Compositions and methods for inhibiting the expression of angiotensinogen (AGT) protein

Abstract

Compositions and methods are provided that inhibit angiotensinogen (AGT) protein expression. In particular, compositions and methods are provided that can be used to reduce AGT gene expression and to treat AGT-related diseases and disorders. AGT dsRNA agents, AGT antisense polynucleotide agents, compositions containing AGT dsRNA agents, and compositions containing AGT antisense polynucleotide agents are provided that can be used to reduce AGT expression in cells and subjects.

Description

[Technical Field]

[0001] Some embodiments of the present invention relate to compositions and methods useful for inhibiting the expression of angiotensinogen (AGT) protein. [Background technology]

[0002] The renin-angiotensin-aldosterone system (RAAS) plays an important role in blood pressure regulation. The RAAS cascade begins with the secretion of renin into the circulation by the juxtaglomerular cells of the kidney. Renin secretion is mediated by the release of Na in the distal tubule. + It is stimulated by several factors, including overloading, beta-sympathetic nerve stimulation, and / or reduced renal blood flow. Active renin in the plasma breaks down angiotensinogen (produced by the liver) into angiotensin I, which is subsequently converted to angiotensin II by circulating and locally expressed angiotensin-converting enzyme (ACE). Most of the action of angiotensin II on the RAAS is exerted by its binding to the angiotensin II type 1 receptor (AT1R), which converts Na + It results in arterial vasoconstriction, tubular and glomerular effects, such as improved regulation of reabsorption or glomerular filtration rate. In addition, other stimuli (such as adrenocorticotropic hormone, antidiuretic hormone, catecholamines, endothelin, and serotonin) and Mg 2+ and K. + Along with the level, AT1R stimulation leads to the release of aldosterone, followed by Na+ in the distal tubule. + and K. + This promotes the excretion of

[0003] For example, dysregulation of the RAAS resulting in excessive angiotensin II production and / or AT1R stimulation can cause hypertension, increase oxidative stress, promote inflammation, and hypertrophy in, for example, the heart, kidneys, and arteries, and can also lead to, for example, left ventricular fibrosis, arterial remodeling, and glomerular sclerosis.

[0004] Hypertension is the most prevalent and manageable disease in developed countries, affecting 20-50% of the adult population. Hypertension is a major risk factor for a variety of diseases, disorders, and conditions, including shortened life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, aneurysms (e.g., aortic aneurysms), peripheral vascular disease, cardiac trauma (e.g., cardiac enlargement or hypertrophy), and other cardiovascular-related diseases, disorders, and / or conditions. Furthermore, hypertension has been found to be a significant risk factor for cardiovascular morbidity and mortality, accounting for or comprising 62% of all strokes and 49% of all heart disease cases.In 2017, guidelines for the diagnosis, prevention, and treatment of hypertension were revised to provide lower blood pressure targets to further reduce the risk of hypertension-related diseases and disorders (e.g., Reboussin et al., Systematic Review for the 2017 ACC / AHA / AAPA / ABC / ACPM / AGS / APhA / ASH / ASPC / NMA / PCNA Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology / American Heart Association Task Force on Clinical Practice Guidelines. J Am Coll Cardiol. 2017 Nov7.pii:S0735-1097(17)41517-8.doi:10.1016 / j.jacc.2017.11.004; and Whelton et al. (2017 ACC / AHA / AAPA / ABC / ACPM / AGS / APhA / ASH / ASPC / NMA / PCNA Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology / American Heart Association Task Force on Clinical Practice Guidelines. J Am Coll Cardiol. 2017 Nov7.pii:S0735-1097(17)41517-8.doi:10.1016 / j.jacc.2017.11.004). Prevention,Detection,Evaluation,and Management of High Blood Pressure in Adults:A Report of the American College of Cardiology / American Heart Association Task Force on Clinical Practice Guidelines.J Am Coll Cardiol.2017 Nov7.pii:S0735-1097(17)41519-1.doi:10.1016 / j.jacc.2017.11.006).

[0005] Despite the large number of antihypertensive drugs available to treat hypertension, in more than two-thirds of subjects, symptoms cannot be controlled with a single antihypertensive drug and two or more antihypertensive drugs selected from different drug classes are required. This further reduces the number of subjects whose blood pressure is controlled due to poor compliance and increased side effects experienced with high drug use.

[0006] Therefore, there is a need in the art for alternative and combination therapies for the treatment of hypertension and other angiotensinogen-related disorders. Summary of the Invention

[0007] According to one aspect of the present invention, a double-stranded ribonucleic acid (dsRNA) agent is provided that inhibits angiotensinogen (AGT) expression, the dsRNA agent comprising a sense strand and an antisense strand, wherein nucleotides 2-18 of the antisense strand comprise a region of complementarity to an AGT RNA transcript, the region of complementarity comprising at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences set forth in Tables 1-4, and optionally comprising a targeting ligand. In some embodiments, the region of complementarity to an AGT RNA transcript comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides that differ by 3 or fewer nucleotides from one of the antisense sequences set forth in Tables 1-4. In certain embodiments, the antisense strand of the dsRNA is at least substantially complementary to a target region of any of SEQ ID NO: 519 and provided in one of Tables 1-4. In some embodiments, the antisense strand of the dsRNA is fully complementary to a target region of any of SEQ ID NO: 519 and provided in one of Tables 1-4. In some embodiments, the dsRNA agent comprises any one of the sense strand sequences shown in Tables 1-4, where the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. In certain embodiments, the dsRNA agent comprises any of the sense strand sequences shown in Tables 1-4, where the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent. In some embodiments, the dsRNA agent comprises any of the sense strand sequences shown in Tables 1-4. In some embodiments, the dsRNA agent comprises any of the sequences shown as duplex sequences in Tables 1-4.

[0008] In some embodiments, the dsRNA agent has the formula (A): 5'-Z1AGCUUGUUUGUGAAACZ2-3' (SEQ ID NO: 656)and a sense strand that differs from Z1 by 0, 1, 2, or 3 nucleotides, wherein Z1 is a nucleotide sequence comprising 0 to 15 nucleotide motifs and Z2 is selected from one of A, U, C, G, or is absent. In certain embodiments, Z1 is a nucleotide sequence comprising 1 to 4 nucleotide motifs. In certain embodiments, Z1 is a nucleotide sequence comprising 1, 2, 3, or 4 nucleotide motifs. In certain embodiments, Z2 is A. In certain embodiments, Z1 is a nucleotide sequence comprising a CACC or GACC motif. In certain embodiments, the Z1 nucleotide sequence comprises one of the following motifs: C, AC, UC, GC, CC, ACC, UCC, GCC, CCC, GACC, AACC, UACC, CACC, CGACC, CCGACC, ACCGACC, AACCGACC, CAACCGACC, CCAACCGACC. (SEQ ID NO: 657) ,UCCAACCGACC (SEQ ID NO: 658) ,UUCCAACCGACC (SEQ ID NO: 659) ,AUUCCAACCGACC (SEQ ID NO: 660) , AAUUCCAACCGACC (SEQ ID NO: 661) or GAAUUCCAACCGACC (SEQ ID NO: 662) In some embodiments, the dsRNA agent has the formula (B): 5'-Z3GUUUCACAAACAAGCUZ4-3' (SEQ ID NO: 663)and an antisense strand that differs by 0, 1, 2, or 3 nucleotides from Z3, wherein Z3 is selected from one of A, U, C, and G, or is absent, and Z4 is a nucleotide sequence comprising 0 to 15 nucleotide motifs. In certain embodiments, Z4 is a nucleotide sequence comprising 1 to 4 nucleotide motifs. In certain embodiments, Z4 is a nucleotide sequence comprising 1, 2, 3, or 4 nucleotide motifs. In certain embodiments, Z3 is U. In certain embodiments, the Z4 nucleotide sequence is selected from nucleotide sequences comprising a GGUC or GGUG motif. In certain embodiments, the Z4 nucleotide sequence is selected from nucleotide sequences comprising the following motifs: G, GU, GC, GA, GG, GGU, GGA, GGC, GGG, GGUG, GGUC, GGUU, GGUA, GGUCG, GGUCGG, GGUCGGU, GGUCGGUU, GGUCGGUUG, GGUCGGUUG. (SEQ ID NO: 664) ,GGUCGGUUGGA (SEQ ID NO: 665) ,GGUCGGUUGGAA (SEQ ID NO: 666) ,GGUCGGUUGGAAU (SEQ ID NO: 667) ,GGUCGGUUGGAAUU (SEQ ID NO: 668) orGGUCGGUUGGAAUUC (SEQ ID NO: 669) In some embodiments, a dsRNA agent comprises a sense strand and an antisense strand, each of which comprises a nucleotide sequence that differs by 0, 1, 2, or 3 nucleotides from Formula (A) and Formula (B) described herein, and optionally includes a targeting ligand. In certain embodiments, the sense strand (A) and antisense strand (B) of a dsRNA agent are each 35 or fewer nucleotides in length. In certain embodiments, the Z1 and Z4 nucleotide motifs are fully or partially complementary. In some embodiments, a dsRNA agent comprises a sequence of Formula (A'): 5'-Z1'CAGCUUGUUUGUGAAACA-3' (SEQ ID NO: 670) and a sense strand that differs by 0, 1, 2, or 3 nucleotides from the (SEQ ID NO: 671)and Z4', wherein Z1' and Z4' each independently comprise a nucleotide motif 0-13 nucleotide sequence. In certain embodiments, Z1' and Z4' each independently comprise a nucleotide motif 1, 2, or 3 nucleotide sequence. In certain embodiments, the Z1' nucleotide sequence comprises one of the following motifs: A, U, G, C, AC, UC, GC, CC, GAC, AAC, UAC, CAC, CGAC, CCGAC, ACCGAC, AACCGAC, CAACCGAC, or GAAUUCCAACCGAC. (SEQ ID NO: 672) The Z4' nucleotide sequence is selected from one of the following motifs: U, C, A, G, GU, GA, GC, GG, GUG, GUC, GUU, GUA, GUCG, GUCGG, GUCGGU, GUCGGUU, GUCGGUUG, or GUCGGUUGGAAUUC. (SEQ ID NO: 673) is selected from one of the following:

[0009] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the antisense strand has the antisense sequence shown below: 5'-UACUCAUUAGAAGAAAGGUG-3' (SEQ ID NO: 162); 5'-UCUUAGACCAAGGAGAAACGG-3' (SEQ ID NO: 163); 5'-UGUUUCACAAACAAGCUGGUC-3' (SEQ ID NO: 167); 5'-UGUUUCACAAACAAGCUGGUG-3' (SEQ ID NO: 523); 5'-UUCGGUUGGAAUUCUUUUUGC-3' (SEQ ID NO: 184); 5'-GUUUCACAAACAAGCUGG-3' (SEQ ID NO: 653); and at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of

[0010] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each of which has the nucleotide sequence shown below: Sense strand: 5'-CACCUUUUCUUCUAAUGAGUA-3' (SEQ ID NO: 65), Antisense strand: 5'-UACUCAUUAGAAGAAAAGGUG-3' (SEQ ID NO: 162) and at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of

[0011] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each of which has the nucleotide sequence shown below: Sense strand: 5'-CCGUUUCUCCUUGGUCUAAGA-3' (SEQ ID NO: 66), Antisense strand: 5'-UCUUAGACCAAGGAGAAACGG-3' (SEQ ID NO: 163) and at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of

[0012] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each of which has the nucleotide sequence shown below: Sense strand: 5'-GACCAGCUUGUUUGUGAAACA-3' (SEQ ID NO: 70), Antisense strand: 5'-UGUUUCACAAACAAGCUGGUC-3' (SEQ ID NO: 167) and at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of

[0013] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each of which has the nucleotide sequence shown below: Sense strand: 5'-CACCAGCUUGUUUGUGAAACA-3' (SEQ ID NO: 522) Antisense strand: 5'-UGUUUCA CA ACAAGCUGGUG-3' (number: 523); and at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each of which has the nucleotide sequence shown below: Sense strand: 5'-GCAAAAAGAAUUCCAACCGAA-3' (SEQ ID NO: 87), Antisense strand: 5'-UUCGGUUGGAAUUCUUUUUGC-3' (SEQ ID NO: 184) and at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of

[0014] In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, each of which has the nucleotide sequence shown below: Sense strand: 5'-CCAGCUUGUUUGUGAAAC-3' (SEQ ID NO: 652), Antisense strand: 5'-GUUUCACAAACAAGCUGG-3' (SEQ ID NO: 653) and at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of

[0015] In some embodiments, the dsRNA agent duplex is selected from the duplexes of any one of AD00158-19-2, AD00158-19-1, AD00158-3, AD00158-1, AD00158-2, AD00158, AD00159, AD00159-1, AD00159-2, AD00159-19-1, AD00159-19-2, AD00163, AD00163-1, AD00163-2, AD00163-19-1, AD00163-19-2, AD00163-3, AD00300-1, AD00300-19-1, and AD00300-19-2 in Table 1.

[0016] In some embodiments, the dsRNA agent duplex is selected from the duplexes of any one of AV01227, AV01228, AV01229, AV01230, AV01231, AV01232, AV01233, AV01234, AV01235, AV01236, AV01237, AV01238, AV01239, AV01240, AV01241, AV01242, AV01243, AV01244, AV01245, AV01246, AV01247, AV01248, AV01249, AV01250, AV01251, AV01252, AV01253, AV01254, AV01255, AV01256, AV01257, or AV01711 in Table 1.

[0017] In some embodiments, the dsRNA agent comprises at least one modified nucleotide. In certain embodiments, all or substantially all of the nucleotides in the antisense strand are modified nucleotides. In some embodiments, the at least one modified nucleotide includes 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2',3'-seconucleotide mimic, locked nucleotide, unlocked nucleic acid (UNA) nucleotide, glycol nucleic acid (GNA) nucleotide, 2'-F-arabinonucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted abasic nucleotide, inverted 2'-OMe nucleotide, inverted 2'-deoxy nucleotide, 2'-amino modified nucleotide, 2'-alkyl modified nucleotide, morpholino nucleotide and 3'-OMe nucleotide, nucleotide containing a 5'-phosphorothioate group, or terminal nucleotide linked to cholesterol derivative or dodecanoic acid bisdecylamide group, 2'-amino modified nucleotide, phosphoramidate or non-natural base-containing nucleotide.

[0018] In some embodiments, these double-stranded ribonucleic acid (dsRNA) agents comprise a sense strand and an antisense strand that is complementary to at least a portion of an mRNA corresponding to a target gene, and the dsRNA agent comprises a nucleotide sequence having an antisense strand represented by formula (C) and a nucleotide sequence having a sense strand represented by formula (D).

[0019] The antisense strand has the formula (C) shown in the 3' to 5' direction: 3'-(N L ) n N M1 N L N M2 N L N F N L N M3 N L N M4 N L N M5 N M6 N L N M7 N M8 N L N F N L -5'comprises the formula (C), and The sense strand has the formula (D), shown in the 5' to 3' direction: 5'-(N' L ) n 'N' L N' L N' L N' N1 N' N2 N' N3 N' N4 N' F N' L N' N5 N' N6 N' L N' L N' L N' L N' L N' L N' L -3' formula (D) Includes:

[0020] In the above formula, N F Each represents a 2'-fluoro-modified nucleotide.M1 , N M2 , N M3 , N M4 , N M5 , N M6 , N M7 and N M8 independently represent a modified or unmodified nucleotide. M1 , N M2 , N M3 , N M4 , N M5 , N M6 , N M7 and N M8 There are only three 2'-fluoro-modified nucleotides, or only one 2'-fluoro-modified nucleotide in N L each independently represents a modified or unmodified nucleotide, wherein the modification is not a 2'-fluoro-modified nucleotide. F Each represents a 2'-fluoro-modified nucleotide. N1 , N' N2 , N' N3 , N' N4 , N' N5 and N' N6 independently represent a modified or unmodified nucleotide. N1 , N' N2 , N' N3 , N' N4 , N' N5 and N' N6 There are only two 2'-fluoro-modified nucleotides in N' L each independently represents a modified or unmodified nucleotide, and the modification is not a 2'-fluoro-modified nucleotide. n and n' can each independently be an integer from 0 to 7.

[0021] In certain embodiments, the nucleotides at positions 2, 7, 12, 14, and 16 (counting from the first pair of nucleotides at the 5' end) of the antisense strand represented by formula (C) in a dsRNA agent are 2'-fluorine-modified nucleotides; and the nucleotides at positions 9, 11, and 13 (counting from the first pair of nucleotides at the 3' end) of the sense strand represented by formula (D) are 2'-fluoro-modified nucleotides. In certain embodiments, the nucleotides at positions NM2, NM3, NM6 of the antisense strand represented by formula (C) are 2'-fluoro-modified nucleotides, and the nucleotides at positions N' of the sense strand represented by formula (D) are 2'-fluoro-modified nucleotides. N3 , N' N5 The nucleotide at position 1 is a 2'-fluoro-modified nucleotide.

[0022] In some embodiments, the dsRNA agent comprises an E-vinyl phosphonate nucleotide at the 5'-end of the guide strand. In certain embodiments, the dsRNA agent comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, the sense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleoside linkage. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages.

[0023] In certain embodiments, all or substantially all nucleotides in the sense strand and antisense strand are modified nucleotides. In some embodiments, the modified sense strand has a modified sense strand sequence shown in Tables 2-4. In some embodiments, the modified antisense strand has a modified antisense strand sequence shown in Tables 2-4.

[0024] In certain embodiments, the sense strand is complementary or substantially complementary to the antisense strand, and the region of complementarity is 16-23 nucleotides in length. In some embodiments, the region of complementarity is 19-21 nucleotides in length. In certain embodiments, the region of complementarity is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0025] In some embodiments, each strand is 40 nucleotides or less in length. In some embodiments, each strand is 30 nucleotides or less in length. In some embodiments, each strand is 25 nucleotides or less in length. In some embodiments, each strand is 23 nucleotides or less in length. In some embodiments, each strand is 4, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0026] In certain embodiments, the dsRNA agent comprises at least one modified nucleotide, and further comprises one or more targeting or linking groups.In some embodiments, one or more targeting groups or linking groups are conjugated to the sense strand.In some embodiments, the targeting group or linking group comprises N-acetyl-galactosamine (GalNAc).In some embodiments, the targeting group has the following structure: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It has.

[0027] In certain embodiments, a dsRNA agent comprises a targeting group conjugated to the 5'-end of the sense strand. In some embodiments, a dsRNA agent comprises a targeting group conjugated to the 3'-end of the sense strand. In some embodiments, the antisense strand comprises an inverted abasic residue at the 3'-end. In certain embodiments, the sense strand comprises one or two inverted abasic residues at the 3'- and / or 5'-end. In some embodiments, the dsRNA agent has two blunt ends. In some embodiments, at least one strand comprises a 3' overhang that is at least 1 nucleotide in length. In some embodiments, at least one strand comprises a 3' overhang that is at least 2 nucleotides in length.

[0028] In certain embodiments, the present invention relates to open nucleic acid (UNA) oligomers for therapeutic use. Unlocked nucleic acids (UNA) are acyclic analogs of RNA in which the bond between the C2' and C3' ribose atoms is broken. The incorporation of UNA has been shown to be well tolerated and, in some cases, enhances the activity of siRNA gene silencing (Meghan A. et al., Locked vs. unlocked nucleic acids (LNA vs. UNA): contrasting structures work towards common therapeutic goals. Chem. Soc. Rev., 2011, 40, 5680-5689).

[0029] UNA is a thermolabile modification, and replacing ribonucleotides with UNA reduces base pair strength and duplex stability. Strategic placement of UNA in the seed region of siRNA antisense strands can reduce off-target activity in microRNA (miRNA)-mediated gene silencing. miRNAs primarily recognize target genes through base pairing between the antisense seed region (positions 2–8 from the end) and the target mRNA for gene silencing. Each miRNA has the potential to regulate multiple genes. siRNA antisense strands loaded by the RNA-induced silencing complex (RISC) may also unintentionally regulate multiple genes via the miRNA-mediated mechanism. Therefore, adding a thermolabile nucleotide such as UNA to the seed region of siRNA can reduce off-target activity (Lam JK, Chow MY, Zhang Y, Leung SW. siRNA Versus miRNA as Therapeutics for Gene Silencing. Mol Ther Nucleic Acids. 2015 Sep 15;4(9):e252. doi:10.1038 / mtna.2015.23. PMID:26372022; PMCID:PMC4877448.). In particular, such RNA oligonucleotides or RNA oligonucleotide complexes contain at least one UNA nucleotide monomer in the seed region (Narendra Vaish et al. Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs. Nucleic Acids Research, 2011, Vol. 39, No. 5 1823-1832).

[0030] Potential advantages of incorporating UNA into RNA oligonucleotides or RNA oligonucleotide complexes according to the technical solution of the present invention include, but are not limited to: 1. Reduction of off-target activity. Adding UNA to the iRNA seed region reduces the base pairing strength of the seed region, thereby reducing potential off-target activity caused by the micro-RNA. 2. Good tolerance of UNA in terms of siRNA activity. In some cases, UNA can cause increased activity.

[0031] Exemplary UNA monomers that can be used in the technical solution include, but are not limited to: [ka] Examples include:

[0032] In some embodiments, the dsRNA agent is a modified duplex selected from any one of the duplexes AD00158-19-2, AD00158-19-1, AD00158-3, AD00158-1, AD00158-2, AD00158, AD00159, AD00159-1, AD00159-2, AD00159-19-1, AD00159-19-2, AD00163, AD00163-1, AD00163-2, AD00163-19-1, AD00163-19-2, AD00163-3, AD00300-1, AD00300-19-1, and AD00300-19-2 in Tables 2-4.

[0033] In some embodiments, the dsRNA agent is a modified duplex selected from any of the duplexes AV01227, AV01228, AV01229, AV01230, AV01231, AV01232, AV01233, AV01234, AV01235, AV01236, AV01237, AV01238, AV01239, AV01240, AV01241, AV01242, AV01243, AV01244, AV01245, AV01246, AV01247, AV01248, AV01249, AV01250, AV01251, AV01252, AV01253, AV01254, AV01255, AV01256, and AV01257 in Tables 2-4.

[0034] According to one aspect of the present invention, there is provided a composition comprising any of the above-mentioned aspects of the dsRNA agent of the present invention.In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier.In some embodiments, the composition further comprises one or more additional therapeutic agents.In certain embodiments, the composition is packaged in a kit, a container, a pack, a dispenser, a pre-filled syringe or a vial.In some embodiments, the composition is formulated for subcutaneous or intravenous (IV) administration.

[0035] According to another aspect of the invention, there is provided a cell comprising an embodiment of any of the above-described dsRNA agent aspects of the invention. In some embodiments, the cell is a mammalian cell, optionally a human cell.

[0036] According to another aspect of the present invention, there is provided a method for inhibiting expression of the AGT gene in a cell, the method comprising: (i) preparing a cell comprising an effective amount of any of the aforementioned dsRNA agents or embodiments of the aforementioned compositions of the present invention. In certain embodiments, the method further comprises: (ii) maintaining the prepared cell for a time sufficient to degrade mRNA transcripts of the AGT gene, thereby inhibiting expression of the AGT gene in the cell. In some embodiments, the cell is a cell in a subject, and the dsRNA agent is administered to the subject subcutaneously. In some embodiments, the cell is a cell in a subject, and the dsRNA agent is administered to the subject intravenously. In certain embodiments, the method further comprises assessing inhibition of the AGT gene after administering the dsRNA agent to the subject, the assessment comprising: (i) determining one or more physiological characteristics of an AGT-associated disease or condition in the subject; and comparing the determined physiological characteristics with baseline pre-treatment physiological characteristics of an AGT-associated disease or condition and / or physiological characteristics of a control for an AGT-associated disease or condition, wherein the comparison indicates the presence or absence of inhibition of expression of the AGT gene in the subject. In some embodiments, the determined physiological characteristic is blood pressure, such as systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAPR). A decrease in blood AGT level and / or blood pressure indicates a decrease in AGT gene expression in the subject.

[0037] According to another aspect of the present invention, there is provided a method for inhibiting AGT gene expression in a subject, the method comprising administering to the subject an effective amount of an embodiment of the dsRNA agent aspect described above or an embodiment of the composition described above. In some embodiments, the dsRNA agent is administered to the subject subcutaneously. In certain embodiments, the dsRNA agent is administered to the subject intravenously. In some embodiments, the method further comprises assessing the inhibition of the AGT gene after administration of the dsRNA agent, the assessment comprising: (i) determining one or more physiological characteristics of an AGT-related disease or condition in the subject; and (ii) comparing the determined physiological characteristics with baseline pre-treatment physiological characteristics of the AGT-related disease or condition and / or physiological characteristics of a control of an AGT-related disease or condition, the comparison indicating the presence or absence of inhibition of expression of the AGT gene in the subject. In some embodiments, the determined physiological characteristic is the level of AGT in the blood; in some embodiments, the determined physiological characteristic is blood pressure, such as systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAPR). A decrease in blood AGT levels and / or blood pressure indicates a decrease in AGT gene expression in the subject.

[0038] According to another aspect of the invention, there is provided a method of treating a disease or condition associated with an AGT protein, comprising administering to a subject an effective amount of any embodiment of the above-described dsRNA agent aspects of the invention or the above-described composition of the invention to inhibit AGT gene expression. In certain embodiments, the AGT-related disorder is selected from: hypertension, high blood pressure, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, treatment-resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, angiopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina, stroke, renal disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), and fetal growth retardation. In some embodiments, the method further comprises administering an additional therapeutic regimen to the subject. In some embodiments, the additional therapeutic regimen comprises treating an AGT-related disease or condition. In certain embodiments, the additional therapeutic regimen comprises: administering to the subject one or more AGT antisense polynucleotides of the invention; administering to the subject a non-AGT dsRNA therapeutic; and effecting a behavioral change in the subject. In some embodiments, the non-AGT dsRNA therapeutic is one of the following additional therapeutic agents, such as a diuretic, angiotensin-converting enzyme (ACE) inhibitor, angiotensin II receptor antagonist, beta-blocker, vasodilator, calcium channel blocker, aldosterone antagonist, alpha-agonist, renin inhibitor, alpha-blocker, peripherally acting adrenergic agonist, selective D1 receptor partial agonist, non-selective alpha-adrenergic antagonist, synthetic steroidal antimineralocorticoid, or a combination of any of the above, and a therapeutic agent for hypertension formulated as a combination of agents.

[0039] In some embodiments, the dsRNA agent is administered subcutaneously to the subject. In certain embodiments, the dsRNA agent is administered IV to the subject. In some embodiments, the method further includes determining the effectiveness of the administered double-stranded ribonucleic acid (dsRNA) agent in the subject. In some embodiments, the means for determining the effectiveness of a treatment in the subject includes: (i) determining one or more physiological characteristics in the subject; and (ii) correlating the determined physiological characteristics with an AGT-related disease or condition, wherein the comparison indicates one or more of the presence or absence, and the level of effectiveness, of the double-stranded ribonucleic acid (dsRNA) agent administered to the subject. In some embodiments, the determined physiological characteristic is the level of AGT in the blood; in some embodiments, the determined physiological characteristic is blood pressure, such as systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAPR). A decrease in the blood AGT level and / or blood pressure indicates the effectiveness of the double-stranded ribonucleic acid (dsRNA) agent administered to the subject.

[0040] According to another aspect of the invention, there is provided a method of reducing the level of AGT protein in a subject compared to the baseline pre-treatment level of AGT protein in the subject, the method comprising administering to the subject an effective amount of any embodiment of the above dsRNA agent aspect of the invention or any embodiment of the above composition of the invention to reduce the level of AGT gene expression. In some embodiments, the dsRNA agent is administered to the subject subcutaneously or IV.

[0041] According to another aspect of the present invention, there is provided a method for modifying physiological characteristics of an AGT-related disease or condition in a subject, compared to a baseline pre-treatment physiological characteristic of the AGT-related disease or condition in the subject, the method comprising administering to the subject an effective amount of any of the above-described dsRNA agent embodiments of the present invention or any of the above-described composition embodiments of the present invention to modify the physiological characteristics of the AGT-related disease or condition in the subject. In some embodiments, the dsRNA agent is administered to the subject subcutaneously or intravenously. In certain embodiments, the physiological characteristic is the level of AGT in the blood; in some embodiments, the determined physiological characteristic is blood pressure, such as systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAPR).

[0042] Array Description The duplexes AD00051 to AD00122-19-2, AD00163-3, AV01227 to AVAV01257, and AV01711 are shown in Table 1, along with their sense strand sequences.

[0043] The duplexes AD00051 to AD00122-19-2, AD00163-3, AV01227 to AVAV01257, and AV01711 are shown in Table 1, along with their antisense strand sequences.

[0044] SEQ ID NO: 519 is human angiotensinogen (AGT) mRNA [NCBI Reference Sequence: NM_001384479.1]: [ka] [ka]

[0045] SEQ ID NO: 520 is mouse angiotensinogen (AGT) mRNA [NCBI Reference Sequence: NM_007428.4] [ka]

[0046] SEQ ID NO: 521 is the cynomolgus monkey angiotensinogen (AGT) mRNA [NCBI Reference Sequence: NM_001283634.1] [ka]

[0047] In the sequences shown in Table 2, chemical modifications are: upper case: 2'-fluoro; lower case: 2'-OMe; thiophosphate: * is shown as:

[0048] In the sequences shown in Table 3, the delivery molecules used in in vivo studies are indicated by "GLO-0" at the 3' end of each sense strand. Chemical modifications are: uppercase: 2'-fluoro; lowercase: 2'-OMe; thiophosphate: * ;Unlocked nucleic acid: UNA; (Note: AD00052, AD00113-AD00260: no UNA; AD00282-AD00301: UNA version).

[0049] In the sequences shown in Table 4, chemical modifications are: upper case: 2'-fluoro; lower case: 2'-OMe; thiophosphate: * ;Invab=inverted abasic; [Brief explanation of the drawings]

[0050] [Figure 1] 1 is a graph showing serum AGT protein levels in cynomolgus monkeys after administration of AD00158-1, AD00158-2, AD00163-1, AD00159-1, and AD00300-1 at 2 mg / kg each. [Figure 2] 1 is a graph showing serum AGT protein levels in cynomolgus monkeys after administration of 10 mg / kg of AD00163-3. [Figure 3] 1 is a graph showing the change in serum SBP in cynomolgus monkeys after administration of 10 mg / kg of AD00163-3. [Figure 4]1 is a graph showing mean blood pressure (MBP) in cynomolgus monkeys after administration of 10 mg / kg of AD00163-3. [Figure 5] 1 is a graph showing diastolic blood pressure (DBP) in cynomolgus monkeys after administration of 10 mg / kg of AD00163-3. DETAILED DESCRIPTION OF THE INVENTION

[0051] Some embodiments of the present invention include RNAi agents capable of inhibiting expression of the angiotensinogen (AGT) gene, such as, but not limited to, double-stranded (ds) RNAi agents. Some embodiments of the present invention also include compositions comprising AGT RNAi agents and methods of using the compositions. The AGT RNAi agents disclosed herein can be attached to a delivery compound for delivery to cells, such as to hepatocytes. Pharmaceutical compositions of the present invention can include at least one dsAGT agent and a delivery compound. In some embodiments of the present invention, the delivery compound is a GalNAc-containing delivery compound. The AGT RNAi agent delivered to a cell can inhibit expression of the AGT gene, thereby reducing AGT protein production in the cell. The dsRNAi agents of the present invention can be used to treat AGT-related diseases and conditions. Examples of such dsRNAi agents include the double-stranded AD00051 to AD00122-19-2 shown in Table 1. In some embodiments, preferred dsRNAi agents include, for example, duplex AD00158, AD00163, AD00159, AD00290, AD00300, or AD00122. In other embodiments, preferred dsRNAi agents include, for example, AD00158-1, AD00158-2, AD00163-1, AD00163-3, AD00159-1, or AD00300-1. In some other embodiments, such dsRNAi agents include duplex variants, such as variants of duplex AD00158, AD00163, AD00163-3, AD00159, AD00290, AD00300, or AD00122.

[0052] In some embodiments of the invention, reducing AGT expression in a cell or a subject treats a disease or condition associated with AGT expression in the cell or subject, respectively. Non-limiting examples of diseases and conditions that can be treated by reducing AGT activity are hypertension, high blood pressure, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, treatment-resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, angiopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina, stroke, renal disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), and fetal growth retardation.

[0053] Described below are compositions and methods for preparing and using compositions containing AGT single-stranded (ssRNA) and double-stranded (dsRNA) agents that inhibit AGT gene expression, as well as compositions and methods for treating diseases and conditions caused by or modulated by AGT gene expression. The term "RNAi" is known in the art and may also be referred to as "siRNA."

[0054] As used herein, the term "RNAi" refers to an agent that comprises RNA and mediates targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. As known in the art, an RNAi target region refers to a contiguous portion of nucleotide sequence in an RNA molecule formed during gene transcription, such as messenger RNA (mRNA), the RNA processing product of a primary transcript. A target portion of a sequence is at least sufficiently long to serve as a substrate for RNAi-directed cleavage at or near this portion. Target sequences can be 8-30 nucleotides in length (inclusive), 10-30 nucleotides in length (inclusive), 12-25 nucleotides in length (inclusive), 15-23 nucleotides in length (inclusive), 16-23 nucleotides in length (inclusive), or 18-23 nucleotides in length (inclusive), including all shorter lengths within each specified range. In some embodiments of the invention, the target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In particular embodiments, the target sequence is 9 to 26 nucleotides in length (inclusive), including all subranges and integers therebetween. For example, and not intended to be limiting, in certain embodiments of the invention, the target sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length and is fully, or at least substantially, complementary to at least a portion of an RNA transcript of the AGT gene. Some aspects of the invention include pharmaceutical compositions comprising one or more AGT dsRNA agents and a pharmaceutically acceptable carrier. In certain embodiments of the present invention, the AGT RNAi described herein inhibits expression of the AGT protein.

[0055] As used herein, "dsRNA agent" refers to a composition comprising an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can degrade or inhibit the translation of a target mRNA transcript. Without wishing to be bound by a particular theory, the dsRNA agent of the present invention can act via an RNA interference mechanism (i.e., induce RNA interference by interacting with the machinery of the RNA interference pathway (RNA-induced silencing complex or RISC) in mammalian cells), or can act via an alternative mechanism or pathway. Methods for silencing genes in plants, invertebrates, and vertebrate cells are well known in the art (see, for example, Sharp et al., Genes Dev. 2001, 15:485; Bernstein, et al., (2001) Nature 409:363; Nykanen, et al., (2001) Cell 107:309; and Elbashir, et al., (2001) Genes Dev. 15:188), the entire disclosures of which are incorporated herein by reference. Gene silencing techniques known in the art can be used in conjunction with the disclosure provided herein to achieve inhibition of AGT expression.

[0056] The dsRNA agent disclosed herein consists of a sense strand and an antisense strand, and includes, but is not limited to, short interfering RNA (siRNA), RNAi agent, microRNA (miRNA), short hairpin RNA (shRNA) and Dicer substrate.The antisense strand of the dsRNA agent described herein is at least partially complementary to the target mRNA, and it is understood in the art that dsRNA duplexes of various lengths can be used to inhibit target gene expression.For example, dsRNAs with duplex structures of 19, 20, 21, 22, and 23 base pairs are known to effectively induce RNA interference (Elbashi et al., EMBO 2001, 20:6877-6888).It is also known in the art that shorter or longer RNA duplex structures are also effective in inducing RNA interference. In certain embodiments of the invention, the AGT dsRNA may comprise at least one strand at least 21 nucleotides in length, or the duplex may have a length based on at least one of the sequences set forth in Tables 1-4, minus 1, 2, or 3 nucleotides, or less. A reduction of 4 nucleotides at one or both ends, respectively, may also be effective compared to the dsRNA set forth in Tables 1-4. In some embodiments of the invention, the AGT dsRNA agent may have a subsequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one or more sequences in Tables 1-4, and differs in its ability to inhibit AGT gene expression at a level of greater than 5%, 10%, 15%, 20%, 25%, or 30% from the level of inhibition produced by a dsRNA comprising the entire sequence (also referred to herein as the "parent" sequence).

[0057] Certain embodiments of the compositions and methods of the present invention include single-stranded RNA in the composition and / or administer single-stranded RNA to a subject. For example, an antisense strand shown in any of Tables 1-4 can be used as or in a composition, which, when administered to a subject, reduces AGT polypeptide activity and / or AGT gene expression in the subject. Tables 1-4 show how the antisense and sense strand cores extend the base sequence of some AGT dsRNA agents. Single-stranded antisense molecules that may be included in certain compositions of the present invention and / or administered in certain methods of the present invention are referred to herein as "single-stranded antisense agents" or "antisense polynucleotide agents." Single-stranded sense molecules that may be included in certain compositions of the present invention and / or administered in certain methods of the present invention are referred to herein as "single-stranded sense agents" or "sense polynucleotide agents." As used herein, the term "base sequence" refers to a polynucleotide sequence without chemical modifications or delivery compounds. For example, the sense strands shown in Table 1 correspond to the corresponding base sequences in Table 3; the corresponding sequences in Table 3 indicate the respective chemical modifications and delivery compounds. The sequences disclosed herein may be assigned identifiers. For example, a single-stranded sense sequence may be identified by a "sense strand SS number"; a single-stranded antisense sequence may be identified by an "antisense strand AS number"; and a duplex comprising a sense strand and an antisense strand may be identified by a "duplex AD number."

[0058] Table 1 includes sense and antisense strands and provides identification numbers for duplexes formed by the sense and antisense strands in the same column of Table 1. In certain embodiments of the present invention, the antisense sequence contains nucleobase u or nucleobase a at its first position. In certain embodiments of the present invention, the antisense sequence contains nucleobase u at its first position. As used herein, the term "matching position" refers to a position in each strand that, in a sense, "pairs" with the nucleobase at position 21 of the antisense strand when the two strands function as a duplex. For example, in a 21-nucleobase sense strand and a 21-nucleobase antisense strand, the nucleobase at position 1 of the sense strand is in the "matching position" with the nucleobase at position 21 of the antisense strand. In another non-limiting example of a 23-nucleobase sense strand and a 23-nucleobase antisense strand, the nucleobase at position 2 of the sense strand is in the matching position with the nucleobase at position 22 of the antisense strand. In another non-limiting example of an 18-nucleobase sense strand and an 18-nucleobase antisense strand, the nucleobase at position 1 of the sense strand is in the matching position with the nucleobase at position 18 of the antisense strand. The nucleobase at position 4 of the sense strand is in a matching position with the nucleobase at position 15 of the antisense strand. Those skilled in the art will understand how to identify matching positions between the sense and antisense strands of a duplex and paired strands.

[0059] The last column in Table 1 indicates the duplex AD number / AV number for duplexes comprising the sense and antisense sequences in the same row of the table. For example, Table 1 discloses a duplex designated "Duplex AD No. AD00051" comprising corresponding sense and antisense strand sequences. Thus, each row of Table 1 identifies duplexes of the invention each comprising the sense and antisense sequences set forth in the same row, with the designated identifier for each duplex indicated at the end of the column.

[0060] In some embodiments of the methods of the invention, an RNAi agent comprising a polynucleotide sequence set forth in Table 1 is administered to a subject. In some embodiments of the invention, the RNAi agent administered to a subject comprises a duplex comprising at least one of the base sequences set forth in Table 1 and comprising 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 sequence modifications. Some embodiments of the methods of the invention further comprise linking the RNAi agent of the polynucleotide sequence set forth in Table 1 to a delivery molecule, a non-limiting example of which is a delivery compound comprising GalNAc.

[0061] [Table 1]

[0062] [Table 2]

[0063] [Table 3]

[0064] [Table 4]

[0065] [Table 5]

[0066] [Table 6]

[0067] [Table 7]

[0068] Table 8

[0069] Table 9

[0070] Table 10

[0071] Table 2 shows the antisense and sense strand sequences of certain chemically modified AGT RNAi agents of the invention. In some embodiments of the invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a cell and / or a subject. In some embodiments of the invention, an RNAi agent having a polynucleotide sequence set forth in Table 2 is administered to a subject. In some embodiments of the invention, the RNAi agent administered to a subject comprises a duplex shown in the first column of Table 2 and includes sequence modifications of the sense and antisense strand sequences shown in the third and sixth columns of the same row in Table 2, respectively. In some embodiments of the invention, the sequences shown in Table 2 can be linked (also referred to herein as conjugates) to compounds capable of delivering the RNAi agent to cells and / or tissues of a subject. A non-limiting example of a delivery compound useful in certain embodiments of the invention is a GalNAc-containing compound. In Table 2, the first column represents the duplex AD number or AV number of the base sequence corresponding to Table 1. For base sequences identified by the AD number of the duplex, not only are the base sequences contained in the sense and antisense strands shown, but the designated chemical modifications are also shown in the same column of Table 2. For example, column 1 of Table 1 shows single-stranded sense and antisense base sequences that together form a duplex identified as duplex AD number AD00051, while column 2 shows duplex AD number AD00051 as a duplex, which includes base sequences AD00051-SS and AD00051-AS and contains chemical modifications in the sense and antisense sequences shown in columns 3 and 6, respectively. The "Sense Strand SS Number" in column 2 of Table 2 is the designated identifier of the sense sequence (including modifications) shown in column 3 of the same row. The "Antisense Strand AS Number" in column 5 of Table 2 is the designated identifier of the antisense sequence (including modifications) shown in column 6.

[0072] [Table 11]

[0073] [Table 12]

[0074] Table 13

[0075] Table 14

[0076] Table 15

[0077] Table 3 shows the antisense and sense strand sequences of certain chemically modified AGT RNAi agents of the invention. In some embodiments of the invention, an RNAi agent shown in Table 3 is administered to a cell and / or a subject. In some embodiments of the invention, an RNAi agent having a polynucleotide sequence set forth in Table 3 is administered to a subject. In some embodiments of the invention, the RNAi agent administered to a subject comprises a duplex identified in the first column of Table 3 and includes the sequence modifications and / or delivery compounds shown in the sense and antisense strand sequences in columns 3 and 6, respectively, of the same row in Table 3. The sequences were used in certain in vivo testing studies described elsewhere herein. In some embodiments of the invention, the sequences shown in Table 3 can be linked (also referred to herein as conjugates) to compounds for delivery, a non-limiting example of which is a GalNAc-containing compound, i.e., the delivery compound identified as "GLX-n" is present on the sense strand in column 3 of Table 3. As used herein, "GLX" is used to mean a "GLS" or "GLO" delivery compound (where "X" can be "S" or "O"), and GLX-n can be any GLS and GLO that can be linked to the 3' or 5' end of an oligonucleotide of the present invention during the synthesis process. As non-limiting examples, GLX-13 and GLX-14 can be linked to the 3' end of an oligonucleotide of the present invention during the synthesis process, and GLX-5 and GLX-15 can be linked to the 5' end of an oligonucleotide of the present invention during the synthesis process. In some embodiments, as used herein and shown in Table 3, "GLX-n" is used to refer to a conjugated GalNAc-containing compound and is any of the compounds GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16.In some implementations, GLO-0 refers to a GalNAc-containing compound disclosed in the prior art as being usable for ligation, including, but not limited to, the GalNAc-containing compounds useful for ligation disclosed in Jayaprakash, et al., (2014) J. Am. Chem. Soc., 136, 16958, all of which are described herein. In some implementations, one skilled in the art will be able to prepare and use the dsRNA compounds of the present invention with conjugated delivery compounds, including, but not limited to, GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, and one of GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, the structures of each of which are provided elsewhere herein. The first column of Table 3 provides the duplex AD number assigned to the sense and antisense sequences in that row of the table. For example, duplex AD number AD00052 is a duplex composed of a sense strand AD00052-SS and an antisense strand AD00052-AS. Each column of Table 3 provides one sense strand and one antisense strand and discloses a duplex composed of the indicated sense and antisense strands. The "Sense Strand SS Number" in the second column of Table 3 is the designated identifier for the sense sequence (including modifications) shown in the third column of the same row. The "Antisense Strand AS Number" in the fifth column of Table 3 is the designated identifier for the antisense sequence (including modifications) shown in the sixth column. The identifier for a particular linked GalNAc-containing GLO compound is designated GLO-0, and it should be understood that another GLO-n or GLS-n compound can be substituted for the compound designated GLO-0, and the resulting compound is also encompassed by embodiments of the methods and / or compositions of the present invention.

[0078] [Table 16]

[0079] Table 17

[0080] Table 18

[0081] Table 19

[0082] Table 20

[0083] Table 21

[0084] Table 4 shows the antisense and sense strand sequences of certain chemically modified AGT RNAi agents of the invention. In some embodiments of the invention, an RNAi agent having a polynucleotide sequence shown in Table 4 is administered to a subject. In some embodiments of the invention, the RNAi agent administered to a subject comprises a duplex identified in a row in column 1 of Table 4 and includes a sequence modification and / or delivery compound shown in the sense and antisense strand sequences in the same row in columns 3 and 6 of Table 4. In some embodiments of the invention, the sequences shown in Table 4 can be linked to a compound capable of delivering the RNAi agent to cells and / or tissues of a subject. A non-limiting example of a delivery compound useful in certain embodiments of the invention is a GalNAc-containing compound. In Table 4, the term "GLX-n" refers to a compound containing GalNAc in the sense strand shown. For example, the terms "GLO-0" and "GLS-5" each represent a different GalNAc-containing compound attached to the sense strand. It should be understood that the compound shown as GLO-0 can be replaced by another GLO-n or GLS-n compound, and the resulting compound is also encompassed by the methods and / or compositions of the invention. Similarly, a compound designated GLS-5 may also be substituted with another of the GLS-n or GLO-n compounds, and the resulting compound is encompassed within the method and / or composition embodiments of the present invention. In Table 4, the compound GLX-n used to represent the conjugated GalNAC-containing compound is the compound GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, the structures of each of which are provided elsewhere herein. The first column of Table 4 represents the duplex AD number corresponding to the duplex shown in Table 3.The duplex AD numbers identify the duplex sequences corresponding to Table 3, and indicate that the sense, antisense, and duplex sequences in Table 4 are identical to the base sequences with the same duplex AD numbers in Table 3, but the sequences and duplexes in Table 4 have different chemical modifications and / or delivery compounds compared to the corresponding sequences and duplexes shown in Table 3. For example, as shown in Table 4, the sequences AD00113-1-SS and AD00113-1-AS and their duplex AD number AD00113-1 have the same base sequences as AD00113-SS (sense), AD00113-AS (antisense), and duplex AD number AD00113 shown in Table 3, respectively, but have chemical modifications and / or delivery compounds as indicated in each table. The first column of Table 4 identifies the duplex AD number; the duplexes identified by numbers in each row contain the sense and antisense strands shown in columns 3 and 6, respectively, in the same row, and contain modifications, each having a GLO- or GLS-delivery compound attached to the 3' or 5' end of the sense strand.

[0085] [Table 22]

[0086] [Table 23]

[0087] [Table 24]

[0088] [Table 25]

[0089] [Table 26]

[0090] [Table 27]

[0091] [Table 28]

[0092] [Table 29]

[0093] mismatch Those skilled in the art know that mismatches can affect the efficacy of a dsRNA, especially when the mismatches are located within the terminal regions of the dsRNA. Certain mismatches, such as those with wobble base pairs G:U and A:C, are better tolerated (Duet et al., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar21;33(5):1671-7. Doi:10.1093 / nar / gki312. Nucleic Acids Res. 2005;33(11):3698). In some embodiments of the methods and compounds of the present invention, an AGT dsRNA agent can contain one or more mismatches relative to an AGT target sequence. In some embodiments, an AGT dsRNA agent of the present invention contains no mismatches. In certain embodiments, an AGT dsRNA agent of the present invention contains one or fewer mismatches. In some embodiments, an AGT dsRNA agent of the present invention contains two or fewer mismatches. In certain embodiments, the AGT dsRNA agent of the invention contains three or fewer mismatches. In some embodiments of the invention, the antisense strand of the AGT dsRNA agent contains a mismatch to the AGT target sequence that is not located in the center of the region of complementarity. In some embodiments, the antisense strand of the AGT dsRNA agent contains one, two, three, four, or more mismatches located within the last 5, 4, 3, 2, or 1 nucleotide from either or both of the 5' or 3' ends of the region of complementarity. Methods described herein and / or known in the art can be used to determine whether an AGT dsRNA agent containing a mismatch to the AGT target sequence is effective in inhibiting expression of the AGT gene.

[0094] Complementarity Unless otherwise specified, as used herein, the term "complementarity" when used to describe the relationship of a first nucleotide sequence (e.g., an AGT dsRNA agent sense strand or a targeting AGT mRNA) to a second nucleotide sequence (e.g., an AGT dsRNA agent antisense strand or a single-stranded antisense polynucleotide) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize with an oligonucleotide comprising the second nucleotide sequence and form a double helix or duplex structure under specified conditions (forming hydrogen bonds between base pairs under mammalian physiological conditions (or similar conditions in vitro)). Other conditions may also apply, such as physiologically relevant conditions that may be encountered in vivo. One of skill in the art will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences based on the ultimate application of the hybridized nucleotides. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the required conditions for hybridization described above are met. The sequence identity or complementarity is independent of the modification.

[0095] For example, a complementary sequence within an AGT dsRNA described herein comprises base pairs of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence throughout the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to each other. In embodiments in which two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, it should be understood that such overhangs are not considered mismatches as determined by complementarity herein. For example, an AGT dsRNA agent comprising one oligonucleotide 19 nucleotides in length and another oligonucleotide 20 nucleotides in length, where the longer oligonucleotide comprises a 19-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, may be referred to as "fully complementary" for purposes described herein. Thus, as used herein, "fully complementary" means that all (100%) of the bases in the contiguous sequence of a first polynucleotide hybridize with the same number of bases in the contiguous sequence of a second polynucleotide. The flanking sequences may include all or part of the first or second nucleotide sequence.

[0096] As used herein, the term "substantially complementary" means that in a hybrid pair of nucleobase sequences, at least about 85% (but not all) of the bases in the contiguous sequence of a first polynucleotide hybridize to the same number of bases in the contiguous sequence of a second polynucleotide. When hybridized, the two sequences contain one or more mismatched base pairs, e.g., at least 1, 2, 3, 4, or 5 mismatched base pairs. The term "substantially complementary" can be used in reference to duplexes of up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs (bp) formed by a first sequence with a second sequence while retaining the ability to hybridize under conditions most relevant to its end use, e.g., inhibition of AGT gene expression via the RISC pathway. The term "partially complementary" may be used herein to refer to a hybridizing pair of nucleobase sequences in which at least 75% (but not all) of the bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide. In some embodiments, "partially complementary" means that at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bases in a contiguous sequence of a first polynucleotide hybridize to the same number of bases in a contiguous sequence of a second polynucleotide.

[0097] "Complementary," "fully complementary," "substantially complementary," and "partially complementary" can be used to refer to base mismatches between the sense strand and antisense strand of an AGT dsRNA agent, between the antisense strand of an AGT dsRNA agent and the sequence of a target AGT mRNA, or between a single-stranded antisense oligonucleotide and the sequence of a target AGT mRNA. It should be understood that an "antisense strand of an AGT dsRNA agent" can refer to the same sequence of an "AGT antisense polynucleotide agent."

[0098] As used herein, the term "substantially identical" or "substantial identity," when referring to a nucleic acid sequence, means that the nucleic acid sequence comprises a sequence having at least about 85% sequence identity to a reference sequence, preferably 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%, or at least 99% sequence identity. The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions where the same nucleic acid base is present in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The invention disclosed herein includes nucleotide sequences that are substantially identical to the sequences disclosed herein (e.g., Tables 1-5). In some embodiments, the nucleotide sequence is identical to, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to, a sequence disclosed herein (e.g., in Tables 1-4).

[0099] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referenced using standard nucleotide nomenclature. As used herein, the term "double-stranded RNA" or "dsRNA" refers to an RNA molecule or sequence comprising a complex of RNAi molecules having a hybridized duplex region comprising two antiparallel and substantially or completely complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to the target AGT RNA. The duplex region can be of any desired length that allows specific degradation of the target AGT RNA by the RISC pathway, but is typically 9-30 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex of 9 to 30 base pairs, the duplex can be any length within this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs, and subranges therein, including, but not limited to, 15 to 30 base pairs, 15 to 26 base pairs; 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs, 18 to 30 base pairs, 18 to 29 base pairs, 19 to 30 base pairs, 20 to 31 base pairs, 21 to 32 base pairs, 22 to 33 base pairs, 23 to 24 base pairs, 24 to 25 base pairs, 25 to 26 base pairs, 26 to 27 base pairs, 27 to 28 base pairs, 28 to 30 base pairs, 29 to 31 base pairs, 30 to 32 base pairs, 31 to 33 base pairs, 32 to 34 base pairs, 33 to 35 base pairs, 34 to 36 base pairs, 35 to 37 base pairs, 36 to 38 base pairs, 37 to 39 base pairs, 38 to 40 base pairs, 40 to 41 base pairs, 41 to 42 base pairs, 42 to 43 base pairs, 43 to 44 base pairs, 44 to 45 base pairs, 45 to 46 base pairs, 46 to 47 base The length can be 26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. AGT dsRNA agents produced in cells by processing with Dicer and similar enzymes are typically in the 19-22 base pair range. One strand of the duplex region of an AGT dsDNA agent contains a sequence that is substantially complementary to a region of the target AGT RNA. The two strands that form the duplex structure can arise from a single RNA molecule with at least one self-complementary region, or can be formed from two or more separate RNA molecules.When the duplex region is formed from a single molecule, the molecule has a duplex structure formed between one strand at the 3' end of a single strand of nucleotides and another strand at the corresponding 5' end (referred to herein as a "hairpin loop"). In some embodiments of the present invention, the hairpin configuration contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more unpaired nucleotides. When the two substantially complementary strands of an AGT dsRNA agent are composed of separate RNA molecules, these molecules do not need to be covalently linked, but can be. When the two strands are covalently linked by means other than a hairpin loop, the linking structure is referred to as a "linker." The term "siRNA" is also used herein to refer to the dsRNA agent described herein.

[0100] In some embodiments of the present invention, an AGT dsRNA agent may comprise sense and antisense sequences with unpaired nucleotides or nucleotide analogs at one or both ends of the dsRNA agent. Ends that do not contain unpaired nucleotides are called "blunt ends" and do not have nucleotide overhangs. When both ends of a dsRNA agent are blunt, the dsRNA is said to be "blunt-ended." In some embodiments of the present invention, when the first end of a dsRNA agent is blunt, in some embodiments, the second end of the dsRNA agent is blunt, and in certain embodiments of the present invention, both ends of an AGT dsRNA agent are blunt.

[0101] In some embodiments of the dsRNA agent of the present invention, the dsRNA does not have one or two blunt ends. In this case, there is at least one unpaired nucleotide at the end of the strand of the dsRNA agent. For example, a nucleotide overhang exists when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA may contain an overhang of at least 1, 2, 3, 4, 5, 6, or more nucleotides. The nucleotide overhang may comprise or consist of a nucleotide / nucleoside analog, such as a deoxynucleotide / nucleoside. In some embodiments, the nucleotide overhang is located on the sense strand of the dsRNA agent, on the antisense strand of the dsRNA agent, or on both ends of the dsRNA agent, and it should be understood that the nucleotides of the overhang may be located at the 5' end, 3' end, or both ends of either the antisense strand or the sense strand of the dsRNA. In certain embodiments of the present invention, one or more nucleotides in the overhang are replaced with a nucleoside thiophosphate.

[0102] As used herein, the term "antisense strand" or "guide strand" refers to the strand of an AGT dsRNA agent that includes a region that is substantially complementary to an AGT target sequence. As used herein, the term "sense strand" or "passenger strand" refers to the strand of an AGT dsRNA agent that contains a region that is substantially complementary to a region of the antisense strand of the AGT dsRNA agent.

[0103] qualification In some embodiments of the present invention, the RNA of an AGT RNAi agent is chemically modified to improve stability and / or to confer one or more other beneficial properties. Nucleic acids in certain embodiments of the present invention can be synthesized and / or modified by methods well known in the art, see, for example, "Current protocols in Nucleic Acid Chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications that may be present in certain embodiments of an AGT dsRNA agent of the present invention include, for example: (a) terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage, etc.), 3'-end modifications (conjugation, DNA nucleotide, inverted linkage, etc.); (b) base modifications, such as substitution with a stabilizing base, a base or base pairing with an expanded partner pool, a missing base (abasic nucleotide), or a conjugated base; (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; and (d) backbone modifications, such as modification or substitution of a phosphodiester bond. Specific examples of RNA compounds useful in certain embodiments of the AGT dsRNA agents, AGT antisense polynucleotides, and AGT sense polynucleotides of the present invention include, but are not limited to, RNAs containing modified backbones or lacking natural internucleoside linkages. As a non-limiting example, RNAs with backbone modifications do not have a phosphorus atom in the backbone. RNAs that do not have a phosphorus atom in their internucleoside backbone can be called oligonucleosides. In certain embodiments of the present invention, modified RNAs have a phosphorus atom in their internucleoside backbone.

[0104] The terms "RNA molecule" or "RNA" or "ribonucleic acid molecule" should be understood to encompass not only RNA molecules expressed or found in nature, but also analogs and derivatives of RNA, including ribonucleotide / molecular analogs or derivatives described herein or known in the art. The terms "molecule" and "ribonucleotide" may be used interchangeably herein. RNA molecules may be modified in the nucleobase structure or ribose-phosphate backbone structure (e.g., as described below), and molecules containing molecular analogs or derivatives must retain the ability to form duplexes. As a non-limiting example, an RNA molecule may also contain at least one modified molecule, such as, but not limited to, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro-modified nucleoside, a 2'-amino-modified nucleoside, a 2'-alkyl-modified nucleoside, a morpholino nucleoside, a phosphoramidate, or a non-natural base-containing nucleoside, or a combination thereof. In some embodiments of the invention, an RNA molecule includes the following number of modified ribonucleosides: at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to the full length of an AGT dsRNA agent molecule. The modifications need not be the same for each of the multiple modified ribonucleosides in such an RNA molecule.

[0105] In some embodiments, dsRNA agents, AGT antisense polynucleotides, and / or AGT sense polynucleotides of the invention can contain one or more independently selected modified nucleotides and / or one or more independently selected non-phosphodiester linkages. As used herein, when used to refer to selected elements, such as modified nucleotides, non-phosphodiester linkages, etc., the term "independently selected" means that two or more selected elements can be identical to each other, but need not be identical to each other. As used herein, a "nucleotide base," "nucleotide," or "nucleobase" refers to a heterocyclic pyrimidine or purine compound that is a standard building block of all nucleic acids and includes the bases that form nucleotides: adenine (a), guanine (g), cytosine (c), thymine (t), and uracil (u). Nucleobases can be further modified to include, but are not limited to, universal bases, hydrophobic bases, promiscuous bases, size-extended bases, and fluorinated bases. As used herein, the terms "ribonucleotide" or "nucleotide" can refer to an unmodified nucleotide, a modified nucleotide, or another moiety. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be substituted by other moieties without significantly altering the base pairing properties of oligonucleotides containing nucleotides bearing such substituted moieties.

[0106] In one embodiment, modified RNAs contemplated for use in the methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form desired duplex structures and enable or mediate specific target degradation via the RISC pathway. In certain embodiments of the invention, AGT RNA interfering agents comprise single-stranded RNAs that interact with a target AGT RNA sequence and direct cleavage of the target AGT RNA.

[0107] Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (such as 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (such as 3'-aminophosphoramidates and aminoalkylphosphoramidates), thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, and boranophosphates (both with normal 3'-5' linkages and their 2'-5' linked analogs, and with reverse polarity, in which adjacent pairs of nucleoside units are linked in a 3'-5' to 5'-3' or 2'-5' to 5'-2' fashion). Various salts, mixed salts, and free acid forms are also included. Methods for forming phosphorus-containing bonds are routinely practiced in the art and can be used to prepare certain modified AGT dsRNA agents, certain modified AGT antisense polynucleotides, and / or certain modified AGT sense polynucleotides of the invention.

[0108] Modified RNA backbones that do not contain phosphorus atoms include structures formed with short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages, morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, methylacetyl and thiomethylacetyl backbones, methylenemethylacetyl and thiomethylacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other moieties with mixed N, O, S, and CH2 components. Methods for producing modified RNA backbones that do not contain a phosphorus atom are routinely practiced in the art and can be used to prepare certain modified AGT dsRNA agents, certain modified AGT antisense polynucleotides, and / or certain modified AGT sense polynucleotides of the invention.

[0109] In certain embodiments of the present invention, RNA mimetics are included in AGT dsRNA, AGT antisense polynucleotides, and / or AGT sense polynucleotides, for example, but not limited to, by substituting novel groups for the sugar and internucleoside linkage (i.e., backbone) of the nucleotide units. In such embodiments, the base units are maintained for hybridization with appropriate AGT nucleic acid target compounds. Such oligomeric compounds, RNA mimetics, which have been shown to have excellent hybridization properties, are called peptide nucleic acids (PNAs). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, specifically an aminoethylglycine backbone. The nucleobases are retained and linked, directly or indirectly, to the aza nitrogen atoms of the amide portion of the backbone. Methods for producing RNA mimetics are commonly practiced in the art, and such methods can be used to produce certain modified AGT dsRNA agents of the present invention.

[0110] Some embodiments of the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, particularly --CH2--NH--CH2-, --CH2--N(CH3)--O--CH2-- [known as a methylene(methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2---- [a natural phosphodiester backbone is represented as --O--P--O--CH2--]. Methods for producing RNAs with phosphorothioate backbones and oligonucleotides with heteroatom backbones are routinely practiced in the art and can be used to produce specific modified AGT dsRNA agents, specific AGT antisense polynucleotides, and / or specific AGT sense polynucleotides of the invention.

[0111] Modified RNAs may also contain one or more substituted sugar moieties. The AGT dsRNAs, AGT antisense polynucleotides, and / or AGT sense polynucleotides of the invention may include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl; where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. An exemplary suitable modification is: O[(CH) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. In other embodiments, the dsRNA comprises at the 2' position one of the following: C1 to C 10lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group used to improve the pharmacokinetic properties of an AGT dsRNA agent, or group used to improve the pharmacodynamic properties of an AGT dsRNA agent, AGT antisense polynucleotide and / or AGT sense polynucleotide, and other groups with similar properties. In some embodiments, the modification comprises 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martinez et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is the group 2'-dimethylaminoethoxyethoxy, i.e., O(CH2)2ON(CH3)2, also known as 2'-DMAOE, as described in the Examples below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2-O-CH2-N(CH2). Methods for producing modified RNAs such as those described are routinely practiced in the art, and such methods can be used to produce certain modified AGT dsRNA agents of the invention.

[0112] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an AGT dsRNA agent, AGT antisense polynucleotide, AGT sense polynucleotide, and / or AGT sense polynucleotide of the invention, particularly the 3' position of the sugar on the 3'-terminal nucleotide, or the 3' position in a 2'-5'-linked AGT dsRNA, AGT antisense polynucleotide, or AGT sense polynucleotide, and the 5' position of the 5'-terminal nucleotide. AGT dsRNA agents, AGT antisense polynucleotides, and / or AGT sense polynucleotides can also have sugar mimetics, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. Methods for producing modified RNAs, such as those described, are routinely practiced in the art and can be used to produce certain modified AGT dsRNA agents, AGT antisense polynucleotides, and / or AGT sense polynucleotides of the invention.

[0113] In some embodiments, AGT dsRNA agents, AGT antisense polynucleotides, and / or AGT sense polynucleotides may contain nucleobase (commonly referred to in the art as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 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-thiocytosan pyrimidines, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-amino ... These include zouracil, 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, 8-azaguanine and 8-azaadenine, 7-azaguanine and 7-azaadenine, and 3-azaguanine and 3-azaadenine.Additional nucleobases that may be included in certain embodiments of the AGT dsRNA agents of the invention are known in the art, see, for example: Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH2008; The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, JL, Ed. John Wiley & Sons, 1990; English et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302; and Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Methods for producing dsRNA, AGT antisense strand polynucleotides, and / or AGT sense strand polynucleotides containing nucleobase modifications and / or substitutions, such as those described herein, are routinely practiced in the art and can be used to produce certain modified AGT dsRNA agents, AGT sense polynucleotides, and / or AGT antisense polynucleotides of the invention.

[0114] Certain embodiments of the AGT dsRNA agents, AGT antisense polynucleotides, and / or AGT sense polynucleotides of the invention include RNAs modified to contain one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide with a modified ribose moiety that contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-end structural conformation. The addition of a locked nucleic acid to an AGT dsRNA agent, AGT antisense polynucleotide, and / or AGT sense polynucleotide of the invention can increase stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O. R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Methods for producing dsRNA agents, AGT antisense polynucleotides, and / or AGT sense polynucleotides containing locked nucleic acids are commonly practiced in the art, and such methods can be used to produce certain modified AGT dsRNA agents of the invention. Certain embodiments of the AGT dsRNA compounds, sense polynucleotides and / or antisense polynucleotides of the invention comprise at least one modified nucleotide, which includes: a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2',3'-seconucleotide mimic, a locked nucleotide, a 2'-F-arabino nucleotide, a 2'-methoxyethyl nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide and a 3'-Ome nucleotide, a nucleotide containing a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or a non-natural base-containing nucleotide.In some embodiments, the AGT dsRNA compound contains an E-vinyl phosphonate nucleotide at the 5' end of the antisense strand (also referred to herein as the guide strand).

[0115] In certain embodiments of the present invention, at least one modified nucleotide is included at the 3' and 5' ends of the AGT dsRNA compound, the sense polynucleotide, and / or the 3' end of the antisense polynucleotide, and the at least one modified nucleotide includes an abasic nucleotide, ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, and an inverted 2'-deoxynucleotide. Those skilled in the art know that the inclusion of an abasic or inverted abasic nucleotide at the end of an oligonucleotide can enhance stability (Czauderna et al. Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res. 2003;31(11):2705-2716. doi:10.1093 / nar / gkg393).

[0116] In certain embodiments of the present invention, AGT dsRNA compounds and antisense polynucleotides comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises an unlocked nucleic acid (UNA) nucleotide and / or a glycol nucleic acid (GNA) nucleotide. Those skilled in the art are aware that UNA and GNA are thermolabile chemical modifications that can significantly improve the off-target profile of siRNA compounds (Janas, et al., Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun 2018;9(1):723. doi:10.1038 / s41467-018-02989-4; Laursen et al., Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo. Mol BioSyst. 2010;6:862-70).

[0117] Another modification that may be included in the RNA of certain embodiments of the AGT dsRNA agents, AGT antisense polynucleotides, and / or AGT sense polynucleotides of the present invention includes one or more ligands, moieties, or conjugates chemically attached to the RNA that enhance one or more characteristics of the AGT dsRNA agent, AGT antisense polynucleotide, and / or AGT sense polynucleotide, respectively. Non-limiting examples of characteristics that may be enhanced include: AGT dsRNA agent, AGT antisense polynucleotide, and / or AGT sense polynucleotide activity, cellular distribution, delivery of the AGT dsRNA agent, pharmacokinetic properties of the AGT dsRNA agent, and cellular uptake of the AGT dsRNA agent. In some embodiments of the present invention, the AGT dsRNA agent includes one or more targeting or linking groups that, in certain embodiments of the present invention, are conjugated to the sense strand. A non-limiting example of a targeting group is a compound containing N-acetyl-galactosamine (GalNAc). The terms "targeting agent," "linking agent," "targeting compound," and "targeting ligand" can be used interchangeably herein. In certain embodiments of the invention, an AGT dsRNA agent comprises a targeting compound conjugated to the 5'-end of the sense strand. In certain embodiments of the invention, an AGT dsRNA agent comprises a targeting compound conjugated to the 3'-end of the sense strand. In some embodiments of the invention, an AGT dsRNA agent comprises a GalNAc-containing targeting group. In certain embodiments of the invention, an AGT dsRNA agent does not comprise a targeting compound conjugated to either or both of the 3'-end and 5'-end of the sense strand. In certain embodiments of the invention, an AGT dsRNA agent does not comprise a GalNAc-containing targeting compound conjugated to either or both of the 5'-end and 3'-end of the sense strand.

[0118] Additional targeting and linking agents are well known in the art, and for example, targeting and linking agents useful in certain embodiments of the present invention include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acids Sci. USA, 1989, 86:6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), thioethers such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. 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 2-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-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), palmitoyl moieties (Mishra et al., Biochim. Biophys.Acta, 1995, 1264:229-237) or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0119] Certain embodiments of compositions comprising an AGT dsRNA agent, an AGT antisense polynucleotide, and / or an AGT sense polynucleotide may include a ligand that alters the distribution, targeting, or other properties of the AGT dsRNA agent. In some embodiments of compositions comprising an AGT dsRNA agent of the invention, for example, the ligand increases affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment, e.g., a cell or organ compartment, a tissue, an organ, or a body region) compared to a species in which such ligand is absent. Ligands useful in the compositions and / or methods of the invention can be naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins), carbohydrates (e.g., dextran, amylopectin, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), or lipids. Ligands can also be recombinant or synthetic molecules, e.g., synthetic polymers such as synthetic polyamino acids or polyamines. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendritic polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0120] The ligand included in the compositions and / or methods of the present invention can comprise a targeting group, non-limiting examples of which are cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, e.g., antibodies that bind to specific cell types such as kidney cells or liver cells. The targeting group can be thyroid stimulating hormone, melanogen, lectins, glycoproteins, surfactant protein A, mucin carbohydrates, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonates, polyglutamic acid, polyaspartic acid, lipids, cholesterol, steroids, bile acids, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptidomimetic.

[0121] Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerin, geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl, palmitic acid, myristic acid, O 3-(oleoyl)lithocholic acid, O3-(oleoyl)cholic acid, dimethoxytrityl or phenoxazine and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocycles, Eu 3+ conjugate), dinitrophenyl, HRP or AP.

[0122] The ligand included in the compositions and / or methods of the invention can be a protein, such as a glycoprotein or peptide, e.g., a molecule with specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a specific cell type, such as cancer cells, endothelial cells, cardiomyocytes, or bone cells. A ligand useful in embodiments of the compositions and / or methods of the invention can be a hormone or hormone receptor. A ligand useful in embodiments of the compositions and / or methods of the invention can be a lipid, a lectin, a carbohydrate, a vitamin, a coenzyme, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. A ligand useful in embodiments of the compositions and / or methods of the invention can be, for example, an agent that increases uptake of an AGT dsRNA agent into a cell by disrupting the cytoskeleton of the cell (e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments). Non-limiting examples of such agents can be: taxon, vincristine, vinblastine, cytochalasin, nocodazole, jasplakinolide, latrunculin A, phalloidin, swinholide A, indanocine and myoservin.

[0123] In some embodiments, the ligand attached to the AGT dsRNA agent of the present invention serves as a pharmacokinetic (PK) modifier. Examples of PK modifiers useful in the compositions and methods of the present invention include, but are not limited to, lipophilic agents, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and aptamers that bind to serum proteins. Oligonucleotides containing many phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides containing multiple phosphorothioate linkages in the backbone, such as oligonucleotides of about 5, 10, 15, or 20 bases, can also be used as ligands in the compositions and / or methods of the present invention.

[0124] AGT dsRNA agent composition In some embodiments of the present invention, the AGT dsRNA agent is in a composition. The compositions of the present invention may include one or more AGT dsRNA agents and, optionally, one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable labels, etc. Non-limiting examples of targeting agents that may be useful in accordance with some embodiments of the methods of the present invention are agents that direct the AGT dsRNA agent of the present invention to and / or within the cells to be treated. The choice of targeting agent will depend on the nature of the AGT-related disease or condition and the target cell type. In a non-limiting example, in some embodiments of the present invention, it is desirable to target the AGT dsRNA agent to and / or within hepatocytes. It should be understood that in some embodiments of the present invention, the therapeutic agent comprises an AGT dsRNA agent together with only a delivery agent, such as a delivery agent containing N-acetylgalactosamine (GalNAc), without an additional linking element. For example, in some embodiments of the invention, an AGT dsRNA agent can be linked to a delivery compound that includes GalNAc, and can be included in a composition containing a pharmaceutically acceptable carrier, and can be administered to a cell or subject in the absence of a detectable label or targeting agent, etc., linked to the AGT dsRNA agent.

[0125] It will be understood that when the AGT dsRNA agents of the invention are administered with and / or linked to one or more delivery agents, targeting agents, labeling agents, etc., one of skill in the art will be able to select and use appropriate agents for use in the methods of the invention. Labeling agents may be used in certain methods of the invention to determine the location of AGT dsRNA agents in cells and tissues, and may be used to identify the cell, tissue, or organ location of therapeutic compositions comprising AGT dsRNA agents administered in the methods of the invention. Means for attaching and using enzyme labels, dyes, radiolabels, etc. are well known in the art. It will be understood that in some embodiments of the compositions and methods of the invention, a labeling agent is linked to one or both of the sense and antisense polynucleotides contained in the AGT dsRNA agent.

[0126] Delivery of AGT dsRNA agents and AGT antisense polynucleotide agents Certain embodiments of the methods of the invention involve the delivery of an AGT dsRNA agent into a cell. As used herein, the term "delivery" means to facilitate or affect cellular uptake or absorption. Absorption or uptake of an AGT dsRNA agent can occur by independent diffusive or active cellular processes, or by the use of delivery agents, targeting agents, etc., that can bind to the AGT dsRNA agents of the invention. Delivery modes suitable for use in the methods of the invention include, but are not limited to, in vivo delivery, in which the AGT dsRNA agent is injected into a tissue site or administered systemically. In some embodiments of the invention, the AGT dsRNA agent is linked to a delivery agent.

[0127] Non-limiting examples of methods that can be used to deliver AGT dsRNA agents to cells, tissues, and / or subjects include: AGT dsRNA-GalNAc conjugates, SAMiRNA technology, LNP-based delivery methods, and naked RNA delivery. These and other delivery methods have been successfully used in the art to deliver RNAi therapeutics in the treatment of various diseases and conditions, including, but not limited to, liver disease, acute intermittent porphyria (AIP), hemophilia, pulmonary fibrosis, etc. Details of various delivery methods are described in publications such as Nikam, R.R. & K.R.Gore (2018) Nucleic Acid Ther, 28(4), 209-224 Aug 2018; Springer A.D. & S.F. Dowdy (2018) Nucleic Acid Ther. Jun1; 28(3): 109-118; Lee, K. et al., (2018) Arch Pharm Res, 41(9), 867-874; and Nair, J.K. et al., (2014) J.Am.Chem.Soc. 136: 16958-16961, the contents of which are incorporated herein by reference.

[0128] Some embodiments of the present invention include the use of lipid nanoparticles (LNPs) to deliver the AGT dsRNA agents of the present invention to cells, tissues, and / or subjects. LNPs are commonly used for in vivo delivery of AGT dsRNA agents, such as AGT dsRNA therapeutic agents. One advantage of using LNPs or other delivery agents is that the stability of the AGT RNA agent is enhanced when delivered to a subject using LNPs or other delivery agents. In some embodiments of the present invention, the LNPs comprise cationic LNPs loaded with one or more AGT RNAi molecules of the present invention. The LNPs containing the AGT RNAi molecules are administered to a subject, and the LNPs and their bound AGT RNAi molecules are taken up by cells via endocytosis, and their presence releases the RNAi trigger molecule, thereby mediating RNAi.

[0129] Another non-limiting example of a delivery agent that can be used in embodiments of the present invention to deliver an AGT dsRNA agent of the present invention to a cell, tissue, and / or subject is a GalNAc-containing agent that is linked to an AGT dsRNA agent of the present invention and delivers the AGT dsRNA agent to a cell, tissue, and / or subject. Examples of certain other GalNAc-containing delivery agents that can be used in certain embodiments of the methods and compositions of the present invention are disclosed in PCT Application WO 2020191183A1. A non-limiting example of a GalNAc targeting ligand that can be used in the compositions and methods of the present invention to deliver an AGT dsRNA agent to a cell is a targeting ligand cluster. Examples of targeting ligand clusters proposed herein are: GalNAc ligands with phosphodiester linkages (GLO) and GalNAc ligands with phosphorothioate linkages (GLS). The term "GLX-n" may be used herein to mean that the conjugated GalNAC-containing compound is any one of compounds GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, the structures of each of which are shown below. In the figures below, the attachment positions of the GalNAc targeting ligands and RNAi agents of the invention are at the far right of each targeting ligand. It is understood that any of the RNAi and dsRNA molecules of the present invention can be linked to GLS-1, GLS-2, GLS-3, GLS-4, GLS-5, GLS-6, GLS-7, GLS-8, GLS-9, GLS-10, GLS-11, GLS-12, GLS-13, GLS-14, GLS-15, GLS-16, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16.The structures of GLO-1 to GLO-16 and GLS-1 to GLS-16 are shown below. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0130] In some embodiments of the present invention, in vivo delivery can also be via a β-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Patent Application Publication No. 2005 / 0281781, the entire contents of which are incorporated herein by reference. AGT RNAi agents can also be introduced into cells ex vivo using methods known in the art, such as electroporation and lipofection. In certain embodiments of the methods of the present invention, AGT dsRNAs are delivered without a targeting agent. These RNAs can be delivered as "naked" RNA molecules. As a non-limiting example, AGT dsRNAs of the present invention can be administered to a subject in a pharmaceutical composition comprising an RNAi agent, rather than a targeting agent (e.g., a GalNAc-targeting compound), to treat an AGT-related disease or condition in the subject, such as hypertension.

[0131] It is understood that in addition to the specific delivery modes described herein, other RNAi delivery modes can be used with the AGT RNAi agent and treatment method embodiments described herein, including, but not limited to, those described herein and those used in the art.

[0132] The AGT dsRNA agents of the invention can be administered to a subject in an amount and manner effective to reduce the level and activity of an AGT polypeptide in a cell and / or subject. In some embodiments of the invention, one or more AGT dsRNA agents are administered to a subject to treat a disease or condition associated with AGT expression and activity. In some embodiments, methods of the invention comprise administering one or more AGT dsRNA agents to a subject in need of such treatment to alleviate a disease or condition associated with AGT expression in the subject. The AGT dsRNA agents or AGT antisense polynucleotide agents of the invention can be administered to reduce AGT expression and / or activity in one or more cells in vitro, ex vivo, and in vivo.

[0133] In some embodiments of the present invention, the level of AGT polypeptide in a cell, and therefore its activity, is reduced by delivering (e.g., introducing) an AGT dsRNA agent or an AGT antisense polynucleotide agent into the cell. Targeting agents and methods can be used to facilitate delivery of the AGT dsRNA agent or AGT antisense polynucleotide agent to a specific cell type, cell subtype, organ, or spatial region within a subject and / or subcellular region within a cell. In certain methods of the present invention, an AGT dsRNA agent is administered alone or in combination with one or more additional AGT dsRNA agents. In some embodiments, two, three, four, or more independently selected AGT dsRNA agents are administered to a subject. In certain embodiments of the present invention, an AGT dsRNA agent is administered to a subject to treat an AGT-related disease or condition in combination with one or more additional therapies for treating an AGT-related disease or condition. Non-limiting examples of additional therapeutic regimens include administration of one or more AGT antisense polynucleotides of the present invention, administration of a non-AGT dsRNA therapeutic agent, and behavioral modification. The additional treatment regimen may be administered at one or more of the following times: before, simultaneously with, and after administration of an AGT dsRNA agent of the invention. As used herein, "simultaneously" means within 5 minutes of time zero, within 10 minutes of time zero, within 30 minutes of time zero, within 45 minutes of time zero, and within 60 minutes of time zero, where "time zero" is understood to be the time at which an AGT dsRNA agent of the invention is administered to a subject. Non-limiting examples of non-AGT dsRNA therapeutic agents are: additional therapeutic agents such as diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta-blockers, vasodilators, calcium channel blockers, aldosterone antagonists, alpha-2 agonists, renin inhibitors, alpha-blockers, peripherally acting adrenergic agonists, selective D1 receptor partial agonists, non-selective alpha-adrenergic antagonists, synthetic, steroidal antimineralcorticoids, or combinations of any of the above, and drugs for treating hypertension formulated into pharmaceutical combinations. Non-limiting examples of behavioral modifications are: dosing regimens, counseling, and exercise therapy.These and other therapeutic agents and behavioral modifications are known in the art and can be used to treat an AGT disease or condition in a subject, and can be administered to a subject in conjunction with one or more AGT dsRNA agents of the invention to treat an AGT disease or condition. An AGT dsRNA agent of the invention administered to a cell or subject to treat an AGT-associated disease or condition can act synergistically with one or more other therapeutic agents or active ingredients, thereby boosting the effectiveness of the one or more other therapeutic agents or active ingredients and / or enhancing the effectiveness of the AGT dsRNA agent in treating an AGT-associated disease or condition.

[0134] Therapeutic methods of the invention involve administration of an AGT dsRNA agent before the onset of an AGT-associated disease or condition, and / or when an AGT-associated disease or condition is present, e.g., at early, intermediate, or late stages of the disease or condition, and any time before or after any of these stages. The methods of the invention can also treat subjects who have been previously treated for an AGT-associated disease or condition with one or more other therapeutic agents and / or therapeutically active ingredients that have been unsuccessful, minimally successful, and / or no longer successful in treating the subject's AGT-associated disease or condition.

[0135] Vector-encoding dsRNA In certain embodiments of the present invention, a vector can be used to deliver an AGT dsRNA agent to a cell. The AGT dsRNA agent transcription unit can be contained in a DNA or RNA vector. The construction and use of such transgene-encoding vectors for delivering sequences to cells and / or subjects is well known in the art. For example, vectors that result in transient expression of AGT dsRNA for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more hours, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks, can be used in the methods of the present invention. The length of transient expression can be determined using conventional methods based on factors such as, but not limited to, the specific vector construct selected and the target cell and / or tissue. Such transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative vectors. The transgene can also be engineered so that it is inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0136] One or more single strands of an AGT dsRNA agent can be transcribed from a promoter on an expression vector. When two separate strands are expressed to produce, for example, dsRNA, two separate expression vectors can be co-introduced into cells using means such as transfection or infection. In certain embodiments, each individual strand of an AGT dsRNA agent of the present invention can be transcribed from a promoter contained on the same expression vector. In certain embodiments of the present invention, an AGT dsRNA agent is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence so that the AGT dsRNA agent has a stem and loop structure.

[0137] Non-limiting examples of RNA expression vectors are DNA plasmids or viral vectors. The expression vectors useful in embodiments of the present invention can be compatible with eukaryotic cells. Eukaryotic expression vectors are commonly used in the art and are available from many commercial sources. The delivery of AGT dsRNA expression vectors can be systemic, for example, by intravenous or intramuscular administration, by administration to target cells that are removed from the subject and reintroduced into the subject, or by any means that allows the introduction of target cells of interest.

[0138] Viral vector systems that can be included in the method embodiments include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors, including, but not limited to, lentiviral vectors, Moloney murine leukemia virus vectors, and the like; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopoxvirus vectors, for example, vaccinia virus vectors or avian poxvirus vectors, such as canary or fowl poxvirus vectors; and (j) helper-dependent or gutless adenovirus vectors. Constructs for recombinant expression of AGT dsRNA agents can contain regulatory elements, such as promoters, enhancers, and the like, which can be selected to provide constitutive or regulated / inducible expression. The use of viral vector systems, promoters, enhancers, and the like, is conventional in the art and can be used in conjunction with the methods and compositions described herein.

[0139] Certain embodiments of the present invention include the use of viral vectors to deliver AGT dsRNA agents to cells.Many adenovirus-based delivery systems are commonly used in the art, for example, to deliver to lung, liver, central nervous system, endothelial cells, and muscle.Non-limiting examples of viral vectors that can be used in the methods of the present invention include: AAV vectors, poxviruses such as vaccinia virus, modified virus Ankara (MVA), NYVAC, or avian poxviruses such as fowl or canary poxvirus.

[0140] Certain embodiments of the present invention include methods of delivering an AGT dsRNA agent to a cell using a vector, which may be a pharmaceutically acceptable vector that does not need to, but may, include a slow-release matrix in which the gene delivery vector is embedded. In some embodiments, the vector for delivering AGT dsRNA can be produced by recombinant cells, and the pharmaceutical compositions of the present invention may include one or more cells that produce the AGT dsRNA delivery system.

[0141] Pharmaceutical compositions containing AGT dsRNA or ssRNA agents Certain embodiments of the invention include the use of pharmaceutical compositions containing an AGT dsRNA agent or an AGT antisense polynucleotide agent and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing an AGT dsRNA agent or an AGT antisense polynucleotide agent can be used in the methods of the invention to reduce AGT gene expression and AGT activity in cells and can be used to treat related diseases or conditions. Such pharmaceutical compositions can be formulated based on the mode of delivery. Non-limiting examples of formulations for delivery modes include compositions formulated for subcutaneous delivery, compositions formulated for systemic administration via parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for intrathecal delivery, and compositions formulated for direct delivery into the brain. Pharmaceutical compositions of the invention can be administered using one or more means to deliver an AGT dsRNA agent or AGT antisense polynucleotide agent to cells, for example: topical administration (e.g., via a transdermal patch); pulmonary administration, e.g., via inhalation or insufflation of a powder or aerosol, such as via a nebulizer; intratracheal, intranasal, epidermal, and transdermal, oral, or parenteral administration. Parenteral administration can be by intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous administration, e.g., via an implanted device; or intracranial administration, e.g., via intraparenchymal, intrathecal, or intraventricular administration. An AGT dsRNA agent or AGT antisense polynucleotide agent can also be delivered directly to a target tissue, such as directly to the liver or directly to the kidney. "Delivering an AGT dsRNA agent" or "delivering an AGT antisense polynucleotide agent" to a cell should be understood to encompass any suitable means of delivering an AGT dsRNA agent or an AGT antisense polynucleotide agent, respectively, expressing an AGT dsRNA agent directly in a cell, and expressing an AGT dsRNA agent from an encoding vector delivered to a cell, or presenting an AGT dsRNA or an AGT antisense polynucleotide agent in a cell. The preparation and use of formulations and means of delivering inhibitory RNA are well known and commonly used in the art.

[0142] As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of an AGT dsRNA agent or AGT antisense polynucleotide agent of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier used to administer a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. This term specifically excludes cell culture media. For orally administered drugs, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, with corn starch and alginic acid being suitable disintegrants. Binders include starch and gelatin, and white lubricants, if present, are usually magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. Agents included in pharmaceutical formulations are further described below. As used herein, terms such as "pharmacologically effective amount," "therapeutically effective amount," and "effective amount" refer to the amount of an AGT dsRNA agent or AGT antisense polynucleotide agent of the present invention that produces the intended pharmacological, therapeutic, or preventative result. For example, if a given clinical treatment is considered effective if it reduces a measurable parameter associated with a disease or disorder by at least 10%, then a therapeutically effective amount of a drug used to treat that disease or condition is the amount required to reduce that parameter by at least 10%. For example, a therapeutically effective amount of an AGT dsRNA agent or AGT antisense polynucleotide agent can reduce AGT polypeptide levels by at least 10%. The pharmaceutical composition may include a duplex-containing dsRNAi agent such as AD00051 to AD00122-19-2, AD00163-3, AV01227 to AVAV01257, and AV01711 shown in Table 1.In some embodiments, preferred dsRNAi agents include, for example, duplex AD00158, AD00163, AD00159, AD00290, AD00300, or AD00122. In other embodiments, preferred dsRNAi agents include, for example, AD00158-1, AD00158-2, AD00163-1, AD00159-1, or AD00300-1. In some other embodiments, such dsRNAi agents include duplex variants, for example, duplex AD00158, AD00163, AD00163-3, AD00159, AD00290, AD00300, or AD00122 variants.

[0143] Effective dose In some aspects, the methods of the invention involve contacting a cell with an effective amount of an AGT dsRNA agent or an AGT antisense polynucleotide agent to reduce AGT gene expression in the contacted cell. Certain embodiments of the methods of the invention involve administering an AGT dsRNA agent or an AGT antisense polynucleotide agent to a subject in an amount effective to reduce AGT gene expression and treat an AGT-related disease or condition in the subject. When used to reduce expression of AGT and / or to treat an AGT-related disease or condition, an "effective amount" is an amount necessary or sufficient to realize a desired biological effect. For example, an effective amount of an AGT dsRNA agent or an AGT antisense polynucleotide agent for treating an AGT-related disease or condition can be the amount necessary to (i) slow or halt the progression of the disease or condition; or (ii) reverse, reduce, or eliminate one or more symptoms of the disease or condition. In some aspects of the invention, an effective amount is the amount of an AGT dsRNA agent or AGT antisense polynucleotide agent that, when administered to a subject in need of treatment for an AGT-related disease or condition, results in a therapeutic response and the disease or condition is prevented and / or treated. According to some aspects of the invention, an effective amount is the amount of an AGT dsRNA agent or AGT antisense polynucleotide agent that, when combined with or co-administered with another therapeutic treatment for an AGT-related disease or condition, results in a therapeutic response and the disease or condition is prevented and / or treated. In some embodiments of the invention, the biological effect of treating a subject with an AGT dsRNA agent or AGT antisense polynucleotide agent of the invention can be the alleviation and / or complete elimination of symptoms caused by an AGT-related disease or condition. In some embodiments of the invention, the biological effect is the complete prevention of an AGT-related disease or condition, as demonstrated, for example, by a diagnostic test indicating that the subject is free of an AGT-related disease or condition. Non-limiting examples of detectable physiological symptoms include reduced lipid accumulation in the liver of a subject following administration of an agent of the invention. Other means known in the art of assessing the status of an AGT-associated disease or condition can be used to determine the effect of the agents and / or methods of the invention on an AGT-associated disease or condition.

[0144] The effective amount of an AGT dsRNA agent or AGT antisense polynucleotide agent that reduces AGT polypeptide activity to a level that treats an AGT-related disease or condition is typically determined in clinical trials, with the effective dose established in blinded studies of a test population versus a control population. In some embodiments, an effective amount is an amount that produces a desired response, such as a reduction in an AGT-related disease or condition in cells, tissues, and / or subjects affected by the disease or condition. Thus, an effective amount of an AGT dsRNA agent or AGT antisense polynucleotide agent for treating an AGT-related disease or condition treatable by reducing AGT polypeptide activity can be an amount that, when administered, reduces the amount of AGT polypeptide activity in a subject to an amount below that which would be present in cells, tissues, and / or subjects not administered the AGT dsRNA agent or AGT antisense polynucleotide agent. In certain aspects of the invention, the level of AGT polypeptide activity and / or AGT gene expression present in cells, tissues, and / or subjects not exposed to or administered an AGT dsRNA agent or AGT antisense polynucleotide agent of the invention is referred to as a "control" amount. In some embodiments of the invention, the subject's control amount is the amount before treatment of the subject; in other words, the level in the subject before administration of the AGT agent can be the subject's control level and can be used for comparison with the level of AGT polypeptide activity and / or AGT gene expression in the subject after administration of the siRNA to the subject. When treating an AGT-related disease or condition, the desired response can be a reduction or elimination of one or more symptoms of the disease or condition in a cell, tissue, and / or subject. The reduction or elimination can be temporary or permanent. It should be understood that the status of an AGT-related disease or condition can be monitored using methods to determine AGT polypeptide activity, AGT gene expression, symptom assessment, clinical testing, etc. In some aspects of the invention, the desired response to treatment of an AGT-related disease or condition is delaying or even preventing the onset of the symptom or condition.

[0145] The effective amount of a compound that reduces the activity of an AGT polypeptide can also be determined by evaluating the physiological effect of administering the AGT dsRNA agent or AGT antisense polynucleotide agent to a cell or subject, such as a reduction in an AGT-related disease or condition after administration. Assays and / or monitoring of symptoms in a subject can be used to determine the effectiveness of an AGT dsRNA agent or AGT antisense polynucleotide agent of the present invention (which can be administered in a pharmaceutical composition of the present invention) and to determine whether there is a response to treatment. A non-limiting example is one or more blood pressure tests known in the art. Another non-limiting example is one or more blood pressure tests known in the art for determining the status of a subject's AGT-related disorder before and after treating the subject with an AGT dsRNA agent of the present invention. In another non-limiting example, the status of an AGT-related disease in a subject is determined using one or more tests known in the art that reduce blood pressure levels. In this example, the disease includes hypertension, and the test is used to determine a reduction in blood pressure levels in the subject before and after treatment with an AGT dsRNA agent of the present invention.

[0146] Some embodiments of the invention include methods of determining the effectiveness of a dsRNA agent or AGT antisense polynucleotide agent of the invention administered to a subject to treat an AGT-associated disease or condition by assessing and / or monitoring one or more "physiological characteristics" of the AGT-associated disease or condition in the subject. Non-limiting examples of physiological characteristics of an AGT-associated disease or condition are serum AGT levels, mean blood pressure, and diastolic blood pressure in the subject. Standard methods for determining such physiological characteristics are known in the art and include, but are not limited to, blood tests, imaging studies, physical examinations, etc.

[0147] It should be understood that the amount of an AGT dsRNA agent or AGT antisense polynucleotide agent administered to a subject may be modified, at least in part, based on such determinants of disease and / or the state and / or physiological characteristics of the condition as determined by the subject. To increase or decrease the amount of an AGT dsRNA agent or AGT antisense polynucleotide agent, the therapeutic dose can be altered, for example, by changing the composition in which the AGT dsRNA agent or AGT antisense polynucleotide agent is administered, by changing the route of administration, or by changing the timing of administration. The effective amount of an AGT dsRNA agent or AGT antisense polynucleotide agent will vary depending on the specific condition being treated, the age and condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent treatment (if any), the specific route of administration, and other factors within the knowledge and professional opinion of a healthy physician. For example, the effective amount may vary depending on the desired level of AGT polypeptide activity and / or AGT gene expression effective in treating an AGT-related disease or condition. Those skilled in the art can empirically determine the effective amount of a particular AGT dsRNA agent or AGT antisense polynucleotide agent used in the methods of the present invention without undue experimentation. By selecting from among the various AGT dsRNA agents or AGT antisense polynucleotide agents of the present invention, along with the teachings provided herein, and taking into account important factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred mode of administration, it is possible to devise an effective prophylactic or therapeutic regimen for effectively treating a specific subject. An effective amount of an AGT dsRNA agent or AGT antisense polynucleotide agent of the present invention used in embodiments of the present invention can be an amount that, when contacted, produces a desired biological effect in cells.

[0148] It will be appreciated that AGT gene silencing can be achieved constitutively or by genome engineering in any cell that expresses AGT and can be determined by an appropriate assay. In some embodiments of the invention, AGT gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% by administering an AGT dsRNA agent of the invention. In some embodiments of the invention, AGT gene expression is reduced by 5-10%, 5-25%, 10-50%, 10-75%, 25-75%, 25-100%, or 50-100% by administering an AGT dsRNA agent of the invention.

[0149] dosage AGT dsRNA agents and AGT antisense polynucleotide agents are delivered in pharmaceutical compositions in dosages sufficient to inhibit expression of the AGT gene. In certain embodiments of the invention, the dose of an AGT dsRNA agent or AGT antisense polynucleotide agent is 0.01-200.0 mg per kilogram of recipient body weight per day, generally 1-50 mg / kg body weight, 5-40 mg / kg body weight, 10-30 mg / kg body weight, 1-20 mg / kg body weight, 1-10 mg / kg body weight, or 4-15 mg / kg body weight per day. For example, Each single administration of a dsRNA agent or AGT antisense polynucleotide agent may be administered in the amount of about 0.01 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2mg / kg, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4mg / kg, 4.1m g / kg, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5mg / kg, 5.1mg / kg, 5 .2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6mg / kg, 6.1mg / kg, 6.2mg / k g, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3m g / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8mg / kg, 8.9mg / kg, 9mg / kg, 9.1mg / kg, 9.2mg / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg, 9.7mg / kg, 9.8mg / kg, 9.9mg / kg, 10mg / kg, 11mg / kg, 12mg / kg, 13mg / kg, 14mg / kg, 15mg / kg, 16mg / kg, 17mg / kg, 18mg / kg, 19mg / kg, 20mg / kg, 21mg / kg, 22m It may be administered at doses ranging from 23mg / kg, 24mg / kg, 25mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg, 36mg / kg, 37mg / kg, 38mg / kg, 39mg / kg, 40mg / kg, 41mg / kg, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, and 49-50mg / kg body weight.

[0150] When determining the dosage and delivery timing of the AGT dsRNA agent of the present invention, various factors can be considered.The absolute amount of the AGT dsRNA agent or AGT antisense polynucleotide agent delivered will vary depending on various factors, such as simultaneous treatment, number of administrations, and individual subject parameters such as age, physical condition, size, and weight.These factors are well known to those skilled in the art and can be addressed by routine experimentation.In some embodiments, a maximum dose, i.e., the highest safe dose according to sound medical judgment, can be used.

[0151] In some embodiments, the methods of the invention can include administering one, two, three, four, five, six, seven, eight, nine, ten, or more doses of an AGT dsRNA agent or an AGT antisense polynucleotide agent to a subject. In some cases, a dose of a pharmaceutical compound (e.g., comprising an AGT dsRNA agent or comprising an AGT antisense polynucleotide agent) can be administered to a subject at least daily, every other day, weekly, biweekly, monthly, etc. Doses can be administered one or more times daily, for example, two, three, four, five, or more times within a 24-hour period. The pharmaceutical compositions of the invention can be administered once daily; alternatively, the AGT dsRNA agent or AGT antisense polynucleotide agent can be administered in two, three, or more subdoses at appropriate intervals throughout the day, or using continuous infusion or sustained-release delivery. In some embodiments of the invention, the pharmaceutical compositions of the invention are administered to a subject one or more times daily, one or more times weekly, one or more times monthly, or one or more times yearly.

[0152] In certain embodiments, the methods of the present invention involve administering a pharmaceutical compound alone, in combination with one or more other AGT dsRNA agents or AGT antisense polynucleotide agents, and / or in combination with other medications or therapeutic activities or dosing regimens administered to a subject suffering from an AGT-related disease or condition. The pharmaceutical compound may be administered in the form of a pharmaceutical composition. The pharmaceutical compositions used in the methods of the present invention are sterile and may contain an amount of an AGT dsRNA agent or AGT antisense polynucleotide agent that reduces the activity of an AGT polypeptide to a level sufficient to produce the desired response in a unit of weight or volume suitable for administration to a subject. The dosage of a pharmaceutical composition containing an AGT dsRNA agent or AGT antisense polynucleotide agent administered to a subject to reduce AGT protein activity can be selected depending on various parameters, particularly the mode of administration used and the subject's condition. Other factors include the required duration of treatment. If the subject responds inadequately to the initial dose, a higher dose can be administered (or the dose can be effectively increased via a different, more localized delivery route) as long as it is tolerated by the patient.

[0153] treatment As used herein, the term "prevention" or "preventing," when used to refer to a disease, disorder, or condition that would benefit from reduced AGT gene expression, means that a subject is less likely to develop symptoms associated with the disease, disorder, or condition, e.g., a condition caused by or related to activation of the renin-angiotensin-aldosterone system (RAAS), such as hypertension. In such a situation, the likelihood of developing hypertension is reduced; for example, prevention is considered effective if an individual has one or more risk factors for hypertension but does not develop hypertension, or only develops less severe hypertension, or does not develop the disease, disorder, or condition, or the onset of symptoms associated with such disease, disorder, or condition is reduced (e.g., by at least 10% on the scale of the disease or condition in a clinical setting), or the onset of symptoms is delayed (e.g., by days, weeks, months, or years) compared to a population with the same risk factors that does not receive the treatment described herein.

[0154] Based on the average of accurately measured sitting blood pressure readings during two or more visits, normotensive subjects had a systolic blood pressure of approximately 90 to 119 mmHg (approximately 12 to 15.9 kPa (kN / m 2 )) and diastolic blood pressure of approximately 60-79 mmHg (approximately 8.0-10.5 kPa (kN / m 2 Subjects with prehypertension have a systolic blood pressure of approximately 120-139 mmHg (approximately 16.1-18.5 kPa (kN / m 2 )) and diastolic blood pressure of approximately 60-79 mmHg (approximately 8.0-10.5 kPa (kN / m 2 )); hypertensive subjects (e.g., Stage I hypertension) have a systolic blood pressure of about 140-159 mmHg (about 18.7-21.2 kPa (kN / m 2 )) and diastolic blood pressure of approximately 90-99 mmHg (approximately 12.0-13.2 kPa (kN / m 2 a hypertensive subject (e.g., stage II hypertension) has a systolic blood pressure of about ≧160 mmHg (about ≧21.3 kPa (kN / m ≧)) and a diastolic blood pressure of about ≧100 mmHg (about ≧13.3 kPa (kN / m ≧)); 2 )).

[0155] In certain embodiments, the angiotensinogen-related disorder is essential hypertension. "Essential hypertension" is the result of environmental or genetic factors (e.g., the result without a clear, underlying medical cause).

[0156] In certain embodiments, the angiotensinogen-related disorder is secondary hypertension. "Secondary hypertension" has an identifiable underlying condition and can have multiple etiologies, including renal, vascular, and endocrine causes, such as renal parenchymal disease (e.g., polycystic kidney disease, glomerular, or interstitial disease), renal vascular disease (e.g., renal artery stenosis, fibromuscular dysplasia), endocrine disorders (e.g., corticosteroid or mineralocorticoid excess, pheochromocytoma, hyper- or hypothyroidism, growth hormone excess, hyperparathyroidism), coarctation of the aorta, or use of oral contraceptives.

[0157] In certain embodiments, the angiotensinogen-related disorder is a hypertensive emergency, such as malignant hypertension and accelerated hypertension. "Accelerated hypertension" refers to a severe elevation in blood pressure (i.e., systolic blood pressure ≥ 180 mmHg or diastolic blood pressure ≥ 110 mmHg) accompanied by direct damage to one or more end organs. Blood pressure must be immediately reduced to prevent further organ damage. "Malignant hypertension" refers to severe hypertension (i.e., systolic blood pressure ≥ 180 mmHg or diastolic blood pressure ≥ 110 mmHg) accompanied by direct damage to one or more end organs and papilledema. Blood pressure must be immediately reduced to prevent further organ damage. End organ damage resulting from uncontrolled blood pressure may include hypertensive encephalopathy, cerebrovascular accident / cerebral infarction, subarachnoid hemorrhage, and / or intracranial hemorrhage. Cardiovascular end-organ damage includes myocardial ischemia / infarction, acute left ventricular dysfunction, acute pulmonary edema, and / or arterial dissection. Other organ systems may also be affected by uncontrolled hypertension, which may lead to acute renal failure / dysfunction, retinopathy, eclampsia, or microangiopathic hemolytic anemia.

[0158] In certain embodiments, the angiotensinogen-related disorder is acute hypertension. "Acute hypertension" refers to a severe increase in blood pressure (i.e., systolic blood pressure of 180 mmHg or greater or diastolic blood pressure of 110 mmHg) without direct damage to one or more organs. Blood pressure can be safely reduced within a few hours.

[0159] In certain embodiments, the angiotensinogen-related disorder is pregnancy-associated hypertension, such as chronic hypertension of pregnancy, gestational hypertension, pre-eclampsia, eclampsia, pre-eclampsia aggravated by chronic hypertension, HELLP syndrome, and pregnancy-induced hypertension (also called transient gestational hypertension, chronic hypertension seen in late pregnancy, and gestational hypertension (PIH)). A subject with "chronic gestational hypertension" is one whose blood pressure exceeds 140 / 90 mmHg before pregnancy or before 20 weeks of pregnancy. "Gestational hypertension" or "pregnancy-induced hypertension" refers to hypertension that develops late in pregnancy (more than 20 weeks of pregnancy) without other features of pre-eclampsia and returns to normal after delivery. "Mild pre-eclampsia" is defined as two episodes of hypertension (blood pressure ≥ 140 / 90 mmHg) occurring at least 6 hours apart in a normotensive woman before 20 weeks of pregnancy, but without evidence of end-organ damage. In subjects with existing essential hypertension, preeclampsia is diagnosed when systolic blood pressure rises by 30 mmHg or diastolic blood pressure rises by 15 mmHg. "Severe preeclampsia" is defined as the presence of one of the following signs or symptoms of preeclampsia: two episodes at least 6 hours apart with a systolic blood pressure of 160 mmHg or more, or a diastolic blood pressure of 110 mmHg or more; proteinuria of more than 5 g collected over 24 hours, or more than 3+ in two random urine samples collected at least 4 hours apart; pulmonary edema or cyanosis; oligourinary output (less than 400 mL in 24 hours); persistent headache, epigastric pain, and / or liver dysfunction; thrombocytopenia, oligohydramnios, fetal growth retardation, or placental abruption. "Eclampsia" is defined as an attack that cannot be attributed to other causes in women with preeclampsia. "HELLP syndrome" (also known as edematous-proteinuric-hypertensive preeclampsia type B) refers to hemolysis, elevated liver enzyme levels, and decreased platelet levels in pregnant subjects.

[0160] In certain embodiments, the angiotensinogen-related disorder is resistant hypertension. "Resistant hypertension" refers to blood pressure that remains above the target value (for example, 140 / 90 mmHg) despite the simultaneous use of three different classes of antihypertensive drugs, one of which is a thiazide diuretic. Subjects who control their blood pressure with four or more drugs are also considered to have resistant hypertension.

[0161] AGT-associated diseases and conditions, in which a decrease in the level and / or activity of an AGT polypeptide is effective in treating the disease or condition, can be treated using the methods and AGT dsRNA agents of the invention to inhibit AGT expression. Examples of diseases and conditions that can be treated with the AGT dsRNA agents or AGT antisense polynucleotide agents of the invention and methods of the invention include, but are not limited to, hypertensive disease, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-associated hypertension, diabetic hypertension, treatment-resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), and fetal growth retardation. Such diseases and conditions may be referred to herein as "AGT-associated diseases and conditions" and "diseases and conditions caused and / or modulated by AGT."

[0162] In certain aspects of the invention, the AGT dsRNA agents or AGT antisense polynucleotide agents of the invention can be administered to a subject one or more times before or after diagnosis of an AGT-related disease or condition. In some aspects of the invention, the subject is suffering from or at risk of developing an AGT-related disease or condition. A subject at risk of developing an AGT-related disease or condition is a subject who has an increased likelihood of developing an AGT-related disease or condition compared to a control risk of developing an AGT-related disease or condition. In some embodiments of the invention, the level of risk is statistically significant compared to the control level of risk. At-risk subjects include, for example, subjects who are or will be subjects with a pre-existing disease and / or genetic abnormality that predisposes the subject to developing an AGT-related disease or condition relative to control subjects without the pre-existing disease or genetic abnormality; subjects with a family and / or personal history of an AGT-related disease or condition; and subjects who have previously received treatment for an AGT-related disease or condition. It is understood that the pre-existing disease and / or genetic abnormality that predisposes a subject to an AGT-associated disease or condition can be a disease or genetic abnormality that, if present, has previously been determined to be associated with an increased likelihood of developing an AGT-associated disease or condition.

[0163] It should be understood that an AGT dsRNA agent or an AGT antisense polynucleotide agent can be administered to a subject based on the individual subject's condition. For example, the health care provided to the subject can assess the AGT level measured in a sample taken from the subject and determine whether it is desirable to reduce the subject's AGT level by administering an AGT dsRNA agent or an AGT antisense polynucleotide agent of the present invention. In a non-limiting example, a biological sample, such as a blood or serum sample, can be taken from the subject, and the subject's AGT level can be determined in the sample. An AGT dsRNA agent or an AGT antisense polynucleotide agent is administered to the subject, a blood or serum sample is taken from the subject after administration, the AGT level is determined using the sample, and the result is compared to the subject's pre-administration (previous) sample. A decrease in the subject's AGT level in the subsequent sample compared to the pre-administration level indicates the effectiveness of the administered AGT dsRNA agent or AGT antisense polynucleotide agent in reducing the subject's AGT level. In one non-limiting example, even if a subject has not been diagnosed with an AGT-related disorder, such as a disorder disclosed herein, blood pressure may be considered a physiological characteristic of the related disorder. Health care providers can monitor changes in a subject's blood pressure as a measure of the effectiveness of an administered AGT dsRNA or AGT antisense polynucleotide agent of the invention.

[0164] Certain embodiments of the methods of the invention include administering a dsRNA agent or AGT antisense polynucleotide agent of the invention to a subject based at least in part on an assessment of changes in one or more physiological characteristics of an AGT-related disease or condition in the subject. For example, in some embodiments of the invention, the effect of a dsRNA agent or AGT antisense polynucleotide agent of the invention administered to a subject can be determined and used to help adjust the amount of a dsRNA agent or AGT antisense polynucleotide agent of the invention subsequently administered to the subject. In one non-limiting example, a dsRNA agent or AGT antisense polynucleotide agent of the invention is administered to a subject, the subject's blood pressure is determined after administration, and based at least in part on the determined level, it is determined whether a larger amount of the dsRNA agent or AGT antisense polynucleotide agent is needed to enhance the physiological effect of the administered agent, such as lowering or further lowering the subject's blood pressure. In another non-limiting example, a dsRNA agent or AGT antisense polynucleotide agent of the invention is administered to a subject, the subject's blood pressure is determined after administration, and based at least in part on the determined level, it is anticipated that a smaller amount of the dsRNA agent or AGT antisense polynucleotide agent will be administered.

[0165] Thus, some embodiments of the invention include assessing changes in one or more physiological characteristics of a subject from before treatment in order to adjust the amount of a dsRNA agent or AGT antisense polynucleotide agent of the invention subsequently administered to the subject. Some embodiments of the methods of the invention include determining one, two, three, four, five, six, or more physiological characteristics of an AGT-related disease or condition; evaluating and / or monitoring the effectiveness of the administered AGT dsRNA agent or AGT antisense polynucleotide agent of the invention; and, optionally, using the determined results to adjust one or more of the dosage, administration schedule, and / or administration frequency of the dsRNA agent or AGT antisense polynucleotide agent of the invention to treat an AGT-related disease or condition in the subject. In some embodiments of the invention, the desired result of administering an effective amount of a dsRNA agent or AGT antisense polynucleotide agent of the invention to a subject is a reduction in the subject's blood pressure compared to the subject's previous blood pressure determined for that subject, or a blood pressure within the normal blood pressure range.

[0166] As used herein, the terms "treatment," "therapeutic," or "treated," when used in reference to an AGT-associated disease or condition, can refer to prophylactic treatment to reduce the likelihood of a subject developing an AGT-associated disease or condition, and can also refer to treatment after a subject has developed an AGT-associated disease or condition to eliminate the AGT-associated disease or condition or to reduce the level of the AGT-associated disease or condition, to prevent the AGT-associated disease or condition from becoming more severe, and / or to slow the progression of the AGT-associated disease or condition in the subject compared to a subject who has not received treatment that reduces AGT polypeptide activity in the subject.

[0167] Certain embodiments of the agents, compositions, and methods of the present invention can be used to inhibit AGT gene expression. As used herein, the terms "inhibit," "silence," "reduce," "downregulate," and "knockdown," with respect to AGT gene expression, refer to altering AGT gene expression by, for example, one or more of the following: when a cell, group of cells, tissue, organ, or subject is exposed to (e.g., treated with) an AGT dsRNA agent or AGT antisense polynucleotide agent of the present invention, the level of RNA transcribed from the gene, the level of expressed AGT activity, and the level of AGT polypeptide, protein, or protein subunit translated from mRNA are reduced in a cell, group of cells, tissue, organ, or subject in which the AGT gene is transcribed, compared to a control level of RNA transcribed from the AGT gene, a control level of expressed AGT activity, or a control level of AGT translated from mRNA, respectively. In some embodiments, the control level is the level in a cell, tissue, organ, or subject that has not been exposed to an AGT dsRNA agent or an AGT antisense polynucleotide agent (e.g., not treated with an AGT dsRNA agent or an AGT antisense polynucleotide agent).

[0168] Administration method Various routes of administration of AGT dsRNA agents or AGT antisense polynucleotide agents can be used in the methods of the present invention. The selection of a particular delivery mode will depend, at least in part, on the specific condition being treated and the dosage required for therapeutic efficacy. Generally, the methods of the present invention can be practiced using any medically acceptable form of administration, and refer to any mode that provides effective therapeutic levels for AGT-related diseases or conditions without producing clinically unacceptable side effects. In some embodiments of the present invention, AGT dsRNA agents or AGT antisense polynucleotide agents can be administered orally, enterally, transmucosally, subcutaneously, and / or parenterally. The term "parenteral" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal, and intrasternal injection or infusion techniques. Other routes include, but are not limited to, intranasal (e.g., via nasogastric tube), transdermal, vaginal, rectal, sublingual, and inhalation. Delivery routes of the present invention include intrathecal, intraventricular, or intracranial delivery. In some embodiments of the invention, an AGT dsRNA agent or an AGT antisense polynucleotide agent can be placed in a slow-release matrix and administered by placing the matrix in a subject. In some aspects of the invention, an AGT dsRNA agent or an AGT antisense polynucleotide agent can be delivered to a subject's cells using nanoparticles coated with a delivery agent that targets specific cells or organelles. A variety of delivery modes, methods, and reagents are known in the art. Non-limiting examples of delivery methods and delivery agents are provided elsewhere herein. In some aspects of the invention, the term "delivery" with respect to an AGT dsRNA agent or an AGT antisense polynucleotide agent can refer to administering one or more "naked" AGT dsRNA agent or AGT antisense polynucleotide agent sequences to a cell or subject. In certain aspects of the invention, "delivery" refers to administering to a cell or subject by transfection, delivering a cell containing an AGT dsRNA agent or an AGT antisense polynucleotide agent, or delivering a vector encoding an AGT dsRNA agent or an AGT antisense polynucleotide agent to a cell and / or subject.Delivery of an AGT dsRNA agent or an AGT antisense polynucleotide agent using transfection can include administration of a vector to a cell and / or a subject.

[0169] In some methods of the invention, one or more AGT dsRNA agents or AGT antisense polynucleotide agents may be administered in the form of a preparation or in a pharmaceutically acceptable solution, which may generally contain salts, buffers, preservatives, compatible carriers, adjuvants, and optionally therapeutic ingredients at pharmaceutically acceptable concentrations. In some embodiments of the invention, the AGT dsRNA agent or AGT antisense polynucleotide agent can be formulated with another therapeutic agent for co-administration. According to the methods of the invention, the AGT dsRNA agent or AGT antisense polynucleotide agent can be administered in the form of a pharmaceutical composition. Typically, a pharmaceutical composition comprises an AGT dsRNA agent or AGT antisense polynucleotide agent and, optionally, a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art. As used herein, a pharmaceutically acceptable carrier refers to a non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient (e.g., the ability of the AGT dsRNA agent or AGT antisense polynucleotide agent to inhibit AGT gene expression in a cell or subject). A variety of methods of administering and delivering dsRNA agents or AGT antisense polynucleotide agents for therapeutic use are known in the art and can be used in the methods of the invention.

[0170] Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Pat. No. 5,211,657, and other carriers are known to those skilled in the art. Such formulations generally contain salts, buffers, preservatives, compatible carriers, and, optionally, other therapeutic agents. When used in pharmaceutical preparations, salts should be pharmaceutically acceptable; however, non-pharmaceutically acceptable salts may be conveniently used to prepare pharmaceutically acceptable salts and are not excluded from the scope of the present invention. Such pharmacological and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Furthermore, pharmaceutically acceptable salts may be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium, or calcium salts.

[0171] Some embodiments of the methods of the invention include administering one or more AGT dsRNA agents or AGT antisense polynucleotide agents directly to a tissue. In some embodiments, the tissue to which the compound is administered is a tissue in which an AGT-related disease or condition exists or may occur, non-limiting examples of which are the liver or kidney. Direct tissue drug delivery can be achieved by direct injection or other means. Many orally delivered compounds naturally enter and pass through the liver and kidney, and some embodiments of the therapeutic methods of the invention include orally administering one or more AGT dsRNA agents to a subject. AGT dsRNA agents or AGT antisense polynucleotide agents can be administered once or multiple times, either alone or in combination with other therapeutic agents. When administered multiple times, the AGT dsRNA agents or AGT antisense polynucleotide agents may be administered by different routes. For example, and not intended to be limiting, the first (or first few) administrations can be administered subcutaneously, and one or more additional administrations can be administered orally and / or systemically.

[0172] For embodiments of the invention in which systemic administration of an AGT dsRNA agent or AGT antisense polynucleotide agent is desired, the AGT dsRNA agent or AGT antisense polynucleotide agent can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Injectable formulations can be in unit dosage forms, such as ampoules or multi-dose containers, with or without added preservatives. AGT dsRNA agent formulations (also known as pharmaceutical compositions) can take the form of suspensions, solutions, or emulsions in oily or aqueous carriers and can contain formulatory agents such as suspending, stabilizing, and / or dispersing agents. Dosage forms for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, such as saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (such as solutions based on Ringer's dextrose solution), and other additives such as preservatives, antimicrobial agents, antioxidants, chelating agents, and inert gases. Other forms of administration, such as intravenous administration, allow for lower dosages. If the subject responds inadequately to the initial dose, a higher dose can be administered (or, alternatively, a more localized delivery route can effectively increase the dosage), as tolerated by the patient. Multiple daily administrations can be used as necessary to achieve appropriate systemic or local levels of one or more AGT dsRNA agents or AGT antisense polynucleotide agents, and to achieve an appropriate reduction in AGT protein activity.

[0173] In other embodiments, the methods of the present invention involve the use of a delivery vehicle, such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation in a recipient, such as a subject. An exemplary biodegradable implant that can be used according to this method is described in WO 95 / 24929 (incorporated herein by reference), which describes a biocompatible, biodegradable matrix for containing biopolymers.

[0174] Both non-biodegradable and biodegradable polymer matrices can be used in the methods of the invention to deliver one or more AGT dsRNA agents or AGT antisense polynucleotide agents to a subject. In some embodiments, the matrix can be biodegradable. The matrix polymer can be a natural or synthetic polymer. The polymer can be selected based on the desired period of release, usually from about a few hours to a year or more. Release periods ranging from a few hours to 3 to 12 months are typically available. The polymer optionally takes the form of a hydrogel, capable of absorbing up to about 90% of its weight in water, and is optionally further crosslinked with multivalent ions or other polymers.

[0175] Typically, in some embodiments of the present invention, AGT dsRNA agents or AGT antisense polynucleotide agents can be delivered by diffusion or degradation of a polymer matrix using a biodegradable implant. Exemplary synthetic polymers for this purpose are well known in the art. Biodegradable and non-biodegradable polymers can be used to deliver AGT dsRNA agents or AGT antisense polynucleotide agents using methods known in the art. Bioadhesive polymers, such as bioerodible hydrogels (H.S. Sawhney, C.P. Pathak and J.A. Hubellin, Macromolecules, 1993, 26, 581-587), can also be used to deliver AGT dsRNA agents or AGT antisense polynucleotide agents for the treatment of AGT-related diseases or conditions. Other suitable delivery systems include sustained-release, delayed-release, or sustained-release delivery systems. Such systems can avoid repeated administration of AGT dsRNA agents or AGT antisense polynucleotide agents, thereby improving convenience for subjects and healthcare professionals. Many types of release delivery systems are available and known to those skilled in the art (see, e.g., U.S. Pat. Nos. 5,075,109, 4,452,775, 4,675,189, 5,736,152, 3,854,480, 5,133,974, and 5,407,686). Additionally, pump-based hardware delivery systems are available, some of which are suitable for implantation.

[0176] The use of long-term sustained-release implants is suitable for prophylactic treatment of subjects experiencing or at risk for recurrent AGT-related diseases or conditions. As used herein, long-term release refers to an implant constructed and arranged to deliver therapeutic levels of an AGT dsRNA agent or AGT antisense polynucleotide agent for at least 10 days, up to 20 days, 30 days, 60 days, 90 days, 6 months, 1 year, or more. Long-term sustained-release implants are well known to those skilled in the art and include some of the release systems described above.

[0177] Therapeutic formulations of AGT dsRNA agents or AGT antisense polynucleotide agents can be prepared for storage and use in the form of lyophilized preparations or aqueous solutions by mixing the molecule or compound having the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer [Remington's Pharmaceutical Sciences 21st edition (2006)]. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and may include: buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., stearyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, and benzyl alcohol, or parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) peptides; serum albumin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0178] Cells, subjects and controls The methods of the present invention can be used with cells, tissues, organs, and / or subjects. In some aspects of the present invention, the subject is a human or a vertebrate mammal, including, but not limited to, a dog, cat, horse, cow, goat, mouse, rat, and primate, such as a monkey. Thus, the present invention can be used to treat AGT-related diseases or conditions in human and non-human subjects. In some aspects of the present invention, the subject can be a farm animal, zoo animal, domesticated, or non-domesticated animal, and the methods of the present invention can be used in veterinary prophylaxis and therapy. In some embodiments of the present invention, the subject is a human, and the methods of the present invention can be used in human prophylactic and therapeutic therapy.

[0179] Non-limiting examples of subjects to which the present invention may be applied include subjects who have been diagnosed with, are suspected of having, or are at risk for a disease or condition associated with higher than desirable levels of AGT expression and / or activity, also referred to as "high AGT expression levels." Non-limiting examples of diseases and conditions associated with higher than desirable levels of AGT expression and / or activity are described elsewhere herein. The methods of the present invention may be applied to subjects who, at the time of treatment, have been diagnosed with a disease or condition, have higher than desirable levels of AGT expression and / or activity, or are considered to be at risk for a disease or condition associated with higher than desirable levels of AGT expression and / or activity. In some embodiments of the present invention, the disease or condition associated with higher than desirable levels of AGT expression and / or activity is an acute disease or condition; in certain embodiments of the present invention, the disease or condition associated with higher than desirable levels of AGT expression and / or activity is a chronic disease or condition.

[0180] In a non-limiting example, the AGT dsRNA agent of the present invention is administered to a patient diagnosed with hypertension, such as essential hypertension, secondary hypertension, or hypertensive emergency (such as malignant and accelerated hypertension, acute hypertension, pregnancy-related hypertension, and treatment-resistant hypertension). The method of the present invention can be applied to subjects who, at the time of treatment, have been diagnosed with a disease or condition, or who have developed or are considered to be at risk of developing a disease or condition.

[0181] In another non-limiting example, the AGT dsRNA agent of the present invention is administered to treat the symptoms or progression of a disease or disorder caused by or associated with activation of the renin-angiotensin-aldosterone system (RAAS), or a disease or disorder in which the RAAS is inactivated. The term "angiotensinogen-related disease" includes diseases, disorders, or conditions that benefit from reduced expression of AGT. Such diseases are generally associated with high blood pressure. Non-limiting examples of angiotensinogen-related disorders include hypertension, e.g., borderline hypertension (also known as prehypertension), essential hypertension (also known as essential or idiopathic hypertension), secondary hypertension (also called non-idiopathic hypertension), isolated systolic or diastolic hypertension, pregnancy-related hypertension (ego, pre-eclampsia, eclampsia, and postpartum pre-eclampsia), diabetic hypertension, resistant hypertension, intractable hypertension, paroxysmal hypertension, renovascular hypertension (also called renal hypertension), Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, Examples of AGT-related disorders include systolic hypertension, labile hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease (such as peripheral vascular disease), diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, sleep apnea, heart failure (e.g., left ventricular systolic dysfunction), myocardial infarction, angina, stroke, renal disease (e.g., chronic kidney disease or diabetic nephropathy, optionally in the setting of pregnancy), renal failure (e.g., chronic renal failure), cognitive impairment (e.g., Alzheimer's disease), and systemic sclerosis (e.g., scleroderma nephropathy). In certain embodiments, the AGT-related disorder includes intrauterine growth retardation (IUGR) or fetal growth retardation.

[0182] Cells to which the present invention can be applied include in vitro, in vivo, and ex vivo cells. The cells may be in a subject, in culture, and / or in suspension, or in any other suitable state or condition. Cells to which the present invention can be applied may be: liver cells, hepatocytes, cardiac cells, pancreatic cells, cardiovascular cells, kidney cells, or other types of vertebrate cells, such as human and non-human mammalian cells. In certain aspects of the invention, cells to which the present invention can be applied are healthy, normal cells that are not known to be diseased cells. In certain embodiments of the invention, the methods and compositions of the invention are applied to liver, hepatocytes, cardiac cells, pancreatic cells, cardiovascular cells, and / or kidney cells. In certain aspects of the invention, the control cells are normal cells; however, it should be understood that in certain circumstances, cells with a disease or condition may also be used as control cells, such as when comparing the results of treated cells with a disease or condition to untreated cells with the disease or condition.

[0183] According to the methods of the present invention, the level of AGT polypeptide activity can be determined and compared to a control level of AGT polypeptide activity. The control can be a predetermined value that can take many forms. It can be a single cutoff value, such as a median or mean value. For example, it can be established based on comparing groups with a normal level of AGT polypeptide and / or AGT polypeptide activity and a group with an increased level of AGT polypeptide and / or AGT polypeptide activity. Another non-limiting example of a comparison group can be a population with one or more symptoms or a diagnosis of an AGT-related disease or condition compared to a population without one or more symptoms or a diagnosis of the disease or condition, or a group of subjects treated with an siRNA treatment of the present invention compared to a group of subjects not treated with an siRNA treatment of the present invention. Typically, the control can be based on apparently healthy normal individuals or apparently healthy normal cells in an appropriate age group. It will be understood that in addition to a predetermined value, a control according to the present invention can be a sample of material tested in parallel with the experimental material. Examples include samples from a control population or control sample produced by manufacturing for testing in parallel with the experimental sample. In some embodiments of the invention, a control may include a cell or subject that has not been exposed to or treated with an AGT dsRNA agent of the invention, in which case the control level of AGT polypeptide and / or AGT polypeptide activity may be compared to the level of AGT polypeptide and / or AGT polypeptide activity in a cell or subject that has been exposed to an AGT dsRNA agent or an AGT antisense polynucleotide agent of the invention.

[0184] In some embodiments of the present invention, the control level can be an AGT polypeptide level determined for a subject, and AGT polypeptide levels determined for the same subject at different time points are compared. In one non-limiting example, the level of AGT is determined in a biological sample collected from a subject not receiving the AGT treatment of the present invention. In some embodiments, the biological sample is a serum sample. The AGT polypeptide level determined in a sample collected from a subject can serve as a baseline or control value for the subject. After one or more administrations of an AGT dsRNA agent in the treatment methods of the present invention, one or more additional serum samples can be collected from the subject, and the AGT polypeptide level in the subsequent sample or samples can be compared to the subject's control / baseline level. Such a comparison can be used to assess the onset, progression, or regression of an AGT-related disease or condition in a subject. For example, a level of AGT polypeptide in a baseline sample taken from a subject that is higher than the level taken from the same subject after administering to the subject an AGT dsRNA agent or AGT antisense polynucleotide agent of the invention indicates regression of an AGT-associated disease or condition and indicates the effectiveness of the administered AGT dsRNA agent of the invention in treating an AGT-associated disease or condition.

[0185] In certain embodiments of the invention, one or more of the AGT polypeptide and / or AGT polypeptide activity levels determined for a subject can serve as a control for subsequent comparison of AGT polypeptide and / or AGT activity levels in the same subject, thereby allowing assessment of changes from a "baseline" AGT polypeptide activity in the subject. Thus, when an initial value is used as a control level for a subject, the initial AGT polypeptide level and / or initial AGT polypeptide activity level can be used to indicate and / or determine the ability of the methods and compounds of the invention to reduce the level of AGT polypeptide and / or AGT polypeptide activity in that subject.

[0186] Using the methods of the invention, AGT dsRNA agents and / or AGT antisense polynucleotide agents of the invention can be administered to a subject. Such dsRNAi agents include, for example, duplexes AD00051 to AD00122-19-2, AD00163-3, AV01227 to AVAV01257, and AV01711 shown in Table 1. In some embodiments, preferred dsRNAi agents include, for example, duplexes AD00158, AD00163, AD00163-3, AD00159, AD00290, AD00300, or AD00122. In other embodiments, preferred dsRNAi agents include, for example, AD00158-1, AD00158-2, AD00163-1, AD00159-1, or AD00300-1. In some other embodiments, such dsRNAi agents include duplex variants, such as duplex AD00158, AD00163, AD00163-3, AD00159, AD00290, AD00300, or AD00122 variants. The effectiveness of the administration and treatment of the present invention can be evaluated by comparing the pre-administration level of AGT polypeptide in a serum sample collected from the subject at a previous time point, or by comparing it to an unexposed control level (e.g., the level of AGT polypeptide in a control serum sample). When administered and treated, the level of AGT polypeptide in a serum sample collected from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. It is understood that both the level of AGT polypeptide and the level of AGT polypeptide activity are related to the level of AGT gene expression. Certain embodiments of the methods of the present invention include administering to a subject an AGT dsRNA and / or AGT antisense agent of the present invention in an amount effective to inhibit AGT gene expression, thereby reducing the level of AGT polypeptide and reducing the level of AGT polypeptide activity in the subject.

[0187] Some embodiments of the present invention involve determining the presence, absence, and / or amount (herein referred to as level) of AGT polypeptide in one or more biological samples taken from one or more subjects. This determination can be used to assess the effectiveness of a treatment method of the invention. For example, the methods and compositions of the present invention can be used to determine the level of AGT polypeptide in a biological sample taken from a subject previously treated with an AGT dsRNA agent and / or an AGT antisense agent of the present invention. A reduction in AGT polypeptide level in a serum sample after administration and treatment of at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the pre-administration level of AGT polypeptide in a serum sample taken from the subject at an earlier time point, or compared to an unexposed control level (e.g., the AGT polypeptide level in a control serum sample), indicates a level of effectiveness of the treatment administered to the subject.

[0188] In some embodiments of the present invention, physiological characteristics of an AGT-related disease or condition determined for a subject can be used as a control result, and determinations of physiological characteristics of the same subject at different time points can be compared to the control result. In one non-limiting example, blood pressure (and / or other physiological characteristics of an AGT disease or condition) can be measured from a subject who has not received any AGT treatment of the present invention and used as a baseline or control value for the subject. After one or more administrations of an AGT dsRNA agent to a subject in a treatment method of the present invention, blood pressure is measured and compared to the subject's control / baseline level, respectively. Such comparisons can be used to assess the onset, progression, or regression of an AGT-related disease or condition in a subject. For example, a baseline blood pressure obtained from a subject that is higher than the blood pressure measured from the same subject after administration of an AGT dsRNA agent or an AGT antisense polynucleotide agent of the present invention to the subject indicates regression of an AGT-related disease or disorder and demonstrates the effectiveness of administering an AGT dsRNA agent of the present invention in treating an AGT-related disease or condition.

[0189] In some embodiments of the present invention, the values ​​determined for a subject for one or more physiological characteristics of an AGT-related disease or disorder can serve as a control value for subsequent comparison of the physiological characteristics of the same subject, allowing for evaluation of changes in the subject's "baseline" physiological characteristics. Thus, an initial physiological profile can be obtained in an individual, the initial physiological profile measured as a control for that subject, and the effects of reducing the level and / or activity of an AGT polypeptide in the individual can be determined using the methods and compounds of the present invention. Using the methods of the present invention, AGT dsRNA agents and / or AGT antisense polynucleotide agents of the present invention can be administered to a subject in an amount effective for treating an AGT disease or condition. The effectiveness of the administration and treatment of the present invention can be evaluated by determining changes in one or more physiological characteristics of an AGT disease or condition. In one non-limiting example, the subject's blood pressure is reduced by at least 0.5 mmHg, 1 mmHg, 2 mmHg, 3 mmHg, 4 mmHg, 5 mmHg, 6 mmHg, 7 mmHg, 8 mmHg, 9 mmHg, 10 mmHg, 11 mmHg, 12 mmHg, 13 mmHg, 14 mmHg, 15 mmHg, 16 mmHg, 17 mmHg, 18 mmHg, 19 mmHg, 20 mmHg or more until the subject's blood pressure is within the normal range when compared to a blood pressure obtained from the subject at a previous time point or when compared to an unexposed control blood pressure.

[0190] Some embodiments of the present invention involve determining the presence, absence, and / or change in physiological characteristics of an AGT-related disease or condition using methods such as, but not limited to: (1) measuring the blood pressure of a subject; (2) assessing physiological characteristics of one or more biological samples taken from one or more subjects; or (3) physical examination of the subject. This determination can be used to assess the effectiveness of the treatment methods of the present invention.

[0191] kit Also within the scope of the present invention are kits containing one or more AGT dsRNA agents and / or AGT antisense polynucleotide agents and instructions for their use in the methods of the present invention. The kits of the present invention may include one or more of an AGT dsRNA agent, an AGT sense polynucleotide, and an AGT antisense polynucleotide agent useful in treating an AGT-related disease or condition. Kits containing one or more AGT dsRNA agents, AGT sense polynucleotides, and AGT antisense polynucleotide agents can be manufactured for use in the therapeutic methods of the present invention. The components of the kits of the present invention may be packaged in aqueous media or in a lyophilized state. The kits of the present invention may include carriers separated for sealing into one or more container means or a series of container means (test tubes, vials, flasks, bottles, syringes, etc.). A first container means or a series of container means may contain one or more compounds, e.g., an AGT dsRNA agent and / or an AGT sense or antisense polynucleotide agent. A second container means or series of container means may contain a targeting agent, labeling agent, delivery agent, or the like, which may be included therein as part of the AGT dsRNA and / or AGT antisense polynucleotide administered in embodiments of the therapeutic methods of the present invention.

[0192] Kits of the invention may also include instructions, typically in written form, that provide guidelines for carrying out the treatment embodied by the kit and for making decisions based on that treatment.

[0193] The following examples are provided to illustrate specific examples of the practice of the present invention and are not intended to limit the scope of the invention. It will be apparent that the present invention is applicable to a variety of compositions and methods. [Example]

[0194] Example 1. Synthesis of RNAi Agents The AGT RNAi agent duplexes shown in Tables 2-4 above were synthesized according to the following general procedure: Sense and antisense siRNA strand sequences were synthesized on an oligonucleotide synthesizer using established solid-phase synthesis based on phosphoramidite chemistry. Oligonucleotide chain propagation was achieved using a four-step cycle: deprotection, condensation, capping, and an oxidation or sulfurization step for each nucleotide addition. Synthesis was performed on a solid support made of porous glass (CPG, 1000 Å). Monomer phosphoramidites were purchased from a commercial supplier. Phosphoramidites bearing GalNAc ligand clusters (GLPA1 and GLPA2 as non-limiting examples) were synthesized according to the procedures described in Examples 2-3 herein. For siRNAs used in in vitro screening (Table 2), synthesis was performed on a 2 μmol scale; for siRNAs for in vivo testing (Tables 3 and 4), synthesis was performed on a 5 μmol or larger scale. When a GalNAc ligand (GLO-0 as a limiting example) was attached to the 3' end of the sense strand, a CPG solid support was used to which the GalNAc ligand was attached. When a GalNAc ligand (GLS-1 or GLS-2, as non-limiting examples) was attached to the 5' end of the sense strand, a GalNAc phosphoramidite (GLPA1 or GLPA2, as non-limiting examples) was used for the final coupling reaction. Trichloroacetic acid (TCA) in 3% dichloromethane was used to deprotect the 4,4'-dimethoxytrityl protecting group (DMT). 5-Ethylthio-1H-tetrazole was used as an activating agent. I2 in THF / Py / HO and phenylacetyl disulfide (PADS) in pyridine / MeCN were used for the oxidation and sulfurization reactions, respectively. After the final solid-phase synthesis step, the solid-support-bound oligomer was cleaved, and the protecting groups were removed by treatment with a 1:1 volume ratio of 40% (wt) aqueous methylamine and 28% ammonium hydroxide solution. The crude mixture was concentrated to synthesize siRNA for in vitro screening. The remaining solid was dissolved in 1.0 M NaOAc, and ice-cold EtOH was added to precipitate the single-stranded product as the sodium salt, which was used for annealing without further purification.To synthesize siRNA for in vivo testing, the crude single-stranded product was further purified by ion-pair reverse-phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide product from IP-RP-HPLC was converted to the sodium salt by dissolving in 1.0 M NaOAc and precipitating with ice-cold EtOH. The sense and antisense strand oligonucleotides were annealed by equimolar complementation in water to form the double-stranded siRNA product, which was lyophilized to yield a fluffy white solid.

[0195] Example 2. Preparation of Intermediate A and Intermediate B As shown in Scheme 1 below, intermediate A was synthesized by treating commercially available galactosamine pentaacetate with trimethylsilyl trifluoromethanesulfonate (TMSOTf) in dichloromethane (DCM). This was followed by glycosylation with Cbz-protected 2-(2-aminoethoxy)ethan-1-ol to give compound II. The Cbz-protecting group was removed by hydrogenation to give intermediate A as the trifluoroacetic acid (TFA) salt. Intermediate B was synthesized using the same scheme, except that Cbz-protected 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol served as the starting material. [ka]

[0196] Scheme 1 To a solution of compound I (20.0 g, 51.4 mmol) in 100 mL of 1,2-dichloroethane (DCE), TMSOTf (17.1 g, 77.2 mmol) was added. The resulting reaction solution was stirred at 60 °C for 2 hours, then at 25 °C for 1 hour. Cbz-protected 2-(2-aminoethoxy)ethan-1-ol (13.5 g, 56.5 mmol) was dissolved in DCE (100 mL), dried over 4 Å powdered molecular sieves (10 g), and added dropwise to the above reaction solution at 0 °C under a N atmosphere. The resulting reaction mixture was stirred at 25 °C for 16 hours under a N atmosphere. The reaction mixture was filtered and washed with saturated NaHCO (200 mL), water (200 mL), and saturated brine (200 mL). The organic layer was dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give the crude product, which was triturated with 2-methyltetrahydrofuran / heptane (5 / 3, v / v, 1.80 L) for 2 hours. The resulting mixture was filtered and dried to give compound II (15.0 g, 50.3% yield) as a white solid.

[0197] To a dried and argon-purged hydrogenation flask, 10% Pd / C (1.50 g) was carefully added, followed by 10 mL of tetrahydrofuran (THF), followed by a solution of compound II (15.0 g, 26.4 mmol) in THF (300 mL) and TFA (trifluoroacetic acid, 3.00 g, 26.4 mmol). The resulting mixture was degassed and purged with H 3 times and stirred under an H 45 psi atmosphere at 25° C. for 3 hours. Thin layer chromatography (TLC, solvent: DCM:MeOH=10:1) indicated complete consumption of compound II. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in anhydrous DCM (500 mL) and concentrated. The process was repeated three times to give intermediate A as a foamy white solid (14.0 g, 96.5% yield). 1H NMR (400MHz DMSO-d6): δ ppm 7.90(d,J=9.29Hz,1H),7.78(br s,3H),5.23(d,J=3.26Hz,1H),4.98(dd,J=11.29,3.26Hz,1H),4.56(d,J=8. 53Hz,1H),3.98-4.07(m,3H),3.79-3.93(m,2H),3.55-3.66(m,5H),2.98(br d,J=4.77Hz,2H),2.11(s,3H),2.00(s,3H),1.90(s,3H),1.76(s,3H).

[0198] Intermediate B was synthesized using a procedure similar to that used to synthesize Intermediate A. 1 H NMR (400MHz DMSO-d6): δ ppm 7.90(br d,J=9.03Hz,4H),5.21(d,J=3.51Hz,1H),4.97(dd,J=11.1Hz,1H),4.54(d,J=8.53Hz,1H),3.98 -4.06(m,3H),3.88(dt,J=10.9Hz,1H),3.76-3.83(m,1H),3.49-3.61(m,9H),2.97(br s,2H),2.10(s,3H),1.99(s,3H),1.88(s,3H),1.78(s,3H).C 20 H 34 N2O 11 Calculated mass: 478.22; Found mass: 479.3 (M+H + ).

[0199] Example 3. Synthesis of GalNAc Ligand Cluster Phosphoramidites GLPA1, GLPA2, and GLPA15 GLPAl and GLPA2 were prepared according to Scheme 2 below. Starting from benzyl-protected propane-1,3-diamine, alkylation with t-butyl 2-bromoacetate gave triester compound I. Removal of the benzyl protecting group by hydrogenation gave secondary amine compound II. Amide coupling with 6-hydroxycaproic acid gave compound III. Treatment with HCl in dioxane then removed the t-butyl protecting group to give triacid compound IV. Amide coupling of triacid compound IV with intermediate A or intermediate B was carried out to give compound Va or Vb. Phosphoramidite GLPA1 or GLPA2 was synthesized by phosphorylating compound Va or Vb using 2-cyanoethyl N,N-diisopropyl chloride phosphoramidite and a catalytic amount of 1H-tetrazole. [ka]

[0200] Scheme 2 N-benzyl-1,3-propanediamine (5.00 g, 30.4 mmol) in dimethylformamide (DMF, 100 mL) was added to t-butyl 2-bromoacetate (23.7 g, 121 mmol), followed by dropwise addition of diisopropylethylamine (DIEA, 23.61 g, 182 mmol). The resulting reaction mixture was stirred at 25-30 °C for 16 h. LCMS indicated complete consumption of N-benzyl-1,3-propanediamine. The reaction mixture was diluted with HO (500 mL) and extracted with EtOAc (500 mL × 2). The combined organics were washed with saturated brine (1 L), dried over anhydrous NaSO, and concentrated under reduced pressure to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether:ethyl acetate 20:1 to 5:1). Compound I was obtained as a colorless oil (12.1 g, 78.4% yield). 1H NMR(400MHz,CDCl3):δ ppm 7.26-7.40(m,5H),3.79(s,2H),3.43(s,4H),3.21(s,2H),2.72(dt,J=16.9,7.34Hz,4H),1.70(quin,J=7.2Hz,2H),1.44-1.50(m,27H).

[0201] A dry hydrogenation bottle was purged with argon three times. Pd / C (200 mg, 10%) was added, followed by MeOH (5 mL), and then compound I (1.00 g, 1.97 mmol) in MeOH (5 mL). The reaction mixture was degassed under vacuum and refilled with H. This process was repeated three times. The mixture was stirred at 25° C. under an H (15 psi) atmosphere for 12 hours. LCMS showed that compound I was completely consumed. The reaction mixture was filtered under reduced pressure under an N atmosphere. The filtrate was concentrated under reduced pressure to give compound II (655 mg, 79.7% yield) as a yellow oil, which was used in the next step without further purification. 1 H NMR (400MHz, CDCl3): δ ppm 3.44(s,4H),3.31(s,2H),2.78(t,J=7.1Hz,2H),2.68(t,J=6.9Hz,2H),1.88(br s,1H),1.69(quin,J=7.03Hz,2H),1.44-1.50(s,27H).

[0202] A mixture of compound II (655 mg, 1.57 mmol), 6-hydroxycaproic acid (249 mg, 1.89 mmol), DIEA (1.02 g, 7.86 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 904 mg, 4.72 mmol), and 1-hydroxybenzotriazole (HOBt, 637 mg, 4.72 mmol) in DMF (6 mL) was degassed and purged with N three times, then stirred under N at 25 °C for 3 h. LCMS showed the desired product. The reaction mixture was diluted with HO (10 mL) and extracted with 20 mL of EtOAc (2 x 10 mL). The organic components were combined, washed with saturated brine (20 mL), dried over anhydrous Na2SO4, filtered, and concentrated to give the crude product, which was purified by silica gel column chromatography (gradient: petroleum ether: ethyl acetate 5:1 to 1:1) to give compound III (650 mg, 77.8% yield) as a yellow oil. 1 H NMR(400MHz,CDCl3):δ ppm 3.90-3.95(s,2H),3.63(t,J=6.40Hz,2H),3.38-3.45(m,6H),2.72(t,J =6.65Hz,2H),2.40(t,J=7.28Hz,2H),1.55-1.75(m,8H),1.44(s,27H).C 27 H 50 Calculated mass of N2O8: 530.36; Found: 531.3 (M+H + ).

[0203] A mixture of compound III (5.5 g, 10.3 mmol) in HCl / dioxane (2 M, 55 mL) was stirred at 25 °C for 3 hours. LCMS showed complete consumption of compound III. The reaction mixture was filtered, washed with EtOAc (50 mL), and dried under reduced pressure to give the crude product, which was dissolved in CHCN (50 mL), and the volatile components were removed under vacuum. This process was repeated three times to give compound IV as a white solid (2.05 g, 54.5% yield). 1H NMR(400MHz,D2O):δ ppm 4.21(s,1H),4.07(d,J=4.5Hz,4H),3.99(s,1H),3.45-3.52(m,3H),3. 42(t,J=6.5Hz,1H),3.32-3.38(m,1H),3.24-3.31(m,1H),2.37(t,J=7 .4Hz,1H),2.24(t,J=7.4Hz,1H),1.99(dt,J=15.5,7.53Hz,1H),1.85-1.94(m,1H),1.85-1.94(m,1H),1.39-1.56(m,4H),1.19-1.31(m,2H).

[0204] A mixture of compound IV (500 mg, 1.05 mmol), intermediate A (2.02 g, 3.67 mmol), DIEA (813 mg, 6.30 mmol), EDCI (704 mg, 3.67 mmol), and HOBt in DMF (10 mL) (496 mg, 3.67 mmol) was degassed and purged with N three times before the mixture was stirred at 25 °C under a N atmosphere for 3 h. LCMS showed the desired product. The reaction mixture was quenched by adding HO (10 mL) and extracted with DCM (10 mL × 2). The combined organic components were extracted with 10% citric acid (20 mL). The aqueous phase was neutralized with saturated NaHCO solution and re-extracted with DCM (10 mL × 2). The organic portion was dried over sodium sulfate, filtered, and concentrated under reduced pressure to give compound Va as a white solid (570 mg, 0.281 mmol, 26.8% yield). 1 H NMR:(400MHz,CDCl3)ppm δ 7.84-8.12(m,3H),6.85-7.15(m,2H),6.66-6.81(m,1H),5.36(br d,J=2.7Hz,3H),5.11-5.27(m,3H),4.63-4.85(m,3H),3.90-4.25(m,18H),3.37-3.75(m,28H),3.15-3.28(m,4H),2.64(br d,J=6.53Hz,2H),2.30-2.46(m,2H),2.13-2.18(m,9H),2.05(s,9H),1.94-2.03(m,18H),1.68(br s,2H),1.45(br s,2H),1.12(br t,J=7.0Hz,2H).

[0205] To a solution of compound Va (260 mg, 0.161 mmol) in anhydrous DCM (5 mL) was added diisopropylammonium tetrazolide (30.3 mg, 0.177 mmol) followed by 3-bis(diisopropylamino)phosphanyloxypropanenitrile (194 mg, 0.645 mmol) dropwise at ambient temperature under N. The reaction mixture was stirred at 20-25 °C for 2 h. LCMS indicated that compound Va was completely consumed. After cooling to -20 °C, the reaction mixture was added to a stirred solution of brine / saturated NaHCO (1:1, 5 mL) at 0 °C. After stirring for 1 min, DCM (5 mL) was added, resulting in layer formation. The organic layer was washed with brine / saturated aqueous NaHCO (1:1, 5 mL), dried over NaSO, filtered, and concentrated to a volume of approximately 1 mL. The remaining solution was added dropwise to 20 mL of methyl t-butyl ether (MTBE) while stirring. This resulted in the precipitation of a white solid. The mixture was centrifuged, and the solid was collected. The solid was redissolved in 1 mL of DCM and precipitated by adding 20 mL of MTBE. The solid was again isolated by centrifugation. The recovered solid was dissolved in anhydrous CH3CN, and the volatile components were removed. This process was repeated two more times to yield the GalNAc ligand phosphoramidite compound GLPA1 (153 mg, 84.4 μmol) as a white solid. 1 H NMR(400MHz,CDCl3):ppm δ 7.71-8.06(m,2H),6.60-7.06(m,3H),5.37(br d,J=3.0Hz,3H),5.18-5.32(m,3H),4.70-4.86(m,3H),3.92-4.25(m,18H),3.42-3.85(m,30H),3.25(m ,4H),2.59-2.75(m,4H),2.27-2.44(m,2H),2.15-2.20(s,9H)2.07(s,9H),1.96-2.03(m,18H),1.65(br s,4H),1.44(br d,J=7.28Hz,2H),1.14-1.24(m,12H). 31 P NMR(CDCl3): ppm δ 147.15.

[0206] The GalNAc ligand phosphoramidite compound GLPA2 was synthesized using the same procedure, except that intermediate-B was used. 1 H NMR(400MHz,CDCl3):ppm δ 7.94-8.18(m,1H),7.69(br s,1H),6.66-7.10(m,3H),5.35(d,J=3.5Hz,3H),5.07-5.25(m,3H),4.76-4 .86(m,3H),4.01-4.31(m,10H),3.91-4.01(m,8H),3.74-3.86(m,4H),3.52- 3.71(m,30H),3.42-3.50(m,6H),3.15-3.25(m,4H),2.52-2.70(m,4H),2.2 2-2.45(m,2H),2.15-2.22(s,9H),2.06(s,9H),1.95-2.03(m,18H),1.77(br s,2H),1.58-1.66(m,4H),1.40(m,2H),1.08-1.24(m,12H). 31 P NMR(CDCl3): ppm δ 147.12.

[0207] To prepare GLPA15, the following Scheme 3 was followed. [ka] To a solution of intermediate compound II (275 g, 660 mmol, 1.00 equiv.) in dichloromethane (2.75 L), triethylamine (133 g, 1.32 mol, 2.00 equiv.) was added, followed by dropwise addition of Cbz-Cl (169 g, 990 mmol, 1.50 equiv.). The reaction solution was stirred at 25 °C for 2 h, and LCMS indicated complete conversion of compound II. The reaction solution was washed sequentially with saturated NaHCO3 (800 mL) solution and saturated brine (500 mL), and the organic phase was dried over anhydrous Na2SO4. After filtration to remove the drying agent, the filtrate was concentrated to dryness. The crude product was subjected to column chromatography (SiO2, PE / EA = 100 / 1 to 5 / 1) to give compound 5 (290 g, 527 mmol, 75.7% yield) as a colorless oil. 1H NMR(400MHz in DMSO-d6):δ ppm 7.23-7.40(m,5H),5.00-5.12(m,2H),3.86-3.95(m,2H),3.23-3.39(m,6H ),2.55-2.67(m,2H),1.56-1.64(m,2H),1.31-1.46(m,27H).MS(ESI)[M+H] + m / z:551.6.

[0208] To compound 5 (145 g, 263 mmol, 1.00 equiv.) was added HCOOH (2.9 L) and the solution was stirred at 60 °C for 12 h, resulting in complete conversion of compound 5 as indicated by LCMS. 1.5 L of toluene and acetonitrile were added to the reaction solution, which was then concentrated to approximately 500 mL under reduced pressure. Toluene / acetonitrile (1:1, approximately 750 mL) was then added before concentrating to approximately 500 mL. This was followed by the addition of acetonitrile (approximately 1000 mL) before concentrating to dryness. The crude product was triturated with 700 mL of acetonitrile at 60 °C for 2 h and then filtered. The solid was collected and dried to give a white solid, compound 6 (105 g, quantitative). 1 H NMR(400MHz in DMSO-d6):δ ppm 7.26-7.40(m,5H),5.02-5.10(m,2H),3.89-4.00(m,2H),3.36-3.45(m,4H),3.24-3.34(m,2H),2.59-2.72(m,2H),1.40(s,2H).MS(ESI)[M+H] + m / z:383.0.

[0209] To a solution of compound 6 (100 g, 261 mmol) and intermediate A (502 g, 915 mmol, 3.50 equiv.) in DMF (1.0 L) was added TBTU (327 g, 1.02 mol, 3.90 equiv.) and triethylamine (212 g, 2.09 mol, 8.00 equiv.). The reaction was carried out at 25 °C for 1 h, and LCMS showed complete conversion of compound 6. The reaction solution was added to 4000 mL of water and extracted with methyl t-butyl ether (2000 mL x 2) to remove impurities. The remaining aqueous phase was extracted with dichloromethane (3000 mL x 2). The dichloromethane phase was washed successively with 10% aqueous citric acid (2000 mL, divided into two washes), saturated NaHCO (2.0 L, divided into two washes), and saturated brine (2.0 L), and then dried over anhydrous NaSO. The filtrate was filtered and concentrated under reduced pressure to give a white solid, compound 8 (260 g, 159 mmol, 60.9% yield). 1 H NMR(400MHz in DMSO-d6):δ ppm 7.99-8.08(m,2H),7.93(br d,J=5.50Hz,1H),7.79-7.86(m,3H),7.26-7.39(m,5H),5.22(d,J=3.13Hz,3H),4.95-5.08(m,5H),4.54(br d,J=8.38Hz,3H),4.03(s,9H),3.81-3.93(m,5H),3.76(br d,J=4.88Hz,3H),3.44-3.62(m,10H),3.34-3.43(m,6H),3.24(br d,J=6.13Hz,7H),3.02-3.09(m,4H),2.40-2.47(m,2H),2.10(s,9H),1.99(s,9H),1.89(s,9H),1.77(s,9H),1.57-1.68(m,2H).MS(ESI)[M+H] + m / z:816.4.

[0210] A 2 L hydrogenation kettle was inerted with argon before dry Pd / C (9 g) was carefully added, followed by MeOH (50 mL) to wet the Pd / C. A solution of compound 8 (90 g, 55.1 mmol, 1.00 equiv.) and trifluoroacetic acid (6.29 g, 55.1 mmol, 1.00 equiv.) in MeOH (850 mL) was then added slowly under an argon atmosphere. The mixture was degassed and purged with H2 three times to obtain a hydrogen atmosphere and stirred at 25 °C for 10 h. LCMS indicated complete conversion of compound 8. The Pd / C was removed by filtration, and the filtrate was concentrated under reduced pressure to give compound 9 (80 g, 90.2% yield). 1 H NMR(400MHz in DMSO-d6):δ ppm 9.12(br s,2H),8.50(br t,J=5.19Hz,1H),8.10(br t,J=5.50Hz,2H),7.85-7.91(m,3H),5.22(d,J=3.25Hz,3H),4.95-5.01(m,3H),4.52 -4.58(m,3H),4.03(s,9H),3.84-3.93(m,3H),3.75-3.83(m,3H),3.39-3.61(m,16H) ,3.23-3.32(m,6H),3.15-3.18(m,3H),2.97-3.05(m,2H),2.54-2.61(m,2H),2.10(s ,9H),2.00(s,9H),1.89(s,9H),1.77-1.80(m,9H),1.70-1.76(m,2H).MS(ESI)[M+H] + m / z:749.3.

[0211] To a solution of compound 9 (270 g, 168 mmol, 1.00 equiv.) in dichloromethane (2.7 L) and glutaric anhydride (28.6 g, 252 mmol, 1.50 equiv.) was added triethylamine (67.8 g, 672 mmol, 4.00 equiv.), and the solution was stirred at 25° C. for 1 hour. LCMS showed complete conversion of compound 9 to compound 11. 4-Hydroxypiperidine (42.4 g, 420 mmol, 2.50 equiv.) and TBTU (107 g, 335 mmol, 2.00 equiv.) were added to the reaction solution, and stirring was continued at 25° C. for 1 hour. LCMS showed complete conversion of compound 11. The reaction was quenched by the slow addition of saturated NH4Cl (3.0 L), the layers were separated, and the aqueous phase was extracted with dichloromethane (2 x 1000 mL) and combined with the previous organic phase. The combined organic phase was washed with a 1:1 mixture of saturated NaHCO3 (aq) and saturated brine (3.0 L), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was dissolved in 1.5 L of dichloromethane and added dropwise to methyl t-butyl ether (7.5 L). During the dropwise addition, a translucent white precipitate gradually formed. The precipitate was filtered under vacuum, and the solid was collected and dried under vacuum to give compound 13 as a white solid (207 g, 72.8% yield). 1 H NMR (400MHz in DMSO-d6): δ ppm 8.05(br d,J=2.00Hz,2H),7.82(br d,J=7.38Hz,3H),5.21(br s,3H),4.98(br d,J=10.26Hz,3H),4.72(br s,1H),4.54(br d,J=7.88Hz,3H),4.03(br s,9H),3.74-3.94(m,9H),3.45-3.71(m,12H),3.40(br s,6H),3.24(br s,7H),3.07(br d,J=14.13Hz,5H),2.91-3.01(m,1H),2.24-2.44(m,5H),2.20(br s,1H),2.10(s,9H),1.96-2.04(m,9H),1.89(br s,9H),1.74-1.81(m,9H),1.51-1.73(m,6H),1.07-1.36(m,3H).MS(ESI)[M+H] + m / z:848.0.

[0212] To a solution of compound 13 (200 g, 118 mmol, 1.00 equiv.) and tetrazolediisopropylammonium (8.08 g, 47.2 mmol, 0.40 equiv.) in dichloromethane (2.0 L) was added 3-bis(diisopropylamino)phosphanyloxypropanenitrile (53.3 g, 177 mmol, 1.50 equiv.). The reaction solution was stirred at 40 °C for 2 h, and LCMS showed complete conversion of compound 13. The reaction solution was washed with a 1:1 mixture of saturated NaHCO and saturated brine (2.0 L) and dried over anhydrous NaSO. The crude product obtained after concentrating the filtrate was dissolved in dichloromethane (1.2 L) and added dropwise to stirred methyl t-butyl ether (6.0 L). The suspension was filtered, and the filter cake was rinsed with t-butyl ether. The solid was collected and dried under vacuum. The product was dissolved in dichloromethane (1.0 L) and concentrated to dryness, and this procedure was repeated four times to remove residual t-butyl ether, yielding GLPA15 (164 g, 73.3% yield). 1 H NMR (400MHz in DMSO-d6): δ ppm 8.05(br d,J=6.50Hz,2H),7.81(br d,J=9.01Hz,3H),5.22(d,J=3.25Hz,3H),4.98(dd,J=11.26,3.25Hz,3H),4.55(br d,J=8.50Hz,3H),4.03(s,9H),3.64-3.97(m,12H),3.55-3.63(m,6H),3.50(br s,5H),3.40(br d,J=6.13Hz,6H),3.17-3.30(m,9H),3.07(br d,J=14.26Hz,4H),2.76(t,J=5.82Hz,2H),2.18-2.47(m,6H),2.10(s,9H),1 .99(s,9H),1.89(s,9H),1.78(s,9H),1.52-1.74(m,6H),1.12-1.19(m,12H). 31 P NMR(DMSO-d6):ppm δ 145.25.MS(ESI)[M+H] + m / z:1895.7.

[0213] In some studies, a method is provided for attaching a targeting group comprising GalNAc (also referred to herein as a GalNAc delivery compound) to the 5' end of a sense strand, the method comprising the use of GalNAc phosphoramidite (GLPA1) in the final coupling step of solid-phase synthesis using a synthetic process such as that used for oligonucleotide chain elongation (i.e., addition of nucleotides to the 5' end of the sense strand) to attach it to the 5' end of the sense strand.

[0214] In some studies, the method of linking a GalNAc-containing targeting group to the 3'-end of a sense strand includes using a GLO-n-containing solid support (CPG).In some studies, the method of linking a GalNAc-containing targeting group to the 3'-end of a sense strand includes linking a GalNAc-containing targeting group to a CPG solid support via an ester bond, and using the resulting CPG with a GalNAc-containing targeting group linked during the synthesis of the sense strand, thereby obtaining a GalNAc-containing targeting group linked to the 3'-end of the sense strand.Other GalNAc phosphoramidite compounds (GLPAn) can also be obtained by using a method similar to that described herein or a method well known in the art after using a reasonably equivalent intermediate, and can be linked to the appropriate position of an siRNA duplex as a targeting group.

[0215] Example 4. In vitro screening of AGT siRNA duplexes Hep3B cells were trypsinized, adjusted to the appropriate density, and then seeded into 96-well plates. Simultaneously with seeding, cells were transfected with test or control siRNA using Lipofectamine RNAiMax (Invitrogen-13778-150) according to the manufacturer's recommendations. siRNAs were tested in triplicate at two concentrations (0.2 nM to 1.0 nM), and control siRNA was tested in triplicate at eight concentrations in three-fold serial dilutions from 4.6 pM to 10 nM.

[0216] 24 hours after transfection, the medium was removed and the cells were collected for RNA extraction. Total RNA was extracted using TRIzol™ Reagent (Invitrogen-15596018) according to the manual.

[0217] cDNA was synthesized using the PrimeScript™ RT Reagent Kit and gDNA Eraser (Perfect Real Time) (TaKaRa-RR047A) according to the manufacturer's instructions. AGT cDNA was detected by qPCR. GAPDH cDNA was detected in parallel with an internal control. PCR was performed as follows: 95°C for 30 seconds, followed by 40 cycles between 95°C for 10 seconds and 60°C for 30 seconds.

[0218] Data analysis Expression in each sample was determined by relative quantification (RQ) using the comparative Ct (ΔΔCt) method, in which the Ct difference (ΔCt) between the target gene and a housekeeping gene (GAPDH) is measured.

[0219] The equation is shown below: ΔCT=Target gene average Ct-GAPDH average Ct ΔΔCT = ΔCT (sample) - ΔCT (random control or Lipofectamine RNAiMax control) Relative quantification of target gene mRNA = 2( -ΔΔCT ) Percent inhibition = (relative quantification of control - relative quantification of sample) / relative quantification of control x 100%

[0220] [Table 30]

[0221] [Table 31]

[0222] Example 5. In vivo testing of AGT siRNA duplexes To evaluate the in vivo activity of AGT siRNA, mice infected with AAV encoding the human AGT gene (4 mice per group) were used. 14 days after siRNA administration, 1 × 10 of an adeno-associated virus 8 (AAV8) vector encoding the human AGT gene was injected. 11 Female C57BL / 6J mice were infected by intravenous injection of viral particles. On day 0, a single dose of 2.5 mg / kg or 3 mg / kg of AGT siRNA or PBS was subcutaneously injected. Blood samples were collected on day 0, before siRNA administration, and at the end of day 7. Human AGT protein concentrations were measured by ELISA assay according to the manufacturer's recommended protocol (IBL America, Human Angiotensinogen ELISA Kit). Percent knockdown was calculated by comparing human AGT mRNA levels in mouse liver (determined by qPCR) or human AGT protein levels in plasma samples on day 7 between the siRNA-treated and PBS-treated groups. The results are shown in Tables 6–9.

[0223] [Table 32]

[0224] [Table 33]

[0225] [Table 34]

[0226] [Table 35]

[0227] [Table 36]

[0228] Example 6. In vivo testing of AGT siRNA duplexes To evaluate the in vivo activity of AGT siRNA, a total of 15 cynomolgus monkeys (13-22 years old, weighing 7-9 kg) were recruited for this study. The animals were randomly divided into five groups of three animals each, and each animal was subcutaneously injected with 2 mg / kg of the test substance. The test substances used correspond to the compounds in Table 4 (AD00158-1, AD00158-2, AD00163-1, AD00159-1, and AD00300-1).

[0229] After an overnight fast, blood was collected on days -14 (pre-dose), -7 (pre-dose), 1 (pre-dose), and post-dose days 8, 15, 22, 29, 43, 57, 64, 71, 78, 85, and 92. Collected blood samples were then allowed to clot at room temperature for at least 30 minutes and then centrifuged at 350 rpm for 10 minutes at 4°C. The collected serum (approximately 1.0 mL) was transferred to two pre-labeled polypropylene screw-cap vials (0.5 mL / vial, one for the ELISA assay and the other for later use) and stored in a -80°C freezer until testing.

[0230] AGT protein levels in serum were determined by ELISA. The percentage remaining compared to AGT levels in the plasma of monkeys on day 1 is shown in Figure 1.

[0231] Example 7. In vitro screening of AGT siRNA duplexes In vitro studies were performed according to the methods of Example 4, and the experimental results are shown in Table 10.

[0232] [Table 37]

[0233] Example 8. In vivo testing of AD00163-3 in an AAV mouse model: After the acclimation period, 12 female C57BL / 6J mice were randomly divided into two groups based on body weight: a model (vehicle) group and an AD00163-3 (1 mg / kg) group. On day 1, each mouse was injected with 1 × 10 AAV-AGT virus into the tail vein. 11 The animal model was established by injecting 100 μL / animal of PBS or AD00163-3 (Table 4) at a volume of 100 μL / animal. On day 15, mice in each group were given PBS or AD00163-3 (Table 4) by subcutaneous injection at a volume of 5 mL / kg. Before administration on day 15, blood was collected from the submandibular vein of each mouse, and serum samples were collected after centrifugation. On day 22, all mice were sacrificed with CO2, whole blood was collected by cardiac puncture, and serum samples were collected after centrifugation. Human AGT protein concentrations were measured by ELISA assay according to the manufacturer's recommended protocol (IBL America, Human Angiotensinogen ELISA Kit). The knockdown percentage was calculated by comparing human-derived AGT protein levels in mouse plasma samples on day 7 between the siRN-treated and PBS-treated groups. The data showed that AD00163-3 (1 mg / kg) treatment significantly reduced human AGT protein expression in mouse serum by 91%.

[0234] Example 9. Testing of AD00163-3 in a Cynomolgus Monkey Model of Spontaneous Hypertension Ten cynomolgus monkeys with hypertension were randomly divided into two groups (five monkeys in each group) and administered either saline or AD00163-3 at 10 mg / kg as shown in Table 4. Blood samples were collected on days -6 and -2 (pre-dose) and on days 2, 7, 14, 21, 28, and 35 after dosing. Serum AGT concentrations were measured by ELISA according to the manufacturer's recommended protocol, and blood pressure was measured using a tailcuff device. As shown in Figures 2 and 3, concomitant with a reduction in serum AGT (98% reduction on day 35 after dosing), a single subcutaneous administration of 10 mg / kg AD00163-3 resulted in a significant reduction in SBP of 28 mmHg on day 35 after dosing (SBP changed from baseline 147 to 119 mmHg), with no significant change in SBP in the control group during the same period (SBP changed from baseline 144 to 145 mmHg). A significant reduction in mean and diastolic blood pressure (MBP and DBP) was also confirmed as shown in Figures 4 and 5, respectively.

[0235] equivalent While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily recognize that various other means and / or structures may be used to perform the functions and / or obtain the results and / or one or more advantages described herein, and that each of these variations and / or modifications is considered to be within the scope of the present invention. More generally, those skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are exemplary, and that the actual parameters, dimensions, materials, and / or configurations will vary depending on the specific application for which the teachings of the present invention are used. Those skilled in the art will recognize, or be able to recognize, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Accordingly, it should be understood that the above-described embodiments are presented by way of example only, and that, within the scope of the appended claims and their equivalents, may be practiced other than as specifically described and claimed. The present invention relates to each individual feature, system, article, material, and / or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and / or methods is included within the scope of the present invention, provided that such features, systems, articles, materials, and / or methods are not mutually inconsistent.

[0236] All definitions and terms used herein should be understood as referencing dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0237] Where no quantitative limitation is used in the specification and claims, this should be understood as "at least one" unless expressly specified to the contrary.

[0238] As used in this specification and in the claims, the phrase "and / or" should be understood to mean "one or both" of the elements so combined, that is, such elements may be combined in certain instances and separately in other instances. In addition to the elements specifically identified by "and / or," other elements, whether related or unrelated to the specifically identified elements, may optionally be present, unless expressly specified to the contrary.

[0239] All references, patents and patent applications and publications mentioned or referred to in this application are incorporated herein by reference in their entirety. The inventions described in the original claims of this application are listed below. [Invention 1] A double-stranded ribonucleic acid (dsRNA) agent that inhibits expression of angiotensinogen (AGT), the dsRNA agent comprising a sense strand and an antisense strand, wherein nucleotides 2-18 of the antisense strand comprise a region of complementarity to an AGT RNA transcript. a dsRNA agent, wherein the region of complementarity comprises at least 15 contiguous nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences set forth in one of Tables 1-4, and optionally comprises a targeting ligand. [Invention 2] 2. The dsRNA agent of claim 1, wherein the region of complementarity to the AGT RNA transcript comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences set forth in one of Tables 1 to 4. [Invention 3] 3. The dsRNA agent according to claim 1 or 2, wherein the antisense strand of the dsRNA is at least substantially complementary to any target region of SEQ ID NO: 519 and provided in any one of Tables 1 to 4. [Invention 4] 4. The dsRNA agent according to invention 3, wherein the antisense strand of the dsRNA is at least fully complementary to any target region of SEQ ID NO: 519 and provided in any one of Tables 1 to 4. [Invention 5] 2. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a sense strand sequence set forth in any one of Tables 1 to 4, and the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. [Invention 6] 2. The dsRNA agent according to invention 1, wherein the dsRNA agent comprises a sense strand sequence set forth in any one of Tables 1 to 4, and the sense strand sequence is completely complementary to the antisense strand sequence in the dsRNA agent. [Invention 7] A dsRNA agent according to Invention 1, comprising an antisense strand sequence as set forth in any one of Tables 1 to 4. [Invention 8] 2. The dsRNA agent according to invention 1, comprising a sequence shown as a double-stranded sequence in any one of Tables 1 to 4. [Invention 9] 2. The dsRNA agent according to claim 1, comprising at least one modified nucleotide. [Invention 10] 2. The dsRNA agent of claim 1, wherein all or substantially all of the nucleotides in the antisense strand are modified nucleotides. [Invention 11] 7. The dsRNA agent of claim 5 or 6, wherein the at least one modified nucleotide comprises: a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide, a 2'-deoxy nucleotide, a 2',3'-seconucleotide mimic, a locked nucleotide, an unlocked nucleic acid (UNA) nucleotide, a glycol nucleic acid (GNA) nucleotide, a 2'-F-arabinonucleotide, a 2'-methoxyethyl nucleotide, an abasic nucleotide, ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, an inverted 2'-deoxy nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, and a 3'-OMe nucleotide, a nucleotide comprising a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidate, or a non-natural base-containing nucleotide. [Invention 12] 11. The dsRNA agent of claim 9 or 10, wherein the E-vinylphosphonate nucleotide is included at the 5' end of the guide strand. [Invention 13] 2. The dsRNA agent of claim 1, comprising at least one phosphorothioate internucleoside linkage. [Invention 14] 2. The dsRNA agent of claim 1, wherein the sense strand comprises at least one phosphorothioate internucleoside linkage. [Invention 15] 2. The dsRNA agent of claim 1, wherein the antisense strand comprises at least one phosphorothioate internucleoside linkage. [Invention 16] 2. The dsRNA agent of claim 1, wherein the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. [Invention 17] 2. The dsRNA agent of claim 1, wherein the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. [Invention 18] 2. The dsRNA agent according to claim 1, wherein all or substantially all nucleotides of said sense and antisense strands are modified nucleotides. [Invention 19] The dsRNA agent according to Invention 1, wherein the modified sense strand has a modified sense strand sequence shown in one of Tables 2 to 4. [Invention 20] The dsRNA agent according to Invention 1, wherein the modified antisense strand has a modified antisense strand sequence shown in one of Tables 2 to 4. [Invention 21] 2. The dsRNA agent according to invention 1, wherein the sense strand is complementary or substantially complementary to the antisense strand, and the region of complementarity is 16 to 23 nucleotides in length. [Invention 22] 22. The dsRNA agent according to claim 21, wherein the region of complementarity is 19 to 21 nucleotides in length. [Invention 23] 2. The dsRNA agent of claim 1, wherein each strand is 30 nucleotides or less in length. [Invention 24] 2. The dsRNA agent of claim 1, wherein each strand is 25 nucleotides or less in length. [Invention 25] 2. The dsRNA agent of claim 1, wherein each strand is 23 nucleotides or less in length. [Invention 26] 2. A dsRNA agent according to claim 1, comprising at least one modified nucleotide and further comprising one or more targeting groups or linking groups. [Invention 27] 27. The dsRNA agent of claim 26, wherein the one or more targeting groups or linking groups are conjugated to the sense strand. [Invention 28] 28. The dsRNA agent of claim 26 or 27, wherein the targeting group or linking group comprises N-acetyl-galactosamine (GalNAc). [Invention 29] Each targeting group has the structure:

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Claims

1. A double-stranded RNA (dsRNA) agent that inhibits the expression of angiotensinogen (AGT), the dsRNA agent comprises a sense strand and an antisense strand; the sense strand comprises the sequence of SEQ ID NO: 526 ((GLS-15) * (Invab) * caccagcuUgUuUgugaaaca * (Invab)), and the antisense strand comprises the sequence of SEQ ID NO: 527 (u * G * uuucAcaaaCaAgCugg * u * g), wherein in each of the sequences of SEQ ID NOs: 526 and 527, each nucleotide in lowercase is 2'-O-methyl (2'-OMe) modified, each nucleotide in uppercase is 2'-fluoro modified, each asterisk (*) represents a phosphorothioate bond, each Invab represents an inverted abasic position, and GLS-15 is 【Chemistry 1】 That is, The dsRNA agent.

2. 10. A composition comprising the dsRNA agent of claim 1 and a pharmaceutically acceptable carrier.

3. 3. The composition of claim 2 packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial.

4. 3. The composition of claim 2, formulated for subcutaneous administration.

5. The composition of claim 2 , wherein the pharmaceutically acceptable carrier comprises a sodium salt.

6. The sense strand has the sequence of SEQ ID NO: 526 ((GLS-15) * (Invab) * caccagcuUgUuUgugaaaca * (Invab)), and the antisense strand is the sequence of SEQ ID NO: 527 (u * G * uuucAcaaaCaAgCugg * u * g), wherein in each of the sequences of SEQ ID NOs: 526 and 527, each nucleotide in lower case is 2'-O-methyl (2'-OMe) modified, each nucleotide in upper case is 2'-fluoro modified, and each asterisk ( * ) indicates a phosphorothioate bond, each Invab indicates an inverted abasic position, and GLS-15 indicates 【Chemistry 2】 2. The dsRNA agent of claim 1, wherein:

7. 10. A composition comprising the dsRNA agent of claim 6 and a pharmaceutically acceptable carrier.

8. The composition of claim 7 , wherein the pharmaceutically acceptable carrier comprises a sodium salt.

9. 8. The composition of claim 7, packaged in a kit, container, pack, dispenser, pre-filled syringe, or vial.

10. 8. The composition of claim 7, formulated for subcutaneous administration.

11. 11. A pharmaceutical composition for use in treating an AGT-related disease or condition, said pharmaceutical composition comprising a dsRNA agent according to claim 1 or 6, or a composition according to any one of claims 2 to 5 and 7 to 10, wherein said pharmaceutical composition is administered in an amount effective to reduce serum AGT levels, thereby treating the AGT-related disease or condition in a subject.

12. 12. The pharmaceutical composition according to claim 11, wherein the AGT-related disease or condition is hypertension.

13. 12. The pharmaceutical composition of claim 11, wherein the AGT related disease or condition is one or more of hypertensive disorders, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated hypertension, systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, treatment-resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, angiopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina, stroke, renal disease, renal failure, systemic sclerosis, intrauterine growth retardation, and fetal growth retardation.

14. i) inhibiting the expression of the AGT gene in a subject, ii) reducing the level of AGT protein in a subject compared to the baseline level of AGT protein in the subject, or iii) altering physiological characteristics of an AGT-related disease or condition in a subject compared to baseline pre-treatment physiological characteristics of an AGT-related disease or condition in the subject; 12. Use of an effective amount of a dsRNA agent according to claim 1 or 6, or a composition according to any one of claims 2 to 5 and 7 to 10, for the manufacture of a medicament, composition, kit or product for treating a disease in a patient with atopic dermatitis.

15. 15. The use according to claim 14, wherein the AGT-related disease or condition is hypertension.

16. 15. The use according to claim 14, wherein the AGT related disease or condition is one or more of hypertensive disorders, hypertension, borderline hypertension, essential hypertension, secondary hypertension, isolated hypertension, systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, treatment-resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, unstable hypertension, hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, angiopathy, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina, stroke, renal disease, renal failure, systemic sclerosis, intrauterine growth retardation, and fetal growth retardation.

17. The use described in claim 14, wherein one or more physiological characteristics of the AGT-related disease or condition are AGT levels in the blood and / or blood pressure.

18. The use described in claim 17, wherein the blood pressure is systolic blood pressure (SBP), diastolic blood pressure (DBP), or mean arterial pressure (MAPR).

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