incorporated herein as SEQ ID NO:3), GENBANK Accession No. AK303755.1 (incorporated herein as SEQ ID NO:4), GENBANK Accession No. AK293507.1 (incorporated herein as SEQ ID NO:5), and GENBANK Accession No. CR606672.1 (incorporated herein as SEQ ID NO:6). In certain embodiments, the antisense compounds described herein target nucleic acid sequences encoding AGT. In certain embodiments, the antisense compounds described herein target any of the sequences of SEQ ID NOs:1-6.

[0241] It is to be understood that the sequences described by each SEQ ID NO in the Examples contained herein are independent of any modifications to a sugar moiety, internucleoside linkage, or nucleobase. Thus, antisense compounds defined by a SEQ ID NO may independently contain one or more modifications to a sugar moiety, internucleoside linkage, or nucleobase. ISIS Numbers (ISIS Antisense compounds described by the formula (I) refer to combinations of nucleobase sequences and motifs.

[0242] In certain embodiments, the target region is a structurally defined region of the target nucleic acid. For example, the target region may include a 3'UTR, a 5'UTR, an exon, an intron, an exon / intron boundary, a coding region, a translation initiation region, a translation termination region, or other defined nucleic acid region. A structurally defined region for AGT can be obtained by accession number from a sequence database such as NCBI, and such information is incorporated herein by reference. In certain embodiments, the target region may include a sequence from a 5' target site of a target segment within the target region to a 3' target site of another target segment within the target region. In certain embodiments, the target region may include at least 8 contiguous nucleobases selected from at least 8 contiguous nucleobases from the 5' end of the antisense compound within the antisense compound (the remaining nucleobases are a contiguous stretch beginning immediately upstream of the 5' end of the antisense compound specifically hybridizable to the target nucleic acid and continuing until the region contains about 8 to about 80 nucleobases). In certain embodiments, the target region may encompass at least 8 contiguous nucleobases selected from at least 8 contiguous nucleobases from the 3' end of the antisense compound within the antisense compound (the remaining nucleobases are a contiguous stretch beginning immediately downstream of the 3' end of the antisense compound that is specifically hybridizable to the target nucleic acid and continuing until the region contains about 8 to about 80 nucleobases). In certain embodiments, the target region includes at least 8 contiguous nucleobases selected from any of SEQ ID NOs: 14-2051, up to 80 nucleobases of the 5' or 3' 8 contiguous nucleobase sequence. To be continued.

[0243] In certain embodiments, a "target segment" is defined as a smaller sub-portion of a target region in a nucleic acid. For example, a target segment can be a sequence of nucleotides in a target nucleic acid that one or more antisense compounds target. "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.

[0244] Targeting includes determining at least one target segment that antisense compound hybridizes to produce desired effect.In certain embodiments, desired effect is the reduction of mRNA target nucleic acid level.In certain embodiments, desired effect is the reduction of the protein level encoded by target nucleic acid or the phenotypic change associated with target nucleic acid.

[0245] A target region may contain one or more target segments. The target segments in a target region may overlap. Alternatively, they may not overlap. In certain embodiments, the target segments in a target region are separated by about 300 or less nucleotides. In certain embodiments, the target segments in a target region are separated by a number of nucleotides, such as 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides, about that number of nucleotides, less than that number of nucleotides, about that number of nucleotides or less, or a range defined by any two of the aforementioned values. In certain embodiments, the target segments in a target region are separated by 5 or less nucleotides or about 5 or less nucleotides on the target nucleic acid. In certain embodiments, the target segments are contiguous. 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 are contemplated.

[0246] Suitable target segments can be in 5'UTR, coding region, 3'UTR, intron, exon or exon / intron boundary.The target segment containing start codon or stop codon is also suitable target segment.Suitable target segments may specifically exclude certain structurally defined regions such as start codon or stop codon.

[0247] Determining suitable target segment can include comparing the sequence of target nucleic acid with other sequences throughout genome.For example, BLAST algorithm can be used to identify the region of similarity between different nucleic acids.This comparison can avoid the selection of antisense compound sequence that can nonspecifically hybridize to sequences other than the selected target nucleic acid (i.e., non-target sequence or off-target sequence).

[0248] The activity of antisense compounds within an active target region (e.g., as defined by the percentage reduction in target nucleic acid levels) may vary. In certain embodiments, a reduction in AGT mRNA levels is indicative of inhibition of AGT expression. A reduction in AGT protein levels is also indicative of inhibition of AGT expression. Additionally, phenotypic changes are indicative of AGT expression. For example, a reduction in fibrosis in a tissue may be indicative of inhibition of AGT expression. As another example, a reduction in hypertension may be indicative of inhibition of AGT expression.

[0249] Hybridization In some embodiments, hybridization occurs between the antisense compounds disclosed herein and AGT nucleic acids. The most common hybridization mechanism involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of nucleic acid molecules.

[0250] Hybridization can occur under a variety of conditions. Stringent conditions are sequence-dependent and depend on the nature and composition of the nucleic acid molecules to be hybridized.

[0251] Methods for determining whether a sequence specifically hybridizes to a target nucleic acid are well known in the art (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 31(1):131-135). rd Ed., 2001). In certain embodiments, the antisense compounds provided herein are specifically hybridizable to an AGT nucleic acid.

[0252] Complementarity An antisense compound and a target nucleic acid are complementary to one another when a sufficient number of nucleobases of the antisense compound are capable of hydrogen bonding with corresponding nucleobases of the target nucleic acid to produce the desired effect (e.g., antisense inhibition of a target nucleic acid, such as an AGT nucleic acid).

[0253] Non-complementary nucleobases between an antisense compound and an AGT nucleic acid are permissible, provided that the antisense compound is still capable of specifically hybridizing to an AGT nucleic acid. In addition, an antisense compound may hybridize across one or more segments of an AGT nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch, or hairpin structure).

[0254] In certain embodiments, the antisense compounds provided herein or designated portions thereof are 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary or at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to an AGT nucleic acid, target region, target segment or designated portion thereof. The percent complementarity between an antisense compound and a target nucleic acid can be determined using routine methods. For example, 18 of 20 nucleobases of an antisense compound are complementary to target region, and therefore the antisense compound that specifically hybridizes is said to have 90% complementarity.In this example, the remaining non-complementary nucleobases can be grouped together or interspersed among complementary nucleobases, and do not need to be contiguous with each other or contiguous with complementary nucleobases.Thus, an 18-nucleobase long antisense compound that has four non-complementary nucleobases flanked by two regions that are completely complementary with target nucleic acid has a total complementarity of 77.8% with the target nucleic acid, and is therefore within the scope of the present invention.

[0255] The percent complementarity of an antisense compound to a region of a target nucleic acid can be routinely determined using the BLAST (Basic Local Alignment Search Tool) 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, for example, using the GAP program (Wisconsin Sequence Analysis Package,Version 8 for Unix,Gen etics Computer Group,University Research Park, Madison Wis.) using default settings.

[0256] In certain embodiments, the antisense compounds provided herein or the designated portions thereof are fully complementary (i.e., 100% complementary) to the target nucleic acid or the designated portions thereof. For example, the antisense compounds can be fully complementary to the AGT nucleic acid or the target region or target segment or target sequence thereof. As used herein, "fully complementary" means that each nucleobase of the antisense compound can form an exact base pair with the corresponding nucleobase of the target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long as the 20 nucleobase portion of the corresponding target nucleic acid is fully complementary to the antisense compound. Fully complementary can also be used in reference to the designated portions of the first and / or 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. A 20 nucleobase portion of a 30 nucleobase oligonucleotide is fully complementary to a target sequence if the target sequence has a corresponding 20 nucleobase portion, each nucleobase of which is complementary to a 20 nucleobase portion of an antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to a target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

[0257] The location of the non-complementary nucleobase may be at the 5'-end or 3'-end of the antisense compound. Alternatively, one or more non-complementary 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, the non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

[0258] In certain embodiments, antisense compounds that are 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleobases in length or up to 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleobases in length contain no more than 4, no more than 3, no more than 2 or no more than 1 non-complementary nucleobase(s) to a target nucleic acid, such as an AGT nucleic acid, or a specified portion thereof.

[0259] In certain embodiments, antisense compounds that are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases in length or up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleobases in length contain 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) to a target nucleic acid, such as an AGT nucleic acid, or a specified portion thereof.

[0260] Antisense compounds provided herein also include those that are complementary to a portion of a target nucleic acid. As used herein, a "portion" refers to a predetermined number of contiguous (i.e., linked) nucleobases within a region or segment of a target nucleic acid. A "portion" can also refer to a predetermined 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, the antisense compounds can be complementary to at least an 8 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more nucleobase portion of a sequence, or a range defined by any two of these values.

[0261] identity The antisense compounds provided herein may also have a certain percent identity with a compound or part thereof represented by a certain nucleotide sequence, sequence number, or specific ISIS number.As used herein, an antisense compound is identical to a sequence disclosed herein if it has the same ability to form nucleic acid base pairs.For example, an RNA containing uracil instead of thymidine in a disclosed DNA sequence is considered to be identical to the DNA sequence, since both uracil and thymidine pair with adenine.Shortened and extended versions of the antisense compounds described herein, as well as compounds with non-identical bases with respect to the antisense compounds provided herein, are also contemplated.The non-identical bases may be adjacent to each other or distributed throughout the antisense compound.The percent identity of an antisense compound is calculated according to the number of bases with identical base pairing with the sequence to be compared.

[0262] In certain embodiments, the antisense compounds or portions thereof are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs or portions thereof disclosed herein.

[0263] qualification A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of a nucleoside is usually a heterocyclic base moiety. A nucleotide is a nucleoside that further comprises a phosphate group covalently linked to the sugar portion of the nucleoside. In the case of nucleosides that contain a pentofuranosyl sugar, the phosphate group can be attached to the 2', 3' or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed by covalently linking adjacent nucleosides to each other to form linear polymeric oligonucleotides. The phosphate groups are commonly referred to within the oligonucleotide structure as forming the internucleoside linkages of the oligonucleotide.

[0264] Modifications to antisense compounds include substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over natural forms because they may possess desirable properties, such as, for example, improved cellular uptake, increased affinity for nucleic acid targets, increased stability in the presence of nucleases, or increased inhibitory activity.

[0265] Chemically modified nucleosides can also be used to increase the binding affinity of shortened or truncated antisense oligonucleotides to their target nucleic acids, and therefore shorter antisense compounds having such chemically modified nucleosides can often achieve equivalent results.

[0266] Modified Internucleoside Linkages The naturally occurring internucleoside linkage in RNA and DNA is a 3'-5' phosphodiester linkage. Antisense compounds having one or more modified internucleoside linkages, i.e., non-naturally occurring internucleoside linkages, may have advantages, such as improved cellular uptake, improved affinity for nucleic acid targets, over antisense compounds having naturally occurring internucleoside linkages. These are often chosen because they have desirable properties such as increased stability in the presence of enzymes and nucleases.

[0267] The oligonucleotide with modified internucleoside linkage includes the internucleoside linkage that holds phosphorus atom and the internucleoside linkage that does not have phosphorus atom.Exemplary phosphorus-containing internucleoside linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate and phosphorothioate.The method of preparing phosphorus-containing and non-phosphorus-containing linkages is well known.

[0268] In certain embodiments, the antisense compound targeted to AGT nucleic acid comprises one or more modified internucleoside linkages.In certain embodiments, at least one of the modified internucleoside linkages is a phosphorothioate linkage.In certain embodiments, each internucleoside linkage of the antisense compound is a phosphorothioate internucleoside linkage.

[0269] modified sugar moiety Antisense compounds of the invention can optionally contain one or more nucleosides in which the sugar group has been modified. Such sugar-modified nucleosides may confer improved nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compound. In certain embodiments, the nucleoside comprises a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (e.g., 5' and 2' substituents, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNAs), modification of the ribosyl ring oxygen atoms with S, N(R) or C(R1)(R2) (where R, R1 and R2 are each independently H, C1-C1, or C2-C3). 12Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO2008 / 101157 published August 21, 2008 for other 5',2'-bis substituted nucleosides disclosed) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2' position (see US Patent Application Publication US2005-0130923 published June 16, 2005), or 5'-substitution of BNAs (see PCT International Application WO2007 / 134181 published November 22, 2007; e.g., LNAs substituted with 5'-methyl or 5'-vinyl groups).

[0270] Examples of nucleosides having modified sugar moieties include nucleosides containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F and 2'-O(CH2)2OCH3 substituents. Substituents at the 2' position include allyl, amino, azido, thio, O-allyl, O-C1-C2 10 Alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), O-CH2-C(=O)-N(R m )(R n ) and O-CH2-C(=O)-N(R l )-(CH2)2-N(R m )(R n )(In the formula, each R l , R m and R n are independently H or substituted or unsubstituted C1-C 10 It may also be selected from the group consisting of alkyl.

[0271] As used herein, "bicyclic nucleoside" refers to a modified nucleoside that contains a bicyclic sugar moiety. Examples of bicyclic nucleic acids (BNAs) include, but are not limited to, nucleosides that contain a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the antisense compounds provided herein contain one or more BNA nucleosides, in which the bridge is of the following formula: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (cEt) and 4'-CH(CHOCH3)-O-2' (and analogs thereof; see U.S. Patent No. 7,399,845 issued July 15, 2008); 4'-C( 4'-CH2-N(OCH3)-2' (and analogs thereof; see PCT / US2008 / 068922, published as WO / 2009 / 006478, published January 8, 2009); 4'-CH2-N(OCH3)-2' (and analogs thereof; see PCT / US2008 / 064591, published as WO / 2008 / 150729, published December 11, 2008); 4'-CH2-ON(CH3)-2' (see U.S. Patent Application Publication US2004-0171570, published September 2, 2004); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C 12 No. 7,427,672, issued Sep. 23, 2008; 4'-CH2-C(H)(CH3)-2' (see, e.g., Zhou, et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof: see PCT / US2008 / 066154, published as WO2008 / 154401, published Dec. 8, 2008).

[0272] Further bicyclic nucleosides have been reported in the open literature (e.g., Srivastava et al., J. Am. Chem. Soc., 2007, 129(26)8362-8379; Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum ... et al.,Curr.Opinion Mol.Ther.,2001,3,239-243;Wahlestedt et al.,Proc.Natl.Acad.Sci.USA,2000,97,5633-5638;Singh et al.,Chem.Commun.,1998,4,455-456;Koshkin et al. al., Tetrahedron,1998,54,3607-3630;Kumar et al.,Bioorg.Med.Chem.Lett.,1998,8,2219-2222;Singh et al. al., J. Org. Chem., 1998, 63, 10035-10039; U.S. Patent Nos. 7,399,845, 7,053,207, 7,034,133, 6,794,499, 6,770,748, 6,670,461, 6,525,191, and 6,268,490; U.S. Patent Application Publication Nos. US2008-0039618, US2007-0287831, and US2004-0171570; U.S. Patent Application Nos. 12 / 129,154, 61 / 09 (See, for example, PCT International Application Nos. WO2007 / 134181, WO2005 / 021570, WO2004 / 106356, WO99 / 14226, and PCT International Application Nos. PCT / US2008 / 068922, PCT / US2008 / 066154, and PCT / US2008 / 064591). Each of the foregoing bicyclic nucleosides can be prepared with one or more stereochemical sugar structures, including, for example, α-L-ribofuranose and β-D-ribofuranose (see PCT International Application No. PCT / DK98 / 00393, published as WO 99 / 14226, published March 25, 1999).

[0273] As used herein, "monocyclic nucleoside" refers to a nucleoside that includes a modified sugar moiety that is not a bicyclic sugar moiety. In certain embodiments, the sugar moiety or sugar moiety analog of the nucleoside can be modified or substituted at any position.

[0274] As used herein, a "4'-2' bicyclic nucleoside" or "4'→2' bicyclic nucleoside" refers to a bicyclic nucleoside containing a furanose ring that contains a bridge connecting two carbon atoms of the furanose ring that connects the 2' and 4' carbon atoms of the sugar ring.

[0275] In certain embodiments, the bicyclic sugar moiety of a BNA nucleoside includes, but is not limited to, compounds having at least one bridge between the 4' and 2' carbon atoms of the pentofuranosyl sugar moiety, including, but not limited to, -[C(R a )(R b )] n -, -C(R a )=C(R b )-, -C(R a )=N-, -C(=NR a )-, -C(=O)-, -C(=S)-, -O-, -Si(R a )2-, -S(=O) x - and -N(R a )-, wherein x is 0, 1 or 2, n is 1, 2, 3 or 4, and each R a and R b are independently H, a protecting group, hydroxyl, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 aryl, heterocyclic group, substituted heterocyclic group, heteroaryl, substituted heteroaryl, C5-C7 alicyclic group, substituted C5-C7 alicyclic group, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1), each J1 and J2 being independently H, C1-C 12Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C5-C 20 Aryl, substituted C5-C 20 Aryl, acyl (C(=O)-H), substituted acyl, heterocyclic group, substituted heterocyclic group, C1-C 12 Aminoalkyl, substituted C1-C 12 It is an aminoalkyl or a protecting group.

[0276] In certain embodiments, the bridge of the bicyclic sugar moiety is -[C(R a )(R b )] n -,-[C(R a )(R b )] n -O-, -C(R a R b )-N(R)-O- or -C(R a R b In certain embodiments, the bridges are 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-ON(R)-2', and 4'-CH2-N(R)-O-2'-, where R is independently H, a protecting group, or a C1-C 12 (alkyl).

[0277] In certain embodiments, bicyclic nucleosides are further defined by their isomeric configuration. For example, nucleosides that contain a 4'-(CH2)-O-2' bridge can be in the α-L or β-D configuration. Previously, α-L-methyleneoxy (4'-CH2-O-2') BNAs have been incorporated into antisense oligonucleotides and have shown antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0278] In certain embodiments, bicyclic nucleosides include those having a 4'→2' bridge, including, but not limited to, α-L-4'-(CH)-O-2', β-D-4'-CH-O-2', 4'-(CH)-O-2', 4'-CH-ON(R)-2', 4'-CH-N(R)-O-2', 4'-CH(CH)-O-2', 4'-CH-S-2', 4'-CH-N(R)-2', 4'-CH-CH(CH)-2', and 4'-(CH)-2', where R is H, a protecting group, or a C-C 12 alkyl).

[0279] In certain embodiments, the bicyclic nucleoside has the formula:

[0280] [ka]

[0281] During the ceremony, Bx is a heterocyclic base moiety; -Q a -Q b -Q c - is -CH2-N(R c )-CH2-, -C(=O)-N(R c )-CH2-, -CH2-ON(R c )-, -CH2-N(R c )-O- or -N(R c )-O-CH2, R c is C1~C 12 is an alkyl or amino protecting group, T a and T b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support.

[0282] In certain embodiments, the bicyclic nucleoside has the formula:

[0283] [ka]

[0284] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support; Z a is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thiol.

[0285] In one embodiment, each of the substituents is independently selected from halogen, oxo, hydroxyl, OJ c , N.J. c J d , S.J. c , N3, OC(=X)J c and N.J. e C(=X)NJ c J d wherein each J is mono- or polysubstituted with substituents independently selected from c , J d and J e are independently H, C1-C6 alkyl or substituted C1-C6 alkyl, and X is O or NJ c It is.

[0286] In certain embodiments, the bicyclic nucleoside has the formula:

[0287] [ka]

[0288] During the ceremony, Bx is a heterocyclic base moiety; T a and T bare each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support; Z b is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl or substituted acyl (C(=O)-).

[0289] In certain embodiments, the bicyclic nucleoside has the formula:

[0290] [ka]

[0291] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support; R d is C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; each q a , q b , q c and q d are independently H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, C1-C6 alkoxyl, substituted C1-C6 alkoxyl, acyl, substituted acyl, C1-C6 aminoalkyl or substituted C1-C6 aminoalkyl.

[0292] In certain embodiments, the bicyclic nucleoside has the formula:

[0293] [ka]

[0294] During the ceremony, Bx is a heterocyclic base moiety; T a and T b are each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support; q a , q b , q e and q f are each independently hydrogen, halogen, or C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C1-C 12 Alkoxy, substituted C1-C 12 Alkoxy, O.J. j , S.J. j , S.O.J. j , SO2J j , N.J. j J k , N3, CN, C(=O)OJ j , C(=O)NJ j J k , C(=O)J j ,OC(=O)N J j J k , N(H)C(=NH)NJ j J k , N(H)C(=O)NJ j J k or N(H)C(=S)NJ j J k and Or q e and q f Together, =C(q g )(q h ) and q g and q h are each independently H, halogen, C1-C12 alkyl or substituted C1-C12 It is an alkyl.

[0295] The synthesis and preparation of adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil bicyclic nucleosides with 4'-CH2-O-2' bridges has been described along with their oligomerization and nucleic acid recognition properties (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). The synthesis of bicyclic nucleosides is also described in WO98 / 39352 and WO99 / 14226.

[0296] Various bicyclic nucleoside analogues with 4'→2' bridging groups such as 4'-CH2-O-2' and 4'-CH2-S-2' have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of oligodeoxyribonucleotide duplexes containing bicyclic nucleosides for use as substrates for nucleic acid polymerases has also been described (Wengel et al., WO99 / 14226). Furthermore, the synthesis of 2'-amino-BNAs, a new conformationally restricted high affinity oligonucleotide analogue, has also been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino-BNAs and 2'-methylamino-BNAs have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

[0297] In certain embodiments, the bicyclic nucleoside has the formula:

[0298] [ka]

[0299] During the ceremony, Bx is a heterocyclic base moiety; T a and T bare each independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety, or a covalent bond to a support; each q i , q j , q k and q l are independently H, halogen, C1-C 12 Alkyl, substituted C1-C 12 Alkyl, C2-C 12 Alkenyl, substituted C2-C 12 Alkenyl, C2-C 12 Alkynyl, substituted C2-C 12 Alkynyl, C1-C 12 Alkoxyl, substituted C1-C 12 Alkoxyl, OJ j , S.J. j , S.O.J. j , SO2J j , N.J. j J k , N3, CN, C(=O)OJ j , C(=O)NJ j J k , C(=O)J j ,OC(=O)NJ j J k , N(H)C(=NH)NJ j J k , N(H)C(=O)NJ j J k or N(H)C(=S)NJ j J k and q i and q j or q l and q k are taken together, =C(q g )(q h ) in which q g and q h are each independently H, halogen, C1-C12 alkyl or substituted C1-C 12 It is an alkyl.

[0300] 4'-(CH2)3-2' bridge and alkenyl analog bridge 4'-CH=CH-CH2- Certain carbocyclic bicyclic nucleosides with 2'-amino groups have been described (Frier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides have also been described along with their oligomerization and biochemical studies (Srivastava et al., al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0301] In certain embodiments, bicyclic nucleosides include, as shown below: (A) α-L-methyleneoxy (4'-CH2-O-2') BNAs, (B) β-D-methyleneoxy (4'-CH2-O-2') BNAs, (C) ethyleneoxy (4'-(CH2)2-O-2') BNAs, (D) aminooxy (4'-CH2-ON(R)-2') BNAs, (E) oxyamino (4'-CH2-N(R)-O-2') BNAs, (F) methyl (methyl) (R) methylene-amino (4'-CH-N(R)-2') BNAs, (I) methyl carbocyclic (4'-CH-CH(CH)-2') BNAs, (J) propylene carbocyclic (4'-(CH)-2') BNAs, and (K) vinyl BNAs.

[0302] [ka]

[0303] In the formula, Bx is a base moiety and R is independently H, a protecting group, C1-C6 alkyl, or C1-C6 alkoxy.

[0304] As used herein, the term "modified tetrahydropyran nucleoside" or "modified THP nucleoside" refers to a nucleoside that has a six-membered tetrahydropyran "sugar" in place of the pentofuranosyl residue in a normal nucleoside, and may also be referred to as a sugar surrogate. Modified THP nucleosides include, but are not limited to, those referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854), or fluoro-HNA (F-HNA), which has a tetrahydropyranyl ring system as shown below.

[0305] [ka]

[0306] In certain embodiments, the sugar substitute is selected from those having the formula:

[0307] [ka]

[0308] During the ceremony, Bx is a heterocyclic base moiety; T3 and T4 are each independently an internucleoside linking group that attaches a tetrahydropyran nucleoside analog to an oligomeric compound, or one of T3 and T4 is an internucleoside linking group that attaches a tetrahydropyran nucleoside analog to an oligomeric compound or oligonucleotide, and the other of T3 and T4 is H, a hydroxyl protecting group, an attached conjugate group, or a 5' or 3' terminal group; q1, q2, q3, q4, q5, q6 and q7 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; One of R1 and R2 is hydrogen and the other is selected from halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, where X is O, S or NJ1, and each J1, J2 and J3 is independently H or C1-C6 alkyl.

[0309] In certain embodiments, q1, q2, q3, q4, q5, q6, and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6, and q7 is methyl. In certain embodiments, THP nucleosides are provided where one of R1 and R2 is F. In certain embodiments, R1 is fluoro, R2 is H, R1 is methoxy, R2 is H, R1 is methoxyethoxy, and R2 is H.

[0310] In certain embodiments, the sugar surrogate has six or more atoms and includes a ring with two or more heteroatoms.For example, nucleosides containing morpholino sugar moieties and their use in oligomeric compounds have been reported (see, for example, Braasch et al., Biochemistry, 2002, 41, 4503-4510 and U.S. Patent Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506).As used herein, the term "morpholino" refers to a sugar surrogate having the formula:

[0311] [ka]

[0312] In certain embodiments, morpholinos may be modified, for example, by adding or changing various substituents to the morpholino structures described above. Such sugar surrogates are also referred to herein as "modified morpholinos."

[0313] Combinations of modifications are also provided, including, but not limited to, 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO2008 / 101157 published August 21, 2008 for other 5',2'-bis substituted nucleosides disclosed) and replacement of the ribosyl ring oxygen atom with S with further substitutions at the 2' position (see U.S. Patent Application Publication US2005-0130923 published June 16, 2005) or 5'-substitution of bicyclic nucleic acids (see PCT International Application WO2007 / 134181 published November 22, 2007; 4'-CH2-O-2' bicyclic nucleosides further substituted at the 5' position with a 5'-methyl or 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides have also been described along with their oligomerization and biochemical studies (see, for example, Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0314] In certain embodiments, the antisense compounds comprise one or more modified cyclohexenyl nucleosides, which are nucleosides having a six-membered cyclohexenyl in place of a pentofuranosyl residue in a naturally occurring nucleoside. Modified cyclohexenyl nucleosides include, but are not limited to, those described in the art (e.g., commonly owned PCT International Application WO2010 / 036696, published April 10, 2010; Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids, 2005, 24(5-7), 993-998). Research,2005,33(8),2452-2463;Robeyns et al.,Acta Crystallographica,Section F:Structural Biology and Crystallization Communications,2005,F61(6),585-586;Gu et al.,Tetrahedron,2004,60(9),2111-2123;Gu et al.,Oligonucleotides,2003,13(6),479-489;Wang et al. (See, e.g., Verbeure et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; PCT Publication No. WO 06 / 047842; and PCT Publication No. WO 01 / 049687; each of which is incorporated herein by reference in its entirety.) A particular modified cyclohexenyl nucleoside has the formula X:

[0315] [ka]

[0316] wherein, independently, for each of said at least one cyclohexenyl nucleoside analog of formula X: Bx is a heterocyclic base moiety; T3 and T4 are each independently an internucleoside linking group that attaches a cyclohexenyl nucleoside analog to an antisense compound; or one of T3 and T4 is an internucleoside linking group that attaches a tetrahydropyran nucleoside analog to an antisense compound, and the other of T3 and T4 is H, a hydroxyl protecting group, an attached conjugate group, or a 5' or 3' terminal group; q1, q2, q3, q4, q5, q6, q7, q8, and q9 are each independently H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl, or other sugar substituents.

[0317] Many other monocyclic, bicyclic and tricyclic ring systems are known in the art and are suitable as sugar surrogates that can be incorporated into the oligomeric compounds provided herein and used to modify nucleosides (see, for example, review article: Leumann, Christian J. Bioorg. & Med. Chem., 2002, 10, 841-854). Such ring systems may be subject to a variety of additional substitutions to further improve their activity.

[0318] As used herein, "2'-modified sugar" refers to a furanosyl sugar modified at the 2' position. In certain embodiments, such modifications include a substituent selected from halide, substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted aminoalkyl, substituted and unsubstituted alkyl, substituted and unsubstituted aryl, and substituted and unsubstituted alkynyl. In certain embodiments, the 2' modification is O[(CH2) n O] m CH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n F, O(CH2) n ONH2, OCH2C(=O)N(H)CH3, and O(CH2) n ON [(CH2) n CH3]2, where n and m are from 1 to about 10. Other 2'-substituents are selected from the following: C1-C 12The substituents may be selected from alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, F, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups that improve the pharmacokinetic properties or pharmacodynamic properties of the antisense compound, and other substituents with similar properties. In certain embodiments, the modified nucleoside comprises a 2'-MOE side chain (Baker et al., J.Biol.Chem., 1997,272,11944-12000). Such 2'-MOE substitutions have been described to improve binding affinity compared to unmodified nucleosides and other modified nucleosides, such as 2'-O-methyl, O-propyl, and O-aminopropyl. Contains 2'-MOE substituent Oligonucleotides have also been shown to be antisense inhibitors of gene expression, with useful characteristics for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

[0319] As used herein, "2'-modified" or "2'-substituted" refers to a nucleoside that includes a sugar that includes a substituent other than H or OH at the 2' position. 2'-modified nucleosides include allyl, amino, azido, thio, O-allyl, O-C1-C2-C3-C4-C5-C6-C7-C8-C9-C10-C11-C12-C13-C14-C15-C16-C17-C18-C29-C28-C39-C47-C48-C59-C60-C19-C29-C30-C29-C31-C27-C32-C19-C28-C33-C29-C34-C45-C46-C19-C29-C35-C47-C48-C19-C29-C36-C37-C48-C49-C49-C59-C60-C19-C29-C38-C49-C49-C59-C60-C19-C29-C31-C29-C32-C49 ...59-C60-C19-C29-C31-C2 10 Alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-ON(R m )(R n ) or O-CH2-C(=O)-N(R m )(R n )(In the formula, each Rm and R n are independently H or substituted or unsubstituted C1-C 10 2'-Modified nucleosides include, but are not limited to, nucleosides that contain a non-bridging 2' substituent such as an aryl group, an aryl group, or an alkyl group. 2'-Modified nucleosides can further contain other modifications, for example, at other positions on the sugar and / or at the nucleobase.

[0320] As used herein, "2'-F" refers to a nucleoside that includes a sugar that includes a fluoro group at the 2' position of the sugar ring.

[0321] As used herein, "2'-OMe" or "2'-OCH3", "2'-O-methyl" or "2'-methoxy" each refer to a nucleoside containing a sugar that includes an -OCH3 group at the 2' position of the sugar ring.

[0322] As used herein, "MOE" or "2'-MOE" or "2'-OCH2CH2OCH3" or "2'-O-methoxyethyl" each refers to a nucleoside containing a sugar that includes a -OCH2CH2OCH3 group at the 2' position of the sugar ring.

[0323] Methods for the preparation of modified sugars are well known to those of skill in the art. Some representative United States patents that teach the preparation of such modified sugars include, but are not limited to, U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,576,427, 5,591,722, 5, Nos. 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,670,633, 5,700,920, 5,792,847, and 6,600,032, and International Application PCT / US2005 / 019219 (filed June 2, 2005), published as WO2005 / 121371 on December 22, 2005, each of which is incorporated by reference in its entirety.

[0324] As used herein, "oligonucleotide" refers to a compound that includes a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides are modified. In certain embodiments, an oligonucleotide includes one or more ribonucleosides (RNA) and / or deoxyribonucleosides (DNA).

[0325] In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified, or a combination thereof) are retained for hybridization with an appropriate nucleic acid target.

[0326] In certain embodiments, the antisense compounds comprise one or more nucleosides having a modified sugar moiety. In certain embodiments, the modified sugar moiety is 2'-MOE. In certain embodiments, the 2'-MOE modified nucleoside is positioned in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4'-CH(CH3)-O-2') bridging group. In certain embodiments, the (4'-CH(CH3)-O-2') modified nucleoside is positioned throughout the wings of a gapmer motif.

[0327] Modified Nucleobases Nucleobase (or base) modifications or substitutions are structurally distinguishable from natural or synthetic unmodified nucleobases, but functionally interchangeable. Both natural and modified nucleobases can participate in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity, or some other beneficial biological property to the antisense compound. Modified nucleobases include synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of antisense compounds to target nucleic acids. For example, 5-methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

[0328] Further unmodified nucleobases include 5-hydroxymethylcytosine, 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-azouracil, 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, especially 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.

[0329] Heterocyclic base moieties also include those in which the purine or pyrimidine base is replaced with other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Particularly useful nucleobases for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

[0330] In certain embodiments, the antisense compound targeted to AGT nucleic acid comprises one or more modified nucleobases.In certain embodiments, the gap widening antisense oligonucleotide targeted to AGT nucleic acid comprises one or more modified nucleobases.In certain embodiments, at least one of the modified nucleobases is 5-methylcytosine.In certain embodiments, each cytosine is 5-methylcytosine.

[0331] Compositions and methods for making pharmaceutical compositions The antisense oligonucleotides may be pharma- ceutically administered for the preparation of a pharmaceutical composition or formulation. It may be mixed with acceptable active or inactive substances. The composition and method for preparing a pharmaceutical composition depends on many criteria, including but not limited to the route of administration, the extent of the disease, or the dose to be administered.

[0332] Antisense compounds targeted to AGT nucleic acids can be utilized in pharmaceutical compositions by combining the antisense compounds with a suitable pharma- ceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include water, e.g., water for injection (WFI). Pharmaceutically acceptable diluents include saline, e.g., phosphate buffered saline (PBS). Water or saline is a suitable diluent for use in parenterally delivered compositions. Thus, in one embodiment, a pharmaceutical composition comprising an antisense compound targeted to AGT nucleic acid and a pharma- ceutically acceptable diluent is utilized in the methods described herein. In certain embodiments, the pharma- ceutically acceptable diluent is water or saline. In certain embodiments, the antisense compound is an antisense oligonucleotide.

[0333] The pharmaceutical composition comprising antisense compounds includes any pharmaceutically acceptable salt, ester or salt of said ester or any other oligonucleotide that can produce (directly or indirectly) biologically active metabolite or its residue when administered to animals, including humans.Thus, for example, the present disclosure also relates to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of said prodrugs and other bioequivalents.Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts.

[0334] The "pharmaceutical acceptable salt" of the compound described herein can be prepared by methods well known in the art.For a general overview of pharmaceutical acceptable salts, see Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use (Wiley-VCH, Weinheim, Germany, 2002).For therapeutic administration to humans, the sodium salt of antisense oligonucleotide is useful and well tolerated.Therefore, in one embodiment, the compound described herein is in the form of sodium salt.

[0335] Prodrugs may include the incorporation of additional nucleosides at one or both ends of the antisense compound that are cleaved by endogenous nucleases in the body to form the active antisense compound.

[0336] dosage In certain embodiments, the pharmaceutical composition is administered according to a dosing regimen (e.g., dose, frequency of administration and duration), which can be selected to achieve a desired effect, which can be, for example, a reduction in AGT or the prevention, reduction, amelioration or delay in progression of a disease, disorder or condition associated with AGT.

[0337] In certain embodiments, the variables of the dosing regimen are adjusted to provide the desired concentration of the pharmaceutical composition in the subject.The "concentration of the pharmaceutical composition" used in relation to the dosing regimen may refer to the compound, oligonucleotide, or active ingredient of the pharmaceutical composition.For example, in certain embodiments, the dosage and administration frequency are adjusted to obtain a tissue concentration or plasma concentration of the pharmaceutical composition sufficient to achieve the desired effect.

[0338] Dosing depends on the severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. It also depends on efficacy and metabolism. In certain embodiments, the dosage is in the dosage range of 0.01 μg to 100 mg or 0.001 mg to 1000 mg per kg of body weight and can be administered daily, weekly, biweekly, monthly, quarterly, semiannually or once or more per year, or once every 2 to 20 years. After successful treatment, the patient may be desired to undergo maintenance therapy to prevent recurrence of the disease state, in which case the oligonucleotide is administered at a maintenance dosage in the dosage range of 0.01 μg to 100 mg or 0.001 mg to 1000 mg per kg of body weight, once or more per day to once every 20 years.

[0339] Administration The compounds or pharmaceutical compositions of the present invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration may be by inhalation (i.e., pulmonary), enteral (i.e., enteric), parenteral or topical administration.

[0340] In certain embodiments, the compounds and compositions described herein are administered parenterally. Parenteral administration includes, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, intraocular, intramuscular, intracranial, intrathecal, intramedullary, intravenous or intratumoral injection or infusion. Parenteral administration also includes intranasal administration.

[0341] In certain embodiments, parenteral administration is by infusion. Infusion can be chronic, or continuous, or short-term, or intermittent. In certain embodiments, the infusion is delivered by a pump.

[0342] In certain embodiments, parenteral administration is by injection. The injection can be delivered by a syringe or a pump. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly into a tissue or organ.

[0343] In certain embodiments, formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds and other pharma- ceutically acceptable carriers or excipients.

[0344] In certain embodiments, the compounds and compositions described herein are administered enterally. Enteral administration includes, but is not limited to, oral, mucosal, intestinal or rectal (e.g., suppository, enema). In certain embodiments, the compound or composition formulation for enteral administration may include, but is not limited to, a pharmaceutical carrier, excipient, powder or granule, microparticle, nanoparticle, suspension or solution in water or non-aqueous media, capsule, gel capsule, sachet, tablet or mini-tablet. Viscosifiers, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In certain embodiments, enteral formulations are those in which the compounds provided herein are administered with one or more penetration enhancers, surfactants and chelators.

[0345] In certain embodiments, administration includes pulmonary administration. In certain embodiments, pulmonary administration includes delivering aerosolized oligonucleotide to the lungs of a subject by inhalation. When a subject inhales aerosolized oligonucleotide, the oligonucleotide distributes to cells of both normal and inflamed lung tissues, including alveolar macrophages, eosinophils, epithelium, vascular endothelium, and bronchiolar epithelium. Suitable devices for delivery of pharmaceutical compositions containing modified oligonucleotides include, but are not limited to, standard nebulizer devices. Further suitable devices include dry powder inhalers or metered dose inhalers.

[0346] In certain embodiments, the pharmaceutical composition is administered to achieve local rather than systemic exposure, for example, pulmonary administration delivers the pharmaceutical composition to the lungs with minimal systemic exposure.

[0347] Conjugated Antisense Compounds In certain embodiments, the oligonucleotide or oligomeric compounds provided herein are modified by the covalent attachment of one or more conjugate groups. In general, the conjugate group modifies one or more properties of the oligonucleotide or oligomeric compound to which it is attached, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. As used herein, "conjugate group" refers to a radical group that includes an atomic group attached to an oligonucleotide or oligomeric compound. In general, the conjugate group modifies one or more properties of the compound to which it is attached, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and / or clearance. Conjugate groups are routinely used in the chemical arts and may include conjugate linkers that covalently attach the conjugate group to the oligonucleotide or oligomeric compound. In certain embodiments, the conjugate group includes a cleavable moiety that covalently attaches the conjugate group to the oligonucleotide or oligomeric compound. In certain embodiments, the conjugate group comprises a conjugate linker and a cleavable moiety that covalently attaches the conjugate group to an oligonucleotide or oligomeric compound. In certain embodiments, the conjugate group has the general formula:

[0348] [ka]

[0349] In the formula, n is 1 to about 3, and when n is 1, m is 0, or when n is 2 or 3, m is 1, j is 1 or 0, k is 1 or 0, and the sum of j and k is at least 1.

[0350] In certain embodiments, n is 1, j is 1, and k is 0. In certain embodiments, n is 1, j is 0, and k is 1. In certain embodiments, n is 1, j is 1, and k is 1. In certain embodiments, n is 1, j is 1, and k is 1. In certain embodiments, n is 2, j is 1, and k is 0. In certain embodiments, n is 2, j is 0, and k is 1. In certain embodiments, n is 2, j is 1, and k is 1. In certain embodiments, n is 3, j is 1, and k is 0. In certain embodiments, n is 3, j is 0, and k is 1. In certain embodiments, n is 3, j is 1, and k is 1.

[0351] Conjugate group is referred to herein as a group that provides a bond to form a covalent bond to an oligomeric compound such as an oligonucleotide. In certain embodiments, the attachment point of the oligomeric compound is the 3'-terminal nucleoside or modified nucleoside. In certain embodiments, the attachment point of the oligomeric compound is the 3'-oxygen atom of the 3'-hydroxyl group of the 3'-terminal nucleoside or modified nucleoside. In certain embodiments, the attachment point of the oligomeric compound is the 5'-terminal nucleoside or modified nucleoside. In certain embodiments, the attachment point of the oligomeric compound is the 5'-oxygen atom of the 5'-hydroxyl group of the 5'-terminal nucleoside or modified nucleoside. In certain embodiments, the attachment point of the oligomeric compound is any reactive site on the nucleoside, modified nucleoside or internucleoside bond.

[0352] As used herein, "cleavable moiety" and "cleavable bond" refer to a cleavable bond or a group of atoms that can be split or broken under certain physiological conditions. In certain embodiments, the cleavable moiety is a cleavable bond. In certain embodiments, the cleavable moiety comprises a cleavable bond. In certain embodiments, the cleavable moiety is a group of atoms. In certain embodiments, the cleavable moiety is selectively cleaved inside a cell or an intracellular compartment such as a lysosome. In certain embodiments, the cleavable moiety is selectively degraded by an endogenous enzyme such as a nuclease. In certain embodiments, the cleavable moiety comprises a group of atoms having one, two, three, four or more than four cleavable bonds.

[0353] In certain embodiments, the conjugate group comprises a cleavable moiety. In certain such embodiments, the cleavable moiety covalently links the oligomeric compound to the conjugate linker. In certain such embodiments, the cleavable moiety covalently links the oligomeric compound to the cell targeting moiety.

[0354] In certain embodiments, the cleavable bond is selected from among amide, polyamide, ester, ether, phosphodiester or both esters, phosphate ester, carbamate, disulfide, or peptide. In certain embodiments, the cleavable bond is a phosphodiester ester. In certain embodiments, the cleavable bond is a phosphodiester ester or both esters. In certain embodiments, the cleavable moiety is a phosphodiester bond between the oligomeric compound and the remaining conjugate group. In certain embodiments, the cleavable moiety comprises a phosphodiester bond located between the oligomeric compound and the remaining conjugate group. In certain embodiments, the cleavable moiety comprises a phosphate or phosphodiester. In certain embodiments, the cleavable moiety is attached to the conjugate linker by either a phosphodiester or phosphorothioate bond. In certain embodiments, the cleavable moiety is attached to the conjugate linker by a phosphodiester bond. In certain embodiments, the conjugate group does not comprise a cleavable moiety.

[0355] In certain embodiments, the cleavable moiety is a cleavable nucleoside or modified nucleoside. In certain embodiments, the nucleoside or modified nucleoside comprises an optionally protected heterocyclic base selected from a purine, a substituted purine, a pyrimidine, or a substituted pyrimidine. In certain embodiments, the cleavable moiety is a nucleoside selected from uracil, thymine, cytosine, 4-N-benzoylcytosine, 5-methylcytosine, 4-N-benzoyl-5-methyl-cytosine, adenine, 6-N-benzoyladenine, guanine, and 2-N-isobutyrylguanine.

[0356] In certain embodiments, the cleavable moiety is a 2'-deoxynucleoside that is linked to either the 3'- or 5'-terminal nucleoside of the oligomeric compound by a phosphodiester bond and covalently linked to the remaining conjugate group by a phosphodiester or phosphorothioate bond. In certain embodiments, the cleavable moiety is a 2'-deoxyadenosine that is linked to either the 3'- or 5'-terminal nucleoside of the oligomeric compound by a phosphodiester bond and covalently linked to the remaining conjugate group by a phosphodiester or phosphorothioate bond. In certain embodiments, the cleavable moiety is a 2'-deoxyadenosine that is linked to the 3'-oxygen atom of the 3'-hydroxyl group of the 3'-terminal nucleoside or modified nucleoside by a phosphodiester bond. In certain embodiments, the cleavable moiety is a 2'-deoxyadenosine that is linked to the 5'-oxygen atom of the 5'-hydroxyl group of the 5'-terminal nucleoside or modified nucleoside by a phosphodiester bond. In certain embodiments, the cleavable moiety is attached to the 2' position of a nucleoside or modified nucleoside of an oligomeric compound. .

[0357] As used herein, "conjugate linker" in the context of a conjugate group refers to a portion of the conjugate group, including any atom or group of atoms that covalently attaches a cell targeting moiety to an oligomeric compound, either directly or through a cleavable moiety. In certain embodiments, the conjugate linker comprises a group selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether (-S-) and hydroxylamino (-ON(H)-). In certain embodiments, the conjugate linker comprises a group selected from alkyl, amino, oxo, amide and ether groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and amide groups. In certain embodiments, the conjugate linker comprises a group selected from alkyl and ether groups. In certain embodiments, the conjugate linker comprises at least one phosphorus linking group. In certain embodiments, the conjugate linker comprises at least one phosphodiester group. In certain embodiments, the conjugate linker comprises at least one neutral linking group.

[0358] In certain embodiments, the conjugate linker is covalently attached to the oligomeric compound. In certain embodiments, the conjugate linker is covalently attached to the oligomeric compound and the branching group. In certain embodiments, the conjugate linker is covalently attached to the oligomeric compound and the tethered ligand. In certain embodiments, the conjugate linker is covalently attached to the cleavable moiety. In certain embodiments, the conjugate linker is covalently attached to the cleavable moiety and the branching group. In certain embodiments, the conjugate linker is covalently attached to the cleavable moiety and the tethered ligand. In certain embodiments, the conjugate linker comprises one or more cleavable bonds. In certain embodiments, the conjugate group does not comprise a conjugate linker.

[0359] As used herein, "branched group" refers to an atomic group having at least three positions capable of forming covalent bonds to two or more tether-ligands and the remainder of the conjugate group. In general, the branched group provides multiple reactive sites for connecting the tethered ligand to the oligomeric compound via a conjugate linker and / or a cleavable moiety. In certain embodiments, the branched group comprises a group selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain embodiments, the branched group comprises a branched aliphatic group comprising a group selected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxylamino groups. In certain such embodiments, the branched aliphatic group comprises a group selected from alkyl, amino, oxo, amide, and ether groups. In certain such embodiments, the branched aliphatic group comprises a group selected from alkyl, amino, and ether groups. In certain such embodiments, the branched aliphatic group comprises a group selected from alkyl, amino, and ether groups. In certain such embodiments, the branched aliphatic group comprises a group selected from alkyl and ether groups. In certain embodiments, the branched group comprises a monocyclic or polycyclic ring system.

[0360] In certain embodiments, the branching group is covalently attached to the conjugate linker. In certain embodiments, the branching group is covalently attached to the cleavable moiety. In certain embodiments, the branching group is covalently attached to each of the conjugate linker and the tethered ligand. In certain embodiments, the branching group comprises one or more cleavable bonds. In certain embodiments, the conjugate group does not comprise a branching group.

[0361] In certain embodiments, the conjugate groups provided herein include a cell targeting moiety having at least one tethered ligand. In certain embodiments, the cell targeting moiety includes two tethered ligands covalently attached to a branching group. In certain embodiments, the cell The targeting moiety comprises three tethered ligands covalently attached to branching groups.

[0362] As used herein, "tether" refers to an atomic group that connects a ligand to the remainder of a conjugate group. In certain embodiments, each tether is a linear aliphatic group that includes one or more groups selected from alkyl groups, substituted alkyl groups, ether groups, thioether groups, disulfide groups, amino groups, oxo groups, amide groups, phosphodiester groups, and polyethylene glycol groups, in any combination. In certain embodiments, each tether is a linear aliphatic group that includes one or more groups selected from alkyl groups, ether groups, thioether groups, disulfide groups, amino groups, oxo groups, amide groups, and polyethylene glycol groups, in any combination. In certain embodiments, each tether is a linear aliphatic group that includes one or more groups selected from alkyl groups, substituted alkyl groups, phosphodiester groups, ether groups, and amino groups, oxo groups, and amide groups, in any combination. In certain embodiments, each tether is a linear aliphatic group that includes one or more groups selected from alkyl groups, substituted alkyl groups, phosphodiester groups, ether groups, and amino groups, oxo groups, and amide groups, in any combination. In certain embodiments, each tether is a linear aliphatic group that includes one or more groups selected from alkyl groups, amino groups, and oxo groups, in any combination. In certain embodiments, each tether is a linear aliphatic group that includes one or more groups selected from alkyl groups and oxo groups, in any combination. In certain embodiments, each tether is a linear aliphatic group that includes one or more groups selected from alkyl and phosphodiester, in any combination. In certain embodiments, each tether includes at least one phosphorus or neutral linking group.

[0363] In certain embodiments, the tether comprises one or more cleavable bonds. In certain embodiments, each tethered ligand is attached to a branching group. In certain embodiments, each tethered ligand is attached to a branching group via an amide group. In certain embodiments, each tethered ligand is attached to a branching group via an ether group. In certain embodiments, each tethered ligand is attached to a branching group via a phosphorus linking group or a neutral linking group. In certain embodiments, each tethered ligand is attached to a branching group via a phosphodiester group. In certain embodiments, each tether is attached to a ligand via either an amide group or an ether group. In certain embodiments, each tether is attached to a ligand via an ether group.

[0364] In certain embodiments, each tether comprises a chain length between the ligand and the branching group of about 8 to about 20 atoms. In certain embodiments, each tether comprises a chain length between the ligand and the branching group of about 10 to about 18 atoms. In certain embodiments, each tether comprises a chain length of about 13 atoms.

[0365] In certain embodiments, the disclosure provides ligands, each of which is covalently attached to the remainder of the conjugate group via a tether. In certain embodiments, each ligand is selected to have affinity for at least one receptor on a target cell. In certain embodiments, the ligand is selected to have affinity for at least one receptor on the surface of a mammalian liver cell. In certain embodiments, the ligand is selected to have affinity for the hepatic asialoglycoprotein receptor (ASGP-R). In certain embodiments, each ligand is a carbohydrate. In certain embodiments, each ligand is independently selected from galactose, N-acetylgalactosamine, mannose, glucose, glucosamine, and fucose. In certain embodiments, each ligand is N-acetylgalactosamine (GalNAc). In certain embodiments, the targeting moiety comprises one to three ligands. In certain embodiments, the targeting moiety comprises three ligands. In certain embodiments, the targeting moiety comprises two ligands. In certain embodiments, the targeting moiety comprises one ligand. In certain embodiments, the targeting moiety comprises one ligand. In certain embodiments, the targeting moiety comprises three N-acetylgalactosamine ligands. In certain embodiments, the targeting moiety comprises two N-acetylgalactosamine ligands. In certain embodiments, the targeting moiety comprises one N-acetylgalactosamine ligand.

[0366] In certain embodiments, each ligand is a carbohydrate, a carbohydrate derivative, a modified carbohydrate, a polyvalent carbohydrate cluster, a polysaccharide, a modified polysaccharide, or a polysaccharide derivative. In certain embodiments, each ligand is an amino sugar or a thio sugar. For example, the amino sugar can be selected from a number of compounds known in the art, such as glucosamine, sialic acid, α-D-galactosamine, N-acetylgalactosamine, 2-acetamido-2-deoxy-D-galactopyranose (GalNAc), 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose (β-muramic acid), 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, and N-sulfo-D-glucosamine and N-glycoloyl-α-neuraminic acid. For example, the thiosugar may be selected from the group consisting of 5-thio-β-D-glucopyranose, methyl 2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose and ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

[0367] In certain embodiments, the conjugate groups provided herein comprise carbohydrate clusters. As used herein, "carbohydrate cluster" refers to a portion of a conjugate group in which two or more carbohydrate residues are attached to a branching group via a tether group (see, e.g., Maier et al., "Synthesis of Antisense Oligonucleotides," for examples of carbohydrate conjugate clusters). Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting,” Bioconjugate Chemistry, 2003,(14):18-29 (incorporated herein by reference in its entirety) or Rensen et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor,” J. Med. Chem. 2004,(47):5798-5808).

[0368] As used herein, "modified carbohydrate" means any carbohydrate that has one or more chemical modifications relative to a naturally occurring carbohydrate.

[0369] As used herein, "carbohydrate derivative" means any compound that can be synthesized using a carbohydrate as a starting material or intermediate.

[0370] As used herein, "carbohydrate" means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative.

[0371] In certain embodiments, conjugate groups are provided in which the cell targeting moiety has the formula:

[0372] [ka]

[0373] In certain embodiments, conjugate groups are provided in which the cell targeting moiety has the formula:

[0374] [ka]

[0375] In certain embodiments, conjugate groups are provided in which the cell targeting moiety has the formula:

[0376] [ka]

[0377] In certain embodiments, the conjugate group has the formula:

[0378] [ka]

[0379] In certain embodiments, the antisense oligonucleotide attached to the conjugate group shown in the formula above has the nucleobase sequence of SEQ ID NO:1914.

[0380] Representative U.S. patents, U.S. patent application publications, and international patent application publications that teach the preparation of some of the above-described conjugate groups, conjugated oligomeric compounds, such as antisense compounds containing conjugate groups, tethers, conjugate linkers, branching groups, ligands, cleavable moieties, and other modifications, include, but are not limited to, U.S. Pat. No. 5,994,517, U.S. Pat. No. 6,300,319, U.S. Pat. No. 6,660,720, U.S. Pat. No. 6,906,182, U.S. Pat. No. 7,262,177, U.S. Pat. No. 7,491,805, U.S. Pat. No. 8,106,022, U.S. Pat. No. 7,723,509, U.S. Pat. No. 2006 / 0148740, U.S. Pat. No. 2011 / 0123520, WO2013 / 033230, WO2014 / 179620, and WO2012 / 037254, each of which is incorporated by reference in its entirety.

[0381] Representative publications that teach the preparation of some of the above-mentioned conjugate groups, conjugated oligomeric compounds, such as antisense compounds containing conjugate groups, tethers, conjugate linkers, branching groups, ligands, cleavable moieties and other modifications include, but are not limited to, BIESSEN et al., "The Cholesterol Derivative of a Triantennary Galactoside with High Affinity for the Hepatic Asialoglycoprotein Receptor: a Potent C holesterol Lowering Agent” J.Med.Chem.(1995)38:1846-1852, BIESSEN et al., “Synthesis of Cluster Galactosides with High Affinity for the Hepatic Asialoglycoprotein Receptor” J.Med.Chem.(1995)38:1538-1546,LEE et al.,“New and more multivalent glyco-ligands for asialoglycoprotein receptor of mammalian hepatocytes efficient”Bioorganic&Medicinal Chemistry(2011)19:2494-2500,RENSEN et al.,“Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo” J.Biol.Chem.(2001)276(40):37577-37584,RENSEN et al.,“Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asialoglycoprotein Receptor” J.Med.Chem.(2004)47:5798-5808,SLIEDREGT et al.,“Design and Synthesis of Novel Amphiphilic Dendritic Galactosides for Selective Targeting of Liposomes to the Hepatic Asialoglycoprotein Receptor” J.Med.Chem.(1999)42:609-618 and Valentijn et al., “Solid-phase synthesis of lysine-based cluster galactosides with high affinity for the Asialoglycoprotein Receptor” Tetrahedron, 1997, 53(2), 759-770, each of which is incorporated herein by reference in its entirety.

[0382] In certain embodiments, conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, and dyes. Specific conjugate groups have been previously described, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), and the like. al., Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipids such as hexadecyl-rac-glycerol or triethyl-ammonium. 1,2-Di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0383] In certain embodiments, the conjugate group comprises an active drug substance, such as aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, benzothiadiazide, chlorothiazide, diazepines, indomethicine, barbiturates, cephalosporins, sulfa drugs, antidiabetics, antibacterial substances or antibiotics.

[0384] Some non-limiting examples of conjugate linkers include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), 4-(N-maleimidomethyl)cyclohexane-1-carboxylate succinimidyl (SMCC), and 6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include, but are not limited to, substituted C1-C 10 Alkyl, substituted or unsubstituted C2-C 10 Alkenyl, or substituted or unsubstituted C2-C 10Alkynyl is included, where a non-limiting list of preferred substituents includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

[0385] Conjugate groups may be attached to one or both termini of the oligonucleotide (terminal conjugate groups) and / or at any internal position.

[0386] In certain embodiments, the conjugate group is at the 3' end of the oligonucleotide of the oligomeric compound. In certain embodiments, the conjugate group is near the 3' end. In certain embodiments, the conjugate is at the 3' end of the oligomeric compound, but before one or more terminal nucleosides. In certain embodiments, the conjugate group is located within the terminal group.

[0387] In certain embodiments, the conjugate group is at the 5' end of the oligonucleotide of the oligomeric compound. In certain embodiments, the conjugate group is near the 5' end.

[0388] In certain embodiments, the modified oligonucleotides targeting AGT described herein further comprise a GalNAc conjugate group. In certain embodiments, the GalNAc conjugate group is 5'-trishexylamino-(THA)-C6 GalNAc3. In certain embodiments, the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate has the formula:

[0389] [ka]

[0390] In certain embodiments, the modified oligonucleotide is linked to the 5'-trishexylamino-(THA)-C6 GalNAc3 conjugate by a cleavable moiety. In certain embodiments, the cleavable moiety is a phosphate group.

[0391] Cell culture and antisense compound treatment The effect of antisense compounds on the level, activity or expression of AGT nucleic acid can be tested in vitro in various cell types. The cell types used for such analysis are available from suppliers (e.g., American Type Culture Collection, Manassas, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD), and the cells are cultured using commercially available reagents (e.g., Invitrogen Life Technologies, Carlsbad, CA) according to the supplier's instructions. Exemplary cell types include, but are not limited to, HepG2 cells, Hep3B cells, Huh7 (hepatocellular carcinoma) cells, primary hepatocytes, A549 cells, GM04281 fibroblasts and LLC-MK2 cells.

[0392] In vitro testing of antisense oligonucleotides Here we describe a method for treating cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

[0393] Generally, cells are treated with antisense oligonucleotides when the cells in culture reach approximately 60-80% confluence.

[0394] One commonly used reagent for introducing 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 obtain the desired final concentration of antisense oligonucleotide and a LIPOFECTIN® concentration that typically ranges from 2-12 ug / mL for 100 nM of antisense oligonucleotide.

[0395] Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE 2000® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with LIPOFECTAMINE 2000® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to obtain the desired final concentration of antisense oligonucleotide and typically 2 μg / mL of 100 nM of antisense oligonucleotide. Obtain a LIPOFECTAMINE 2000® concentration in the range of ∼12 ug / mL.

[0396] Another reagent used to introduce antisense oligonucleotides into cultured cells includes Cytofectin® (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides are mixed with Cytofectin® in OPTI-MEM® 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to obtain the desired final concentration of antisense oligonucleotide and a Cytofectin® concentration that typically ranges from 2-12 ug / mL for 100 nM antisense oligonucleotide.

[0397] Another reagent used to introduce antisense oligonucleotides into cultured cells includes Oligofectamine™ (Invitrogen Life Technologies, Carlsbad, CA). Antisense oligonucleotides are mixed with Oligofectamine™ in Opti-MEM® 1 reduced serum medium (Invitrogen Life Technologies, Carlsbad, CA) to obtain the desired oligonucleotide concentration, with the ratio of Oligofectamine™ to oligonucleotide being approximately 0.2-0.8 μL per 100 nM.

[0398] Another reagent used to introduce antisense oligonucleotides into cultured cells includes FuGENE6 (Roche Diagnostics Corp., Indianapolis, Ind.). Antisense oligomeric compounds are mixed with FuGENE6 in 1 mL of serum-free RPMI to obtain the desired oligonucleotide concentration, with the ratio of FuGENE6 to oligomeric compound being 1-4 μL per 100 nM.

[0399] Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation (Sambrook and Russell in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor laboratory Press, Cold Spring Harbor, New York. 2001).

[0400] Cells are treated with antisense oligonucleotides by standard techniques. Typically, cells are harvested 16-24 hours after antisense oligonucleotide treatment, at which point RNA or protein levels of the target nucleic acid are measured by methods well known in the art and described herein (Sambrook and Russell in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor Laboratory Press, 2003). Press, Cold Spring Harbor, New York. 2001. Generally, when treatments are performed in multiple replicates, data are expressed as the average of the replicates.

[0401] The concentration of antisense oligonucleotide used varies with different cell lines. The method of determining the optimal concentration of antisense oligonucleotide for a particular cell line is well known in the art (Sambrook and Russell in Molecular Cloning. A Laboratory Manual. Third Edition. Cold Spring Harbor laboratory Press, Cold Spring Harbor, New York. 2001). When transfected using LIPOFECTAMINE2000®, Lipofectin or Cytofectin, antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM. When transfected using electroporation, antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM.

[0402] RNA isolation RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. RNA isolation methods are well known in the art (Sambrook and Russell, Molecular Cloning: A Laboratory Manual). Manual,3 rd Ed., 2001). RNA is prepared using methods well known in the art, for example, using TRIZOL® Reagent (Invitrogen, Carlsbad, Calif.) following the manufacturer's recommended protocol.

[0403] Analysis of inhibition of target levels or expression Inhibition of AGT nucleic acid levels or expression can be assayed by a variety of methods well known in the art (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3 rd Ed., 2001). For example, target nucleic acid levels can be quantified, for example, by Northern blot analysis, competitive polymerase chain reaction (PCR) or quantitative real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. RNA isolation methods are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be performed using commercially available ABI PRISM® 7600, 7700 or 7900 Sequence Detection Kits. This can be conveniently performed using the PCR amplification system (available from PE-Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions.

[0404] Quantitative real-time PCR analysis of target RNA levels Quantitation of target RNA levels can be performed by quantitative real-time PCR using the ABI PRISM® 7600, 7700 or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to the manufacturer's instructions. Quantitative real-time PCR methods are well known in the art.

[0405] Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction to generate complementary DNA (cDNA), which is then used as a substrate for real-time PCR amplification. The RT and real-time PCR reactions are carried out sequentially in the same sample well. The RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, CA). The RT and real-time PCR reactions are carried out by methods well known to those skilled in the art.

[0406] The amount of gene (or RNA) target obtained by real-time PCR is normalized using the expression level of a gene whose expression is constant, such as Cyclophilin A, or by quantifying total RNA using RIBOGREEN® (Invitrogen, Inc., Carlsbad, CA). Cyclophilin A expression is quantified by real-time PCR, either simultaneously with the target, multiplexed, or separately. Total RNA is quantified using RIBOGREEN® RNA quantification reagent (Invitrogen, Inc., Eugene, OR). RIBOGREEN® RNA quantification reagent (Invitrogen, Inc., Eugene, OR) The method of RNA quantification by EEN® is taught by Jones, LJ et al. (Analytical Biochemistry, 1998, 265, 368-374). RIBOGREEN® fluorescence is measured using a CYTOFLUOR® 4000 instrument (PE Applied Biosystems).

[0407] Probes and primers are designed to hybridize to AGT nucleic acids. Methods for designing probes and primers for real-time PCR are well known in the art and can include the use of software such as PRIMER EXPRESS® software (Applied Biosystems, Foster City, Calif.).

[0408] Protein level analysis Antisense inhibition of AGT nucleic acid can be assessed by measuring AGT protein levels. AGT protein levels can be assessed or quantified by a variety of methods well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assay, protein activity assay (e.g., caspase activity assay), immunohistochemistry, immunocytochemistry, or fluorescence-activated cell sorting (FACS) (Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 30(1):131-135). rd Ed., 2001). Antibodies against the targets can be identified and obtained from a variety of commercial sources or can be prepared by conventional monoclonal or polyclonal antibody production techniques well known in the art.

[0409] In vivo testing of antisense compounds Antisense compounds, such as antisense oligonucleotides, are tested in animals to assess their ability to inhibit the expression of AGT and to cause phenotypic changes, such as reducing hypertension in vivo. Testing can be performed in normal animals or experimental disease models. For administration to animals, antisense oligonucleotides are formulated in a pharma- ceutically acceptable diluent, such as sterile water for injection or phosphate-buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of the dose and frequency of administration of antisense oligonucleotides depends on factors such as the route of administration and the weight of the animal. In one embodiment, after a period of treatment with antisense oligonucleotides, RNA is isolated from liver tissue and changes in AGT nucleic acid expression are measured. Changes in AGT protein levels can be measured directly. Changes in AGT expression can also be measured by determining the level of inhibition of the RAAS pathway. RAAS pathway-related diseases, disorders, and / or conditions can be used as markers for determining the level of AGT inhibition.

[0410] Specific Indications Certain embodiments of the present invention provide compounds, compositions, and methods for reducing AGT levels using the compounds and compositions.In certain embodiments, the present invention provides compounds, compositions, and methods for treating a subject using the compounds and compositions, comprising administering to the subject a therapeutically effective amount of the compounds or compositions.In certain embodiments, the subject has or is at risk of having a RAAS pathway-related disease, disorder, or condition.In certain embodiments, the compound or composition comprises an antisense compound.

[0411] In certain embodiments, administration of a therapeutically effective amount of an antisense compound targeted to an AGT nucleic acid is accompanied by monitoring the subject's serum or tissue levels of AGT to determine the subject's response to the antisense compound. The subject's response to administration of the antisense compound is used by a physician to determine the amount and duration of therapeutic intervention.

[0412] In certain embodiments, administration of antisense compounds targeted to AGT nucleic acid results in a reduction of AGT expression of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99% or 100% or a range defined by any two of these values. In certain embodiments, administration of antisense compounds targeted to AGT nucleic acid results in inhibition of RAAS pathway of at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99% or 100% or a range defined by any two of these values. In certain embodiments, administration of antisense compounds targeted to AGT nucleic acid results in a change in RAAS pathway-related disease, disorder, pathology, symptom or marker (e.g., hypertension or organ damage). In certain embodiments, administration of an AGT antisense compound increases or reduces a RAAS-related disease, disorder, condition, symptom or marker by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99% or 100%, or a range defined by any two of these values.

[0413] In certain embodiments, pharmaceutical compositions comprising antisense compounds targeted to AGT are used to prepare medicaments for reducing AGT levels. In certain embodiments, pharmaceutical compositions comprising antisense compounds targeted to AGT are used to prepare medicaments for treating patients suffering from or susceptible to RAAS-related diseases, disorders or conditions.

[0414] In certain embodiments, the reduction of AGT levels in a subject results in the treatment, amelioration, prevention, slowing of progression or slowing of onset of a disease, condition or disorder. In certain embodiments, the disease, condition or disorder is shortened life span, hypertension, hypertensive emergency (i.e., malignant hypertension), renal disease (e.g., chronic kidney disease, polycystic kidney disease), pre-eclampsia, Marfan syndrome, stroke, cardiac disease (e.g., myocardial infarction, heart failure, congestive heart failure, valvular heart disease), vascular aneurysm, abdominal aneurysm, peripheral arterial disease, organ damage, pulmonary arterial hypertension, obesity, metabolic syndrome, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) and RAAS-related disease, disorder and / or condition or symptoms thereof. In certain embodiments, the hypertension is non-treatment-resistant hypertension or treatment-resistant hypertension. In certain embodiments, the vascular aneurysm is an aortic aneurysm. In certain embodiments, the organ damage is myocardial hypertrophy or fibrosis of an organ or tissue. In certain embodiments, the organ is the heart, liver or kidney, and the tissue is derived from the heart, liver or kidney.

[0415] In certain embodiments, the reduction of AGT level in a subject results in the treatment, improvement, prevention, slowing of progression or slowing of onset of RAAS pathway-related disease, disorder or condition.In certain embodiments, the RAAS pathway-related disease, disorder or condition is shortened life span, hypertension, hypertensive emergency (i.e., malignant hypertension), renal disease (e.g., chronic kidney disease, polycystic kidney disease), pre-eclampsia, Marfan syndrome, stroke, heart disease (e.g., myocardial infarction, heart failure, congestive heart failure, valvular heart disease), vascular aneurysm, abdominal aneurysm, peripheral arterial disease, organ damage, pulmonary arterial hypertension, obesity, metabolic syndrome, NASH, NAFLD and other RAAS-related disease, disorder and / or condition or symptoms thereof.In certain embodiments, the hypertension is non-treatment-resistant hypertension or treatment-resistant hypertension.In certain embodiments, the vascular aneurysm is aortic aneurysm.In certain embodiments, the organ damage is myocardial hypertrophy or fibrosis of organ or tissue. In certain embodiments, the organ is the heart, liver or kidney, and the tissue is derived from the heart, liver or kidney.

[0416] In certain embodiments, compounds, compositions and methods are provided for modulating symptoms or markers of a disease, disorder and / or condition. In certain embodiments, the marker is: The condition may be selected from one or more of shortened life span, hypertension, hypertensive emergency (i.e., malignant hypertension), renal disease (e.g., chronic renal disease, polycystic kidney disease), pre-eclampsia, Marfan syndrome, stroke, cardiac disease (e.g., myocardial infarction, heart failure, congestive heart failure, valvular heart disease), vascular aneurysm, abdominal aneurysm, peripheral arterial disease, organ damage and other RAAS-related diseases, disorders and / or conditions or symptoms thereof.

[0417] Certain combination therapies In certain embodiments, the first agent comprising the antisense compound provided herein is co-administered with one or more second agents.In certain embodiments, the antisense compound is an antisense oligonucleotide.In certain embodiments, the antisense oligonucleotide is a modified oligonucleotide.

[0418] In certain embodiments, such second agent is designed to treat the same RAAS pathway-related disease, disorder or condition as the first agent described herein. In certain embodiments, such second agent is designed to treat a different disease, disorder or condition than the first agent described herein. In certain embodiments, such second agent is designed to treat an undesirable side effect of one or more pharmaceutical compositions described herein. In certain embodiments, such first agent is designed to treat an undesirable side effect of the second agent. In certain embodiments, the second agent is co-administered with the first agent to treat an undesirable side effect of the first agent. In certain embodiments, the second agent is co-administered with the first agent to provide a combined or additive effect. In certain embodiments, the second agent is co-administered with the first agent to provide a synergistic effect.

[0419] In certain embodiments, the co-administration of the first agent and the second agent allows the use of lower doses than the doses required to achieve the therapeutic or prophylactic effects when these agents are administered as independent therapies.In certain embodiments, the dose of the co-administered second agent is the same as the dose that the second agent is administered when it is administered alone.In certain embodiments, the dose of the co-administered second agent is higher than the dose that the second agent is administered when it is administered alone.

[0420] In certain embodiments, the first agent and one or more second agents are administered simultaneously. In certain embodiments, the first agent and one or more second agents are administered at different times. In certain embodiments, the first agent and one or more second agents are prepared together in a single pharmaceutical formulation. In certain embodiments, the first agent and one or more second agents are prepared separately.

[0421] In certain embodiments, the second agent includes, but is not limited to, certain treatments to reduce hypertension, dietary modifications, lifestyle modifications, antifibrotic and antihypertensive agents, such as RAAS inhibitors, endothelin receptor antagonists, neprilysin inhibitors, diuretics, calcium channel antagonists, sympathetic receptor blockers, sympathomimetics, and vasodilators.

[0422] Examples of treatments that can reduce hypertension include, but are not limited to, renal denervation and baroreceptor activation therapy.

[0423] Examples of RAS or RAAS inhibitors include ACE inhibitors (e.g., captopril, enalapril, fosinopril, lisinopril, perindopril, quinapril, ramipril, trandolapril, and benazepril), angiotensin II receptor antagonists (e.g., candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, and valsartan), renin inhibitors (e.g., aliskiren), aldosterone receptor antagonists (e.g., aldosterone receptor antagonists), and aldosterone receptor blockers (e.g., aldosterone receptor antagonists). These include, but are not limited to, vasoconstrictor antagonists (e.g., eplerenone, spironolactone, and finerenone).

[0424] Examples of endothelin receptor antagonists include ambrisentan, sitaxsentan, atrasentan, BQ-123, zibotentan, bosentan, macitentan, and tezosentan.

[0425] Examples of neprilysin inhibitors include sacubitril and omapatrilat.

[0426] Examples of diuretics include loop diuretics (e.g., bumetanide, ethacrynic acid, furosemide, torasemide), thiazide diuretics (e.g., epitizide, hydrochlorothiazide, chlorothiazide, and bendroflumethiazide), thiazide-like diuretics (e.g., indapamide, chlorthalidone, and metolazone), and potassium-sparing diuretics (e.g., amiloride, triamterene, and spironolactone).

[0427] Examples of calcium channel antagonists include dihydropyridines (eg, amlodipine, felodipine, isradipine, lercanidipine, nicardipine, nifedipine, nimodipine, and nitrendipine) and non-dihydropyridines (eg, diltiazem and verapamil).

[0428] Examples of sympathetic receptor blockers include beta blockers (e.g., atenolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, and timolol), alpha blockers (e.g., doxazosin, phentolamine, indoramin, phenoxybenzamine, prazosin, terazosin, and tolazoline), and mixed alpha + beta blockers (e.g., bucindolol, carvedilol, and labetalol).

[0429] Examples of vasodilators include sodium nitroprusside and hydralazine and its derivatives. Examples of sympathomimetics include alpha-2 agonists (e.g., clonidine, guanabenz, methyldopa, and moxonidine). Further examples of antihypertensives include guanethidine, reserpine, and the like.

[0430] A second agent may be used in combination with the therapeutic compounds described herein to alleviate a disease, disorder and / or condition, such as hypertension, organ damage, etc.

[0431] Specific Compounds Preferred antisense compounds with beneficial properties that improve their use as human therapeutics are shown in the examples herein.For the sake of brevity, only the tests that contribute to the selection of preferred antisense compounds are described.For ease of reference, a limited summary of examples is provided below.

[0432] Over 2000 antisense compounds containing MOE-containing and / or cEt-containing gapmer motifs targeting human AGT have been designed. Example 1 shows representative single-dose inhibition data for the effect of over 2000 potent antisense compounds on human AGT mRNA tested in HepG2 cells.

[0433] Of the more than 2000 antisense compounds tested in vitro by single dose, over 160 antisense compounds were selected for testing in dose-dependent inhibition studies to determine the 50% inhibitory concentration (IC 50 ) was determined (Example 2).

[0434] Based on in vitro dose-response studies, as described in the exemplary studies in Example 3 More than 50 antisense compounds were selected for single-dose efficacy and tolerability testing in human AGT transgenic (huAGT tg) mice. Of the 50+ antisense compounds, approximately 14 antisense compounds were further selected for dose-response and tolerability testing in huAGT tg mice (Example 4).

[0435] Nine antisense compounds that exhibited significant efficacy and tolerability in huAGT mice were selected for further testing: viscosity assay (Example 5); testing in CD1 mice (Example 6) and Sprague-Dawley rats (Example 7) to evaluate the tolerability of the antisense compounds; testing in monkey hepatocytes to test the cross-species efficacy of inhibiting monkey AGT (Example 8); and testing in cynomolgus monkeys to evaluate efficacy and tolerability (Example 9). The antisense compounds in the test described in Example 9 were tested in cynomolgus monkeys, but because the cynomolgus monkey AGT sequence was not available for comparison with the sequence of the antisense compounds, the sequence of the antisense compounds was compared to that of the closely related rhesus monkey (Example 8).

[0436] Based on the extensive characterization of the nine antisense compounds, the sequence of antisense compound ISIS654472 (parent compound) was selected for further testing (Example 10). Six antisense compounds were designed with the sequence of parent compound ISIS654472, but with different chemical modifications and GalNAc conjugates. The six newly designed compounds were administered to CD1 mice (Example 10) and Sprague-Dawley rats (Example 11) to test the tolerability in these animal models. Among the six GalNAc conjugated antisense compounds, compound ISIS757456 was selected to be tested in huAGT mice and compared with parent antisense compound ISIS654472. ISIS757456 showed an 8-fold improvement in potency compared with non-conjugated compound ISIS654472.

[0437] Thus, provided herein are antisense compounds that have one or more of the optional characteristics that make them useful for use as therapeutic agents. In certain embodiments, provided herein are antisense compounds that include modified oligonucleotides described herein that are targeted to or can specifically hybridize to a region of a nucleotide sequence selected from any of SEQ ID NOs: 1-6.

[0438] In certain embodiments, certain antisense compounds described herein are effective because they are effective in inhibiting AGT expression. In certain embodiments, the compound or composition inhibits AGT by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

[0439] In certain embodiments, certain antisense compounds described herein have in vitro IC 50 is effective since it is less than 20 μM, less than 10 μM, less than 8 μM, less than 5 μM, less than 2 μM, less than 1 μM, less than 0.9 μM, less than 0.8 μM, less than 0.7 μM, less than 0.6 μM or less than 0.5 μM (as described in Example 2).

[0440] In certain embodiments, certain antisense compounds described herein have an in vivo 50% effective dose (ED 50 ) is ≦10mpk / week, ≦9mpk / week, ≦8mpk / week, ≦7mpk / week, ≦6mpk / week, ≦5mpk / week, ≦4mpk / week, ≦3mpk / week, ≦2mpk / week, or ≦1mpk / week. In certain embodiments, preferred antisense compounds, such as the antisense compound ISIS 757456, have an ED of ≦3mpk / week, as shown in Example 12. 50 has.

[0441] In certain embodiments, certain antisense compounds described herein are effective because they have a viscosity of less than 40 cP, less than 35 cP, less than 30 cP, less than 25 cP, less than 20 cP, less than 15 cP, or less than 12 cP, as described in Example 5. Oligonucleotides having a viscosity of more than 40 cP are less than optimal.

[0442] In certain embodiments, certain antisense compounds described herein are well tolerated, as demonstrated by the in vivo tolerability measurements described in the Examples.In certain embodiments, certain antisense compounds described herein are well tolerated, as demonstrated by having an increase in ALT and / or AST levels of 3-fold, 2-fold, or 1.5-fold or less relative to saline-treated animals.

[0443] In certain embodiments, certain antisense compounds described herein exhibit inhibitory efficacy of greater than 50% in transgenic mice, an ED of ≦5 mpk / week, 50 , a viscosity of less than 40 cP, and a no more than 3-fold increase in ALT and / or AST.

[0444] In certain embodiments, ISIS757456 (SEQ ID NO: 1914) is preferred. This compound was found to be a potent inhibitor in AGT transgenic mice and a well-tolerated antisense compound in CD-1 mice. Mice showed up to a 3-fold increase in ALT and / or AST levels relative to saline-treated animals. ED in huAGT transgenic mice 50 was ≦3mpk / week. EXAMPLES

[0445] Non-Limiting Disclosure and Incorporation by Reference While certain compounds, compositions and methods described herein have been specifically described according to certain embodiments, the following examples are provided solely to illustrate, but not to limit, the compounds described herein. Each of the references cited in this application is incorporated herein by reference in its entirety.

[0446] Example 1: Antisense inhibition of human angiotensinogen (AGT) in HepG2 cells More than 2000 antisense oligonucleotides targeting human AGT nucleic acid were designed and tested for their in vitro effects on AGT mRNA in a series of experiments with similar culture conditions. The results of representative antisense oligonucleotides are shown in the table below.

[0447] The newly designed chimeric antisense oligonucleotides in the table below were designed as MOE and / or cEt containing gapmers. The MOE containing oligonucleotides have a central gap segment comprising 2'-deoxynucleosides, with wing segments arranged in the 5' and 3' directions. At least one nucleoside in the 5' wing segment and / or one nucleoside in the 3' wing segment has a 2'-MOE sugar modification. The cEt containing oligonucleotides have a central gap segment comprising 2'-deoxynucleosides, with wing segments arranged in the 5' and 3' directions. At least one nucleoside in the 5' wing segment and / or one nucleoside in the 3' wing segment has a cEt sugar modification. In some cases, oligonucleotides were designed to contain both MOE and cEt. The MOE and cEt containing oligonucleotides have a central gap segment comprising 2'-deoxynucleosides, with wing segments arranged in the 5' and 3' directions. At least one nucleoside in the 5' wing segment and / or one nucleoside in the 3' wing segment has a cEt sugar modification. The oligonucleotide has wing segments disposed at the 5' and 3' orientations, and at least one nucleoside in the 5' wing segment and / or one nucleoside in the 3' wing segment has an MOE and / or a cEt sugar modification.

[0448] The "Chemical Structure" column describes the sugar modification of each oligonucleotide. "k" indicates a cEt sugar modification, "d" indicates a deoxyribose, and "e" indicates an MOE modification. The internucleoside linkages in each gapmer are phosphorothioate (P=S) linkages. All cytosine residues in each gapmer are 5-methylcytosine.

[0449] "Start site" indicates the 5'-most nucleoside in the human gene sequence to which the gapmer is targeted. "End site" indicates the 3'-most nucleoside in the human gene sequence to which the gapmer is targeted. Each gapmer shown in the table below targets either the human AGT mRNA, shown herein as SEQ ID NO: 1 (GENBANK Accession No. NM_000029.3), and / or the human AGT genomic sequence, shown herein as SEQ ID NO: 2 (GENBANK Accession No. NT_167186.1, truncated from nucleotides 24354000 to 24370100).

[0450] Table 1 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 4500 nM of antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 (forward sequence CCCTGATGGGAGCCAGTGT, shown herein as SEQ ID NO: 8; reverse sequence AGCAGGGAGAAGCCCTTCA, shown herein as SEQ ID NO: 9; and probe sequence CCCTGGCTTTCAACACCTACGTCCACTX, shown herein as SEQ ID NO: 10, where X is a fluorescent label) was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as % inhibition of AGT relative to untreated control cells.

[0451] [Table 2-1]

[0452] [Table 2-2]

[0453] [Table 2-3]

[0454] [Table 2-4]

[0455] Table 2 shows the percentage of AGT mRNA inhibition by additional gapmer oligonucleotides. HepG2 cells cultured at a density of about 20,000 cells / well were transfected with 4,000 nM of antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage of AGT inhibition relative to untreated control cells.

[0456] [Table 3-1]

[0457] [Table 3-2]

[0458] [Table 3-3]

[0459] [Table 3-4]

[0460] [Table 3-5]

[0461] [Table 3-6]

[0462] [Table 3-7]

[0463] [Table 3-8]

[0464] [Table 3-9]

[0465] [Table 3-10]

[0466] [Table 3-11]

[0467] Table 3 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 500 nM antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage inhibition of AGT relative to untreated control cells.

[0468] [Table 4-1]

[0469] [Table 4-2]

[0470] [Table 4-3]

[0471] [Table 4-4]

[0472] [Table 4-5]

[0473] [Table 4-6]

[0474] [Table 4-7]

[0475] [Table 4-8]

[0476] [Table 4-9]

[0477] [Table 4-10]

[0478] Table 4 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 1000 nM of antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage inhibition of AGT relative to untreated control cells.

[0479]

Table 5-1

[0480]

Table 5-2

[0481]

Table 5-3

[0482]

Table 5-4

[0483]

Table 5-5

[0484]

Table 5-6

[0485]

Table 5-7

[0486]

Table 5-8

[0487]

Table 5-9

[0488] Table 5 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 1000 nM antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage inhibition of AGT relative to untreated control cells.

[0489] [Table 6-1]

[0490] [Table 6-2]

[0491] [Table 6-3]

[0492] [Table 6-4]

[0493] [Table 6-5]

[0494] [Table 6-6]

[0495] [Table 6-7]

[0496] Table 6 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 1000 nM of antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS4039 (forward sequence GGACAAGGTGGAGGTCTCA, shown herein as SEQ ID NO: 11; reverse sequence AGATCCTGCAGCACCAGTTG, shown herein as SEQ ID NO: 12; and probe sequence ATGAAGAAACTATCTCCCCGGACCATCCAX, shown herein as SEQ ID NO: 13, where X is a fluorescent label) was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as % inhibition of AGT relative to untreated control cells.

[0497] [Table 7-1]

[0498] [Table 7-2]

[0499] [Table 7-3]

[0500] Table 7 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 1000 nM antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS4039 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage inhibition of AGT relative to untreated control cells.

[0501] [Table 8-1]

[0502] [Table 8-2]

[0503] [Table 8-3]

[0504] [Table 8-4]

[0505] [Table 8-5]

[0506] [Table 8-6]

[0507] Table 8 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 4000 nM of antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage inhibition of AGT relative to untreated control cells.

[0508] [Table 9-1]

[0509] [Table 9-2]

[0510] [Table 9-3]

[0511] [Table 9-4]

[0512] [Table 9-5]

[0513] [Table 9-6]

[0514] [Table 9-7]

[0515] Table 9 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 4000 nM antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS4039 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage inhibition of AGT relative to untreated control cells.

[0516] [Table 10-1]

[0517] [Table 10-2]

[0518] [Table 10-3]

[0519] [Table 10-4]

[0520] Table 10 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 4000 nM antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage inhibition of AGT relative to untreated control cells.

[0521]

Table 11-1

[0522]

Table 11-2

[0523]

Table 11-3

[0524]

Table 11-4

[0525]

Table 11-5

[0526]

Table 11-6

[0527]

Table 11-7

[0528]

Table 11-8

[0529]

Table 11-9

[0530]

Table 11-10

[0531] [Table 11-11]

[0532] [Table 11-12]

[0533] [Table 11-13]

[0534] [Table 11-14]

[0535] [Table 11-15]

[0536] Table 11 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 500 nM of antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are presented as % inhibition of AGT relative to untreated control cells.

[0537] [Table 12-1]

[0538] [Table 12-2]

[0539] [Table 12-3]

[0540] Table 12 shows the percentage of AGT mRNA inhibition by antisense oligonucleotides. HepG2 cells cultured at a density of about 20,000 cells / well were transfected with 3,000 nM of antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage of AGT inhibition relative to untreated control cells.

[0541] [Table 13-1]

[0542] [Table 13-2]

[0543] [Table 13-3]

[0544] [Table 13-4]

[0545] [Table 13-5]

[0546] Table 13 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 4000 nM antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage inhibition of AGT relative to untreated control cells.

[0547] [Table 14-1]

[0548] [Table 14-2]

[0549] [Table 14-3]

[0550] [Table 14-4]

[0551] [Table 14-5]

[0552] [Table 14-6]

[0553] [Table 14-7]

[0554] [Table 14-8]

[0555] [Table 14-9]

[0556] Table 14 shows AGT mRNA inhibition in HepG2 cells cultured at a density of 20,000 cells / well and transfected with 4000 nM antisense oligonucleotides using electroporation. After a treatment period of about 24 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as the percentage inhibition of AGT relative to untreated control cells.

[0557] [Table 15-1]

[0558] [Table 15-2]

[0559] [Table 15-3]

[0560] [Table 15-4]

[0561] [Table 15-5]

[0562] [Table 15-6]

[0563] [Table 15-7]

[0564] [Table 15-8]

[0565] [Table 15-9]

[0566] [Table 15-10]

[0567] [Table 15-11]

[0568] [Table 15-12]

[0569] Example 2: Dose-dependent antisense inhibition of human angiotensinogen (AGT) in HepG2 cells Among more than 2000 antisense oligonucleotides designed and tested in the in vitro single dose assay described in Example 1, several antisense oligonucleotides exhibiting significant inhibition of AGT mRNA were selected and further tested at various doses in HepG2 cells. The results of exemplary antisense oligonucleotides tested in several series of experiments are shown in the table below.

[0570] Cells were plated at a density of 20,000 cells / well and transfected using electroporation with antisense oligonucleotides at concentrations of 0.406 μM, 0.813 μM, 1.63 μM, 3.25 μM, 6.5 μM, and 13.0 μM, as set forth in Table 15 below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and AGT mRNA was analyzed. AGT levels were measured by quantitative real-time PCR. mRNA levels were measured using human primer probe set RTS3721. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are shown as % inhibition of AGT relative to untreated control cells. The 50% inhibitory concentration (IC 50 ) are also shown. AGT mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide-treated cells.

[0571] [Table 16]

[0572] Cells were cultured at a density of 20,000 cells / well and transfected using electroporation with antisense oligonucleotides at 39.1 nM, 156.3 nM, 625.0 nM, 2500 nM and 10,000 nM concentrations as set forth in Table 16 below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are presented as % inhibition of AGT relative to untreated control cells. Results are the average of duplicate experiments. The 50% inhibitory concentration (IC 50 ) are also shown. AGT mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide-treated cells.

[0573] [Table 17]

[0574] Cells were cultured at a density of 20,000 cells / well and transfected using electroporation with antisense oligonucleotides at 312.5 nM, 625 nM, 1250 nM, 2500 nM and 5000 nM concentrations as set forth in Tables 17 and 18 below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are presented as % inhibition of AGT relative to untreated control cells. Results are the average of duplicate experiments. The 50% inhibitory concentration (IC 50 ) are also shown. AGT mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide-treated cells.

[0575] [Table 18-1]

[0576] [Table 18-2]

[0577] [Table 19]

[0578] Cells were cultured at a density of 20,000 cells / well and transfected using electroporation with antisense oligonucleotides at 37 nM, 111 nM, 333 nM, 1,000 nM and 3,000 nM concentrations as set forth in Table 19 below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are presented as % inhibition of AGT relative to untreated control cells. Results are the average of duplicate experiments. The 50% inhibitory concentration (IC 50 ) are also shown. AGT mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide-treated cells.

[0579] [Table 20]

[0580] Cells were cultured at a density of 20,000 cells / well and transfected using electroporation with antisense oligonucleotides at concentrations of 12.3 nM, 37 nM, 111 nM, 333 nM, 1,000 nM and 3,000 nM, as specified in Table 20 below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are presented as % inhibition of AGT relative to untreated control cells. Results are the average of duplicate experiments. The 50% inhibitory concentration (IC 50 ) are also shown. AGT mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide-treated cells.

[0581] [Table 21]

[0582] Cells were cultured at a density of 20,000 cells / well and transfected using electroporation with antisense oligonucleotides at 0.33 μM, 1.0 μM, 3.0 μM and 9.0 μM concentrations as set forth in Table 21 below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are presented as % inhibition of AGT relative to untreated control cells. The 50% inhibitory concentration (IC) of each oligonucleotide was calculated. 50 ) are also shown. AGT mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide-treated cells.

[0583] [Table 22-1]

[0584] [Table 22-2]

[0585] Cells were cultured at a density of 20,000 cells / well and transfected using electroporation with antisense oligonucleotides at 0.44 μM, 1.33 μM, 4.0 μM and 12.0 μM concentrations as set forth in Table 22 below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and AGT mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3721 was used to measure mRNA levels. AGT mRNA levels were adjusted according to total RNA content measured by RIBOGREEN®. Results are presented as % inhibition of AGT relative to untreated control cells. The 50% inhibitory concentration (IC) of each oligonucleotide was calculated.50 ) are also shown. AGT mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide-treated cells.

[0586] [Table 23-1]

[0587] [Table 23-2]

[0588] Example 3: Tolerability and efficacy of single-dose treatment with antisense oligonucleotides targeting human AGT in a transgenic mouse model We created a transgenic (Tg) mouse model "huAGT" and evaluated the efficacy of antisense oligonucleotides in this huAGT Tg model. AGT antisense oligonucleotides selected from in vitro studies were evaluated in huAGT mice.

[0589] The huAGT transgenic mice were housed on a 12-hour light / dark cycle and fed regular rat chow ad libitum. The animals were acclimated for at least 7 days in the research facility before the start of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2μ filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

[0590] Procedure #1 Female 10-week-old transgenic huAGT mice were divided into groups of 4 mice each. Eight groups received weekly subcutaneous injections of antisense oligonucleotides at a dose of 20 mg / kg for 2.5 weeks (3 treatments). One group of mice received weekly subcutaneous injections of PBS for 2.5 weeks. This saline-injected group served as a control group to compare with the oligonucleotide-treated groups.

[0591] RNA Analysis, Treatment #1 On day 17, total RNA was extracted from the liver and kidney of transgenic mice for real-time PCR analysis and measurement of human AGT mRNA expression. Results are shown as % inhibition relative to PBS control, normalized by RIBOGREEN®. As shown in Table 23, most antisense oligonucleotide treatments resulted in a significant reduction in human AGT mRNA compared to PBS control.

[0592] [Table 24]

[0593] Plasma Chemistry Markers, Treatment #1 To assess the effects of antisense oligonucleotides on liver and kidney function, plasma levels of aminotransferases, total bilirubin, and blood urea nitrogen (BUN) were measured using a clinical chemistry automated analyzer (Hitachi Olympus AU400e, Melville, NY). The results are shown in Table 24. Antisense oligonucleotides that caused changes in the levels of any of the liver or kidney function markers beyond the range expected for the antisense oligonucleotide were excluded from further study.

[0594] [Table 25]

[0595] Body and organ weights, treatment #1 The transgenic mice were weighed on day 15. The average body weight for each group is shown in the table below. Liver, spleen and kidney weights were measured at the end of the study and are shown in Table 25. Antisense oligonucleotides that produced any changes in organ weights beyond the range expected for the antisense oligonucleotide were excluded from further studies.

[0596] [Table 26]

[0597] Procedure #2 Groups of two huAGT mice each received subcutaneous injections of antisense oligonucleotides at a dose of 25 mg / kg / week for two weeks. One group of huAGT mice received subcutaneous injections of PBS to serve as a control group for comparison with the oligonucleotide-treated groups.

[0598] RNA Analysis, Treatment #2 On day 10, total RNA was extracted from the liver of transgenic mice for real-time PCR analysis and measurement of human AGT mRNA expression. Results are shown as percent inhibition relative to the PBS control, averaged for each group of two mice and normalized by RIBOGREEN®. As shown in Table 26, treatment with most antisense oligonucleotides significantly reduced the expression of human AGT mRNA compared to the PBS control. This resulted in a significant decrease.

[0599] [Table 27]

[0600] Plasma Chemistry Markers, Treatment #2 To evaluate the effect of antisense oligonucleotides on liver function, plasma levels of aminotransferases were measured using a clinical chemistry automated analyzer (Hitachi Olympus AU400e, Melville, NY). Results were averaged for each group of two mice and are shown in Table 27. Antisense oligonucleotides that caused changes in the levels of any of the liver function markers beyond the range expected for the antisense oligonucleotide were excluded from further study.

[0601] [Table 28]

[0602] Body and Organ Weights, Treatment #2 Body weights of huAGT mice in all treatment groups were measured on days 1 and 8. On day 10, animals were sacrificed and their livers were removed and weighed. Results were averaged for each group of 2 mice and are shown in Table 28. Antisense oligonucleotides that produced any changes in organ weights beyond the range expected for the antisense oligonucleotide were excluded from further studies.

[0603] [Table 29]

[0604] Procedure #3 Groups of two huAGT mice each received subcutaneous injections of antisense oligonucleotides at a dose of 25 mg / kg / week for two weeks. A group of four huAGT mice received subcutaneous injections of PBS and served as a control group to which the oligonucleotide treatment groups were compared.

[0605] RNA Analysis, Treatment #3 On day 10, total RNA was extracted from the liver of transgenic mice for real-time PCR analysis and measurement of human AGT mRNA expression. Results are shown as % inhibition relative to PBS control, averaged for each group of 2 mice and normalized by RIBOGREEN®. As shown in Table 29, most antisense oligonucleotide treatments resulted in a significant reduction in human AGT mRNA compared to PBS control.

[0606] [Table 30]

[0607] Plasma Chemistry Markers, Treatment #3 To evaluate the effect of antisense oligonucleotides on liver function, plasma levels of aminotransferases were measured using a clinical chemistry automated analyzer (Hitachi Olympus AU400e, Melville, NY). Results were averaged for each group of two mice and are shown in Table 30. Antisense oligonucleotides that caused changes in the levels of any of the liver function markers beyond the range expected for the antisense oligonucleotide were excluded from further study.

[0608] [Table 31]

[0609] Body and organ weights, treatment #3 Body weights of huAGT mice in all treatment groups were measured on days 1 and 8. On day 10, animals were sacrificed and their livers were removed and weighed. Results were averaged for each group of 2 mice and are shown in Table 31. Antisense oligonucleotides that produced any change in weight beyond the range expected for the antisense oligonucleotide were excluded from further studies.

[0610] [Table 32]

[0611] Procedure #4 Male 6-week-old transgenic huAGT mice were divided into groups of 3-4 mice each. Eight groups received weekly subcutaneous injections of antisense oligonucleotides at a dose of 5 mg / kg for 2 weeks. One group of mice received weekly subcutaneous injections of PBS for 2 weeks. This saline-injected group served as a control group for comparison with the oligonucleotide-treated groups.

[0612] RNA Analysis, Treatment #4 On day 17, total RNA was extracted from the liver and kidney of transgenic mice for real-time PCR analysis and measurement of human AGT mRNA expression. Results are shown as % inhibition relative to PBS control, normalized by RIBOGREEN®. As shown in Table 32, most antisense oligonucleotide treatments resulted in a significant reduction in human AGT mRNA compared to PBS control.

[0613] [Table 33]

[0614] Plasma Chemistry Markers, Treatment #4 On day 15, to assess the effect of antisense oligonucleotides on liver and kidney function, plasma levels of aminotransferases, total bilirubin, and blood urea nitrogen (BUN) were measured using a clinical chemistry automated analyzer (Hitachi Olympus AU400e, Melville, NY). The results are shown in Table 33. Antisense oligonucleotides that caused changes in the levels of any of the liver function markers beyond the range expected for the antisense oligonucleotide were excluded from further study.

[0615] [Table 34]

[0616] Body and organ weights, treatment #4 The body weights of the transgenic mice were measured on days 1, 8, and 13. The average weights for each group are shown below. The weights of the liver, spleen and kidneys were also measured on day 15 and are shown in Table 34. Antisense oligonucleotides that produced any change in weight beyond the range expected for the antisense oligonucleotide were excluded from further studies.

[0617] [Table 35]

[0618] Example 4: Tolerability and efficacy of repeated dose treatment with antisense oligonucleotides targeting human AGT in a transgenic mouse model Selected AGT antisense oligonucleotides from the single-dose study in huAGT transgenic mice were further evaluated in dose-response studies in huAGT transgenic mice.

[0619] The huAGT transgenic mice were housed on a 12-hour light / dark cycle and fed regular rat chow ad libitum. The animals were acclimated for at least 7 days in the research facility before the start of the experiment. Antisense oligonucleotides (ASOs) were prepared in buffered saline (PBS) and sterilized by filtering through a 0.2 micron filter. Oligonucleotides were dissolved in 0.9% PBS for injection.

[0620] Procedure #1 For a four-point dose-response study, male huAGT mice were divided into 37 groups of 4 mice each. Thirty-six groups received subcutaneous injections of antisense oligonucleotides at doses of 5, 10, 25, and 50 mg / kg / week for 2.5 weeks (a total of 3 doses). One group of huAGT mice received subcutaneous injections of saline to serve as a control group for comparison with the oligonucleotide-treated groups.

[0621] RNA Analysis, Treatment #1 On day 17, huAGT mice were sacrificed and total RNA was extracted from liver and kidney for real-time PCR analysis and measurement of human AGT mRNA expression. RT-PCR results are shown as the mean percent inhibition relative to saline-treated controls, normalized by RIBOGREEN®. As shown in Table 35, the selected antisense oligonucleotides Treatment with nucleotides resulted in a significant decrease in human AGT mRNA compared to saline controls.

[0622] [Table 36-1]

[0623] [Table 36-2]

[0624] Body and organ weights, treatment #1 Body weights of huAGT mice in all treatment groups were measured on days 1, 8 and 15 of the study. Results were averaged for each group of mice and are shown in Table 36.

[0625] [Table 37-1]

[0626] [Table 37-2]

[0627] Procedure #2 For another 4-point dose-response study, five potent antisense oligonucleotides targeting human AGT (ISIS numbers 620003, 654451, 654472, 654691 and 654999) were selected from the previous study and compared with ISIS 568637, which was potent in vitro and also potent and well tolerated in a single-dose study in huAGT transgenic mice. In this study, huAGT mice were divided into 25 groups of 3 mice each. These groups received 2 injections of antisense oligonucleotides subcutaneously over a 10-day period at doses of 1, 4, 10 and 40 mg / kg. One group of 3 huAGT mice received a subcutaneous injection of saline as a control group to compare with the oligonucleotide-treated groups.

[0628] RNA Analysis, Treatment #2 On day 10, antisense oligonucleotide-treated huAGT mice were sacrificed, and total RNA was extracted from liver and kidney for real-time PCR analysis and measurement of human AGT mRNA expression. Results are shown as the mean percent inhibition of mRNA relative to the PBS control group, normalized by RIBOGREEN®. As shown in Table 37, treatment with antisense oligonucleotides resulted in a significant reduction in human AGT mRNA compared to saline control.

[0629] [Table 38]

[0630] Plasma Chemistry Markers, Treatment #2 On day 10, to assess the effect of antisense oligonucleotides on liver and kidney function, plasma levels of aminotransferases, bilirubin, and BUN were measured using a clinical chemistry automated analyzer (Hitachi Olympus AU400e, Melville, NY). The results are shown in Table 38.

[0631] [Table 39]

[0632] Procedure #3 Five potent human AGT-targeted antisense oligonucleotides (ISIS numbers 568637, 594622, 594624, 594625 and 594627) were selected from a previous dose-response study for a three-point dose-response study. In this study, huAGT mice were divided into 16 groups of 3 mice each. These groups received two subcutaneous injections of antisense oligonucleotides at doses of 1, 5 and 15 mg / kg over a one-week period. One group of 3 huAGT mice received subcutaneous saline injections and served as a control group to compare with the oligonucleotide-treated groups.

[0633] RNA Analysis, Treatment #3 On day 8, total RNA was extracted from the liver and kidney of transgenic mice for real-time PCR analysis and measurement of human AGT mRNA expression. Results are shown as % inhibition relative to PBS control, normalized by RIBOGREEN®. As shown in Table 39, most antisense oligonucleotide treatments resulted in a significant reduction in human AGT mRNA compared to the PBS control.

[0634] [Table 40]

[0635] Plasma Chemistry Markers, Treatment #3 On day 8, to assess the effect of antisense oligonucleotides on liver and kidney function, plasma levels of aminotransferases, total bilirubin, and blood urea nitrogen (BUN) were measured using a clinical chemistry automated analyzer (Hitachi Olympus AU400e, Melville, NY). The results are shown in Table 40.

[0636] [Table 41]

[0637] Body and organ weights, treatment #3 The transgenic mice were weighed on days 1, 8 and 13, with the average for each group being shown in the table below. The liver, spleen and kidneys were also weighed on day 15, and are shown in Table 41.

[0638] [Table 42]

[0639] Example 5: Viscosity evaluation of nine potent AGT-targeting antisense oligonucleotides The viscosity of nine antisense oligonucleotides was measured in order to screen for and eliminate antisense oligonucleotides with a viscosity greater than 40 cP, which would be too viscous for administration to any subject.

[0640] Antisense oligonucleotides (32-35 mg) were weighed into a glass vial, 120 μL of water was added, and the antisense oligonucleotides were dissolved in solution by heating the vial at 50° C. A portion of the preheated sample (75 μL) was pipetted into a microviscometer (Cambridge). The temperature of the microviscometer was set to 25° C., and the viscosity of the sample was measured. Another portion of the preheated sample (20 μL) was pipetted into 10 mL of water for UV reading at 85° C., 260 nM (Cary UV instrument). The results are shown in Table 42. The results show that the antisense oligonucleotides tested do not exceed a viscosity of 40 cP.

[0641] [Table 43]

[0642] Example 6: Tolerance of nine potent human AGT-targeted antisense oligonucleotides (ASOs) in CD1 mice CD1® mice (Charles River, MA) are a versatile mouse model commonly used for safety and efficacy studies. Mice were treated with selected antisense oligonucleotides from the studies described above and assessed for changes in the levels of various plasma chemical markers.

[0643] The nine antisense oligonucleotides identified in the above examples were tested for tolerability in CD1 mice. Mice were divided into groups of four mice per group and subcutaneously injected with 50 mg / kg of antisense oligonucleotide twice a week for six weeks (a dose of 100 mg / kg / week). A group of male CD1 mice was subcutaneously injected with PBS twice a week for six weeks. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.

[0644] Body and organ weights The ASO-treated CD1 mice were weighed weekly. On day 43, the mice were sacrificed and organs were removed and weighed. Body and organ weights in grams (g) at the end of the study are shown in Table 43.

[0645] [Table 44]

[0646] Plasma Chemistry Markers To assess the effects of oligonucleotides on liver and kidney function, plasma levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), bilirubin, creatinine and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, NY). The results were averaged for each group and are shown in Table 44 for each item.

[0647] [Table 45]

[0648] In a separate study, the antisense compounds ISIS 568637, 594622, 594624, 594625 and 594627 were also tested in CD1 mice, but exhibited some tolerability problems and this study was terminated early.

[0649] Example 7: Tolerability of nine potent human AGT-targeted antisense oligonucleotides (ASOs) in Sprague-Dawley rats The Sprague-Dawley (SD) rat is a versatile model used for safety and efficacy evaluation. SD rats were treated with nine antisense oligonucleotides selected from the studies described in the above examples and evaluated for changes in the levels of various plasma chemical markers.

[0650] treatment Male SD rats were housed under a 12-hour light / dark cycle and fed Purina normal rat chow ad libitum. Rats were divided into groups of 4 rats per group and each group was subcutaneously injected with 100 mg / kg / week for 6 weeks. Rats were euthanized 48 hours after the last dose and organs and plasma were collected for further analysis.

[0651] organ weight The liver, spleen and kidney weights of rats treated with antisense oligonucleotides were measured at the end of the study. Body and organ weights in grams are shown in Table 45.

[0652] [Table 46]

[0653] Liver and kidney function To evaluate the effects of the nine antisense oligonucleotides on liver and kidney function, plasma levels of ALT (alanine aminotransferase), AST (aspartate aminotransferase), albumin, BUN, creatinine, and bilirubin were measured using a clinical chemistry automated analyzer (Hitachi Olympus AU400e, Melville, NY). Total urinary protein and urinary creatinine levels were also measured, and the ratio of total urinary protein to creatinine (P / C ratio) was calculated.

[0654] The results for each group were averaged and are shown in Table 46 for each item.

[0655] [Table 47]

[0656] histology Microscopic examination of the livers and kidneys of rats treated with antisense oligonucleotides revealed no significant adverse findings related to treatment.

[0657] In a separate study, the antisense compounds ISIS 568637, 594622, 594624, 594625 and 594627 were also tested in SD rats, but exhibited some tolerability problems and this study was prematurely terminated.

[0658] Example 8: Efficacy of nine potent human AGT-targeted antisense oligonucleotides (ASOs) in cynomolgus monkey hepatocytes At the time this study was undertaken, the cynomolgus genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database, so cross-reactivity with cynomolgus gene sequences could not be confirmed. Instead, the sequences of the antisense oligonucleotides used in cynomolgus were compared to rhesus sequences for complementarity. Antisense oligonucleotides with complementarity to rhesus sequences are expected to be fully cross-reactive with cynomolgus sequences as well.

[0659] The human antisense oligonucleotides tested had up to three mismatches with the rhesus genomic sequence (Genbank Accession No. NW_001109259.1 truncated from nucleotides 16090000-16106000, shown herein as SEQ ID NO: 7). The greater the complementarity between the human oligonucleotide and the rhesus sequence, the more likely the human oligonucleotide will be able to cross-react with rhesus and cyno sequences. The start and end sites of each oligonucleotide relative to SEQ ID NO: 7 are shown in Table 47. "Start site" indicates the 5'-most nucleotide in the rhesus gene sequence to which the gapmer is targeted.

[0660] Nine antisense oligonucleotides that showed significant inhibition of AGT mRNA and were tolerated in previous studies were selected and seeded in cryopreserved individual male primary cynomolgus monkey hepatocytes. Various doses were tested. These nine potent antisense oligonucleotides are listed in the table below.

[0661] [Table 48]

[0662] Cynomolgus monkey primary hepatocytes were cultured at a density of 35,000 cells / well and transfected using electroporation with antisense oligonucleotides at 0.156 μM, 0.313 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5.0 μM, 10.0 μM and 20.0 μM concentrations as set forth in Table 48 below. After a treatment period of approximately 24 hours, cells were washed, lysed and RNA was isolated. Monkey AGT mRNA levels were measured by quantitative real-time PCR using primer probe set RTS4039. AGT was expressed according to total RNA content measured by RIBOGREEN®. mRNA target levels were adjusted and results are shown as % inhibition of AGT relative to untreated control cells.

[0663] [Table 49]

[0664] In antisense oligonucleotide-treated cells, most monkey AGT mRNA levels were significantly reduced in a dose-dependent manner.

[0665] Example 9: Efficacy of antisense oligonucleotides targeting human AGT in cynomolgus monkeys In a 12-week dose-response study, cynomolgus monkeys were treated with nine antisense oligonucleotides selected from the studies described in the Examples above. The efficacy and tolerability of the antisense oligonucleotides, as well as their pharmacokinetic profiles in the liver and kidney, were evaluated.

[0666] treatment Prior to the study, the monkeys were placed in isolation and observed daily for general health during the study. The monkeys were 2-4 years old and weighed 2-4 kg. Ten groups of five male cynomolgus monkeys were randomly assigned to each group to receive subcutaneous injections of either antisense oligonucleotide or PBS. The monkeys received 40 mg / kg / week of antisense oligonucleotide once a week for 12 weeks (monkeys received two loading doses of 40 mg / kg on weeks 1 and 2) for a total of 15 doses. A control group of cynomolgus monkeys was injected with PBS in a similar manner and served as the control group.

[0667] Monkeys were observed twice daily for signs of illness or distress during the study. Animals experiencing more than temporary or minor pain or distress due to treatment, trauma, or disease were treated with approved analgesics or pain-relieving medications by veterinary staff in consultation with the study director. Animals in poor health or potentially moribund condition requiring further monitoring and possible euthanasia were identified. At the end of the 12-week study, monkeys were sacrificed and organs removed. The protocols described in the examples were approved by the Institutional Animal Care and Use Committee (IACUC).

[0668] Body and organ weights Body weights were assessed weekly and no significant effect of the antisense oligonucleotides on body weight was observed. Body weights were measured on day 77 and organ weights were measured on day 79 and are shown below in Table 49.

[0669] [Table 50]

[0670] Pharmacodynamics During the study, plasma, serum, and urine were collected for analysis. To evaluate the effect of nucleotides on liver and kidney function, plasma levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase), BUN, and bilirubin were measured using a clinical chemistry automated analyzer (Hitachi Olympus AU400e, Melville, NY) on Day 79. As shown in Table 50, no significant effect was observed on ALT, AST, BUN, and bilirubin.

[0671] [Table 51]

[0672] Furthermore, no significant changes in ECG, blood pressure, plasma electrolytes, proteinuria, inflammatory responses (eg, CRP levels) or renal accumulation were observed. Overall, the antisense oligonucleotides were well tolerated.

[0673] RNA analysis At the end of the study, RNA was extracted from the liver and kidney of the monkeys for real-time PCR analysis to measure AGT mRNA expression. Primer probe set RTS4039 was used. The results of each group were averaged and shown as % mRNA inhibition relative to PBS control, normalized by RIBOGREEN®. As shown in Table 51, treatment with antisense oligonucleotides produced different effects on AGT mRNA levels.

[0674] [Table 52]

[0675] Example 10: Tolerance of GalNAc-conjugated antisense oligonucleotides in CD-1 mice A leading candidate (ISIS 654472) 5-10-5 fully phosphorothioate MOE gapmer was selected from the above studies and used as the basis for the design of six 5'-trishexylamino-(THA)-C6 GalNAc3 (also known as "GalNAc")-conjugated 5-10-5 MOE gapmers with the same nucleotide sequence but different backbone structures as listed in Table 52 below. "s" is a phosphorothioate internucleoside linkage. "o" is a phosphodiester internucleoside linkage. "A" is an adenine nucleobase. "mC" is a 5-methylcytosine nucleobase. "G" is a guanine nucleobase. "T" is a thymine nucleobase. "e" indicates MOE modification. "d" indicates deoxyribose.

[0676] [Table 53]

[0677] For a three-point dose-response study, 16 groups of 4 CD1 mice each were subcutaneously injected with 10 mg / kg / week of GalNAc-conjugated antisense oligonucleotide for 4 weeks. One group of mice was subcutaneously injected with PBS twice a week for 6 weeks. The weights of ASO-treated CD1 mice were measured weekly. 48 hours after the last dose, mice were euthanized and organs and plasma were harvested for further analysis. Plasma and urine were collected, and plasma levels of aminotransferases, bilirubin, and BUN were measured using a clinical chemistry autoanalyzer (Hitachi Olympus AU400e, Melville, NY) to evaluate the effects of antisense oligonucleotides on liver and kidney function. At the end of the experiment, the liver, kidneys, and spleen were removed and weighed. The results were averaged for each group and are shown in Table 53.

[0678] [Table 54]

[0679] Example 11: Tolerance of GalNAc-conjugated ASO in SD rats Twenty-eight male SD rats were divided into seven groups of four rats per group. The rats were subcutaneously injected with PBS (untreated control) or 10 mg / kg / week of GalNAc-conjugated antisense oligonucleotides for four weeks.

[0680] Plasma and urine were collected and analyzed using a clinical chemistry autoanalyzer (Hitachi Olympus AU400e, Melville, NY) to assess the effects of antisense oligonucleotides on liver and kidney function. At the end of the experiment, the liver, kidneys, and spleen were removed and weighed.

[0681] Results are expressed as the average of four animals in each group and are shown in Table 54.

[0682] [Table 55]

[0683] Example 12: Dose-response comparison of unconjugated and GalNAc-conjugated antisense oligonucleotides in male and female huAGT mice As described in the previous example, huAGT mice are useful for testing the efficacy of antisense oligonucleotides. A dose-response comparison was performed between parent 5-10-5MOE gapmer (ISIS654472) and GalNAc-conjugated compound (ISIS757456) with the same sequence.As shown in Table 55, GalNAc-conjugated antisense oligonucleotides are 8 times more potent than non-conjugated antisense oligonucleotides.

[0684] [Table 56]

Claims

1. A compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 2250 to 2337 of SEQ ID NO:1, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to SEQ ID NO:

1.

2. A compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 8 contiguous nucleobases complementary to an equal length portion of nucleobases 2281 to 2300 of SEQ ID NO:1, wherein said nucleobase sequence of said modified oligonucleotide is at least 80% complementary to SEQ ID NO:

1.

3. 3. The compound of claim 1 or 2, wherein the modified oligonucleotide consists of 15-30, 15-25, 15-24, 16-24, 17-24, 18-24, 19-24, 20-24, 19-22, 20-22, 16-20, 16 or 20 linked nucleosides.

4. 3. The compound of claim 1 or 2, wherein the modified oligonucleotide comprises a nucleobase sequence comprising a portion of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleobases complementary to an equal length portion of SEQ ID NO:

1.

5. 10. The compound of any one of the preceding claims, wherein the nucleobase sequence of the modified oligonucleotide is at least 85%, at least 90%, at least 95% or 100% complementary to SEQ ID NO:

1.

6. A compound comprising a modified oligonucleotide comprising 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases of the nucleobase sequence of SEQ ID NOs: 14-2051.

7. 12-30 linked nucleosides and SEQ ID NO: 46, 53-54, 61, 68, 76, 83, 85, 93, 96-97, 109, 127, 129-130, 132, 134-15, 137-39, 142, 163-172, 180-184, 186, 189, 234, 236, 238-239, 267, 313, 411, 452, 463-470, 475-47 8, 480, 500-503, 512, 517-518, 524-526, 654, 689, 702, 725-726, 728, 738, 779, 786-787, 800, 808, 810-811, 825, 865, 868, 889, 894, 903, 905, 909, 954, 966, 1011, 1015, 1021, 1024, 1080, 1085, 1258-1259, 1261-1262, 1293-1294, 1299, 1325, 1470, 1472-1473, 1522, 1542, 1604, 1623-1624, 1667, 1670, 1682-1683, 1687, 1700, 1703-1704, 1708, 1714, 1716, 1719-1720, 1724-1726, 1729-1730, 1827, 1936, 1843- At least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 of the nucleic acid base sequences of 1844, 1846, 1886, 1893-1894, 1914, 1923, 1925, 1932, 1979, 1986, 1988, 1990, 2003, 2015, 2018, 2020, 2027-2028, 2035, 2037, 2039, and 2044 , at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases.

8. 20. A compound comprising a modified oligonucleotide comprising 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases of the nucleobase sequences of SEQ ID NOs: 238, 1714, 1719, 1893-1894, 1914, 1923, 1925, 2003.

9. A compound, comprising a modified oligonucleotide comprising 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or 20 consecutive nucleobases of the nucleobase sequence of SEQ ID NO:1914.

10. 2. A compound according to any one of the preceding claims which is single-stranded.

11. 2. A compound according to any one of the preceding claims which is double-stranded.

12. 2. A compound according to any one of the preceding claims, wherein at least one internucleoside linkage is a modified internucleoside linkage.

13. 13. The compound of claim 12, wherein at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.

14. 13. The compound of claim 12, wherein each modified internucleoside linkage is a phosphorothioate internucleoside linkage.

15. 2. The compound of any one of the preceding claims, wherein the modified oligonucleotide comprises at least one modified sugar.

16. 16. The compound of claim 15, wherein at least one modified sugar is a bicyclic sugar.

17. At least one modified sugar is 2'-O-methoxyethyl, constrained ethyl (cEt), 3'-fluoro-HNA or 4'-(CH ２ ) ｎ The compound of claim 15, comprising an -O-2' bridge, where n is 1 or 2.

18. 2. The compound according to any one of the preceding claims, wherein at least one nucleoside comprises a modified nucleobase.

19. 19. The compound of claim 18, wherein the modified nucleobase is 5-methylcytosine.

20. the modified oligonucleotide consists of 12 to 30 linked nucleosides; a. a gap segment consisting of a plurality of linked deoxynucleosides; b. a 5' wing segment consisting of a plurality of linked nucleosides; c. a 3' wing segment consisting of a plurality of linked nucleosides; the gap segment is disposed between the 5' wing segment and the 3' wing segment, and each nucleoside of each wing segment comprises a modified sugar. The compound according to any one of claims 1 to 5.

21. 21. The compound of claim 20, wherein the modified oligonucleotide consists of 16 to 20 linked nucleobases.

22. 21. The compound of claim 20, wherein the modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage.

23. 21. The compound of claim 20, wherein the modified oligonucleotide further comprises a modified nucleobase.

24. the modified oligonucleotide comprising: a. a gap segment consisting of 10 linked deoxynucleosides; b. a 5' wing segment consisting of 3 to 5 linked nucleosides; c. a 3' wing segment consisting of 3 to 5 linked nucleosides; consisting of 16 to 20 linked nucleosides, 21. The compound of claim 20, wherein the gap segment is disposed between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, each internucleoside linkage comprises a phosphorothioate linkage, and each cytosine residue is a 5-methylcytosine.

25. The following formula: mCes Aes mCes Aes Aes Ads mCds Ads Ads Gds mCds Tds Gds Gds Tds mCes Ges Ges Tes Te (SEQ ID NO: 1914); (In the formula, A = adenine, mC=5'-methylcytosine G = guanine, T = thymine; e=2′-O-methoxyethyl modified nucleoside; d=2′-deoxynucleoside, and s = phosphorothioate internucleoside linkage A compound comprising a modified oligonucleotide according to

26. 2. A compound according to any one of the preceding claims, further comprising a conjugate group.

27. 27. The compound of claim 26, wherein the conjugate group is a GalNAc moiety.

28. A modified oligonucleotide consisting of 20 linked nucleosides having a nucleobase sequence comprising at least 8 consecutive nucleobases of SEQ ID NO: 1914, said modified oligonucleotide comprising: a. a gap segment consisting of 10 linked deoxynucleosides; b. a 5' wing segment consisting of 5 linked nucleosides; c. a 3' wing segment consisting of 5 linked nucleosides; d. a GalNAc conjugate; the gap segment is disposed between the 5' wing segment and the 3' wing segment, each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar, each internucleoside linkage comprises a phosphorothioate linkage, and each cytosine residue is a 5-methylcytosine.

29. A modified oligonucleotide according to the formula: 【Chemistry 1】

30. 13. A composition comprising a compound or modified oligonucleotide according to any one of the preceding claims, or a salt thereof, and a pharma- ceutically acceptable carrier or diluent.

31. 13. A composition comprising a compound or a modified oligonucleotide according to any one of the preceding claims for use in therapy.

32. 13. A composition comprising a compound or a modified oligonucleotide according to any one of the preceding claims for use in the preparation of a medicament.

33. 10. A compound or modified oligonucleotide according to any one of the preceding claims for use in reducing AGT in a cell, tissue, organ or animal.

34. 10. A compound or modified oligonucleotide according to any one of the preceding claims for use in treating, preventing, ameliorating or delaying progression of a disease, disorder or condition associated with a RAAS pathway associated disease, disorder and / or condition in an animal.

35. 35. The compound or modified oligonucleotide of claim 34, wherein the disease, disorder or condition is hypertension.

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