ANGIOTENSINOGEN (AGT) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

Abstract

To provide double-stranded ribonucleic acid (RNAi) agents targeting the angiotensinogen (AGT) gene.SOLUTION: There is provided an RNAi agent for inhibiting expression of AGT in a cell, where the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, the sense strand comprising at least 15 contiguous nucleotides differing by 3 or less nucleotides from a nucleotide sequence having a specific sequence, the antisense strand comprising at least 15 contiguous nucleotides differing by 3 or less nucleotides from a nucleotide sequence having another specific sequence, where substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and where the sense strand form a conjugate with a ligand attached at the 3'-terminus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 001,731, filed May 22, 2014, and U.S. Provisional Patent Application No. 62 / 047978, filed September 9, 2014, the entire contents of each of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on May 18, 2015 is referred to as 121301-01320_SL.txt and is 318,688 bytes in size. [Background technology]

[0003] Background of the Invention The renin-angiotensin-aldosterone system (RAAS) plays an important role in regulating blood pressure. The RAAS cascade begins with the release of renin into the circulation by juxtaglomerular cells in the kidney. Renin secretion is stimulated by several factors, including distal tubular Na+ reabsorption, β-sympathetic nerve stimulation, and / or reduced renal perfusion. Active renin in plasma cleaves angiotensinogen (produced in the liver) to angiotensin I, which then circulates and is converted to angiotensin II by locally expressed angiotensin-converting enzyme (ACE). Most of the effects of angiotensin II in the RAAS are exerted through its binding to the angiotensin II type 1 receptor (AT1R), resulting in arterial vasoconstriction, tubular effects, and glomerular effects, such as increased Na+ reabsorption or regulation of the glomerular filtration rate. In addition, AT1R stimulation, along with other stimuli such as adrenocorticotropic hormone, antidiuretic hormone, catecholamines, endothelin, serotonin, and Mg2+ and K+ concentrations, induces the release of aldosterone, which in turn promotes the excretion of Na+ and K+ in the distal tubules of the kidney.

[0004] For example, dysregulation of the RAAS, resulting in excessive angiotensin II production and / or AT1R stimulation, can result in hypertension, which can lead to increased oxidative stress, promoted inflammation, hypertrophy and fibrosis in, for example, the heart, kidney, and pre-existing arteries, and can result in, for example, left ventricular fibrosis, arterial remodeling, and glomerulosclerosis.

[0005] Hypertension is one of the most prevalent and controllable diseases in developed countries, affecting 20 to 50% of the adult population. It is a major risk factor for a variety of diseases, disorders, and conditions, such as reduced life expectancy, chronic kidney disease, stroke, myocardial infarction, heart failure, aneurysms (e.g., aortic aneurysms), peripheral arterial disease, heart failure (e.g., heart enlargement or hypertrophy), and other cardiovascular-related diseases, disorders, and / or conditions. In addition, hypertension has been shown to be a significant risk factor for cardiovascular morbidity, accounting for or contributing to 62% of all cases of stroke and 49% of all cases of heart disease.

[0006] Despite the large number of antihypertensive drugs available for treating hypertension, more than two-thirds of subjects are not controlled with a single antihypertensive drug and require two or more antihypertensive agents selected from different drug classes, which further reduces the number of subjects with controlled blood pressure because compliance and side effects increase with increasing medication.

[0007] Thus, there is a need in the art for alternative and combination therapies for subjects with angiotensinogen-related disorders. Summary of the Invention

[0008] Summary of the Invention The present invention provides iRNA compositions that affect RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of the angiotensinogen (AGT) gene, which can be present in a cell, e.g., a cell in a subject, such as a human.

[0009] The present invention also provides methods and therapies for treating subjects with disorders that may benefit from inhibition or reduction of AGT gene expression, e.g., angiotensinogen-related disorders such as hypertension, using iRNA compositions that have the effect of cleaving the AGT gene via RNA-induced silencing complex (RISC)-mediated RNA transcripts to inhibit AGT gene expression.

[0010] Thus, in one aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of angiotensinogen (AGT), comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO: 2, wherein substantially all of the nucleotides in the sense strand and substantially all of the nucleotides in the antisense strand are modified nucleotides, and the sense strand is complexed with a ligand attached at its 3' end. In one embodiment, all of the nucleotides in the sense strand and all of the nucleotides in the antisense strand are modified nucleotides.

[0011] In another aspect, the invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of angiotensinogen (AGT), comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand is selected from the group consisting of nucleotides 2801-2101; 803-843; 834-859; 803-859; 803-875; 834-875; 847-875; 1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1621; 2035-2144; 2075-2144; 2076-2145; 2077-2146; 2078-2147; 2079-2148; 2080-2184; 2081-2184; 2082-2184; 2083-2184; 2084-2184; 2085-2184; 2086-2184; 2087-2184; 2088-2184; 2089-2190; 2090-2190; 2191-2191; 2192-2192; 2193-2194; 2195-2195; 2196-2196; 2200-2200; 2201-2201; 2202-2202; 2203-2204; 2204-2204; 2205-2206; 2206-2207; 0-2144;2070-2103;2201-2223;2227-2360;2227-2304;2290-2318;2304-2350;2304-2326;2320-2342;2333-2360;2333-2358;485-503;517-535;56 0-578;635-653;803-821;814-832;822-840;825-843;834-852;837-855;841-859;855-873;967-985;1247-1265;1248-1266;1249-1267;1251-1269 ;1253-1271;1566-1584;1570-1588;1572-1590;1574-1592;1584-1602;1587-1605;1591-1609;1592-1610;1595-1613;1601-1619;1602-1620;1605 -1623;1729-1747;1738-1756;1739-1757;1741-1769;1767-1785;1810-1828;1827-1845;1880-1989;1892-1914;1894-1914;1894-2012;2035-2053 ;2046-2064;2057-2075;2070-2088;2072-2090;2078-2096;2078-2107;2078-2011;2080-2098;2081-2099;2081-2104;2081-2011;2082-2100;2084 -2102;2084-2011;2090-2108;2100-2118;2111-2129;2124-2142;2125-2143;2167-2185;2179-2197;2201-2219;2202-2220;2203-2221;2204-2222;2227-2245;2230-2248;2234-2252;2244-2264;2255-2273;2266-2284;2268-2286;2270-2288;2279-2297;2281-2299;2283-2301;2284-2302;2285-2303;2286-2304;2288-2306;2290-2308;2291-2309;2291-2311;2291-2318;2291-2315;2292-2310;2294-2312;2296-2314;2299-2317;2304-2322;2304-2329;2306-2324;2307-2325;2309-2327;2309-2329;2309-2342;2309-2350;2309-2358;2314-2332;2316-2334;2317-2335;2320-2338;2321-2339;2323-2341;2325-2343;2326-2344;2328-2346;2329-2347;2331-2349;2333-2351;2334-2352;2335-2353;2339-2357;2340-2358;Alternatively, a double-stranded RNAi agent is provided, comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from 2341-2359, and the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotides at corresponding positions in the nucleotide sequence of SEQ ID NO: 2, such that the antisense strand is substantially complementary to the at least 15 consecutive nucleotides in the sense strand. In any embodiment, substantially all nucleotides in the sense strand are modified nucleotides. In other embodiments, substantially all nucleotides in the antisense strand are modified nucleotides. In still other embodiments, substantially all nucleotides in both strands are modified nucleotides. In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In one embodiment, the sense strand is complexed with a ligand attached at the 3' end. In one embodiment, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from nucleotides 2801-2101 of the nucleotide sequence of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotides at corresponding positions in the nucleotide sequence of SEQ ID NO: 2 such that the antisense strand is substantially complementary to the at least 15 contiguous nucleotides in the sense strand;

[0012] In one aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of angiotensinogen (AGT), comprising a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand is selected from the group consisting of nucleotides 803-843; 834-859; 803-859; 1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1621; 2035-2144; 2070-2144; 2070-2103; 2201-2223; 2227-2327 of the nucleotide sequence of SEQ ID NO:1. 60;2227-2304;2290-2318;2304-2350;2304-2326;2320-2342;2333-2360;2333-2358;485-503;517-535;560-578;635-653;803-821;814-832;822 -840;825-843;834-852;837-855;841-859;855-873;967-985;1247-1265;1248-1266;1249-1267;1251-1269;1253-1271;1566-1584;1570-1588;15 72-1590;1574-1592;1584-1602;1587-1605;1591-1609;1592-1610;1595-1613;1601-1619;1602-1620;1605-1623;1729-1747;1738-1756;1739-1 757;1741-1769;1767-1785;1810-1828;1827-1845;1880-1989;1894-2012;2035-2053;2046-2064;2057-2075;2070-2088;2072-2090;2078-2096;2 080-2098;2081-2099;2082-2100;2084-2102;2090-2108;2100-2118;2111-2129;2124-2142;2125-2143;2167-2185;2179-2197;2201-2219;2202- 2220;2203-2221;2204-2222;2227-2245;2230-2248;2234-2252;2244-2264;2255-2273;2266-2284;2268-2286;2270-2288;2279-2297;2281-2299;2283-2301;2284-2302;2285-2303;2286-2304;2288-2306;2290-2308;2291-2309;2292-2310;2294-2312;2296-2314;2299-2317;2304-2322;2306- 2324;2307-2325;2309-2327;2314-2332;2316-2334;2317-2335;2320-2338;2321-2339;2323-2341;2325-2343;2326-2344;2328-2346;2329-2347; The present invention provides a double-stranded ribonucleic acid (RNAi) agent comprising at least 15 consecutive nucleotides that differ by no more than three nucleotides from 2331-2349; 2333-2351; 2334-2352; 2335-2353; 2339-2357; 2340-2358; or 2341-2359, and the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than three nucleotides from the nucleotides at corresponding positions in the nucleotide sequence of SEQ ID NO: 2, such that the antisense strand is substantially complementary to the at least 15 consecutive nucleotides in the sense strand. In some embodiments, substantially all nucleotides in the sense strand are modified nucleotides. In other embodiments, substantially all nucleotides in the antisense strand are modified nucleotides. In still other embodiments, substantially all nucleotides in both strands are modified nucleotides. In one embodiment, the sense strand is complexed with a ligand attached at the 3' end.

[0013] In one embodiment, the sense and antisense strands comprise a region of complementarity comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences listed in any of Tables 3, 4, 7, 8, 11, 13 and 15. For example, in any embodiment, the sense and antisense strands may be duplexed with one another to form the following: AD-52433.1, AD-52438.1, AD-52439.1, AD-52445.1, AD-52449.1, AD-52451.1, AD-52456.1, AD-52457.1, AD-52462.1, AD-52463.1, AD-52469.1, AD-52474.1, AD-55976.1, AD-55978.1, AD-55979.1, AD-55980.1, AD-55981, AD-55982, AD-55983, AD-55984, AD-55985, AD-55986, AD-55987, AD-55988, AD-55989, AD-55990, AD-55991, AD-55992, AD-55993, AD-55994, AD-55995, AD-55996, AD-55997, AD-559980, AD-559999, AD-55990, AD-55991, AD-55992, AD-55993, AD-55994, AD-55995, AD-55996, AD-55997, AD-55998, AD-55999, AD-55900, AD-55999, AD-55901, AD-559902, AD-55991, AD-55992, AD-55993, AD-55994, AD-55995, AD-55996, AD-55997, AD D-55981.1, AD-55982.1, AD-55983.1, AD-55984.1, AD-55987.1, AD-55988.1, AD-55989.1, AD-55990.1, AD-55991.1, AD-55994.1, AD-55995.1, AD-55996.1, AD-55999.1, AD-56000.1, AD-56001.1, AD-56002.1, AD-56003.1, AD-56006.1, AD-56007.1, AD-56008.1, AD-56009.1, AD-56011.1, AD-56012.1, AD-56013.1, AD-56016.1, AD-56017.1, AD-56019.1, AD-56020.1, AD-56021.1, AD-56022.1 , AD-56024.1, AD-56026.1, AD-56027.1, AD-56029.1, AD-56030.1, AD-56031.1, AD-56032.1, AD-56035.1, AD-56039.1, AD-56041.1 , AD-56043.1, AD-56044.1, AD-56047.1, AD-56048.1, AD-56051.1, AD-56053.1, AD-56054.1, AD-56059.1, AD-56062.1, AD-56065. 1, AD-56066.1, AD-60770.1, AD-60771.1, AD-60776.1, AD-60777.1, AD-60778.1, AD-60779.1, AD-60780.1, AD-60781.1, AD-60783.1. AD-67923.1, AD-67924.1, AD-67925.1, AD-67926.1, AD-67935.1, AD-67965.1, AD-67994.1, AD-67995.1, AD-67996.1, AD-68017.1, AD-68022.1, AD- and AD-68035.1, AD-68036.1, AD-68037.1, AD-68084.2, AD-68085.2, AD-68086.2, AD-68087.2, AD-68090.2, AD-68091.2, AD-68092.2, AD-68093.2, AD-68116.1, AD-68117.1, AD-68118.1, AD-68124.1, AD-68125.1, or AD-68126.1, comprising a region of complementarity comprising at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences of AD-68035.1, AD-68036.1, AD-68037.1, AD-68084.2, AD-68085.2, AD-68086.2, AD-68087.2, AD-68090.2, AD-68091.2, AD-68092.2, AD-68093.2, AD-68116.1, AD-68117.1, AD-68118.1, AD-68124.1, AD-68125.1, or AD-68126.1. In any embodiment, the sense and antisense strands are duplexed as follows: AD-52433.1, AD-52438.1, AD-52439.1, AD-52445.1, AD-52449.1, AD-52451.1, AD-52456.1, AD-52457.1, AD-52462.1, AD-52463.1, AD-52469.1, AD-52474.1, AD-55976.1, AD-55978.1, AD-55979.1, AD-55980.1, AD-55981.1, AD-55982.1, AD-55983.1, AD-55984.1, AD-55985.1, AD-55986.1, AD-55987.1, AD-55988.1, AD-55989.1, AD-55990.1, AD-55991.1, AD-55992.1, AD-55993.1, AD-55994.1, AD-55995.1, AD-55996.1, AD-55997.1, AD-55998.1, AD-559 ... D-55987.1, AD-55988.1, AD-55989.1, AD-55990.1, AD-55991.1, AD-55994.1, AD-55995.1, AD-55996.1, AD-55999.1, AD-56000.1, AD-56001.1, AD-56 002.1; AD-56003.1;1, AD-56020.1, AD-56021.1, AD-56022.1, AD-56024.1, AD-56026.1, AD-56027.1, AD-56029.1, AD-56030.1, AD-56031.1, AD-5 6032.1, AD-56035.1, AD-56039.1, AD-56041.1, AD-56043.1, AD-56044.1, AD-56047.1, AD-56048.1, AD-56051.1, AD-56053.1 , AD-56054.1, AD-56059.1, AD-56062.1, AD-56065.1, AD-56066.1, AD-60770.1, AD-60771.1, AD-60776.1, AD-60777.1, AD-60 778.1, AD-60779.1, AD-60780.1, AD-60781.1, AD-60783.1, AD-60784.1, AD-60785.1, AD-60788.1, AD-60789.1, AD-60791.1, AD-60793.1, AD-60795.1, AD-60798.1, AD-60801.1, AD-67903.1, AD-67906.1, AD-67923.1, AD-67924.1, AD-67925.1, AD-679 26.1, AD-67935.1, AD-67965.1, AD-67994.1, AD-67995.1, AD-67996.1, AD-68017.1, AD-68022.1, AD-68035.1, AD-68036.1, A and any one of AD-68037.1, AD-68084.2, AD-68085.2, AD-68086.2, AD-68087.2, AD-68090.2, AD-68091.2, AD-68092.2, AD-68093.2, AD-68116.1, AD-68117.1, AD-68118.1, AD-68124.1, AD-68125.1, or AD-68126.1, comprising a region of complementarity that includes at least 15 contiguous nucleotides of the region of complementarity.

[0014] In one embodiment, the antisense strand comprises a region of complementarity comprising at least 15 contiguous unmodified nucleotides that differ by no more than three nucleotides from the antisense nucleotide sequence of AD-67327 (5'-AUUAGAAGAAAAGGUGGGAGACU-3'; SEQ ID NO: 537). In another embodiment, the region of complementarity consists of the antisense unmodified nucleotide sequence of AD-67327 (5'-AUUAGAAGAAAAGGUGGGAGACU-3'; SEQ ID NO: 537). In one embodiment, the dsRNA comprises a sense strand consisting of the nucleotide sequence 5'-UCUCCCACCUUUUCUUCUAAU-3' (SEQ ID NO: 499) and an antisense strand consisting of the nucleotide sequence 5'-UUAGAAGAAAAGGUGGGAGACU-3' (SEQ ID NO: 537). In one embodiment, a double-stranded RNAi agent includes the modified nucleotide sequence of AD-67327 (5'-uscsucccAfcCfUfUfuucuucuaau-3'; SEQ ID NO: 1037 and 5'-asUfsuagAfagaaaagGfuGfggagascsu-3'; SEQ ID NO: 1038).

[0015] In certain embodiments, the modified nucleotide(s) are independently selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, nucleotides containing a 5'-phosphorothioate group, and terminal nucleotides linked to a cholesteryl ester derivative or a dodecanoic acid bisdecylamide group. In further embodiments, the modified nucleotides are selected from the group consisting of 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy-modified nucleotides, locked nucleotides, non-locked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-allyl modified nucleotides, 2'-hydroxyl modified nucleotides, morpholino nucleotides, phosphoramidates, and non-natural base containing nucleotides.

[0016] In another embodiment of the double-stranded RNAi agent, at least one strand comprises a 3' overhang of at least one nucleotide, hi another embodiment, at least one strand comprises a 3' overhang of at least two nucleotides.

[0017] In another aspect, the invention provides an RNAi agent, e.g., a double-stranded ribonucleic acid (RNAi) agent, that can inhibit expression of angiotensinogen (AGT) in a cell, wherein the double-stranded RNAi agent comprises a sense strand that is complementary to an antisense strand, wherein the antisense strand comprises a region that is complementary to a portion of an mRNA encoding AGT, and each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by Formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q’ 5'(III) wherein i, j, k, and l each independently represent 0 or 1; p, p', q and q' are each independently 0-6; each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; each N b and N b' represents an oligonucleotide sequence containing 0-10 nucleotides that are independently either modified or unmodified, or a combination thereof; each n p , n p ',n q and n q ' (each of which may or may not be present) independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides; N b The modification in N is different from the modification in Y. b the modification in ' is different from the modification in Y', and The sense strand is complexed with at least one ligand. The present invention provides a double-stranded RNAi agent represented by the formula:

[0018] In one embodiment, i is 0 and j is 0; i is 1 and j is 1; both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; k is 1 and l is 1; both k and l are 0; or both k and l are 1.

[0019] In one embodiment, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

[0020] In one embodiment, the YYY motif occurs at or proximal to the cleavage site on the sense strand.

[0021] In another embodiment, the Y'Y'Y' motif occurs at positions 11, 12 and 13 from the 5' end of the antisense strand.

[0022] In one embodiment, Y' is 2'-O-methyl.

[0023] In one embodiment, formula (III) is represented by formula (IIIa): Sense: 5' n p -N a -YYY-N a -n q 3' Antisense: 3' n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 5'(IIIa).

[0024] In another embodiment, formula (III) is represented by formula (IIIb): Sense: 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' Antisense: 3' n p’ -N a’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(IIIb) [In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1-5 modified nucleotides. It is expressed as:

[0025] In yet another embodiment, formula (III) is represented by formula (IIIc): Sense: 5' n p -N a -XXX-N b -YYY-N a -n q 3' Antisense: 3' n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5'(IIIc) [In the formula, each N b and N b' independently represents an oligonucleotide sequence containing 1-5 modified nucleotides. It is expressed as:

[0026] In a further embodiment, formula (III) has formula (IIId): Sense: 5' n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' Antisense: 3' n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5'(IIId) [In the formula, each N b and N b ' independently represents an oligonucleotide sequence containing 1-5 modified nucleotides, and each N a and N a ' independently represent an oligonucleotide sequence containing 2-10 modified nucleotides. It is expressed as:

[0027] In one embodiment, the double-stranded region is 15-30 nucleotide pairs in length. In another embodiment, the double-stranded region is 17-23 nucleotide pairs in length. In yet another embodiment, the double-stranded region is 17-25 nucleotide pairs in length. In a further embodiment, the double-stranded region is 23-27 nucleotide pairs in length. In another embodiment, the double-stranded region is 19-21 nucleotide pairs in length. In another embodiment, the double-stranded region is 19-23 nucleotide pairs in length. In another embodiment, the double-stranded region is 21-23 nucleotide pairs in length. In yet another embodiment, each strand has 15-30 nucleotides. In yet another embodiment, each strand has 19-30 nucleotides.

[0028] In one embodiment, the modification in the nucleotide is selected from the group consisting of LNA, UNA, CRN, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.In another embodiment, the modification in the nucleotide is 2'-O-methyl modification or 2'-fluoro modification.

[0029] In one embodiment, the ligand is one or more GalNAc derivatives attached via a bivalent linker or a trivalent branched linker. [ka] is.

[0030] In one embodiment, the ligand is attached to the 3' end of the sense strand.

[0031] In another embodiment, the RNAi agent is: [ka] [In the formula, X is O or S.] In certain embodiments, X is O.

[0032] In one embodiment, the agent of the invention further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0033] In a further embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3'-end of one strand. In another embodiment, the strand is the antisense strand. In another embodiment, the strand is the sense strand.

[0034] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5'-end of one strand. In another embodiment, the strand is the antisense strand. In a further embodiment, the strand is the sense strand.

[0035] In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of one strand, hi another embodiment, the strand is the antisense strand.

[0036] In another embodiment, the double-stranded RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In a further embodiment, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end of the sense strand.

[0037] In one embodiment, the base pair at one position of the 5' end of the antisense strand of the double strand is an AU base pair. In another embodiment, the Y nucleotide comprises a 2'-fluoro modification. In a further embodiment, the Y' nucleotide comprises a 2'-O-methyl modification.

[0038] In one embodiment, p'>0. In another embodiment, p'=2. In a further embodiment, q'=0, p=0, q=0, and the p' overhanging nucleotides are complementary to the target mRNA. In a further embodiment, q'=0, p=0, q=0, and the p' overhanging nucleotides are not complementary to the target mRNA.

[0039] In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0040] In another embodiment, at least one n pIn a further embodiment, all n' are linked to adjacent nucleotides via phosphorothioate bonds. p ' is linked to the adjacent nucleotide via a phosphorothioate bond.

[0041] In another embodiment, the RNAi agent is selected from the group of RNAi agents listed in any of Tables 3, 4, 7, 8, 11, 13, and 15.

[0042] In one aspect, the present invention provides double-stranded ribonucleic acid (RNAi) agents for inhibiting the expression of angiotensinogen (AGT). The double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5' terminus and substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' terminus and two phosphorothioate internucleotide linkages at the 3' terminus, and wherein the sense strand is conjugated to one or more GalNAc derivatives attached to the 3' terminus via a divalent linker or a trivalent branched linker.

[0043] In one embodiment, every nucleotide of the sense strand and every nucleotide of the antisense strand comprises a modification.

[0044] In another aspect, the invention provides an RNAi agent, e.g., a double-stranded ribonucleic acid (RNAi) agent, that can inhibit expression of AGT (angiotensinogen) in a cell, comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding AGT, wherein each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by Formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q’ 5'(III) wherein i, j, k, and l each independently represent 0 or 1; p, p', q and q' are each independently 0-6; each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 0-10 nucleotides that are independently either modified or unmodified, or a combination thereof; each n p , n p ',n q and n q ' (each of which may or may not be present) independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications in three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification in N is different from the modification in Y. b the modification in ' is different from the modification in Y', and The sense strand is complexed with at least one ligand. The present invention provides a double-stranded RNAi agent represented by the formula:

[0045] In yet another aspect, the invention provides an RNAi agent, e.g., a double-stranded ribonucleic acid (RNAi) agent, that can inhibit expression of angiotensinogen (AGT) in a cell, comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding AGT, wherein each strand is about 14 to about 30 nucleotides in length, and wherein the double-stranded RNAi agent has a structure represented by Formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q’ 5'(III) wherein i, j, k, and l each independently represent 0 or 1; each n p , n p ',n q and n q ' (each of which may or may not be present) independently represents an overhanging nucleotide; p, q, and q' are each independently 0-6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond, each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 0-10 nucleotides, which are independently either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications in three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification in N is different from the modification in Y. b the modification in ' is different from the modification in Y', and The sense strand is complexed with at least one ligand. The present invention provides a double-stranded RNAi agent represented by the formula:

[0046] In a further aspect, the invention provides an RNAi agent, e.g., a double-stranded ribonucleic acid (RNAi agent), that can inhibit expression of angiotensinogen (AGT) in a cell, comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding AGT, and each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by Formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-Nb -(ZZZ) j -N a -n q 3' Antisense: 3' n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q’ 5'(III) wherein i, j, k, and l each independently represent 0 or 1; each n p , n p ',n q and n q ' (each of which may or may not be present) independently represents an overhanging nucleotide; p, q, and q' are each independently 0-6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond, each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 0-10 nucleotides, which are independently either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications in three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification in N is different from the modification in Y. bthe modification in ' is different from the modification in Y', and The sense strand is complexed with at least one ligand, where the ligand is one or more GalNAc derivatives attached via a bivalent linker or a trivalent branched linker. The present invention provides a double-stranded RNAi agent represented by the formula:

[0047] In another aspect, the invention provides an RNAi agent, e.g., a double-stranded ribonucleic acid (RNAi) agent, that can inhibit expression of angiotensinogen (AGT) in a cell, comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding AGT, and each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has a structure represented by Formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' n p '-N a '-(X'X'X') k -N b '-Y'Y'Y'-N b '-(Z'Z'Z') l -N a '-n q’ 5'(III) wherein i, j, k, and l each independently represent 0 or 1; each n p , n p ',n q and n q ' (each of which may or may not be present) independently represents an overhanging nucleotide; p, q, and q' are each independently 0-6; n p '>0 and at least one n p' is attached to the adjacent nucleotide via a phosphorothioate bond, each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; each N b and N b ' represents an oligonucleotide sequence containing 0-10 nucleotides, which are independently either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent a motif of three identical modifications in three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N b The modification in N is different from the modification in Y. b the modification in ' is different from the modification in Y', and the sense strand comprises at least one phosphorothioate bond; The sense strand is complexed with at least one ligand, where the ligand is one or more GalNAc derivatives attached via a bivalent linker or a trivalent branched linker. The present invention provides a double-stranded RNAi agent represented by the formula:

[0048] In yet another aspect, the invention provides an RNAi agent, e.g., a double-stranded ribonucleic acid (RNAi), that can inhibit expression of angiotensinogen (AGT) in a cell, comprising a sense strand complementary to an antisense strand, wherein the antisense strand comprises a region complementary to a portion of an mRNA encoding AGT, and each strand is about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by Formula (III): Sense: 5' n p -N a -YYY-N a -n q3' Antisense: 3' n p '-N a '-Y'Y'Y'-N a '-n q’ 5'(IIIa) [In the formula, each n p , n p ',n q and n q ' (each of which may or may not be present) independently represents an overhanging nucleotide; p, q, and q' are each independently 0-6; n p '>0 and at least one n p ' is attached to the adjacent nucleotide via a phosphorothioate bond, each N a and N a ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; YYY and Y'Y'Y' each independently represent a motif of three identical modifications in three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; the sense strand comprises at least one phosphorothioate linkage, and The sense strand is complexed with at least one ligand, where the ligand is one or more GalNAc derivatives attached via a bivalent linker or a trivalent branched linker. The present invention provides a double-stranded RNAi agent represented by the formula:

[0049] In another aspect, the invention provides a double-stranded RNAi agent comprising an RNAi agent listed in any one of Tables 3, 4, 7, 8, 11, 13, and 15.

[0050] In another aspect, the present invention provides a composition comprising a modified antisense polynucleotide drug, wherein the drug is capable of inhibiting expression of angiotensinogen (AGT) in a cell and comprises a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 3, 4, 7, 8, 11, 13, and 15, and the polynucleotide is about 14 to about 30 nucleotides in length.

[0051] The invention also provides cells, vectors, host cells and pharmaceutical compositions comprising the double-stranded RNAi agents of the invention.

[0052] In one embodiment, the cell comprises a double-stranded RNAi agent.

[0053] In certain embodiments, a composition comprising a double-stranded RNAi agent or modified antisense polynucleotide agent is administered using a pharmaceutical composition.

[0054] In one embodiment, the pharmaceutical composition of the invention comprises a liquid formulation, such as XTC or MC3.

[0055] In a preferred embodiment, the double-stranded RNAi agent is administered in a solution. In some embodiments, the double-stranded RNAi agent is administered in an unbuffered solution. In another embodiment, the unbuffered solution is saline or water. In another embodiment, the double-stranded RNAi agent is administered with a buffer. In yet another embodiment, the buffer comprises an acetate buffer, a citrate buffer, a prolamin, a carbonate buffer, or a phosphate buffer, or any combination thereof. In some embodiments, the buffer is phosphate-buffered saline (PBS).

[0056] In another aspect, the invention provides a method for inhibiting expression of angiotensinogen (AGT) in a cell, the method including contacting the cell with a double-stranded RNAi agent, a pharmaceutical composition, a composition comprising a modified antisense polynucleotide agent, or a vector comprising an RNAi agent, and maintaining the cells so produced for a period of time sufficient to allow degradation of mRNA transcripts of the AGT gene, thereby inhibiting expression of the AGT gene in the cell.

[0057] In one embodiment, the cell is present in a subject. In a further embodiment, the subject is a human. In a further embodiment, the subject is suffering from an angiotensinogen-related disorder.

[0058] In one embodiment, AGT expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

[0059] In one embodiment, the sense strand comprises nucleotides 2801-2101; 803-843; 834-859; 803-859; 803-875; 834-875; 847-875; 1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1624; 1584-1621; 2035-2144; 2070-2144; 2070-2103; 2201-2223; 2227-2360; 2227-2304; 2290-2318; 2304-2350; 2304-2326; 2320-2327; 2321-2328; 2322-2329; 2323-2330; 2324-2331; 2325-2332; 2326-2333; 2327-2334; 2328-2335; 2329-2336; 2329-2338; 2330-2331; 2331-2332; 2332-2333; 2333-2334; 2334-2335; 2335-2336; 2335-2338; 2335-2339; 2336-2339; 2337-2340; 2337-2340; 2338-2341; 2338-2342; 2338- 342;2333-2360;2333-2358;485-503;517-535;560-578;635-653;803-821;814-832;822-840;825-843;834-852;837-855;841-859;855-873;967- 985;1247-1265;1248-1266;1249-1267;1251-1269;1253-1271;1566-1584;1570-1588;1572-1590;1574-1592;1584-1602;1587-1605;1591-1609;1 592-1610;1595-1613;1601-1619;1602-1620;1605-1623;1729-1747;1738-1756;1739-1757;1741-1769;1767-1785;1810-1828;1827-1845;1880- 1989;1892-1914;1894-1914;1894-2012;2035-2053;2046-2064;2057-2075;2070-2088;2072-2090;2078-2096;2078-2107;2078-2011;2080-2098; 2081-2099;2081-2104;2081-2011;2082-2100;2084-2102;2084-2011;2090-2108;2100-2118;2111-2129;2124-2142;2125-2143;2167-2185;2179- 2197;2201-2219;2202-2220;2203-2221;2204-2222;2227-2245;2230-2248;2234-2252;2244-2264;2255-2273;2266-2284;2268-2286;2270-2288;2279-2297;2281-2299;2283-2301;2284-2302;2285-2303;2286-2304;2288-2306;2290-2308;2291-2309;2291-2311;2291-2318;2291-2315;2292-2310;2294-2312;2296-2314;2299-2317;2304-2322;2304-2329;2306-2324;2307-2325;2309-2327;2309-2329;2309-2342; 2309-2350;2309-2358;2314-2332;2316-2334;2317-2335;2320-2338;2321-2339;2323-2341;2325-2343;2326-2344;2328-2346;2329-2347;2331-2349;2333-2351;2334-2352;2335-2353;2339-2357;2340-2358; or at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from 2341-2359.

[0060] In another aspect, the invention provides a method of treating a subject having an angiotensinogen (AGT)-related disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded RNAi agent, a composition comprising a modified antisense polynucleotide agent, or a pharmaceutical composition comprising a double-stranded RNAi agent, thereby treating the subject.

[0061] In another aspect, the invention provides a method of treating a subject having an angiotensinogen (AGT)-related disorder, comprising subcutaneously administering to the subject a therapeutically effective amount of a double-stranded ribonucleic acid (RNAi agent), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, and wherein substantially all of the nucleotides in the antisense strand are 2'-O-methyl and 2'-fluoro modifications, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'-terminus, substantially all nucleotides of the sense strand comprise modifications selected from the group consisting of 2'-O-methyl modifications and 2'-fluoro modifications, the sense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus, and the sense strand is conjugated to one or more GalNAc derivatives attached to the 3'-terminus via a bivalent linker or a trivalent branched linker.

[0062] In one embodiment, every nucleotide of the sense strand and every nucleotide of the antisense strand comprises a modification.

[0063] In another aspect, the invention provides a method of treating a subject having an angiotensinogen (AGT)-related disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded ribonucleic acid (RNAi agent), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand is selected from the group consisting of nucleotides 2801-2101; 803-843; 834-859; 803-859; 803-875; 834-875; 847-875; 1247-1271; 1566-1624; 1570-1624; 1584-1684 of the nucleotide sequence of SEQ ID NO:1. 24;1584-1624;1584-1621;2035-2144;2070-2144;2070-2103;2201-2223;2227-2360;2227-2304;2290-2318;2304-2350;2304-2326;2320-2342;2 333-2360;2333-2358;485-503;517-535;560-578;635-653;803-821;814-832;822-840;825-843;834-852;837-855;841-859;855-873;967-985;12 47-1265;1248-1266;1249-1267;1251-1269;1253-1271;1566-1584;1570-1588;1572-1590;1574-1592;1584-1602;1587-1605;1591-1609;1592-1 610;1595-1613;1601-1619;1602-1620;1605-1623;1729-1747;1738-1756;1739-1757;1741-1769;1767-1785;1810-1828;1827-1845;1880-1989;1 892-1914;1894-1914;1894-2012;2035-2053;2046-2064;2057-2075;2070-2088;2072-2090;2078-2096;2078-2107;2078-2011;2080-2098;2081- 2099;2081-2104;2081-2011;2082-2100;2084-2102;2084-2011;2090-2108;2100-2118;2111-2129;2124-2142;2125-2143;2167-2185;2179-2197;2201-2219;2202-2220;2203-2221;2204-2222;2227-2245;2230-2248;2234-2252;2244-2264;2255-2273;2266-2284;2268-2286;2270-2288;2279-2297;2281-2299;2283-2301;2284-2302;2285-2303;2286-230 4;2288-2306;2290-2308;2291-2309;2291-2311;2291-2318;2291-2315;2292-2310;2294-2312;2296-2314;2299-2317;2304-2322;2304-2329;2306-2324;2307-2325;2309-2327;2309-2329;2309-2342;2309-2 350;2309-2358;2314-2332;2316-2334;2317-2335;2320-2338;2321-2339;2323-2341;2325-2343;2326-2344;2328-2346;2329-2347;2331-2349;2333-2351;2334-2352;2335-2353;2339-2357;2340-2358;or, The present invention provides a method of treatment in which the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from 2341-2359, and the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from the nucleotides at corresponding positions in the nucleotide sequence of SEQ ID NO: 2, such that the antisense strand is substantially complementary to the at least 15 consecutive nucleotides in the sense strand. In some embodiments, substantially all nucleotides in the sense strand are modified nucleotides. In other embodiments, substantially all nucleotides in the antisense strand are modified nucleotides. In still other embodiments, substantially all nucleotides in both strands are modified nucleotides. In one embodiment, all nucleotides in the sense strand and all nucleotides in the antisense strand are modified nucleotides. In one embodiment, the sense strand is complexed with a ligand attached at the 3' end.

[0064] In another aspect, the invention provides a method of treating a subject having an angiotensinogen (AGT)-related disorder, comprising administering to the subject a therapeutically effective amount of a double-stranded ribonucleic acid (RNAi agent), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand is selected from the group consisting of nucleotides 803-843; 834-859; 803-859; 1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1621; 2035-2144 of the nucleotide sequence of SEQ ID NO:1. 2070-2144;2070-2103;2201-2223;2227-2360;2227-2304;2290-2318;2304-2350;2304-2326;2320-2342;2333-2360;2333-2358;485-503;517-53 5;560-578;635-653;803-821;814-832;822-840;825-843;834-852;837-855;841-859;855-873;967-985;1247-1265;1248-1266;1249-1267;1251 -1269;1253-1271;1566-1584;1570-1588;1572-1590;1574-1592;1584-1602;1587-1605;1591-1609;1592-1610;1595-1613;1601-1619;1602-162 0;1605-1623;1729-1747;1738-1756;1739-1757;1741-1769;1767-1785;1810-1828;1827-1845;1880-1989;1894-2012;2035-2053;2046-2064;20 57-2075;2070-2088;2072-2090;2078-2096;2080-2098;2081-2099;2082-2100;2084-2102;2090-2108;2100-2118;2111-2129;2124-2142;2125-2 143;2167-2185;2179-2197;2201-2219;2202-2220;2203-2221;2204-2222;2227-2245;2230-2248;2234-2252;2244-2264;2255-2273;2266-2284;2268-2286;2270-2288;2279-2297;2281-2299;2283-2301;2284-2302;2285-2303;2286-2304;2288-2306;2290-2308;2291-2309;2292-2310;2294-2312;229 6-2314;2299-2317;2304-2322;2306-2324;2307-2325;2309-2327;2314-2332;2316-2334;2317-2335;2320-2338;2321-2339;2323-2341;2325-2343;2326-2 2344; 2328-2346; 2329-2347; 2331-2349; 2333-2351; 2334-2352; 2335-2353; 2339-2357; 2340-2358; or 2341-2359, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than three nucleotides from the nucleotides at corresponding positions in the nucleotide sequence of SEQ ID NO: 2, such that the antisense strand is substantially complementary to the at least 15 contiguous nucleotides in the sense strand. In some embodiments, substantially all of the nucleotides in the sense strand are modified nucleotides. In other embodiments, substantially all of the nucleotides in the antisense strand are modified nucleotides. In yet other embodiments, substantially all of the nucleotides in both strands are modified nucleotides. In one embodiment, the sense strand is complexed with a ligand attached at the 3' end.

[0065] In one embodiment, the subject is a human.

[0066] In one embodiment, the angiotensinogen-related disorder is hypertension, borderline hypertension, primary hypertension, secondary hypertension, hypertensive emergency, hypertensive urgency, 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, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disorders, diabetic nephropathy, diabetic retinal disease, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, endocardial fibrosis, Cushing's syndrome and other glucocorticoid excess states including chronic steroid therapy, pheochromocytoma, reninoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, kidney disease, renal failure, systemic sclerosis, intrauterine growth retardation (IUGR), and fetal growth restriction.

[0067] In another embodiment, the angiotensinogen-related disorder is selected from the group consisting of hypertension, hypertensive heart disease, hypertensive nephropathy, pregnancy-related hypertension, atherosclerosis, arteriosclerosis, chronic kidney disease, glomerulosclerosis, aortic stenosis, aortic aneurysm, endocardial fibrosis, Cushing's syndrome and other glucocorticoid excess states including chronic steroid therapy, pheochromocytoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, stroke, diabetes mellitus, renal failure, and systemic sclerosis.

[0068] In one embodiment, the angiotensinogen-related disorder is pregnancy-associated hypertension (e.g., pregnancy-induced hypertension, preeclampsia, and eclampsia), and administration of an iRNA of the invention to a subject results in a decrease in maternal blood pressure; a decrease in maternal albuminuria; an increase in uteroplacental unit weight; an increase in fetal weight; a normalization of the fetal brain:liver ratio; a decrease in AGT mRNA expression in the maternal liver and no significant decrease in hAGT mRNA expression in the placenta; an increase in overall placental size; an increase in chorioplacental size; no significant change in placental corpus cavernosum size; a decrease in the ratio of sFLT1:PLGF mRNA expression in the maternal kidney; a decrease in the ratio of serum sFLT1:PLGF levels; and / or a decrease in the level of agonistic autoantibodies to AT1.

[0069] In one embodiment, the double-stranded RNAi agent is administered at a dose of about 0.01 mg to about 10 mg or about 0.5 mg to about 50 mg per kg of body weight. In a preferred embodiment, the double-stranded RNAi agent is administered at a dose of about 10 mg, about 30 mg, or about 3.0 mg per kg of body weight. In one embodiment, the double-stranded RNAi agent is administered at a dose of about 10 mg per kg of body weight. In one embodiment, the double-stranded RNAi agent is administered at a dose of about 0.5 mg per kg of body weight twice a week. In another embodiment, the double-stranded RNAi agent is administered at a dose of about 10 mg per kg of body weight every other week. In another embodiment, the double-stranded RNAi agent is administered at a dose of about 0.5-1.0 mg per kg of body weight once a week. In another embodiment, the RNAi agent is administered at a dose of about 0.1 mg to about 5.0 mg per kg of body weight approximately once a week, once a month, once every two months, or once a quarter (i.e., once every three months).

[0070] In one embodiment, the double-stranded RNAi agent is administered subcutaneously or intravenously.

[0071] In one embodiment, the RNAi agent is administered in two or more doses.

[0072] In one embodiment, the RNAi agent is administered at an interval selected from the group consisting of once about every 12 hours, once about every 24 hours, once about every 48 hours, once about every 72 hours, and once about every 96 hours.

[0073] In one embodiment, the RNAi agent is administered twice a week.

[0074] In one embodiment, the RNAi agent is administered every other week.

[0075] In some embodiments, the RNAi agent is administered once a month.

[0076] In some embodiments, the RNAi agent is administered once every two months.

[0077] In some embodiments, the RNAi agent is administered quarterly (i.e., once every three months).

[0078] In yet another embodiment, the method further comprises administering to the subject an additional therapeutic agent, in some embodiments, the additional therapeutic agent is selected from the group consisting of a diuretic, an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor antagonist, a beta-blocker, a vasodilator, a calcium channel blocker, an aldosterone antagonist, an alpha-agonist, a renin inhibitor, an alpha-blocker, a peripherally acting adrenergic agonist, a selective D1 receptor partial agonist, a non-selective alpha-adrenergic antagonist, a synthetic steroidal antimineralocorticoid, or a combination of any of the above, and an antihypertensive agent formulated as a combination drug. [Brief explanation of the drawings]

[0079] [Figure 1] FIG. 1 is a schematic diagram of the renin-angiotensin-aldosterone system (RAAS) including representations of various time points in the system that have been targeted for therapeutic intervention (adapted from Zaman, et al. (2002) Nat Rev Drug Disc 1:621). [Figure 2]Figure 2A is a graph showing the decrease in mean arterial blood pressure and Figure 2B is a graph showing the decrease in serum albumin in pregnant transgenic rats after administration of AD-60771. [Figure 3] Figure 3A is a graph showing increased uteroplacental unit weight after maternal administration of AD-60771, demonstrating fetal improvement after maternal administration of AD-60771. Figure 3B is a graph showing fetal weight gain after maternal administration of AD-60771, demonstrating fetal improvement after maternal administration of AD-60771. Figure 3C is a graph showing normalization of fetal brain:liver ratio after maternal administration of AD-60771, demonstrating fetal improvement after maternal administration of AD-60771. [Figure 4] Figure 4A is a graph showing the reduction of hAGT mRNA in the maternal liver after administration of AD-60771, demonstrating that iRNA does not cross the placental barrier. Figure 4B is a graph showing no significant reduction of hAGT mRNA in the placenta, demonstrating that iRNA does not cross the placental barrier. [Figure 5] FIG. 5 is a graph showing tissue exposure of maternal liver, placenta, and fetal liver to AD-60771. [Figure 6-1] Figure 6A shows the placenta of a pregnant wild-type rat stained for cytokeratin immunohistochemically, Figure 6B shows the placenta of a treated pregnant PE rat stained for cytokeratin immunohistochemically, and Figure 6C shows the placenta of a pregnant PE rat treated with AD-60771 stained for cytokeratin immunohistochemically. [Figure 6-2]Figure 6D is a graph showing the size of the mesometrial triangle in pregnant PE rats treated with AD-60771 and untreated pregnant PE rats. Figure 6E is a graph showing the size of the corpus cavernosum in pregnant PE rats treated with AD-60771 and untreated pregnant PE rats. Figure 6F is a graph showing the size of the placenta in pregnant PE rats treated with AD-60771 and untreated pregnant PE rats. Figure 6G is a graph showing the size of the labyrinth in pregnant PE rats treated with AD-60771 and untreated pregnant PE rats. [Figure 7] Figure 7A is a graph showing the reduction in mRNA levels of the anti-angiogenic factor sFLT1 in maternal kidneys after administration of AD-60771. Figure 7B is a graph showing the reduction in mRNA levels of the angiogenic factor PLGF in maternal kidneys after administration of AD-60771. Figure 7C is a graph showing the reduction in mRNA levels of the anti-angiogenic factor sFLT1 in placenta after maternal administration of AD-60771. Figure 7D is a graph showing the reduction in mRNA levels of the angiogenic factor PLGF in placenta after maternal administration of AD-60771. [Figure 8] Figure 8 is a graph showing the reduction in AT1-AA levels in PE rats administered AD-60771, as assessed by the effect of AT1-AA isolated from control and pregnant PE rats on the spontaneous beating rate of neonatal rat cardiomyocytes. [Figure 9] Figure 9A is a graph showing the reduction in serum angiotensin II (Ang2-10) levels in pregnant PE rats treated with AD-60771 compared to non-pregnant PE rats and pregnant control Sprague-Dawley rats. Figure 9B is a graph showing the reduction in serum human AGT (hAGT) and rat AGT (rAGT) levels in pregnant PE rats treated with AD-60771 compared to non-pregnant PE rats and pregnant control Sprague-Dawley rats. DETAILED DESCRIPTION OF THE INVENTION

[0080] Detailed Description of the Invention The present invention provides iRNA compositions that effect RNA-induced silencing complex (RISC)-mediated cleavage of an RNA transcript of the angiotensinogen (AGT) gene, which may be present in a cell, e.g., a cell in a subject, such as a human.

[0081] The present invention also provides methods for treating subjects with disorders that would benefit from inhibiting or reducing expression of the AGT gene, e.g., angiotensinogen-related disorders such as hypertension or pregnancy-associated hypertension, using iRNA compositions that cause RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the AGT gene.

[0082] iRNAs of the invention include those having a length of about 30 nucleotides or less, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25 , 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotide long region (which region is substantially complementary to at least a portion of an mRNA transcript of the AGT gene) (antisense strand). In some embodiments, the iRNAs of the present invention include an RNA strand (antisense strand) that has a region of at least 19 consecutive nucleotides that is substantially complementary to at least a portion of the mRNA transcript of the AGT gene, and can be longer, for example, up to 66 nucleotides in length, for example, 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides in length. These iRNAs with longer antisense strands include a second RNA strand (sense strand) of 20-60 nucleotides in length, in which the sense and antisense strands form a duplex of 18-30 consecutive nucleotides. The use of these iRNAs enables targeted degradation of the mRNA of the corresponding gene (AGT gene) in mammals. In particular, very low doses of the iRNAs of the present invention can significantly inhibit the expression of the corresponding gene (AGT gene) specifically and efficiently via RNA interference (RNAi).Using in vitro and in vivo assays, the present inventors have demonstrated that iRNAs targeting the angiotensinogen gene can mediate RNAi, resulting in significant inhibition of AGT expression and alleviating symptoms associated with angiotensinogen-related disorders, such as pregnancy-related hypertension (e.g., pregnancy-induced hypertension, preeclampsia, and eclampsia). Thus, methods and compositions comprising these iRNAs are useful for treating subjects with angiotensinogen-related disorders, such as hypertension.

[0083] The detailed description below also describes methods for making and using compositions containing iRNA that inhibit expression of the angiotensinogen gene, as well as compositions, uses, and methods for treating subjects with diseases and disorders that may benefit from inhibiting and / or reducing the expression of AGT.

[0084] I. Definition In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that when values ​​or ranges of parameters are given, it is intended that values ​​and ranges intermediate to the given values ​​are also intended to be part of the present invention.

[0085] The singular forms "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the term. For example, "an element" means one element or more than one element, e.g., a plurality of elements.

[0086] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to."

[0087] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless expressly stated otherwise.

[0088] The term "about" is used herein to mean within the general range accepted in the art. As used herein, "angiotensinogen," used interchangeably with the term "AGT," refers to the well-known gene and polypeptide, also known in the art as serpin peptidase inhibitor, clade A, member 8; alpha-1 antiproteinase; antitrypsin; SERPINA8; angiotensin I; serpin A8; angiotensin II; alpha-1 antiproteinase angiotensinogen; antitrypsin; pre-angiotensinogen 2; ANHU; serine proteinase inhibitor; and cysteine ​​proteinase inhibitor.

[0089] The term "AGT" includes human AGT (the amino acid and its complete coding sequence can be found, for example, in GenBank Accession No. GI:188595658 (NM_000029.3; SEQ ID NO:1)); cynomolgus monkey AGT (the amino acid and its complete coding sequence can be found, for example, in GenBank Accession No. GI:90075391 (AB170313.1; SEQ ID NO:3)); mouse (Mus musculus) AGT (the amino acid and its complete coding sequence can be found, for example, in GenBank Accession No. GI:113461997 (NM_007428.3; SEQ ID NO:5)); and rat AGT (Rattus norvegicus) AGT (the amino acid and its complete coding sequence can be found, for example, in GenBank Accession No. GI:51036672 (NM_134432; SEQ ID NO:7)).

[0090] Further examples of AGT mRNA sequences are readily available using publicly available databases such as GenBank, UniProt, OMIM, and the Macaca Genome Project website.

[0091] As used herein, the term "AGT" also refers to naturally occurring DNA sequence variants of the AGT gene, such as single nucleotide polymorphisms (SNPs) in the AGT gene. Representative SNPs can be found in the available dbSNP database (www.ncbi.nlm.nih.gov / projects / SNP / snp_ref.cgi-geneId=183). Non-limiting examples of sequence variations within the AGT gene include those described, for example, in U.S. Patent No. 5,589,584 (the entire contents of which are incorporated herein by reference). For example, sequence variations within the AGT gene include C → T at position −532 (relative to the transcription start site); G → A at position −386; G → A at position −218; C → T at position −18; G → A and A → C at positions −6 and −10; C → T (untranslated) at position +10; C → T at position +521 (T174M); T → C at position +597 (P199P); T → C at position +704 (M235T; e.g., Reference SNP (refSNP) Cluster See also Report:rs699, available at www.ncbi.nlm.nih.gov / SNP); A → G (Y248C) at position +743; C → T (N271N) at position +813; G → A (L339L) at position +1017; C → A (L359M) at position +1075; and / or G → A (V388M) at position +1162.

[0092] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the AGT gene, including mRNA that is the product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence can be at least long enough to act as a substrate for iRNA-directed cleavage at or proximal to the portion of the nucleotide sequence of an mRNA molecule formed during transcription of the AGT gene.

[0093] The target sequence can be about 9-36 nucleotides in length, for example, about 15-30 nucleotides in length. For example, the target sequence can be about 15-30 nucleotides in length, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 1 It can be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the invention.

[0094] 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.

[0095] "G," "C," "A," "T," and "U" generally refer to nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or surrogate replacement moieties, as described in more detail below (see, e.g., Table 2). Those skilled in the art will appreciate that guanine, cytosine, adenine, and uracil can be substituted with other moieties without substantially altering the base-pairing properties of oligonucleotides containing nucleotides having such replacement moieties. For example, but not limited to, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be substituted in the nucleotide sequence of a dsRNA of the present invention by, for example, a nucleotide containing inosine. In another example, adenine and cytosine anywhere in the oligonucleotide may be substituted with guanine and uracil, respectively, to form a GU wobble base pair with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods of the present invention.

[0096] The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interference agent," used interchangeably herein, are agents comprising RNA, as those terms are defined herein, that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. iRNAs effect sequence-specific degradation of mRNAs through a process known as RNA interference (RNAi). iRNAs modulate, e.g., inhibit, expression of AGT, e.g., in cells, e.g., cells within a subject, such as a mammalian subject.

[0097] In one embodiment, the RNAi agent of the present invention comprises a single-stranded RNA, such as an AGT target mRNA sequence, that interacts with the target RNA sequence to cleave the target RNA. Regardless of theory, it is believed that long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer (a ribonuclease III-like enzyme) processes dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect, the present invention relates to single-stranded RNA (siRNA) generated within a cell, which promotes the formation of a RISC complex to promote the silencing of a target gene, i.e., the AGT gene. Thus, the term "siRNA" is also used herein to refer to the above-mentioned RNAi.

[0098] In any embodiment, RNAi agents can be single-stranded siRNA (ssRNAi) introduced into cells or organisms to inhibit target mRNA. Single-stranded RNAi agents bind to RISC endonuclease, Argonaute 2, and then cleave target mRNA. Single-stranded siRNAs are generally 15-30 nucleotides and chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894 (the entire contents of which are incorporated herein by reference). Any of the antisense nucleotide sequences described herein can be chemically modified as described herein or by the method described in Lima et al., (2012) Cell 150:883-894, and used as single-stranded siRNA.

[0099] In another embodiment, an "iRNA" for use in the compositions, uses, and methods of the invention is double-stranded RNA, and is referred to herein as a "double-stranded RNAi agent," a "double-stranded RNA (dsRNA) molecule," a "dsRNA agent," or a "dsRNA." The term "dsRNA" refers to a complex of ribonucleic acid molecules having a double-stranded structure comprising two antiparallel and substantially complementary nucleic acid strands, said to have "sense" and "antisense" orientations relative to the target RNA, i.e., the AGT gene. In certain embodiments of the invention, the double-stranded RNA (dsRNA) induces degradation of the target RNA, e.g., mRNA, via a post-transcriptional gene silencing mechanism, referred to herein as RNA interference or RNAi.

[0100] Generally, the majority of nucleotides in each strand of a dsRNA molecule are ribonucleotides; however, as detailed herein, each or both strands may contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, "RNAi agents" may include ribonucleotides with chemical modifications, and RNAi agents may contain substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase. Thus, the term modified nucleotide includes, for example, the substitution, addition, or removal of a functional group or atom, an internucleoside linkage, a sugar moiety, or a nucleobase. Modifications suitable for use in the agents of the present invention include all types of modifications described herein or known in the art. All such modifications used in siRNA-type molecules are encompassed by the term "RNAi agent" for purposes of this specification and claims.

[0101] The majority of nucleotides in each strand of a dsRNA molecule may be ribonucleotides, although, as detailed herein, each or both strands may contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Additionally, as used herein, "RNAi agents" may include ribonucleotides with chemical modifications, and RNAi agents may contain substantial modifications at multiple nucleotides. As used herein, the term "modified nucleotide" refers to a nucleotide that independently has a modified sugar moiety, a modified internucleotide linkage, and / or a modified nucleobase. Thus, the term modified nucleotide includes, for example, the substitution, addition, or removal of a functional group or atom, an internucleoside linkage, a sugar moiety, or a nucleobase. Modifications suitable for use in the agents of the present invention include all types of modifications described herein or known in the art. All such modifications used in siRNA-type molecules are encompassed by the term "RNAi agent" for purposes of this specification and claims.

[0102] The double-stranded region can be of any length that allows for specific degradation of the desired target RNA via the RISC pathway, and can range from about 9 to 36 base pairs in length, e.g., about 15 to 30 base pairs in length, e.g., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, e.g., about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-19, 20-21, 20-22, 20-23, 20-24, 20-25, 20-26 ... 7, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-2 The length may range from 0, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the invention.

[0103] The two strands forming the double-stranded structure can be different parts of one larger RNA molecule, or they can be separate RNA molecules. When the two strands are part of one larger molecule and are therefore connected by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of the corresponding other strand that forms the double-stranded structure, the connected RNA strands are called "hairpin loops." The hairpin loop may contain at least one unpaired nucleotide. In some embodiments, the hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleotides.

[0104] When the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules are not necessary, but can be covalently linked.When the two strands are covalently linked by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of the corresponding other strand that forms a double-stranded structure, the linked structure is called a "linker".RNA strands can have the same or different number of nucleotides.The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs that exist in the double strand.In addition to the double-stranded structure, RNAi can also contain one or more nucleotide overhangs.

[0105] In any embodiment, the RNAi agent of the present invention is a dsRNA, each strand of which contains 19-23 nucleotides that interact with a target RNA sequence, such as the AGT gene. Regardless of theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer (a ribonuclease III-like enzyme) processes dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0106] In one embodiment, the RNAi agent of the present invention is a 24-30 nucleotide dsRNA that interacts with a target RNA sequence, such as an AGT target mRNA sequence, resulting in the cleavage of the target RNA. Regardless of theory, long double-stranded RNA introduced into cells is degraded into siRNA by a type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer (a ribonuclease III-like enzyme) processes dsRNA into short interfering RNAs of 19-23 base pairs with a characteristic two-base 3' overhang (Bernstein, et al., (2001) Nature 409:363). Then, the siRNA is incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within RISC cleave the target, inducing silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0107] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the double-stranded structure of an iRNA, such as a dsRNA. For example, a nucleotide overhang exists when the 3'-end of one strand of a dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleotide. Alternatively, the overhang may contain at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang may contain nucleotide / nucleoside analogs, including deoxyribonucleotides / nucleosides. The overhang(s) may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of the overhang may be present on the 5'-end, the 3'-end, or both ends of either the antisense strand or the sense strand of the dsRNA.

[0108] In one embodiment, the antisense strand of the dsRNA has 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging at the 3' and / or 5' ends. In one embodiment, the sense strand of the dsRNA has 1 to 10 nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, overhanging at the 3' and / or 5' ends. In any embodiment, the overhang on the sense strand or antisense strand or both strands can be 10 nucleotides or more in length, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In any embodiment, the extended overhang is on the sense strand of the duplex. In any embodiment, the extended overhang is present at the 3' end of the sense strand of the duplex. In any embodiment, the extended overhang is present at the 5' end of the sense strand of the duplex. In any embodiment, the extended overhang is present on the antisense strand of the double strand.In any embodiment, the extended overhang is present at the 3'-end of the antisense strand of the double strand.In any embodiment, the extended overhang is present at the 5'-end of the antisense strand of the double strand.In any embodiment, one or more nucleotides in the overhang are replaced with thiophosphate nucleosides.

[0109] "Blunt" or "blunt end" refers to the absence of unpaired nucleotides at the ends of a double-stranded RNAi agent, i.e., the absence of nucleotide overhangs. A "blunt ended" RNAi agent is a dsRNA that is double-stranded throughout its entire length, i.e., has no nucleotide overhangs at either end of the molecule. RNAi agents of the invention include RNAi agents that have nucleotide overhangs at one end (i.e., agents with one end that is an overhang and the other that is blunt) or that have nucleotide overhangs at both ends.

[0110] The term "antisense strand" or "guide strand" refers to the strand of an iRNA, e.g., a dsRNA, that includes a region that is substantially complementary to a target sequence, e.g., an AGT mRNA. As used herein, the term "region of complementarity" refers to a region on the antisense strand that is substantially complementary to a sequence, such as a target sequence, as defined herein, e.g., an AGT nucleotide sequence. If the region of complementarity is not perfectly complementary to the target sequence, mismatches can occur in internal or terminal regions of the molecule. Generally, mismatches are most tolerated in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the iRNA.

[0111] As used herein, the term "sense strand" or "passenger strand" refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0112] As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region includes three bases immediately adjacent to the cleavage site at one end of the cleavage site. In some embodiments, the cleavage region includes two bases immediately adjacent to the cleavage site at one end of the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bounded by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13.

[0113] As used herein, unless otherwise specified, the term "complementary," when used to describe a first nucleotide sequence in the context of a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize to an oligonucleotide or polynucleotide comprising the second nucleotide sequence under specified conditions to form a double-stranded structure, as would be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, such as 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiological conditions that may occur in an organism, may be applied. One of skill in the art can determine the optimal combination of conditions for testing the complementarity of two sequences depending on the end use of the hybridized nucleotides.

[0114] Complementary sequences within an iRNA, such as within a dsRNA described herein, involve base pairing between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence across the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" to one another. However, when a first sequence is referred to herein as "substantially complementary" with respect to a second sequence, the two sequences may be perfectly complementary, or they may form one or more, but generally no more than 5, 4, 3, or 2, mismatched base pairs upon hybridization of up to 30 base pairs of the double strand, while retaining the ability to hybridize under conditions most appropriate for their end use, such as inhibiting gene expression via the RISC pathway. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches in determining complementarity. For example, a dsRNA containing one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide contains a 21 nucleotide sequence that is perfectly complementary to the shorter oligonucleotide, can still be described as "perfectly complementary" for purposes described herein.

[0115] As used herein, "complementary" sequences may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from unnatural and modified nucleotides, so long as the above requirements regarding their hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogsteen base pairs.

[0116] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" may be used in reference to base matching between the sense and antisense strands of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as may be understood from the context in which they are used.

[0117] As used herein, a polynucleotide that is "substantially complementary to at least a portion" of a messenger RNA (mRNA) means a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding AGT). For example, a polynucleotide is complementary to at least a portion of an AGT mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding AGT.

[0118] Thus, in some embodiments, the antisense strand polynucleotides described herein are fully complementary to the target AGT sequence. In other embodiments, the antisense strand polynucleotides described herein are substantially complementary to the target AGT sequence, and comprise a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, or about 95% complementary, over its entire length, to the nucleotide sequence of SEQ ID NO:1, or a region equivalent to a fragment of SEQ ID NO:1.

[0119] In one embodiment, an RNAi agent of the invention comprises a sense strand that is substantially complementary to an antisense strand polynucleotide and is complementary to a target AGT sequence, wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is at least about 80% complementary, e.g., about 85%, about 90%, or about 95% complementary, over its entire length to the nucleotide sequence of SEQ ID NO:2, or a region equivalent to a fragment of SEQ ID NO:2.

[0120] Generally, the majority of the nucleotides in each strand are ribonucleotides, although, as described in detail herein, one or both strands may also contain one or more non-ribonucleotides, such as deoxyribonucleotides and / or modified nucleotides. Furthermore, "iRNA" includes ribonucleotides with chemical modifications. Such modifications can include all types of modifications described herein or known in the art. Any such modifications used in iRNA molecules are encompassed by "iRNA" for purposes of this specification and claims.

[0121] In one aspect of the present invention, the agent for use in the methods and compositions of the present invention is a single-stranded antisense RNA molecule that inhibits target mRNA by antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence in the target mRNA. Single-stranded antisense oligonucleotides can inhibit translation by base pairing with mRNA in a stoichiometric manner and physically interfering with the translational machinery (see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355). The single-stranded antisense RNA molecule can be about 15 to about 30 nucleotides in length and have a sequence complementary to the target sequence. For example, the single-stranded antisense RNA molecule can comprise a sequence that is at least about 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from any one of the antisense sequences described herein.

[0122] As used herein, the term "inhibit" is used interchangeably with "reduce," "silencing," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0123] As used herein, the phrase "inhibiting the expression of AGT" includes the inhibition of expression of any AGT gene (e.g., mouse AGT gene, rat AGT gene, monkey AGT gene, or human AGT gene), as well as variants or mutants of the AGT gene that encode the AGT protein.

[0124] "Inhibition of expression of the AGT gene" includes any level of inhibition of the AGT gene, such as at least partial suppression of expression of the AGT gene, such as at least about 20% inhibition. In any embodiment, the inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% inhibition.

[0125] The expression of AGT gene can be evaluated based on any variable level related to AGT gene expression, such as AGT mRNA level or AGT protein level.Inhibition can be evaluated by the absolute or relative level reduction of one or more of these variables compared with control level.The control level can be any type of control level used in the art, such as pre-administration baseline level, or the level measured from the same subject, cell or sample that is untreated or treated with a control (for example, buffer only control or non-active drug control).

[0126] In one embodiment, at least partial suppression of AGT gene expression is assessed by a decrease in the amount of AGT mRNA isolated from or detectable in a first cell or group of cells in which the AGT gene is transcribed and treated to inhibit AGT gene expression, compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but is not treated. The degree of inhibition can be expressed using the following:

number

[0127] As used herein, the phrase "contacting a cell with an RNAi agent," such as dsRNA, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell with an iRNA in vitro or contacting a cell with an iRNA in vivo. Contacting can be direct or indirect. Thus, for example, the individual performing the method may physically contact the RNAi agent with the cell, or may place the RNAi agent in a situation that subsequently allows or causes contact with the cell.

[0128] For example, cell contact in vitro can be carried out by incubating cells with RNAi agent.For example, cell contact in vivo can be carried out by injecting RNAi agent into the tissue where cell is located or nearby, or for example, by injecting the agent into another area, such as bloodstream or subcutaneous space, so that RNAi agent can reach the tissue where cell is located after contacting.For example, RNAi agent can contain and / or be conjugated with ligand such as GalNAc3, which guides RNAi agent to target site, such as liver.The combination of in vitro and in vivo contact methods is also possible.For example, cell can be contacted with RNAi agent in vitro, and then transplanted into subject.

[0129] In one embodiment, contacting a cell with an iRNA includes "introducing the iRNA" or "delivering the iRNA to a cell" by facilitating or enabling uptake or absorption into the cell. Absorption or uptake of the iRNA can occur through unassisted diffusion or active cellular processes, or by auxiliary agents or devices. Introduction of the iRNA into a cell can be in vitro and / or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be achieved by 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). In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Additional methods are described herein below and / or known in the art.

[0130] The term "lipid nanoparticle" or "LNP" refers to a vehicle comprising a lipid layer that encapsulates a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which the iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0131] As used herein, a "subject" is an animal such as a mammal, including a primate (human, non-human primate, such as monkeys and chimpanzees), a non-primate (such as a cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale), or a bird (e.g., a duck or goose). In one embodiment, the subject is a human, such as a human being treated for or assessed for a disease, disorder, or condition that would benefit from reduced AGT expression; a human being at risk for a disease, disorder, or condition that would benefit from reduced AGT expression; a human having a disease, disorder, or condition that would benefit from reduced AGT expression; and / or a human being treated for a disease, disorder, or condition that would benefit from reduced AGT expression as described herein.

[0132] As used herein, the term "treat" or "treatment" refers to one or more conditions associated with unwanted AGT expression, whether detectable or undetectable, such as angiotensin II type 1 receptor activation (AT1R) (e.g., hypertension, chronic kidney disease, stroke, myocardial infarction, heart failure, aneurysm, peripheral arterial disease, heart disease, increased oxidative stress, e.g., increased superoxide production, inflammation, vasoconstriction, sodium and water retention, potassium and magnesium depletion, renin suppression, etc.). alleviation or amelioration of symptoms of pregnancy-associated hypertension (e.g., pre-eclampsia and eclampsia), symptoms associated with malignant hypertension, symptoms associated with hyperaldosteronism, including, but not limited to, increased cardiac contractility (e.g., increased cardiac arrhythmias, stimulation of plasminogen activator inhibitor 1 (PAI1), activation of the sympathetic nervous system, and increased endothelin secretion), intrauterine growth restriction (IUGR) or fetal growth restriction; reduction in the degree of undesirable AT1R activation; stabilization (i.e., not worsening) of the state of chronic AT1R activation; "Treatment" refers to beneficial or desired results, including, but not limited to, amelioration or mitigation of undesired AT1R activation (e.g., hypertension, chronic kidney disease, stroke, myocardial infarction, heart failure, aneurysm, peripheral arterial disease, heart disease, increased oxidative stress, e.g., increased superoxide production, inflammation, vasoconstriction, sodium and water retention, potassium and magnesium depletion, renin secretion inhibition, muscle hypertrophy and smooth muscle hypertrophy, increased collagen synthesis, vascular stimulation, myocardial and renal fibrosis, increased cardiac contractile rate and force, altered heart rate, e.g., increased arrhythmias, stimulation of plasminogen activator inhibitor 1 (PAI1), activation of the sympathetic nervous system, and increased endothelin secretion). "Treatment" can also mean prolonging survival as compared to expected survival without treatment.

[0133] The term "lower," in the context of AGT levels or disease markers and symptoms in a subject, refers to a statistically significant reduction in such levels. The reduction can be, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more, preferably to a level recognized as being within the normal range for individuals without such disorder.

[0134] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder, or condition that would benefit from reduced expression of the AGT gene, refers to a reduction in the likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition, such as hypertension, chronic kidney disease, stroke, myocardial infarction, heart failure, aneurysm, peripheral arterial disease, heart disease, increased oxidative stress, e.g., increased superoxide production, inflammation, vasoconstriction, sodium and water retention, potassium and magnesium deficiency, renin suppression, muscle hypertrophy and smooth muscle hypertrophy, increased collagen synthesis, vascular stimulation, myocardial and renal fibrosis, increased cardiac contractility and force, altered heart rate, e.g., increased arrhythmias, stimulation of plasminogen activator inhibitor 1 (PAI1), activation of the sympathetic nervous system, and symptoms of undesirable AT1R activation, such as increased endothelin secretion. For example, the likelihood of developing hypertension is reduced, for example, if an individual with one or more hypertension risk factors does not develop hypertension, or develops less severe hypertension compared to a population with the same risk factors but not receiving the treatment described herein. Failure to develop a disease, disorder, or condition, or a reduction in the onset of symptoms associated with such a disease, disorder, or condition (e.g., a reduction of at least about 10% in a clinically recognized measure of the disease or disorder), or a delayed onset of symptoms (e.g., by days, weeks, months, or years) is considered effective prevention.

[0135] As used herein, the term "angiotensinogen-related disease" or "AGT-related disease" refers to a disease or disorder caused by or associated with activation of the renin-angiotensin-aldosterone system (RAAS), or a disease, disorder, or condition that responds to or progresses due to RAAS activation. The term "angiotensinogen-related disease" includes diseases, disorders, or conditions that would benefit from reduced AGT expression. Such diseases are commonly associated with hypertension. Non-limiting examples of angiotensinogen-related disorders include hypertension, e.g., borderline hypertension (also known as prehypertension), primary hypertension (also known as essential or idiopathic hypertension), secondary hypertension (also known as non-essential hypertension), hypertensive emergency (also known as malignant hypertension), hypertensive urgency, isolated systolic or diastolic hypertension, pregnancy-related hypertension (e.g., pre-eclampsia, eclampsia, and postpartum pre-eclampsia), diabetic hypertension, treatment-resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension (also known as renal hypertension), Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disorders ( In some embodiments, the AGT-related disease includes intrauterine growth retardation (IUGR) or uterine growth restriction (EGFR). In some embodiments, the AGT-related disease includes intrauterine growth retardation (IUGR) or uterine growth restriction. In some embodiments, the AGT-related disease includes intrauterine growth retardation (IUGR) or uterine growth restriction.

[0136] Based on the average of seated blood pressure measurements taken suitably during two or more visits, subjects with normotensive blood pressure have a blood pressure of about 90-119 mmHg (about 12-15.9 kPa (kN / m 2 )) systolic blood pressure and approximately 60-79 mmHg (approximately 8.0-10.5 kPa (kN / m 2 )), and subjects with prehypertension are those with a diastolic blood pressure of approximately 120-139 mmHg (approximately 16.1-18.5 kPa (kN / m 2 )) systolic blood pressure and approximately 60-79 mmHg (approximately 8.0-10.5 kPa (kN / m 2 )), and subjects with hypertension (e.g., stage I hypertension) have a diastolic blood pressure of about 140-159 mmHg (about 18.7-21.2 kPa (kN / m 2 )) systolic blood pressure and approximately 90-99 mmHg (approximately 12.0-13.2 kPa (kN / m 2 )), and subjects with hypertension (e.g., stage II hypertension) have a diastolic blood pressure of about ≧160 mmHg (about ≧21.3 kPa (kN / m 2 )) systolic blood pressure and approximately ≥ 100 mmHg (approximately ≥ 13.3 kPa (kN / m 2 )). Subjects with type 1 or type 2 diabetes or renal disease and a blood pressure of 130 / 80 mmHg or higher are considered to have hypertension.

[0137] In one embodiment, the angiotensinogen-related disorder is primary hypertension. "Primary hypertension" is the result of environmental and genetic causes (e.g., the result of no apparent underlying disease cause).

[0138] In one embodiment, the angiotensinogen-related disorder is secondary hypertension. "Secondary hypertension" has an identifiable underlying disease that can have multiple etiologies, including renal, vascular, and endocrine causes, such as parenchymal renal 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), aortic stenosis, or oral contraceptive use.

[0139] In one embodiment, the angiotensinogen-related disorder is a hypertensive emergency, such as malignant hypertension and progressive hypertension. "Progressive hypertension" refers to a severe increase in blood pressure (i.e., a systolic pressure of 180 mmHg or a diastolic pressure of 110 mmHg or greater) that involves direct damage to one or more end organs. Blood pressure must be immediately reduced to prevent further organ damage. "Malignant hypertension" refers to a severe increase in blood pressure (i.e., a systolic pressure of 180 mmHg or a diastolic pressure of 110 mmHg or greater) that involves direct damage to one or more end organs and papilledema. Blood pressure must be immediately reduced to prevent further organ damage. Neural end organ damage caused by uncontrolled blood pressure can include hypertensive encephalopathy, cerebrovascular accident / cerebral infarction, subarachnoid hemorrhage, and / or intracranial hemorrhage. Cardiovascular end organ damage can include myocardial ischemia / infarction, acute left ventricular dysfunction, acute pulmonary edema, and / or aortic 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.

[0140] In one embodiment, the angiotensinogen-related disorder is a hypertensive crisis. A "hypertensive crisis" is a severe increase in blood pressure (i.e., equal to or greater than 180 mmHg systolic or 110 mmHg diastolic) without direct damage to one or more organs. Blood pressure can be safely lowered within a few hours.

[0141] In one embodiment, the angiotensinogen-related disorder is pregnancy-associated hypertension, e.g., chronic hypertension of pregnancy, gestational hypertension, preeclampsia, eclampsia, chronic hypertension plus preeclampsia, HELLP syndrome, and gestational hypertension (also known as transient hypertension of pregnancy, chronic hypertension identified in the second half of pregnancy, and pregnancy-induced hypertension (PIH)). A subject with "chronic hypertension of pregnancy" is one who had a blood pressure greater than 140 / 90 mmHg before pregnancy or before 20 weeks of pregnancy. "Gestational hypertension" or "gestational hypertension" refers to hypertension that develops late in pregnancy (>20 weeks) without other features of preeclampsia, followed by normalization of blood pressure after delivery. "Mild preeclampsia" is defined as the presence of two episodes of high blood pressure (blood pressure ≥ 140 / 90 mmHg) at least 6 hours apart, without evidence of end-organ damage, in a woman who was normotensive before 20 weeks of pregnancy. In subjects with a history of essential hypertension, preeclampsia is diagnosed when systolic blood pressure increases by 30 mmHg or diastolic blood pressure increases by 15 mmHg. "Severe preeclampsia" is defined as the presence of one of the following symptoms or signs in the presence of preeclampsia: systolic blood pressure of 160 mmHg or higher, or diastolic blood pressure of 110 mmHg or higher, occurring twice at least 6 hours apart; urinary protein of 5 g or more in a 24-hour collection or 3+ or higher in two random urine samples collected at least 4 hours apart; pulmonary edema or cyanosis; decreased urine output (<400 mL in 24 hours); persistent headache; epigastric pain and / or liver dysfunction; thrombocytopenia; oligohydramnios; fetal growth restriction; or placental abruption. "Eclampsia" is defined as seizures in women with preeclampsia that cannot be attributed to other causes. "HELLP syndrome" (also known as edema-proteinuria-hypertension of pregnancy type B) is hemolysis, elevated liver enzymes, and low platelet levels in pregnant subjects.

[0142] In one embodiment, the angiotensinogen-related disorder is resistant hypertension. "Resistant hypertension" is blood pressure that remains above target (e.g., 140 / 90 mmHg) despite the simultaneous use of three antihypertensive drugs from different classes, one of which is a thiazide diuretic. Subjects whose blood pressure is controlled with four or more drugs are also considered to have resistant hypertension.

[0143] As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that, when administered to a subject with an angiotensinogen-related disorder, is sufficient to result in treatment of the disorder (e.g., by reducing, ameliorating, or maintaining an existing disorder or one or more symptoms of the disorder). A "therapeutically effective amount" may vary depending on the RNAi agent, the method of agent administration, the disorder and its severity, and the medical history, age, weight, family history, genetic makeup, type of prior or concomitant therapeutic agent, if any, and other personal characteristics of the subject being treated.

[0144] As used herein, a "prophylactically effective amount" is intended to include an amount of iRNA sufficient, when administered to a subject with an angiotensinogen-related disorder, to result in prevention or amelioration of the disease or one or more symptoms of the disease in a subject who is predisposed to the disease, i.e., who is more susceptible to the disease than the general population due to one or more factors, such as age, weight, or pregnancy. Amelioration of the disease includes slowing the course of the disease or reducing the severity of later-onset disease. A "prophylactically effective amount" may vary depending on the iRNA, the method of drug administration, the degree of disease risk, and the patient's medical history, age, weight, family history, genetic makeup, prior or concomitant therapeutic agents, if any, and other personal characteristics.

[0145] A "therapeutically effective amount" or "prophylactically effective amount" also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any therapeutic agent. The iRNAs used in the methods of the invention may be administered in amounts sufficient to produce a reasonable benefit / risk ratio applicable to such treatments.

[0146] The phrase "pharmaceutically acceptable" is used herein to mean compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit / risk ratio.

[0147] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmacologically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricants, magnesium talc, calcium or zinc stearate, or steric acid, etc.), or solvent encapsulating material that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject being treated. Some examples of materials that can act as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) cellulose acetate; (1) polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL, and LDL; and (22) other non-toxic, compatible substances used in pharmaceutical formulations.

[0148] As used herein, the term "sample" includes similar fluid, cell, or tissue collections isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples include samples from tissues, organs, or localized areas. For example, samples may be derived from specific organs, organ parts, or fluids, or cells within these organs. In some embodiments, samples may be derived from the liver (e.g., the entire liver or specific parts of the liver, or specific types of liver cells, such as hepatocytes). In some embodiments, a "sample derived from a subject" refers to blood or plasma taken from a subject.

[0149] II. iRNAs of the Invention The present invention provides iRNA that inhibits AGT gene expression. In one embodiment, the iRNA drug comprises a double-stranded ribonucleic acid (dsRNA) molecule that inhibits AGT gene expression in cells, such as cells in a subject, such as a mammal, such as a human, with an angiotensinogen-related disorder, e.g., hypertension. The dsRNA comprises an antisense strand having a complementary region that is complementary to at least a portion of the mRNA formed during AGT gene expression. The complementary region is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell that expresses the AGT gene, the iRNA inhibits the expression of the AGT gene (e.g., a human, primate, non-primate, or avian AGT gene) by at least about 10%, as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry techniques.

[0150] dsRNA comprises two complementary RNA strands, which hybridize to form a double-stranded structure under the conditions in which dsRNA is used. One strand of dsRNA (antisense strand) comprises a complementary region, which is substantially complementary to the target sequence, and is generally completely complementary. The target sequence can be derived from the sequence of mRNA formed during the expression of the AGT gene. The other strand (sense strand) comprises a region complementary to the antisense strand, so that when combined under suitable conditions, the two strands hybridize to form a double-stranded structure. As described elsewhere herein and known in the art, the complementary sequence of dsRNA can also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

[0151] Generally, the double-stranded structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19- 29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23 or 21-22 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also contemplated as part of the invention.

[0152] Similarly, the region complementary to the target sequence can be 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also contemplated as being part of the invention.

[0153] In some embodiments, the dsRNA is about 15 to about 20 nucleotides in length, about 25 to about 30 nucleotides in length, or about 15 to about 23 nucleotides in length. Generally, the dsRNA is long enough to act as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21-23 nucleotides can act as a substrate for Dice. As those skilled in the art will recognize, the target region of an RNA targeted for cleavage is most often a portion of a larger RNA molecule, which is often an mRNA molecule. Where applicable, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is long enough to be a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0154] Those skilled in the art will appreciate that a double-stranded region of, for example, about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-2 It is also recognized that a double-stranded region of 1, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs is the primary functional portion of a dsRNA. Thus, in one embodiment, an RNA molecule or RNA molecule complex having a double-stranded region of more than 30 base pairs is a dsRNA, provided that it is processed into, for example, a 15-30 base pair functional double-strand that targets a desired RNA for cleavage. Thus, those skilled in the art will recognize that, in one embodiment, an miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful for targeting AGT expression is not produced in the target cell by cleavage of a larger dsRNA.

[0155] The dsRNA described herein may further comprise one or more single-stranded nucleotide overhangs, such as 1, 2, 3, or 4 nucleotides. dsRNAs with at least one nucleotide overhang may have unexpectedly superior inhibitory properties compared to their blunt-end counterparts. The nucleotide overhang may comprise or consist of a nucleotide / nucleoside analog, including a deoxyribonucleotide / nucleoside. The overhang(s) may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of the overhang may be present on the 5'-end, 3'-end, or both ends of either the antisense strand or the sense strand of the dsRNA. As described herein, overhangs extending up to 30 nucleotides in length are also contemplated in various embodiments of the present invention.

[0156] dsRNA can be synthesized by standard methods known in the art using an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems®, Inc., as discussed further below.

[0157] The iRNA compounds of the present invention can be prepared using a two-step method.First, the individual strands of the double-stranded RNA molecule are prepared separately.Then, each strand is annealed.The individual strands of the siRNA compounds can be prepared using solution phase or solid phase organic synthesis or both.Organic synthesis has the advantage that it can easily prepare oligonucleotide strands that comprise unnatural or modified nucleotides.The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0158] In one aspect, the dsRNA of the present invention comprises at least two nucleotide sequences: a sense sequence and an antisense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 3, 4, 7, 8, 11, 13, and 15, and the antisense strand corresponding to the sense strand is selected from the group of sequences provided in any one of Tables 3, 4, 7, 8, 11, 13, and 15. In this aspect, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to an mRNA sequence generated during AGT gene expression. Thus, in this aspect, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described as the sense strand in any one of Tables 3, 4, 7, 8, 11, 13, and 15, and the second oligonucleotide is described as the antisense strand corresponding to the sense strand in any one of Tables 3, 4, 7, 8, 11, 13, and 15. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

[0159] Although some of the sequences in Tables 3, 4, 7, 8, 11, 13, and 15 are described as modified and / or conjugated sequences, it is understood that the RNA of the iRNA of the invention, e.g., the dsRNA of the invention, can comprise any one of the sequences set forth in Tables 3, 4, 7, 8, 11, 13, and 15 unmodified, unconjugated, and / or modified and / or conjugated differently than described herein.

[0160] In another aspect, a double-stranded ribonucleic acid (dsRNA) of the invention for inhibiting expression of angiotensinogen comprises, consists essentially of, or consists of a sense strand and an antisense strand, wherein the sense strand comprises the nucleotide sequence of a sense strand of Tables 3, 4, 7, 8, 11, 13, and 15, and the antisense strand comprises the nucleotide sequence of the corresponding antisense strand of Tables 3, 4, 7, 8, 11, 13, and 15.

[0161] Those skilled in the art are well aware that dsRNA with a double-stranded structure of about 20-23 base pairs, such as 21 base pairs, is believed to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888).However, other skilled artisans have found that shorter or longer RNA double-stranded structures can also be similarly effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the above-mentioned embodiment, due to the nature of the oligonucleotide sequences provided in any one of Tables 3, 4, 7, 8, 11, 13 and 15, the dsRNA described herein can comprise at least one strand that is at least 21 nucleotides long. It can be reasonably expected that shorter duplexes having one of the sequences in any one of Tables 3, 4, 7, 8, 11, 13, and 15, in which only a few nucleotides are deleted from one or both termini, may be similarly effective compared to dsRNAs, as described above. Thus, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences in any one of Tables 3, 4, 7, 8, 11, 13, and 15, which have an ability to inhibit AGT gene expression that differs by no more than about 5, 10, 15, 20, 25, or 30% from a dsRNA containing the full-length sequence, are intended to be within the scope of the present invention.

[0162] Additionally, the RNAs provided in any one of Tables 3, 4, 7, 8, 11, 13, and 15 identify a site(s) in the AGT transcript that is / are highly susceptible to RISC-mediated cleavage. Accordingly, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a specific site in an RNA transcript if it promotes cleavage of the transcript anywhere within that site. Such iRNAs generally comprise approximately 15 contiguous nucleotides from one of the sequences provided in any one of Tables 3, 4, 7, 8, 11, 13, and 15, linked to additional nucleotide sequences from regions adjacent to the selected sequence in the AGT gene.

[0163] Target sequences are generally approximately 15-30 nucleotides in length, although there is wide variability in the suitability of specific sequences within this range to induce cleavage of any given target RNA. While the various software packages and guidelines described herein provide guidance for identifying optimal target sequences for any given gene target, an experimental approach can also be taken in which a "window" or "mask" of a predetermined size (for example, 21 nucleotides) is placed, either physically or figuratively (including, for example, computationally), in the target RNA sequence to identify sequences within a size range that can serve as target sequences. By moving the sequence "window" one nucleotide upstream or downstream of the initial target sequence position, subsequent potential target sequences can be identified until a complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis of the identified sequences and testing (using assays described herein or known in the art) to identify optimally functioning sequences, can identify RNA sequences that best mediate inhibition of target gene expression when targeted with an iRNA drug. Thus, while the sequences identified in, for example, any one of Tables 3, 4, 7, 8, 11, 13 and 15 represent effective target sequences, it is contemplated that further optimization of inhibitory efficiency may be achieved by progressively "moving the window" one nucleotide upstream or downstream of the given sequence to identify sequences with equivalent or better inhibitory properties.

[0164] It is further contemplated that further optimization of any sequence identified, for example, in any one of Tables 3, 4, 7, 8, 11, 13, and 15, can be achieved by systematically adding or removing nucleotides to create longer or shorter sequences and testing these created sequences by moving a longer or shorter window of size upstream or downstream of the target RNA from that position. Again, combining this approach of creating new target candidates with testing the effectiveness of iRNAs based on these target sequences in inhibition assays known in the art and / or described herein can lead to further improvements in inhibition efficiency. Still further, such optimized sequences can be adjusted to further optimize the molecule as an expression inhibitor (e.g., increased serum stability or circulating half-life, increased thermostability, enhanced transmembrane delivery, targeting to specific locations or cell types, increased interaction with silencing pathway enzymes, increased release from endosomes, etc.), for example, by introducing modified nucleotides described herein or known in the art, adding or modifying overhangs, or other modifications known in the art and / or described herein.

[0165] The iRNAs described herein may contain one or more mismatches with the target sequence. In one embodiment, the iRNAs described herein contain three or fewer mismatches. When the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the mismatches are not located in the center of the complementary region. When the antisense strand of an iRNA contains mismatches with the target sequence, it is preferable that the mismatches are limited to the last five nucleotides from either the 5' or 3' end of the complementary region. For example, a 23-nucleotide iRNA drug strand complementary to a region of the AGT gene generally does not contain any mismatches within the central 13 nucleotides. Using methods described herein or known in the art, it is possible to determine whether an iRNA containing mismatches with the target sequence is effective in inhibiting AGT gene expression. Examining the effectiveness of iRNAs with mismatches in inhibiting AGT gene expression is important, especially when a particular complementary region of the AGT gene is known to have polymorphic sequence variation within the population.

[0166] III. Modified iRNAs of the Invention In one embodiment, the RNA, e.g., dsRNA, of an iRNA of the invention is unmodified and does not contain chemical modifications and / or linkages, e.g., those known in the art and described herein. In another embodiment, the RNA, e.g., dsRNA, of an iRNA of the invention is chemically modified to enhance stability or other beneficial properties. In any embodiment of the invention, substantially all nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all nucleotides of an iRNA of the invention are modified iRNA of the invention, where "substantially all nucleotides modified" means that the majority, but not all, of the nucleotides are unmodified and may contain no more than 5, 4, 3, 2, or 1 unmodified nucleotides.

[0167] Nucleic acids as defined herein can be synthesized and / or modified by methods established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA (incorporated herein by reference). Modifications include, for example, terminal modifications such as 5'-end modifications (phosphorylation, conjugated linkage, inverted linkage) or 3'-end modifications (conjugated linkage, DNA nucleotide, inverted linkage, etc.); base modifications such as substitution with a stabilizing base, a destabilizing base, or a base that base-pairs with an elongated repertoire of partners, base removal (abasic nucleotide), or conjugated base; sugar modifications (e.g., at the 2' or 4' position) or sugar substitution; and / or backbone modifications, including modification or replacement of phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or RNAs that do not contain natural internucleoside linkages. The RNA with modified backbone particularly includes those that do not have phosphorus atom in backbone.For the purpose of this specification, and as sometimes referred to in the art, the modified RNA that does not have phosphorus atom in their internucleoside backbone is also considered to be oligonucleoside.In some embodiments, modified iRNA can have phosphorus atom in its internucleoside backbone.

[0168] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and boranophosphates with reverse polarity, in which adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included.

[0169] Representative U.S. patents that teach the preparation of such phosphorus-containing linkages include U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,133; No. 1; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,5 19,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172,209; No. 6, 239,2 No. 65; No. 6,277,603; No. 6,326,199; No. 6,346,614; No. 6,444,423; No. 6,531,590; No. 6,534,639; No. 6,6 Nos. 08,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent RE39464 (the entire contents of which are incorporated herein by reference).

[0170] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (some formed from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, those with amide backbones, and others with mixed N, O, S, and CH2 moieties.

[0171] Representative United States patents which teach the preparation of the above oligonucleosides include U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; Nos. 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439 (the entire contents of which are incorporated herein by reference).

[0172] In other embodiments, suitable RNA mimics are considered for use in iRNA, where both the sugar and internucleoside linkages, i.e., the backbone of the nucleotide unit, are replaced with new groups. The base unit is maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262 (the entire contents of which are incorporated herein by reference). Further suitable PNA compounds for use in the iRNA of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0173] Some embodiments described herein include oligonucleosides having phosphorothioate backbones, and in particular, heteroatom backbones of the aforementioned U.S. Pat. No. 5,489,677, which are --CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- (also known as methylene(methylimino) or MMI group), --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- (where the phosphodiester backbone is represented as --O--P--O--CH2--) and the aforementioned U.S. Pat. No. 5,602,240, which are amide backbones. In certain embodiments, the RNA described herein has the morpholino backbone structure of US Pat. No. 5,034,506, supra.

[0174] Modified RNAs can also contain one or more substituted sugar moieties. For example, iRNAs, such as the dsRNAs described herein, can 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 or alkynyl. Exemplary suitable modifications include 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 includes one of the following at the 2' position: C1 to C 10Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group that improves the pharmacokinetic properties of iRNA, or group that improves the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification comprises 2'-methoxyethoxy (2'-O-CHCHOCH, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification includes the 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, i.e., O(CH)ON(CH), group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH-O-CH-N(CH). Further exemplary modifications include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both the R and S isomers of these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0175] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications are also possible at other positions on the RNA of an iRNA, specifically at the 3' position of the sugar on the 3'-terminal nucleotide, or in 2'-5'-linked dsRNA, and at the 5' position of the 5'-terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of the above modified sugar structures include U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,42 Nos. 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and U.S. Patent No. 5,700,920 (certain of which are co-inventors with the present application), the entire contents of each of which are incorporated herein by reference.

[0176] iRNAs can also contain nucleobase (often simply 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 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-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thiouracil (pseudouracil); 5-isopropyl ... Other synthetic and natural nucleobases include uracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo, specifically 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-daazaadenine; and 3-deazaguanine and 3-deazaadenine.Further examples of nucleobases include those disclosed in U.S. Patent No. 3,687,808; those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6 to 1.2°C (Sanghvi, YS, Crooke, ST, and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0177] Representative United States patents that teach the preparation of the above-mentioned specific modified nucleobases as well as other modified nucleobases include the above-mentioned U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; Nos. 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088 (the entire contents of each of which are incorporated herein by reference).

[0178] The RNA of iRNA can also be modified to contain one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with modified ribose moieties, in which the ribose moiety contains an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce non-specific (off-target) effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0179] In some embodiments, the oligonucleotides of the present invention contain one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNAs are nucleic acids with an unlocked, acyclic structure in which one of the sugar bonds is removed to form an unlocked "sugar" residue. As an example, UNAs also include monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar is removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, both of which are incorporated herein by reference).

[0180] The RNA of an iRNA can also be modified to contain one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In some embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in certain embodiments, an agent of the present invention can contain one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, in which the ribose moiety comprises an additional bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucleotide containing a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of a locked nucleic acid to siRNA has been shown to increase siRNA stability in serum and reduce non-specific effects (off-target effects) (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present invention include, but are not limited to, nucleosides containing a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom. In any embodiment, the antisense polynucleotide drug of the present invention includes one or more bicyclic nucleosides containing a 4'-to-2' bridge.Examples of such 4' to 2' bridged bicyclic nucleosides include 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also known as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs; see, e.g., U.S. Pat. No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and analogs; see, e.g., U.S. Pat. No. 8,222,222). 78,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-ON(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C12 alkyl, or a protecting group) (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the above are incorporated herein by reference.

[0181] Additional representative U.S. patents and published U.S. patent applications that teach the preparation of locked nucleic acid nucleotides include the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427 ,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; U.S. Patent Application Publication No. 2008 / 0039618; and U.S. Patent Application Publication No. 2009 / 0012281 (the entire contents of each of which are incorporated herein by reference).

[0182] Any of the above bicyclic nucleosides can be prepared with one or more sugar stereochemical configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0183] The RNA of an iRNA can also be modified to contain one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety containing a 4'-CH(CH3)-O-2' bridge. In one embodiment, a constrained ethyl nucleotide in the S conformation is referred to herein as an "S-cEt."

[0184] The iRNA of the present invention may also contain one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and C5' carbons of ribose. The CRNs lock the ribose ring into a stable conformation, increasing the affinity of hybridization to mRNA. The linker is long enough to position the oxygen optimally for stability and affinity, reducing puckering of the ribose ring.

[0185] Representative publications that teach the preparation of any of the above CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383; and PCT Publication WO2013 / 036868 (the entire contents of each of which are incorporated herein by reference).

[0186] One or more of the nucleotides of an iRNA of the invention can also comprise a hydroxymethyl-substituted nucleotide. A "hydroxymethyl-substituted nucleotide" is an acyclic 2'-3'-seco-nucleotide, also referred to as a "non-locked nucleic acid" ("UNA") modification.

[0187] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Application Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020 (the entire contents of each of which are incorporated herein by reference).

[0188] Potential stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in WO 2011 / 005861.

[0189] Other modifications of the nucleotides of the iRNAs of the invention include a 5' phosphate or 5' phosphate mimic, e.g., a 5'-terminal phosphate or phosphate mimic, on the antisense strand of the RNAi agent. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0157511 (the entire contents of which are incorporated herein by reference).

[0190] A. Modified iRNAs Containing Motifs of the Invention In any aspect of the invention, double-stranded RNAi agents of the invention include agents having chemical modifications disclosed, for example, in U.S. Provisional Patent Application No. 61 / 561,7108, filed November 18, 2011, or PCT / US2012 / 065691, filed November 16, 2012, the entire contents of each of which are incorporated herein by reference.

[0191] As shown in this specification and provisional application No. 61 / 561,710 or PCT application publication No. PCT / US2012 / 065691, excellent results can be obtained by introducing one or more motifs of three identical modifications into three consecutive nucleotides in the sense strand and / or antisense strand of RNAi agent, particularly at or near the cleavage site.In some embodiments, the sense strand and antisense strand of RNAi agent can be completely modified by other methods.The introduction of these motifs, if present, will interrupt the modification pattern of the sense and / or antisense strand.If necessary, the RNAi agent can be complexed with, for example, a GalNAc-derived ligand on the sense strand.The obtained RNAi agent shows excellent gene silencing activity.

[0192] More specifically, it has been surprisingly discovered that when the sense and antisense strands of a double-stranded RNAi agent are fully modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or proximal to the cleavage site of at least one strand of the RNAi agent, the gene silencing activity of the RNAi agent is significantly enhanced.

[0193] Thus, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the angiotensinogen (AGT) gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can be in the range of 12-30 nucleotides in length. For example, each strand can be between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

[0194] The sense strand and antisense strand generally form double-stranded RNA ("dsRNA"), also referred to herein as an "RNAi agent." The double-stranded region of an RNAi agent can be 12-30 nucleotide pairs in length. For example, the double-stranded region can be 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

[0195] In one embodiment, the RNAi agent may include one or more overhang regions and / or capping groups at the 3'-end, 5'-end, or both ends of one or both strands. The overhangs can be 1-6 nucleotides in length, for example, 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be longer than the other strand, or can result in two strands of the same length arranged alternately. The overhangs can form mismatches with the target mRNA, or can be complementary to the targeted gene sequence, or can be another sequence. As described herein, extended overhangs up to 30 nucleotides in length are also contemplated in various embodiments of the present invention. The first and second strands can also be linked, for example, by additional bases to form a hairpin or by other non-basic linkers.

[0196] In one embodiment, the nucleotides in the overhang region of an RNAi agent can each independently be modified or unmodified nucleotides, including, but not limited to, 2'-sugar modifications such as 2-F, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be the overhang sequence at either end of either strand. The overhang can form a mismatch with the target mRNA, or it can be complementary to the targeted gene sequence, or it can be a different sequence.

[0197] The 5'- or 3'-overhang of the sense strand, antisense strand, or both strands of an RNAi agent may be phosphorylated. In some embodiments, the overhang region(s) comprise two nucleotides with phosphorothioate between them, wherein the two nucleotides may be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, the antisense strand, or both strands. In one embodiment, the 3'-overhang is present in the antisense strand. In one embodiment, the 3'-overhang is present in the sense strand.

[0198] RNAi drugs can only contain a single overhang, which can enhance the interference activity of RNAi without affecting its overall stability.For example, the single-stranded overhang can be located at the 3'-end of the sense strand or at the 3'-end of the antisense strand.RNAi can also have a blunt end located at the 5'-end of the antisense strand (or the 3'-end of the sense strand) or vice versa.Generally, the antisense strand of RNAi has a nucleotide overhang at the 3'-end, and the 5'-end is blunt.Without being bound by theory, the asymmetric blunt ends at the 5'-end of the antisense strand and the 3'-end overhang of the antisense strand favor the guide strand being loaded into the RISC process.

[0199] In one embodiment, the RNAi agent is a blunt-ended 19-nucleotide long strand, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end.

[0200] In another embodiment, the RNAi agent is a 20-nucleotide long strand that is blunt at both ends, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

[0201] In yet another embodiment, the RNAi agent is a blunt-ended 21-nucleotide long strand, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5' end.

[0202] In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5' end; and the antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, wherein one end of the RNAi agent is blunt, while the other end comprises a two-nucleotide overhang. Preferably, the two-nucleotide overhang is at the 3' end of the antisense strand.

[0203] When a two-nucleotide overhang is at the 3'-end of the antisense strand, two phosphorothioate internucleotide bonds may be present between the terminal three nucleotides, where two of the three nucleotides are overhanging nucleotides and the third nucleotide is the next paired nucleotide of the overhanging nucleotide. In one embodiment, the RNAi agent further has two phosphorothioate internucleotide bonds between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand. In one embodiment, all nucleotides of the sense and antisense strands of the RNAi agent, including nucleotides that are part of a motif, are modified nucleotides. In one embodiment, each residue is independently modified with 2'-O-methyl or 3'-fluoro, for example, in an alternating motif. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc3).

[0204] In one embodiment, an RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, and wherein positions 1 through 23, starting from the 5'-terminal nucleotide (position 1) of the first strand, comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length, and beginning from the 3'-terminal nucleotide, comprises at least 8 ribonucleotides at positions that pair with positions 1 through 23 of the sense strand to form a duplex; wherein at least the 3'-terminal nucleotide of the antisense strand is unpaired with the sense strand, and up to 6 consecutive 3'-terminal nucleotides are unpaired with the sense strand, thus forming a 3' single-stranded overhang of 1-6 nucleotides; and wherein the 5'-end of the antisense strand is 10-66 nucleotides in length that are unpaired with the sense strand. the sense strand comprises 30 consecutive nucleotides, thereby forming a single-stranded 5' overhang of 10-30 nucleotides; wherein at least the 5'- and 3'-terminal nucleotides of the sense strand base-pair with nucleotides of the antisense strand when the sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense and antisense strands; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the length of the antisense strand to reduce target gene expression when the double-stranded nucleic acid is introduced into a mammalian cell; and the sense strand comprises at least one motif of three 2'-F modifications on three consecutive nucleotides, wherein at least one motif occurs at or proximal to the cleavage site. The antisense strand comprises at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or proximal to the cleavage site.

[0205] In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the RNAi agent comprises a first strand at least 25 nucleotides in length and at most 29 nucleotides in length, and a second strand at most 30 nucleotides in length having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; wherein the 3' end of the first strand and the 5' end of the second strand form a blunt end, and the second strand is 1-4 nucleotides longer at its 3' end than the first strand, wherein the double-stranded region is at least 25 nucleotides in length, and the second strand is sufficiently complementary to the target RNA along at least 19 nucleotides of the length of the second strand to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, wherein dicer cleavage of the RNAi agent preferentially produces siRNA comprising the 3' end of the second strand, thereby reducing target gene expression in a mammal. Optionally, the RNAi agent further comprises a ligand.

[0206] In one embodiment, the sense strand of the RNAi agent includes at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

[0207] In one embodiment, the antisense strand of the RNAi agent may also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or proximal to the cleavage site in the antisense strand.

[0208] For RNAi agents having a double-stranded region 17-23 nucleotides in length, the cleavage sites of the antisense strand are generally around positions 10, 11, and 12 from the 5' end. Thus, the three identical modification motifs can occur at positions 9, 10, 11 of the antisense strand; positions 10, 11, 12; positions 11, 12, 13; positions 12, 13, 14; or positions 13, 14, 15, counting from the first nucleotide from the 5' end of the antisense strand, or counting from the first paired nucleotide in the double-stranded region from the 5' end of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the double-stranded region of the RNAi from the 5' end.

[0209] The sense strand of RNAi agent can comprise at least one motif of three identical modifications on three consecutive nucleotides at the site of strand breakage; and antisense strand can have at least one motif of three identical modifications on three consecutive nucleotides at or near the site of strand breakage.When sense strand and antisense strand form dsRNA duplex, the sense strand and antisense strand can be aligned so that one motif of three nucleotides on sense strand and one motif of three nucleotides on antisense strand have at least one nucleotide overlap, that is, at least one of the three nucleotides of the motif in sense strand and at least one of the three nucleotides of the motif in antisense strand form base pairs.Alternatively, at least two nucleotides can overlap, or all three nucleotides can overlap.

[0210] In one embodiment, the sense strand of an RNAi agent may contain two or more motifs of three identical modifications on three consecutive nucleotides. The first motif occurs at or near the cleavage site of the strand, and the other motifs may be wing modifications. The term "wing modification" as used herein refers to a motif that occurs in another part of the strand separated from the motif at or near the cleavage site of the same strand. The wing modifications are adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are directly adjacent to each other, the chemical properties of the motifs are different from each other, and when the motifs are separated by one or more nucleotides, the chemical properties may be the same or different. Two or more wing modifications may be present. For example, when two wing modifications are present, each wing modification may occur at one end of the first motif, at or near the cleavage site, or on either side of the lead motif.

[0211] Like the sense strand, the antisense strand of an RNAi agent can contain two or more motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or proximal to the site of strand cleavage. The antisense strand can also contain one or more wing modifications in the same alignment as the wing modifications that may be present on the sense strand.

[0212] In one embodiment, wing modifications on the sense or antisense strand of an RNAi agent generally do not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0213] In another embodiment, wing modifications of the sense or antisense strand of an RNAi agent generally do not include the first one or two paired nucleotides in the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0214] When the sense and antisense strands of an RNAi agent each include at least one wing modification, the wing modifications may be at the same end of the double-stranded region and may have an overlap of 1, 2, or 3 nucleotides.

[0215] When the sense and antisense strands of an RNAi agent each include at least two wing modifications, the sense and antisense strands may be aligned such that each two modifications from one strand are at one end of a double-stranded region with an overlap of 1, 2, or 3 nucleotides; each two modifications from one strand are at the other end of a double-stranded region with an overlap of 1, 2, or 3 nucleotides; and two modifications from one strand are on either side of a lead motif with an overlap of 1, 2, or 3 nucleotides in the double-stranded region.

[0216] In one embodiment, all nucleotides of the sense and antisense strands of an RNAi agent, including nucleotides that are part of a motif, may be modified. Each nucleotide may be modified with the same or different modifications, which may include one or more modifications of one or both of the non-linked phosphate oxygens and / or one or more of the linking phosphate oxygens; modifications of components of the ribose sugar, such as the 2'-hydroxyl of the ribose sugar; wholesale substitution of the phosphate moiety with a "dephosphorylated" linker; modifications or substitutions of naturally occurring bases; and substitutions or modifications of the ribose-phosphate backbone.

[0217] Considering nucleic acids as polymers of subunits, many modifications occur at positions that are repeated within nucleic acids, such as base modifications, or phosphate moieties, or non-linked Os in phosphate moieties. In some cases, modifications can occur at all target positions within a nucleic acid, but in many cases, this is not the case. For example, modifications can occur only at the 3'- or 5'-terminal positions, or only in terminal regions, such as the terminal nucleotide or the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only within the double-stranded region of RNA or only within the single-stranded region of RNA. For example, phosphorothioate modifications at non-linked O positions can occur only at one or both ends, or only in terminal regions, such as the terminal nucleotide or the last 2, 3, 4, 5, or 10 nucleotides of the chain, or can occur in double-stranded and single-stranded regions, especially at the ends. The 5'-end or end can be phosphorylated.

[0218] For example, to enhance stability, the single-stranded overhang, e.g., the 5' or 3' overhang, or both, can contain specific bases in the overhang or modified nucleotides or nucleotide substitutes. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang can be modified, e.g., with the modifications described herein. Modifications can include, for example, the use of modifications at the 2' position of the ribose sugar, with modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications in place of the ribosugar of the nucleobase, and modifications at the phosphate group, e.g., phosphorothioate modifications. The overhang does not need to be homologous to the target sequence.

[0219] In one embodiment, each residue of sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl or 2'-fluoro.Strands can contain two or more modifications.In one embodiment, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.

[0220] At least two different modifications are generally present on the sense and antisense strands. These two modifications may be 2'-O-methyl or 2'-fluoro modifications, for example.

[0221] In one embodiment, N a and / or N b includes an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each modification occurring at alternating nucleotides on one strand. Alternating nucleotides can refer to one every other nucleotide or one every three nucleotides, or similar patterns. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif could be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0222] The types of modifications included in the alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of nucleotide modification, the alternation pattern, i.e., the modification of every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities of modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...", etc.

[0223] In one embodiment, RNAi agents of the present invention include those in which the modification pattern of the alternating motif on the sense strand is shifted relative to the modification pattern of the alternating motif on the antisense strand. The shift can be such that the modification groups of the nucleotides of the sense strand correspond to different modification groups of the nucleotides of the antisense strand, or vice versa. For example, when a sense strand pairs with an antisense strand in a dsRNA duplex, the alternating motif in the sense strand can begin with "ABABAB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from the 5'-3' end of the strand within the double-stranded region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from the 5'-3' end of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from the 5'-3' end of the strand within the double-stranded region, resulting in a complete or partial shift in the modification pattern between the sense and antisense strands.

[0224] In one embodiment, RNAi agents include those in which a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications initially on the sense strand has a shift relative to a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications initially on the antisense strand, i.e., 2'-O-methyl modified nucleotides on the sense strand base pair with 2'-F modified nucleotides on the antisense strand, and vice versa. Position 1 of the sense strand may begin with a 2'-F modification, and position 1 of the antisense strand may begin with a 2'-O-methyl modification.

[0225] The introduction of one or more motifs of three identical modifications on three consecutive nucleotides into sense strand and / or antisense strand interrupts the original modification pattern present in sense strand and / or antisense strand.This interruption of the modification pattern of sense strand and / or antisense strand by the introduction of one or more motifs of three identical modifications on three consecutive nucleotides into sense strand and / or antisense strand surprisingly enhances the gene silencing activity of target gene.

[0226] In one embodiment, when a motif of three identical modifications on three consecutive nucleotides is introduced in either strand, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, a portion of a sequence containing a motif may be a YYYN b ..." (where "Y" represents a modification of a motif of three identical modifications on three consecutive nucleotides, and "N a " and "N b " represents a modification of the next nucleotide of the motif "YYY" that is different from the modification of Y, and N a and N b may be the same or different modifications). a and / or N b may be present or absent when wing modifications are present.

[0227] RNAi agents can further comprise at least one phosphorothioate internucleotide bond or methylphosphonate internucleotide bond.Phosphorothioate internucleotide bond or methylphosphonate internucleotide bond modification can occur in any nucleotide at any position of the sense strand or antisense strand or both strands.For example, internucleotide bond modification can occur at every nucleotide on the sense strand and / or antisense strand; each internucleotide bond modification can occur in an alternating pattern on the sense strand and / or antisense strand; or the sense strand or antisense strand can contain both internucleotide bond modifications in an alternating pattern.The alternating pattern of internucleotide bond modification on the sense strand can be the same or different from that of the antisense strand, and the alternating pattern of internucleotide bond modification on the sense strand can be shifted compared with the alternating pattern of internucleotide bond modification on the antisense strand.In one embodiment, double-stranded RNAi agents comprise 6-8 phosphorothioate internucleotide bonds. In one embodiment, the antisense strand contains two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand contains at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0228] In one embodiment, RNAi comprises phosphorothioate internucleotide bond or methylphosphonate internucleotide bond modification in overhang region.For example, overhang region can comprise two nucleotides with phosphorothioate internucleotide bond or methylphosphonate internucleotide bond between two nucleotides.Internucleotide bond modification can also make overhang nucleotide to be linked with the terminal paired nucleotide in double-stranded region.For example, at least 2, 3, 4 or all overhang nucleotides can be linked through phosphorothioate internucleotide bond or methylphosphonate internucleotide bond, and if necessary, there can be additional phosphorothioate internucleotide bond or methylphosphonate internucleotide bond that connects overhang nucleotide to the next paired nucleotide of overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide bonds between the terminal three nucleotides, where two of the three nucleotides are overhang nucleotides and the third is the next paired nucleotide of overhang nucleotide. These terminal three nucleotides may be at the 3' end of the antisense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and / or the 5' end of the sense strand.

[0229] In one embodiment, the two nucleotide overhang is at the 3'-end of the antisense strand, and two phosphorothioate internucleotide bonds are present between the terminal three nucleotides, where two of the three nucleotides are overhanging nucleotides and the third nucleotide is the next paired nucleotide to the overhanging nucleotide. Optionally, the RNAi agent may further have two phosphorothioate internucleotide bonds between the terminal three nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0230] In one embodiment, the RNAi agent may contain mismatch(es) with the target, mismatch(es) within the duplex, or a combination thereof. Mismatches may occur in the overhang region or the duplex region. Base pairs may be ranked based on their tendency to promote dissociation or melting (e.g., for the free energy of association or dissociation of a particular pairing, the simplest method is to examine each pair individually, although neighboring or identical analyses can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I = inosine). Mismatches, such as non-standard or non-standard pairings (described elsewhere herein), are preferred over standard pairings (A:T, A:U, G:C); and pairings involving universal bases are preferred over standard pairings.

[0231] In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs in the double-stranded region from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, as well as mismatch pairs, e.g., non-canonical pairings or pairings other than canonical pairings or pairings involving universal bases, that promote dissociation of the antisense strand at the 5' end of the duplex.

[0232] In one embodiment, the nucleotide at position 1 in the double-stranded region from the 5' end of antisense strand is selected from the group consisting of A, dA, dU, U and dT.Alternatively, at least one of the first 1, 2 or 3 base pairs in the double-stranded region from the 5' end of antisense strand is an AU base pair.For example, the first base pair in the double-stranded region from the 5' end of antisense strand is an AU base pair.

[0233] In another embodiment, the nucleotide at the 3' end of the sense strand is deoxythymine (dT). In another embodiment, the nucleotide at the 3' end of the antisense strand is deoxythymine (dT). In one embodiment, there is a short sequence of deoxythymine nucleotides, for example, two dT nucleotides on the 3' end of the sense strand and / or antisense strand.

[0234] In one embodiment, the sense strand sequence may be represented by formula (I): 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' (I) During the ceremony: i and j are each independently 0 or 1; p and q are each independently 0-6; N a are each independently an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two different modified nucleotides; N b are each independently an oligonucleotide sequence containing 0-10 modified nucleotides; n p and n q each independently represents an overhanging nucleotide; wherein Nb and Y do not have the same modification; and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably, YYY are all 2'-F modified nucleotides.

[0235] In one embodiment, N a and / or N b includes qualification of alternative patterns.

[0236] In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when an RNAi agent has a double-stranded region 17-23 nucleotides in length, the YYY motif can occur at or near the cleavage site of the sense strand, counting from the 5' end, from the nucleotide at position 1, or, optionally, counting from the 5' end, from the paired nucleotide at position 1 within the double-stranded region (e.g., at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12, or 11, 12, 13).

[0237] In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. Thus, the sense strand can be represented by the formula: 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' (Ib); 5' n p -N a -XXX-N b -YYY-N a -n q 3' (Ic); or 5' n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' (Id).

[0238] When the sense strand is represented by formula (Ib), N b indicates an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a can each independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0239] When the sense strand is represented by formula (Ic), N bindicates an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a can each independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0240] When the sense strand is represented by formula (Id), N b each independently represent an oligonucleotide sequence containing 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6. a can each independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 of the modified nucleotides. Each of X, Y and Z may be the same or different from each other.

[0241] In other embodiments, i is 0, j is 0, and the sense strand is represented by the formula: 5' n p -N a -YYY- N a -n q 3' (Ia). When the sense strand is represented by formula (Ia), N a can each independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0242] In one embodiment, the antisense strand sequence of the RNAi can be represented by formula (II): 5' n q’ -N a '-(Z'Z'Z') k -N b '-Y'Y'Y'-N b '-(X'X'X') l -N' a -n p '3' (II) During the ceremony: k and l are each independently 0 or 1; p' and q' are each independently 0-6; N a ' each independently represent an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two different modified nucleotides; N b ' each independently represent an oligonucleotide sequence containing 0-10 modified nucleotides; n p ' and n q’ each independently represents an overhanging nucleotide; where N b ' and Y' do not have the same modification; and X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0243] In one embodiment, N a ' and / or N b ' includes qualification of alternative patterns.

[0244] The Y'Y'Y' motif occurs at or near the cleavage site of the antisense strand. For example, when an RNAi agent has a double-stranded region 17-23 nucleotides in length, the Y'Y'Y' motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, counting from the nucleotide at position 1 from the 5' end, or, if necessary, counting from the paired nucleotide at position 1 from the 5' end within the double-stranded region. Preferably, the Y'Y'Y' motif occurs at positions 11, 12, 13.

[0245] In one embodiment, the Y'Y'Y' motif is all 2'-OMe modified nucleotides.

[0246] In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1. Thus, the antisense strand can be represented by the formula: 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N a '-n p’ 3' (IIb); 5' n q’ -N a '-Y'Y'Y'-N b '-X'X'X'-n p’ 3' (IIc);or 5' n q’ -N a '-Z'Z'Z'-N b '-Y'Y'Y'-N b '-X'X'X'-N a '-n p’ 3' (IId).

[0247] When the antisense strand is represented by formula (IIb), N b ’ indicates an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a ' each independently indicate an oligonucleotide sequence containing 2-20, 2-15, or 2-10 of this modified nucleotide.

[0248] When the antisense strand is represented by formula (IIc), N b N' indicates an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. a ' each independently designate an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0249] When the antisense strand is represented by formula (IId), N b N' each independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. aEach N' independently represents an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. b is 0, 1, 2, 3, 4, 5 or 6.

[0250] In other embodiments, k is 0, l is 0, and the antisense strand can be represented by the formula: 5' n p’ -N a’ -Y'Y'Y'- N a’ -n q’ 3' (Ia).

[0251] When the antisense strand is represented by formula (IIa), N a ' each independently designate an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. Each of X', Y' and Z' may be the same or different from each other.

[0252] Each nucleotide of sense strand and antisense strand can be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl or 2'-fluoro.For example, each nucleotide of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.X, Y, Z, X', Y' and Z' can each specifically represent 2'-O-methyl modification or 2'-fluoro modification.

[0253] In one embodiment, the sense strand of an RNAi agent can include a YYY motif occurring at positions 9, 10, and 11 of the strand, counting from the nucleotide at position 1 from the 5' end, or, optionally, counting from the paired nucleotide at position 1 in the double-stranded region from the 5' end, when the double-stranded region is 21 bases long, where Y is a 2'-F modification. The sense strand can further include a XXX motif or a ZZZ motif as a wing modification at the opposite end of the double-stranded region, where XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0254] In one embodiment, the antisense strand may include a Y'Y'Y' motif occurring at positions 11, 12, 13 of the strand, counting from the 5'-end from the nucleotide at position 1, or, optionally, counting from the 5'-end from the paired nucleotide at position 1 in the double-stranded region, where Y' represents a 2'-O-methyl modification. The antisense strand may further include an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the opposite end of the double-stranded region, where X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0255] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any one of formulas (IIa), (IIb), (IIc) and (IId), respectively.

[0256] Thus, an RNAi agent for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, and the RNAi duplex is represented by the following formula (III): Sense: 5' n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense: 3' np ’ -N a ’ -(X'X'X') k -N b ’ -Y'Y'Y'-N b ’ -(Z'Z'Z') l -N a ’ -n q ’ 5' (III) During the ceremony: i, j, k, and l are each independently 0 or 1; p, p', q and q' are each independently 0-6; N a and N a ’ each independently represent an oligonucleotide sequence containing 0-25 modified nucleotides, each sequence containing at least two different modified nucleotides; N b and N b ’ each independently represents an oligonucleotide sequence containing 0-10 modified nucleotides; where n p ',n p , n q ' and n q (each of which may be present or absent) each independently represent an overhanging nucleotide; and XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucleotides.

[0257] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; or k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0258] Examples of combinations of sense and antisense strands that form RNAi duplexes include the following formulas: 5' n p -N a -YYY-N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y'-N a ’ n q ’ 5' (IIIa) 5' n p -N a -YYY-N b -ZZZ-N a -n q 3' 3' n p ’ -N a ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a ’ n q ’ 5' (IIIb) 5' n p -N a -XXX-N b -YYY-N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-Na ’ -n q ’ 5' (IIIc) 5' n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' 3' n p ’ -N a ’ -X'X'X'-N b ’ -Y'Y'Y'-N b ’ -Z'Z'Z'-N a -n q ’ 5' (IIId).

[0259] When the RNAi agent has formula (IIIa), N a each independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0260] When the RNAi agent has formula (IIIb), N b N each independently represent an oligonucleotide sequence containing 1-10, 1-7, 1-5, or 1-4 modified nucleotides. a each independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0261] When the RNAi agent has formula (IIIc), N b , N b N' each independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a each independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides.

[0262] When the RNAi agent has formula (IIId), Nb , N b N' each independently represents an oligonucleotide sequence containing 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. a , N a ’ N each independently represent an oligonucleotide sequence containing 2-20, 2-15, or 2-10 modified nucleotides. a , N a ', N b and N b ’ each independently includes an alternative pattern of modifications.

[0263] Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc) and (IIId) may be the same as or different from each other.

[0264] When an RNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may be base-paired to one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides are base-paired to the corresponding Y' nucleotide; or all three of the Y nucleotides are base-paired to the corresponding Y' nucleotide.

[0265] When an RNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may be base-paired with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides are base-paired with a corresponding Z' nucleotide; or all three Z nucleotides are base-paired with a corresponding Z' nucleotide.

[0266] When an RNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides may be base-paired to one of the X' nucleotides, alternatively, at least two of the X nucleotides are base-paired to a corresponding X' nucleotide; or all three X nucleotides are base-paired to a corresponding X' nucleotide.

[0267] In one embodiment, the modification of a Y nucleotide is different from the modification of a Y' nucleotide, the modification of a Z nucleotide is different from the modification of a Z' nucleotide, and / or the modification of an X nucleotide is different from the modification of an X' nucleotide.

[0268] In one embodiment, when the RNAi agent has formula (IIId), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification. In another embodiment, when the RNAi agent has formula (IIId), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p In yet another embodiment, when the RNAi agent has formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage. a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p In another embodiment, when the RNAi agent has formula (IIId), N' is linked to the adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives via a divalent linker or a trivalent branched linker (see below). a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p' is linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives via a divalent linker or a trivalent branched linker.

[0269] In one embodiment, when the RNAi agent has formula (IIIa), N a The modification is a 2'-O-methyl modification or a 2'-fluoro modification, p '>0 and at least one n p ' is linked to adjacent nucleotides via phosphorothioate linkages, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives via a divalent linker or a trivalent branched linker.

[0270] In one embodiment, the RNAi agent is a multimer comprising at least two double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the double strands are connected by a linker. The linker is cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the double strands can target the same gene or two different genes; or each of the double strands can target the same gene at two different target sites.

[0271] In one embodiment, the RNAi agent is a multimer comprising three, four, five, six or more double strands represented by formula (III), (IIIa), (IIIb), (IIIc) and (IIId), wherein the double strands are connected by a linker. The linker is cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the double strands can target the same gene or two different genes; or each of the double strands can target the same gene at two different target sites.

[0272] In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at one or both of the 5' and 3' ends, and optionally complexed with a ligand. Each agent can target the same gene or two different genes; or each agent can target the same gene at two different target sites.

[0273] Various documents describe the multimeric RNAi agent that can be used in the method of the present invention.Such documents include WO2007 / 091269, US Patent No. 7,858,769, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887 and WO2011 / 031520 (their entire contents are incorporated herein by reference).

[0274] As described in more detail below, RNAi agents that include one or more carbohydrate moieties attached to them can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety can be attached to a modified subunit of an RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand is attached. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose-replacement modified subunit (RRMS). The cyclic carrier can be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms can be a heteroatom, such as nitrogen, oxygen, or sulfur. The cyclic carrier can be a monocyclic ring system or can contain two or more rings, for example, a fused ring. The cyclic carrier can be a fully saturated ring system, or it can contain one or more double bonds.

[0275] The ligand may be attached to the polynucleotide via a carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tethering attachment point." As used herein, "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally refers to a bond available for incorporation of the carrier into the backbone and suitable therefor, e.g., a phosphate ester or modified phosphate, e.g., a sulfur-containing backbone, of a ribonucleic acid. In some embodiments, a "tethering attachment point" (TAP) refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or heteroatom (different from the atom providing the backbone attachment point), to which a selected moiety is attached. The moiety may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is linked to the cyclic carrier by an intervening tether. Thus, cyclic carriers often contain a functional group, such as an amino group, or generally provide a bond suitable for incorporation or tethering of another compound, such as a ligand, to the constituent ring.

[0276] RNAi agent can be linked to ligand through carrier, where carrier can be cyclic or acyclic group.Preferably, cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and decalin.Preferably, acyclic group is selected from serinol skeleton or diethanolamine skeleton.

[0277] In any particular embodiment, an RNAi agent for use in the methods of the invention is an agent selected from the group of agents set forth in any one of Tables 3, 4, 7, 8, 11, 13, and 15. These agents may further comprise a ligand.

[0278] IV. Ligand-conjugated iRNA Another modification of the RNA of the iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties, or complexes that enhance the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. 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, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), and the like. 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); such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-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 & Nucleotide, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moieties (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237); or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0279] In one embodiment, the ligand changes the distribution, targeting or life span of the iRNA drug into which it is incorporated.In a preferred embodiment, the ligand provides improved affinity for a selected target, such as a molecule, a cell or cell type, a compartment, such as a subcellular or organ compartment, a tissue or organ or region of the body, for example, compared to a chemical species in which such a ligand is absent.Preferred ligands do not participate in double-stranded pairing in double-stranded nucleic acid.

[0280] Ligands can include natural substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolized) 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 polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, polyamine quaternary salts, or alpha helical peptides.

[0281] The ligand can also include a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid, or protein, e.g., an antibody, that binds to a specific cell type, such as a kidney cell. The targeting group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetylglucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptidomimetic.

[0282] Other examples of ligands include dyes, intercalators (e.g., acridine), cross-linkers (e.g., psoralene, 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)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithium, and the like. Examples of suitable cleavage inhibitors include cleavage inhibitors (e.g., oleic acid, O3-(oleoyl)cholenoic 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, Eu3+ conjugates of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0283] Ligands can be proteins, such as glycoproteins; peptides, such as molecules with specific affinity for co-ligands; or antibodies, such as antibodies that bind to specific cell types, such as liver cells. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, and non-peptide species, such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0284] The ligand can be a substance, such as a drug, that can increase the uptake of an iRNA agent into a cell, for example, by disrupting the cytoskeleton of the cell, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0285] In some embodiments, the ligand attached to the iRNA described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides comprising multiple phosphorothioate linkages are also known to bind to serum proteins. Therefore, short oligonucleotides, such as oligonucleotides of about 5, 10, 15, or 20 bases comprising multiple phosphorothioate linkages in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands) for the present invention. In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0286] Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of oligonucleotides bearing pendant reactive functional groups, such as those derived from the attachment of a binding molecule onto an oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with a commercially available ligand, a ligand synthesized with any of a variety of protecting groups, or a ligand having a binding moiety attached thereto.

[0287] The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made by well-known solid-phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including Applied Biosystems (Foster City, Calif.). Additionally or alternatively, any other means for such synthesis known in the art may be used. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0288] In the ligand-conjugated oligonucleotides and sequence-specific linked nucleosides containing ligand molecules of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already containing a linking moiety, ligand-nucleotide or nucleoside-conjugate precursors already bearing a ligand molecule, or building blocks containing non-nucleoside ligands.

[0289] When using a nucleotide conjugate precursor that already has a linking moiety, synthesis of the sequence-specific linked nucleoside is generally completed, and then a ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0290] A. Lipid Complex In one embodiment, the ligand or complex is a lipid or lipid-based molecule.Such lipid or lipid-based molecule preferably binds to serum protein, for example, human serum albumin (HSA).HSA-binding ligand allows the distribution of complex to target tissue, for example, non-renal target tissue of the body.For example, the target tissue can be the liver, including liver parenchymal cells.Other molecules that can bind to HSA can also be used as ligand.For example, naproxen or aspirin can be used.Lipid or lipid-based ligand can (a) increase the degradation resistance of complex, (b) increase the targeting or transport to target cell or cell membrane, and / or (c) can be used to regulate the binding of serum protein, for example, HSA.

[0291] Lipid-based ligands can be used for inhibition, e.g., to control the binding of the conjugate to a target tissue. For example, lipids or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and therefore less likely to be cleared from the body. Lipids or lipid-based ligands that bind less strongly to HSA can be used to target the conjugate to the kidney.

[0292] In a preferred embodiment, the lipid-based ligand binds to HSA. Preferably, it binds to HSA with sufficient affinity so that the conjugate preferably distributes to non-renal tissues. However, the affinity is preferably not so strong that HSA ligand binding cannot be reversed.

[0293] In another preferred embodiment, the lipid-based ligand binds weakly or not at all to HSA, such that the conjugate preferably distributes to the kidney. Other moieties that target kidney cells can also be used in place of or in addition to the lipid-based ligand.

[0294] In another aspect, the ligand is a moiety, such as a vitamin, that is taken up by target cells, such as proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, such as malignant or non-malignant types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include B vitamins, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells, such as liver cells. Also included are HSA and low-density lipoprotein (LDL).

[0295] B. Cell-penetrating agents In another aspect, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. When the agent is a peptide, it can be modified, including peptidylmimetic, invertomer, non-peptide or pseudo-peptide bond, and the use of D-amino acids. The helical agent is preferably an α-helical agent having a lipophilic phase and a lipophobic phase.

[0296] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. The attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNAs, such as by facilitating cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0297] The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a crosslinked peptide. Alternatively, the peptide moiety can contain a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 9). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 10)) can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, capable of transporting a number of polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12)) have been shown to function as delivery peptides. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage-display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991). For cell targeting purposes, an example of a peptide or peptidomimetic that is linked to a dsRNA drug through an incorporated monomer unit is an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimetic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications that increase stability or induce conformational properties. Any of the following structural modifications can be used:

[0298] The RGD peptide used in the compositions and methods of the present invention can be linear or cyclic, and can be modified, for example, by glycosylation or methylation, to facilitate targeting to specific tissue(s).RGD-containing peptides and peptidomimetics can include D-amino acids and synthetic RGD mimics.In addition to RGD, other moieties that target integrin ligands can be used.Preferred complexes of this ligand target PECAM-1 or VEGF.

[0299] A "cell-penetrating peptide" can penetrate cells, such as microbial cells, e.g., bacterial or fungal cells, or mammalian cells, e.g., human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or cecropin P1), disulfide bond-containing peptides (e.g., α-defensins, β-defensins, or bactenecins), or peptides containing only one or two key amino acids (e.g., PR-39 or indolicidin). Cell-penetrating peptides can also contain a nuclear localization signal (NLS). For example, a cell-penetrating peptide can be a bipartite amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0300] C. Carbohydrate Complex In some embodiments of the compositions and methods of the present invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated iRNAs are advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic applications, as described herein. As used herein, "carbohydrate" refers to a compound that is either a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom; or a compound having as its component a carbohydrate moiety composed of one or more monosaccharide units, each having at least six carbon atoms (which may be linear, branched, or cyclic) with an oxygen, nitrogen, or sulfur atom bonded to each carbon atom. Exemplary carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Particular monosaccharides include AGT and sugars above (e.g., AGT, C6, C7, or C8); di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0301] In one embodiment, the carbohydrate complex used in the compositions and methods of the present invention is a monosaccharide. In one embodiment, the monosaccharide is represented by Formula II [ka] The compound is N-acetylgalactosamine, as shown below.

[0302] In another embodiment, the carbohydrate complexes used in the compositions and methods of the present invention are selected from the group consisting of: [ka] [ka]

[0303] [ka] [ka]

[0304] [ka] [ka]

[0305] Other exemplary carbohydrate conjugates for use in the embodiments described herein include: [ka] (Formula XXIII) wherein one of X or Y is an oligonucleotide and the other is hydrogen. These include, but are not limited to:

[0306] In certain embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and / or a cell-penetrating peptide.

[0307] Additional carbohydrate conjugates (and linkers) suitable for use in the present invention include those described in PCT Publications WO2014 / 179620 and WO2014 / 179627, the entire contents of which are incorporated herein by reference.

[0308] D. Linker In certain embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide by a variety of linkers, which can be cleavable or non-cleavable.

[0309] The term "linker" or "linking group" means an organic moiety that joins two parts of a compound, for example, by covalently bonding the two parts of the compound.Linkers are generally a direct bond, or an atom such as oxygen or sulfur, a unit such as NR, C(O), C(O)NH, SO, SO, SONH, or a unit such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylhetero ... Alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkyl and alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, etc., comprise a chain of atoms, wherein one or more methylenes can be interrupted or terminated by O, S, S(O), SO, N(R), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle (where R is hydrogen, acyl, aliphatic or substituted aliphatic).In one embodiment, the linker is about 1-24 atoms, 2-24 atoms, 3-24 atoms, 4-24 atoms, 5-24 atoms, 6-24 atoms, 6-18 atoms, 7-18 atoms, 7-17 atoms, 8-17 atoms, 6-16 atoms, 7-16 atoms, or 8-16 atoms.

[0310] A cleavable tether is one that is sufficiently stable outside a cell but is cleaved upon entry into a target cell to release the two moieties tethered by the linker. In preferred embodiments, the cleavable tether is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold or more, or at least about 100-fold more rapidly in the target cell, or under first standard conditions (e.g., which may be selected to mimic or correspond to intracellular conditions), than in the subject's blood, or under second standard conditions (e.g., which may be selected to mimic or correspond to conditions found in blood or serum).

[0311] Cleavable linking groups are susceptible to cleavage agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleavage agents are more common in cells than in serum or blood, or are found at higher levels or activity. Examples of such degradable agents include oxidizing or reducing enzymes or reducing agents such as mercaptans present in cells, which can degrade redox-cleavable linking groups by reduction, and are selective for specific substrates or do not have substrate specificity; esterase; endosomes or substances that can create an acidic environment, such as those that produce a pH of 5 or less; enzymes that act as general acids, peptidases (which can be substrate specific), and phosphatases, which can hydrolyze or degrade acid-cleavable linking groups.

[0312] Cleavable linkers, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from approximately 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable linkers that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand intracellularly or to a desired compartment of the cell.

[0313] The linker can comprise a cleavable linking group that can be cleaved by a specific enzyme.The type of cleavable linking group incorporated into the linker can depend on the target cell.For example, a ligand that targets the liver can be linked to a cationic lipid through a linker that comprises an ester group.Liver cells are rich in esterase, and therefore the linker is more efficiently cleaved in liver cells than in cell types that are not rich in esterase.Other cell types that are rich in esterase include lung, renal cortex, and testicular cells.

[0314] Linkers containing peptide bonds can be used in targeting peptidase-rich cell types such as liver cells and synovial cells.

[0315] In general, the suitability of a candidate cleavable linker can be evaluated by testing the ability of a degradable substance (or condition) to cleave the candidate linker. It may also be desirable to test candidate cleavable linkers for their ability to resist cleavage in blood or upon contact with other non-target tissues. Thus, if a first condition is selected to indicate cleavage in target cells and a second condition is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum, the relative susceptibility to cleavage between the first and second conditions can be determined. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in a whole animal. It may be useful to perform initial evaluation in a cell-free or culture condition and confirm with further evaluation in a whole animal. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0316] i. Redox-cleavable linker In one embodiment, the cleavable linker is a redox-cleavable linker that is cleaved upon reduction or oxidation. One example of a reductively cleavable linker is a disulfide linker (-SS-). To determine whether a candidate cleavable linker is a suitable "reductively cleavable linker" or suitable for use with, for example, a particular iRNA moiety and a particular targeting agent, one skilled in the art can rely on the methods described herein. For example, candidates can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate observed in cells, such as target cells. Candidates can also be evaluated under conditions selected to mimic blood or serum conditions. A candidate compound is cleaved at a maximum of about 10% in blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times more rapidly in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic intracellular media compared to conditions selected to mimic extracellular media.

[0317] ii. Phosphate-based cleavable linkers In another embodiment, the cleavable linker comprises a phosphate-based cleavable linker. The phosphate-based cleavable linker can be cleaved by a substance that degrades or hydrolyzes the phosphate group. An example of a substance that cleaves phosphate groups in a cell is an enzyme such as an intracellular phosphatase. Examples of phosphate-based linking groups are -OP(O)(ORk)-O-, -OP(S)(ORk)-O-, -OP(S)(SRk)-O-, -SP(O)(ORk)-O-, -OP(O)(ORk)-S-, -SP(O)(ORk)-S-, -OP(S)(ORk)-S-, -SP(S)(ORk)-O-, -OP(O)(Rk)-O-, -OP(S)(Rk)-O-, -SP(O)(Rk)-O-, -SP(S)(Rk)-O-, -SP(O)(Rk)-S-, -OP(S)(Rk)-S-. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. A preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0318] iii. Acid-cleavable linking group In another embodiment, the cleavable linker comprises an acid-cleavable linker. An acid-cleavable linker is a linker that is cleaved under acidic conditions. In a preferred embodiment, the acid-cleavable linker is cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0 or less) or by a substance, such as an enzyme, that can act as a general acid. Within a cell, certain low-pH organelles, such as endosomes and lysosomes, may provide a cleavage environment for the acid-cleavable linker. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and amino acid esters. Acid-cleavable groups may have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, when the carbon is bonded to the oxygen of the ester (alkoxy group), the linker is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates may be evaluated using methods similar to those described above.

[0319] iv. Ester-based linking groups In another embodiment, the cleavable linker comprises an ester-based cleavable linker. Ester-based cleavable linkers are cleaved in cells by enzymes such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0320] v. Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linker. Peptide-based cleavable linkers are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linkers are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to give rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linkers have the general formula -NHCHRAC(O)NHCHRBC(O)-, where R and R are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0321] In one embodiment, the iRNA of the present invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrates conjugated to linkers in the compositions and methods of the present invention include: [ka] [ka] wherein one of X or Y is an oligonucleotide and the other is hydrogen. These include, but are not limited to:

[0322] In any embodiment of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives attached via a bivalent or trivalent branched linker.

[0323] In one embodiment, the dsRNA of the present invention is represented by the following formulas (XXXII) to (XXXV): [ka] (In the formula, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B, and q5C independently represent 0 to 20 occurrences each, and the repeat units may be identical or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 4A , T 5B , T 5C is, each independently for each occurrence, absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B , Q 5C is, independently for each occurrence, absent, alkylene, substituted alkylene, wherein one or more methylenes may be interrupted or terminated by one or more of O, S, S(O), SO, N(RN), C(R')=C(R''), C≡C, or C(O); R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5Cis independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, [ka] or heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand; i.e., independently for each occurrence, a monosaccharide (e.g., GalNAc), a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide; and R a is H or an amino acid side chain). The trivalent conjugated GalNAc derivative is conjugated to a bivalent or trivalent branched linker selected from the group of structures represented by formula (XXXV): [ka] (In the formula, L 5A , L 5B and L 5C represents a monosaccharide such as a GalNAc derivative) The present invention is particularly useful for use with RNAi agents to inhibit the expression of target genes such as

[0324] Examples of suitable divalent and trivalent branched linker groups for conjugation to GalNAc derivatives include, but are not limited to, the structures shown above as Formulas II, VII, XI, X, and XIII.

[0325] Representative United States patents that teach the preparation of RNA complexes include U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,51 2,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4, 835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5 ,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5, Nos. 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022 (the entire contents of each of which are incorporated herein by reference).

[0326] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the above modifications may be incorporated in a single compound, or even in a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

[0327] "Chimeric" iRNA compounds or "chimeras," in the context of the present invention, are iRNA compounds, preferably dsRNA, that contain two or more chemically distinct regions, each composed of at least one monomer unit, i.e., nucleotides in the case of dsRNA compounds. These iRNAs generally contain at least one region in which the RNA has been modified to confer on the iRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid. Additional regions of the iRNA may serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. As a result, comparable results are often obtained with shorter iRNAs when chimeric dsRNAs are used compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0328] In some cases, the RNA of an iRNA may be modified with a non-ligand group. Numerous non-ligand molecules have been conjugated to iRNAs to enhance their activity, cellular distribution, or intracellular uptake, and procedures for performing such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. A typical conjugation protocol involves the synthesis of RNA bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the conjugating molecule using an appropriate coupling or activating reagent. The conjugation reaction can be carried out in solution phase while the RNA is still bound to the solid support, or following cleavage of the RNA. Purification of the RNA conjugate by HPLC generally affords a pure conjugate.

[0329] V. Delivery of iRNA of the Invention Delivery of an iRNA of the invention to a cell, such as a cell in a subject, such as a human subject (e.g., a subject in need thereof, such as a subject with an angiotensin-related disease or condition), can be achieved in several different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention, either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, such as a dsRNA, to the subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and induce expression of the iRNA. These alternatives are discussed further below.

[0330] Generally, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the present invention (see, for example, Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and International Publication WO94 / 02595, the entire contents of which are incorporated herein by reference). For in vivo delivery, factors to consider for delivering iRNA molecules include, for example, the biological stability of the delivered molecule, prevention of nonspecific effects, and accumulation of the delivered molecule in the target tissue. Nonspecific effects of iRNA can be minimized by local administration, such as direct injection or implantation into tissue or topical administration of a formulation. Local administration at the treatment site maximizes the local concentration of the substance; limits exposure of systemic tissues that may otherwise be harmed by or degrade the substance to agents; and allows for the administration of a lower total dose of iRNA molecules. Several studies have demonstrated successful gene product knockdown when iRNAs are administered locally. For example, intraocular delivery of VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ., et al(2004)Retina 24:132-138) and by subretinal injection in mice (Reich, SJ., et al(2003)Mol.Vis.9:210-216) has both been shown to inhibit neovascularization in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice reduced tumor volume (Pille, J., et al(2005)Mol.Ther.11:267-274) and prolonged the survival of tumor-bearing mice (Kim, WJ., et al(2006)Mol.Ther.14:343-350; Li, S., et al(2007)Mol.Ther.15:515-523).RNA interference can be delivered to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, GT., et al (2004) Neuroscience 129:521-528; Thakker, ER., et al (2004) Proc. Natl. Acad. Sci. USA 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lung by intranasal administration (Howard, KA., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al. (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al. (2005) Nat. Med. 11:50-55) have demonstrated successful local delivery. To administer iRNA systemically to treat disease, the RNA can be modified or delivered using a drug delivery system; both methods act to prevent rapid degradation of dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or pharmaceutical carrier can also enable targeting of iRNA compositions to target tissues, avoiding undesirable nonspecific effects. iRNA molecules can be modified by chemical attachment of lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. For example, systemic injection of iRNAs directed against ApoB conjugated to lipophilic cholesterol moieties into mice resulted in apoB mRNA knockdown in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of iRNAs to aptamers has been shown to suppress tumor growth and mediate tumor regression in mouse models of prostate cancer (McNamara, JO., et al (2006) Nat. Biotechnol. 24:1005-1015).In another embodiment, iRNA can be delivered using drug delivery systems such as nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate the binding of iRNA molecules (negatively charged) and also enhance their interaction with the negatively charged cell membrane, allowing for efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers can be bound to iRNA or induced to form vesicles or micelles that encase iRNA (see, for example, Kim SH., et al. (2008) Journal of Controlled Release 129(2):107-116). The formation of vesicles or micelles further prevents degradation of iRNA when administered systemically. Methods for making and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, DR., et al (2003) J. Mol. Biol 327:761-766; Verma, UN., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entireties).Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNA include DOTAP (Sorensen, D.R., et al (2003), supra; Verma, U.N., et al (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T.S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P.Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M.E., et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D.A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, for systemic administration, the iRNA is complexed with cyclodextrin. Methods for administering iRNA and cyclodextrin and pharmaceutical compositions are found in U.S. Pat. No. 7,427,605, the entire contents of which are incorporated herein by reference.

[0331] A. Vectors Encoding iRNAs of the Invention iRNAs targeting the AGT gene can be expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International Publication No. WO 00 / 22113; Conrad, International Publication No. WO 00 / 22114; and Conrad, U.S. Patent No. 6,054,299). Expression varies depending on the specific construct used and the target tissue or cell type and can be transient (hours to weeks) or persistent (weeks to months or longer). These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrative or non-integrative vectors. Transgenes can also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0332] Individual iRNA strands or strands can be transcribed from the promoter on expression vector.When expressing two separate strands to produce, for example, dsRNA, two separate expression vectors can be simultaneously introduced into target cells (for example, by transfection or infection).Alternatively, the promoters located on the same expression plasmid can transcribe the individual strands of dsRNA.In one embodiment, dsRNA is expressed as an inverted repeat polynucleotide that is linked by a linker polynucleotide sequence, so that dsRNA has a stem-loop structure.

[0333] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for expressing iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, preferably vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from many commercial sources. Typically, such vectors are provided containing convenient restriction enzyme recognition sites for inserting the desired nucleic acid fragment. Delivery of the iRNA expression vector can be by systemic administration, such as intravenous or intramuscular administration, or by administration to target cells explanted from the patient and then reintroduced into the patient, or any other means that allows for introduction into the desired target cells.

[0334] iRNA expression plasmids can be transfected into target cells as a complex with a cationic lipid carrier (e.g., Oligofectamine) or a non-cationic lipid-based carrier (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdown, targeting different regions of a target RNA over a period of one week or more, are also contemplated by the present invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be indicated by a reporter, such as a fluorescent marker like green fluorescent protein (GFP). Stable transfection into cells ex vivo can be ensured using markers that provide transfected cells with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

[0335] Viral vector systems that can be used with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including, but not limited to, lentivirus 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 orthopox, e.g., vaccinia virus vectors, or avipox, e.g., canarypox or fowlpox; and (j) helper-dependent or gutless adenoviruses. Replication-defective viruses may also be advantageous. Different vectors may or may not integrate into the cellular genome. The constructs may optionally contain viral sequences for transfection. Alternatively, the constructs may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA generally require regulatory elements, such as promoters, enhancers, etc., to ensure iRNA expression in target cells. Other aspects of vectors and constructs considered are described in more detail below.

[0336] Vectors useful for delivering iRNA contain sufficient regulatory elements (promoters, enhancers, etc.) for expression of the iRNA in the desired target cells or tissues. Regulatory elements can be selected to provide for either constitutive or regulated / inducible expression.

[0337] The expression of iRNA can be precisely regulated using inducible regulatory sequences that are sensitive to specific physiological regulators, such as circulating glucose levels or hormones (Docherty et al., 1994, FASEB J.8:20-24).Such inducible expression systems suitable for controlling dsRNA expression in cells or mammals include, for example, regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-β-D1-thiogalactopyranoside (IPTG).Those skilled in the art can select suitable regulatory sequences / promoter sequences based on the intended use of the iRNA transgene.

[0338] Viral vectors containing nucleic acid sequences encoding iRNAs can be used. For example, retroviral vectors can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for correct packaging of the viral genome and integration into host cell DNA. The nucleic acid sequences encoding iRNAs are cloned into one or more vectors, which facilitates delivery of the nucleic acid to a patient. More details regarding retroviral vectors can be found in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells, for example, to create stem cells that are more resistant to chemotherapy. Other publications illustrating the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, HIV-based vectors described in U.S. Patent Nos. 6,143,520; 5,665,557; and 5,981,276, which are incorporated herein by reference.

[0339] Adenoviruses are also contemplated for use in delivering iRNAs of the present invention. Adenoviruses are particularly attractive vehicles for delivering genes to, for example, respiratory epithelium. Adenoviruses naturally infect respiratory epithelium, causing a mild disease. Other targets for adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993), present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994), demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelium of rhesus monkeys. Other examples of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94 / 12649; and Wang, et al., Gene Therapy 2:775-783 (1995). Suitable AV vectors for expressing iRNAs featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

[0340] Adeno-associated virus (AAV) vectors can also be used to deliver iRNAs of the invention (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146). In one embodiment, the iRNAs can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector, e.g., with either the U6 or H1 RNA promoter, or the cytomegalovirus (CMV) promoter. AAV vectors suitable for expressing the dsRNA featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors to target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher KJ et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Patent Nos. 5,252,479; 5,139,941; International Publication No. WO94 / 13788; and International Publication No. WO93 / 24641 (the entire disclosures of which are incorporated herein by reference).

[0341] Another viral vector suitable for delivering the iRNA of the invention is a vaccinia virus, e.g., an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, or a poxvirus, e.g., an avipox, e.g., fowlpox or canarypox.

[0342] The tropism of viral vectors can be modified, if desired, by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting capsid proteins from different viruses. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, etc. AAV vectors can be engineered to target different cells by expressing different capsid protein serotypes; see, e.g., Rabinowitz JE et al. (2002), J Virol 76:791-801, the entire disclosure of which is incorporated herein by reference.

[0343] The vector pharmaceutical preparation can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0344] VI. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and formulations containing the iRNAs of the present invention. In one aspect, the present invention provides a pharmaceutical composition comprising the iRNA described herein and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing iRNA are useful for treating diseases or disorders associated with AGT gene expression or activity. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration by parenteral delivery, e.g., by subcutaneous (SC) or intravenous (IV) delivery. Another example is a composition formulated for direct delivery into the brain parenchyma, e.g., by intracerebral infusion, such as continuous pump infusion. Pharmaceutical compositions of the present invention may be administered at a dosage sufficient to inhibit AGT gene expression. Generally, suitable doses of the iRNAs of the present invention range from about 0.001 to about 200.0 milligrams per kilogram of recipient body weight per day, generally from about 1 to 50 mg per kilogram of body weight per day. For example, the dsRNA can be administered at about 0.01 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 3 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg per single dose.

[0345] For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0346] In another embodiment, the dsRNA is administered at a dose of about 0.1 to about 50 mg / kg, about 0.25 to about 50 mg / kg, about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kg, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.1 to about 45 mg / kg, about 0.25 to about 45 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / kg, about 1.5 to about 45 mg / kg to about 45 mg / kg, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 45 mg / kg, about 10 to about 45 mg / kg, about 15 to about 45 mg / kg, about 20 to about 45 mg / kg, about 20 to about 45 mg / kg, about 25 to about 45 mg / kg, about 25 to about 45 mg / kg, about 30 to about 45 mg / kg , about 35 to about 45 mg / kg, about 40 to about 45 mg / kg, about 0.1 to about 40 mg / kg, about 0.25 to about 40 mg / kg, about 0.5 to about 40 mg / kg, about 0.75 to about 40 mg / kg, about 1 to about 40 mg / kg, about 1.5 to about 40 mg / kg, about 2 to about 40 mg / kg, about 2.5 to about 40 mg / kg, about 3 to about 40 mg / kg, about 3.5 to about 40 mg / kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.1 to about 30 mg / kg, about 0.25 to about 30 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.5 to about 3 0 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.1 to about 20 mg / kg, about 0.25 to about 20 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of this invention.

[0347] For example, the dsRNA may be about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0348] In another embodiment, the dsRNA is administered in a dose range of about 0.5 to about 50 mg / kg, about 0.75 to about 50 mg / kg, about 1 to about 50 mg / kg, about 1.5 to about 50 mg / kg, about 2 to about 50 mg / kg, about 2.5 to about 50 mg / kg, about 3 to about 50 mg / kg, about 3.5 to about 50 mg / kg, about 4 to about 50 mg / kg, about 4.5 to about 50 mg / kg, about 5 to about 50 mg / kg, about 7.5 to about 50 mg / kg, about 10 to about 50 mg / kg, about 15 to about 50 mg / kg, about 20 to about 50 mg / kg, about 25 to about 50 mg / kg, about 26 to about 50 mg / kg, about 27 to about 50 mg / kg, about 28 to about 50 mg / kg, about 29 to about 50 mg / kg, about 30 to about 50 mg / kg, about 31 to about 50 mg / kg, about 32 to about 50 mg / kg, about 33 to about 50 mg / kg, about 34 to about 50 mg / kg, about 35 to about 50 mg / kg, about 36 to about 50 mg / kg, about 37 to about 50 mg / kg, about 38 to about 50 mg / kg, about 39 to about 50 mg / kg, about 40 to about 50 mg / kg, about 41 to about 50 mg / kg, about 42 to about 50 mg / kg, about 43 to about 50 mg / kg, about 44 to about 50 mg / kg, about 45 to about 50 mg / kg, about 46 to about 50 mg / kg, about 47 to about 50 mg / 5 to about 50 mg / kg, about 25 to about 50 mg / kg, about 30 to about 50 mg / kg, about 35 to about 50 mg / kg, about 40 to about 50 mg / kg, about 45 to about 50 mg / kg, about 0.5 to about 45 mg / kg, about 0.75 to about 45 mg / kg, about 1 to about 45 mg / kg, about 1.5 to about 45 mg / kg, about 2 to about 45 mg / kg, about 2.5 to about 45 mg / kg, about 3 to about 45 mg / kg, about 3.5 to about 45 mg / kg, about 4 to about 45 mg / kg, about 4.5 to about 45 mg / kg, about 5 to about 45 mg / kg, about 7.5 to about 4 5mg / kg, about 10 to about 45mg / kg, about 15 to about 45mg / kg, about 20 to about 45mg / kg, about 20 to about 45mg / kg, about 25 to about 45mg / kg, about 25 to about 45mg / kg, about 30 to about 45mg / kg, about 35 to about 45mg / kg, about 40 to about 45mg / kg, about 0.5 to about 40mg / kg, about 0.75 to about 40mg / kg, about 1 to about 40mg / kg, about 1.5 to about 40mg / kg, about 2 to about 40mg / kg, about 2.5 to about 40mg / kg, about 3 to about 40mg / kg, about 3.5 to about 40mg / kg kg, about 4 to about 40 mg / kg, about 4.5 to about 40 mg / kg, about 5 to about 40 mg / kg, about 7.5 to about 40 mg / kg, about 10 to about 40 mg / kg, about 15 to about 40 mg / kg, about 20 to about 40 mg / kg, about 20 to about 40 mg / kg, about 25 to about 40 mg / kg, about 25 to about 40 mg / kg, about 30 to about 40 mg / kg, about 35 to about 40 mg / kg, about 0.5 to about 30 mg / kg, about 0.75 to about 30 mg / kg, about 1 to about 30 mg / kg, about 1.5 to about 30 mg / kg, about 2 to about 30 mg / kg, about 2.5 to about 30 mg / kg, about 3 to about 30 mg / kg, about 3.5 to about 30 mg / kg, about 4 to about 30 mg / kg, about 4.5 to about 30 mg / kg, about 5 to about 30 mg / kg, about 7.5 to about 30 mg / kg, about 10 to about 30 mg / kg, about 15 to about 30 mg / kg, about 20 to about 30 mg / kg, about 20 to about 30 mg / kg, about 25 to about 30 mg / kg, about 0.5 to about 20 mg / kg, about 0.75 to about 30 mg / kg The dsRNA is administered at a dose of about 20 mg / kg, about 1 to about 20 mg / kg, about 1.5 to about 20 mg / kg, about 2 to about 20 mg / kg, about 2.5 to about 20 mg / kg, about 3 to about 20 mg / kg, about 3.5 to about 20 mg / kg, about 4 to about 20 mg / kg, about 4.5 to about 20 mg / kg, about 5 to about 20 mg / kg, about 7.5 to about 20 mg / kg, about 10 to about 20 mg / kg, or about 15 to about 20 mg / kg. In one embodiment, the dsRNA is administered at a dose of about 10 mg / kg to about 30 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0349] For example, a subject may receive, for example, a subcutaneous or intravenous injection of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.925, 0. .95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6 .1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16 A single therapeutic dose of iRNA may be administered, such as about 0.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0350] In some embodiments, a subject receives, for example, a dose of about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.92 5, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 ,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10,10.5,11,11.5,12,12.5,13,13.5,14,14.5,15,15.5,16,16 In some embodiments, multiple therapeutic doses of iRNA are administered, such as at about 0.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg. Multiple dose regimens can include administering a therapeutic amount of iRNA daily, for example, for 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more.

[0351] In other embodiments, the subject receives, for example, subcutaneously or intravenously, about 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, 0.275, 0.3, 0.325, 0.35, 0.375, 0.4, 0.425, 0.45, 0.475, 0.5, 0.525, 0.55, 0.575, 0.6, 0.625, 0.65, 0.675, 0.7, 0.725, 0.75, 0.775, 0.8, 0.825, 0.85, 0.875, 0.9, 0.92 5, 0.95, 0.975, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6 ,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8,8.1,8.2,8.3,8.4,8.5,8.6,8.7,8.8,8.9,9,9.1,9.2,9.3,9.4,9.5,9.6,9.7,9.8,9.9,10,10.5,11,11.5,12,12.5,13,13.5,14,14.5,15,15.5,16,16 Repeated therapeutic doses of the iRNA are administered, such as at about 0.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg / kg. Repeated dose regimens can include administering the iRNA therapeutic amount periodically, such as every other day, every third day, every fourth day, twice a week, weekly, every other week, or monthly. In any embodiment, the iRNA is administered about once a month to about once a quarter (ie, about once every three months).

[0352] After the initial treatment regimen, the treatments can be administered less frequently.

[0353] In any embodiment, for example, when the compositions of the invention comprise a dsRNA and lipids described herein, the subject receives a dose of from about 0.01 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 5 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 0.2 mg / kg to about 5 mg / kg, or about 0. 2mg / kg to about 10mg / kg, about 0.3mg / kg to about 5mg / kg, about 0.3mg / kg to about 10mg / kg, about 0.4mg / kg to about 5mg / kg, about 0.4mg / kg to about 10mg / kg, about 0.5mg / kg to about 5mg / kg, about 0.5mg / kg to about 10mg / kg, about 1mg / kg to about 5mg / kg, about 1mg / kg to about 10mg / kg, about 1.5mg / kg to about 5mg / kg, about 1.5mg / kg to about 10mg / kg kg to about 10 mg / kg, about 2 mg / kg to about 2.5 mg / kg, about 2 mg / kg to about 10 mg / kg, about 3 mg / kg to about 5 mg / kg, about 3 mg / kg to about 10 mg / kg, about 3.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 5 mg / kg, about 4.5 mg / kg to about 5 mg / kg, about 4 mg / kg to about 10 mg / kg, about 4.5 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg A therapeutic amount of iRNA may be administered, such as about 5.5 mg / kg to about 10 mg / kg, about 6 mg / kg to about 10 mg / kg, about 6.5 mg / kg to about 10 mg / kg, about 7 mg / kg to about 10 mg / kg, about 7.5 mg / kg to about 10 mg / kg, about 8 mg / kg to about 10 mg / kg, about 8.5 mg / kg to about 10 mg / kg, about 9 mg / kg to about 10 mg / kg, or about 9.5 mg / kg to about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0354] For example, the dsRNA may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, The compound may be administered at a dose of 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or about 10 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention.

[0355] In any embodiment of the invention, for example, when a double-stranded RNAi agent comprises a modification (e.g., one or more motifs of three identical modifications on three consecutive nucleotides) (including those containing such motifs at or proximal to the cleavage site of the agent), six phosphorothioate linkages, and a ligand, such an agent may be administered at a concentration of from about 0.01 to about 0.5 mg / kg, from about 0.01 to about 0.4 mg / kg, from about 0.01 to about 0.3 mg / kg, from about 0.01 to about 0.2 mg / kg, from about 0.01 to about 0.1 mg / kg, from about 0.01 mg / kg to about 0.09 mg / kg, , about 0.01 mg / kg to about 0.08 mg / kg, about 0.01 mg / kg to about 0.07 mg / kg, about 0.01 mg / kg to about 0.06 mg / kg, about 0.01 mg / kg to about 0.05 mg / kg, about 0.02 to about 0.5 mg / kg, about 0.02 to about 0.4 mg / kg, about 0.02 to about 0.3 mg / kg, about 0.02 to about 0.2 mg / kg, about 0.02 to about 0.1 mg / kg, about 0.02 mg / kg to about 0.09 mg / kg, about 0.02 mg / kg to about 0.08 mg / kg, about 0.02 mg / kg to about 0.07 mg / kg, about 0 0.02mg / kg to about 0.06mg / kg, about 0.02mg / kg to about 0.05mg / kg, about 0.03 to about 0.5mg / kg, about 0.03 to about 0.4mg / kg, about 0.03 to about 0.3mg / kg, about 0.03 to about 0.2mg / kg, about 0.03 to about 0.1mg / kg, about 0.03mg / kg to about 0.09mg / kg, about 0.03mg / kg to about 0.08mg / kg, about 0.03mg / kg to about 0.07mg / kg, about 0.03mg / kg to about 0.06mg / kg, about 0.03mg / kg to about 0.05mg / kg, about 0.04mg / kg to about 0.5 mg / kg, about 0.04 to about 0.4 mg / kg, about 0.04 to about 0.3 mg / kg, about 0.04 to about 0.2 mg / kg, about 0.04 to about 0.1 mg / kg, about 0.04 mg / kg to about 0.09 mg / kg, about 0.04 mg / kg to about 0.08 mg / kg, about 0.04 mg / kg to about 0.07 mg / kg, about 0.04 mg / kg to about 0.06 mg / kg, about 0.05 to about 0.5 mg / kg, about 0.05 to about 0.4 mg / kg, about 0.05 to about 0.3 mg / kg, about 0.05 to about 0.2 mg / kg, about 0.05 to about 0.The RNAi agent is administered at a dose of about 1 mg / kg, about 0.05 mg / kg to about 0.09 mg / kg, about 0.05 mg / kg to about 0.08 mg / kg, or about 0.05 mg / kg to about 0.07 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also contemplated as part of the invention; for example, an RNAi agent may be administered to a subject at a dose of about 0.015 mg / kg to about 0.45 mg / kg.

[0356] For example, an RNAi agent, e.g., an RNAi agent in a pharmaceutical composition, can have a concentration of about 0.01 mg / kg, 0.0125 mg / kg, 0.015 mg / kg, 0.0175 mg / kg, 0.02 mg / kg, 0.0225 mg / kg, 0.025 mg / kg, 0.0275 mg / kg, 0.03 mg / kg, 0.0325 mg / kg, 0.035 mg / kg, 0.03 75mg / kg, 0.04mg / kg, 0.0425mg / kg, 0.045mg / kg, 0.0475mg / kg, 0.05mg / kg, 0.0525mg / kg, 0. 055mg / kg, 0.0575mg / kg, 0.06mg / kg, 0.0625mg / kg, 0.065mg / kg, 0.0675mg / kg, 0.07mg / kg, 0. 0725mg / kg, 0.075mg / kg, 0.0775mg / kg, 0.08mg / kg, 0.0825mg / kg, 0.085mg / kg, 0.0875mg / kg , 0.09mg / kg, 0.0925mg / kg, 0.095mg / kg, 0.0975mg / kg, 0.1mg / kg, 0.125mg / kg, 0.15mg / kg, 0. The compound may be administered at a dose of 175 mg / kg, 0.2 mg / kg, 0.225 mg / kg, 0.25 mg / kg, 0.275 mg / kg, 0.3 mg / kg, 0.325 mg / kg, 0.35 mg / kg, 0.375 mg / kg, 0.4 mg / kg, 0.425 mg / kg, 0.45 mg / kg, 0.475 mg / kg, or about 0.5 mg / kg. Values ​​and ranges intermediate to the recited values ​​are also intended to be part of the invention.

[0357] The pharmaceutical composition may be administered intravenously for a period of time such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about 25 minutes or more. Administration may be repeated periodically, for example, once a week or every other week (i.e., once every two weeks) for one, two, three, four, or more months. After the initial treatment regimen, the treatment agent may be administered less frequently. For example, after administration once a week or once every two weeks for three months, administration may be repeated once a month for six months or a year or more.

[0358] The pharmaceutical composition may be administered by subcutaneous administration.

[0359] The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more subdoses at appropriate intervals throughout the day, or even via continuous infusion or delivery via a controlled-release formulation. In such cases, the amount of iRNA contained in each subdose must be correspondingly smaller to achieve the total daily dose. The dosage unit can also be formulated for delivery over several days, for example, using a conventional sustained-release formulation that provides sustained release of the iRNA over several days. Sustained-release formulations are well known in the art and are particularly useful for delivering the agent of the present invention to a specific site where the agent can be used. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. A higher dose can be administered initially (i.e., a loading dose), followed by lower doses administered over a sustained period.

[0360] In other embodiments, a single administration of the pharmaceutical composition may be continued over a prolonged period, with subsequent doses administered no more than 3, 4, or 5 days apart, or no more than 1, 2, 3, or 4 weeks apart. In some embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered once a week. In other embodiments of the invention, a single dose of the pharmaceutical composition of the invention is administered twice a month. In any embodiment, the iRNA is administered about once a month to about once a quarter (i.e., about once every three months).

[0361] The pharmaceutical composition is administered indefinitely to a subject whose blood pressure is elevated, for example due to obesity, or when a cause of high levels of AGT exists, for example, during gestational hypertension.

[0362] Those skilled in the art will understand that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Effective dosages and in vivo half-lives of the individual iRNAs encompassed by the present invention can be determined using conventional procedures or based on in vivo testing using appropriate animal models, as described elsewhere herein.

[0363] The pharmaceutical compositions of the present invention can be administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be topical (e.g., via a transdermal patch), pulmonary administration, for example, by inhalation or insufflation of powders or aerosols, including with a nebulizer; intratracheal, intranasal, transepithelial and transdermal administration, oral administration, or parenteral administration. Parenteral administration can include intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal administration, for example, via an implanted device; or intracranial administration, for example, intraparenchymal, intrathecal, or intraventricular administration.

[0364] The iRNA can be delivered in a manner that targets a specific tissue, such as the liver (e.g., hepatocytes of the liver).

[0365] Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder, or oily bases, thickeners, and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful. Suitable topical formulations include those in which the iRNA featured in the present invention is mixed with a topical delivery agent, such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidylcholine DMPC, distearolyphosphatidylcholine), anionic (e.g., dimyristoylphosphatidylglycerol DMPG), and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). The iRNA featured in the present invention can be encapsulated in or complexed with liposomes, particularly cationic liposomes. Alternatively, the iRNA can be complexed with lipids, particularly cationic lipids. Suitable fatty acids and esters include arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1-20 Topical formulations include, but are not limited to, alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof. Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0366] A. iRNA Formulations Comprising Membrane-Like Molecular Assemblies The iRNAs used in the compositions and methods of the present invention can be formulated for delivery within membrane-like molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one or more bilayers. Liposomes include unilamellar and multilamellar vesicles, which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material separates the aqueous interior from the aqueous exterior, which generally does not contain the iRNA composition, but may in some cases. Liposomes are useful for transporting and delivering active ingredients to their site of action. Because the liposomal membrane is structurally similar to biological membranes, upon application of liposomes to tissues, the liposomal bilayer fuses with the bilayer of the cell membrane. As liposome fusion with the cell proceeds, the internal aqueous contents, including the iRNA, are delivered into the cell, where the iRNA can specifically bind to the target RNA and mediate iRNA activity. In some cases, liposomes are also specifically targeted, for example, to direct iRNA to a particular cell type.

[0367] Liposomes containing iRNA agents can be prepared by various methods. In one example, the lipid components of the liposomes are dissolved in a detergent to form micelles with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid complexes. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholic acid, CHAPS, octylglucoside, deoxycholic acid, and lauroyl sarcosine. The iRNA agent preparation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the iRNA agent, condensing around the iRNA agent to form liposomes. After condensation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of the iRNA agent.

[0368] If necessary, a carrier compound, for example, to aid in condensation, can be added during the condensation reaction by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to aid in condensation.

[0369] Methods for making stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as structural components of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation is described by Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; Bangham, et al. Biophys. Acta 557:9, 1979;Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978;Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984;Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of suitable size for use as delivery vehicles include sonication and a combination of freeze-thawing and extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). When consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted for embedding iRNA drug preparations within liposomes.

[0370] Liposomes are divided into two broad classes: cationic liposomes, which are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes; the positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized inside endosomes; the acidic pH inside the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0371] pH-sensitive or negatively charged liposomes do not complex with nucleic acids but rather encapsulate them. Because both nucleic acids and lipids have similar charges, repulsion occurs rather than complexation. Nevertheless, some nucleic acid is encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected within the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0372] One major type of liposome composition contains phospholipids in addition to naturally occurring phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are primarily formed from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0373] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185; 5,171,678; International Publication No. WO 94 / 00569; International Publication No. WO 93 / 24640; International Publication No. WO 91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0374] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have been studied to test their efficacy in delivering drugs to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine A into the dermis of mouse skin. The results suggested that such nonionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al. STP Pharma. Sci., 1994, 4(6) 466).

[0375] Liposomes also include "sterically stabilized" liposomes, which term, as used herein, refers to liposomes comprising one or more specialized lipids which, when incorporated into the liposome, result in enhanced circulation life compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is (A) monosialoganglioside G M1or (B) derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without being bound by any particular theory, it is believed in the art that the increased circulation half-life of sterically stabilized liposomes containing at least gangliosides, sphingomyelin, or PEG-derivatized lipids is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0376] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., 1987, 507, 64) describe the use of monosialoganglioside G M1 reported the ability of galactocerebroside sulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings were elaborated upon by Gabizon et al. (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). U.S. Patent No. 4,837,028 and WO 88 / 04924, both to Allen et al., report the ability of (1) sphingomyelin, and (2) ganglioside G M1 or galactocerebroside sulfate esters. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0377] In one embodiment, cationic liposomes are used.Cationic liposomes have the advantage of being able to fuse with cell membranes.Non-cationic liposomes cannot fuse with plasma membranes effectively, but can be taken up by macrophages in vivo and can be used to deliver iRNA drugs to macrophages.

[0378] Additional advantages of liposomes include: liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water- and lipid-soluble drugs; and liposomes can protect iRNA drugs encapsulated in their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in preparing liposome formulations are the lipid surface charge, vesicle size and liquid volume of the liposomes.

[0379] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids in the plasma membrane of tissue culture cells, resulting in iRNA drug delivery (see, e.g., Felgner, PL et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use in combination with DNA).

[0380] The DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with phospholipids to form DNA-complexed vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells. It comprises positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the net charge on the resulting complex is also positive. The positively charged complexes thus prepared spontaneously adhere to negatively charged cell surfaces and fuse with the plasma membrane, efficiently delivering functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moiety is attached by an ester rather than an ether bond.

[0381] Other reported cationic lipid compounds include those conjugated to a variety of moieties, including carboxyspermine conjugated to one of two lipid types, such as compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

[0382] Another cationic lipid complex involves derivatizing lipids with cholesterol ("DC-Chol") in combination with DOPE and formulated into liposomes (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, produced by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California), and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO98 / 39359 and WO96 / 37194.

[0383] Liposomal formulations are particularly suitable for topical administration, and liposomes offer several advantages over other formulations. These advantages include reduced side effects associated with high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the possibility of administering iRNA drugs intradermally. In some implementations, liposomes are used to deliver iRNA drugs to epithelial cells and enhance the penetration of iRNA drugs into skin tissues, such as the skin. For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been demonstrated (e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, vol. 259-265; Mannino, RJ and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276, 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, RM and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, CY and Huang, L., Proc. (See Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0384] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have been studied to determine their utility in delivering drugs to the skin. Nonionic liposome formulations containing Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs into the dermis of mouse skin. Such formulations containing iRNA agents are useful for treating skin diseases.

[0385] Liposomes containing iRNA can be made highly deformable. Such deformability can allow the liposome to penetrate pores smaller than the average diameter of the liposome. For example, transferosomes are a type of deformable liposome. Transferosomes can be made by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing iRNA drugs can be delivered subcutaneously, for example, via infection, to deliver iRNA drugs to keratinocytes in the skin. To pass through intact mammalian skin, lipid vesicles must pass through a series of pores, each less than 50 nm in diameter, under the influence of an appropriate transdermal gradient. Furthermore, due to their lipid properties, these transferosomes can self-optimize (e.g., adapt to the shape of skin pores), self-repair, frequently reach their targets without fragmentation, and are often self-loading.

[0386] Other formulations to which the present invention may be applied are described in U.S. Provisional Patent Application No. 61 / 018,616, filed January 2, 2008; U.S. Provisional Patent Application No. 61 / 018,611, filed January 2, 2008; U.S. Provisional Patent Application No. 61 / 039,748, filed March 26, 2008; U.S. Provisional Patent Application No. 61 / 047,087, filed April 22, 2008, and U.S. Provisional Patent Application No. 61 / 051,528, filed May 8, 2008. PCT application PCT / US2007 / 080331, filed October 3, 2007, also describes formulations to which the present invention may be applied.

[0387] Transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so highly deformable that they can easily penetrate pores smaller than droplets. Transfersomes can adapt to the environment in which they are used; for example, they self-optimize (adapt to the shape of skin pores), self-repair, often reach their targets without fragmentation, and are often self-loading. To create transfersomes, a surface edge activator, usually a surfactant, can be added to standard liposome compositions. Transfersomes have been used to deliver serum albumin to the skin. Transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0388] Surfactants have a wide range of applications in formulations such as emulsions (including microemulsions) and liposomes. The most common method of classifying and ranking the properties of the many different surfactant types, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means of classifying different surfactants used in formulations (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0389] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceutical and cosmetic products and can be used over a wide pH range. Their HLB values ​​generally range from 2 to approximately 18, depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most common component of the nonionic surfactant class.

[0390] If surfactant molecule has negative charge when dissolved or dispersed in water, surfactant is classified as anionic.Anionic surfactants include carboxylates such as soap, acyl lactylate, acyl amide of amino acid, sulfate esters such as alkyl sulfate and ethoxylated alkyl sulfate, sulfonates such as alkyl benzene sulfonate, acyl isethionate, acyl taurate and acyl sulfosuccinate, and acyl phosphate.The most important components of anionic surfactant class are alkyl sulfate and soap.

[0391] If the surfactant molecule has a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic.Cationic surfactants include quaternary ammonium salts and ethoxylated amines.Quaternary ammonium salts are the most commonly used components in this class.

[0392] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phospholipids.

[0393] The use of surfactants in pharmaceutical preparations and emulsions has been reviewed (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0394] The iRNA used in the methods of the present invention can also be provided in a micellar formulation. A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that the hydrophobic portions of the molecules all face inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. If the environment is hydrophobic, the opposite arrangement will exist.

[0395] Mixed micelle formulations suitable for transdermal delivery include siRNA compositions, alkali metal C8-C 22The mixed micelle may be prepared by mixing an aqueous solution of alkyl sulfate and a micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, hyaluronic acid pharmaceutically acceptable salts, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleate, monolaurate, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and their analogs, polidocanol alkyl ethers and their analogs, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof. The micelle-forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Mixed micelles can be formed regardless of the mixing of the components, but vigorously mixing can be used to provide smaller micelles.

[0396] In one method, a first micelle composition is prepared, containing an siRNA composition and at least an alkali metal alkyl sulfate.The first micelle composition is then mixed with at least three micelle-forming compounds to form a mixed micelle composition.In another method, a micelle composition is prepared by mixing an siRNA composition, an alkali metal alkyl sulfate, and at least one micelle-forming compound, followed by adding the remaining micelle-forming compounds with vigorous mixing.

[0397] Phenol and / or m-cresol may be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added along with the micelle-forming components. An isotonicity agent, such as glycerin, may also be added after the mixed micelle composition is formed.

[0398] To deliver the micelle formulation as a spray, the formulation can be placed in an aerosol dispenser, and the dispenser can be loaded with a propellant. The propellant under pressure is in liquid form in the dispenser. The ratio of components is adjusted so that the aqueous phase and the propellant phase are one, i.e., one phase. If there are two phases, the dispenser must be shaken before dispersing a portion of the contents, for example, through a metered valve. The dispensed dose of the pharmaceutical is sprayed from the metered valve in a fine spray.

[0399] Propellants include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In an optional embodiment, HFA 134a (1,1,1,2-tetrafluoroethane) may be used.

[0400] The specific concentrations of the essential components can be determined by relatively simple experimentation. For absorption through the oral cavity, it is often desirable to increase the dose, e.g., at least two or three times, that for administration through injection or through the gastrointestinal tract.

[0401] B. Lipid particles For example, an iRNA, such as a dsRNA, of the present invention may be fully encapsulated in a lipid formulation, such as an LNP, or other nucleic acid-lipid particle.

[0402] As used herein, the term "LNP" refers to a stable nucleic acid-lipid particle. LNPs generally contain a cationic lipid, a non-cationic lipid, and a lipid (e.g., a PEG-lipid conjugate) that prevents particle aggregation. LNPs exhibit long circulating life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site), making them extremely useful for systemic applications. Examples of LNPs include "pSPLPs" containing encapsulated condensing agent-nucleic acid conjugates, as described in International Publication No. WO 00 / 03683. The particles of the present invention generally have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially nontoxic. Additionally, when present in the nucleic acid-lipid particles of the present invention, the nucleic acid is resistant to nuclease degradation in aqueous solution. Nucleic acid-lipid particles and methods for preparing them are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120; and WO 96 / 40964.

[0403] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to dsRNA ratio) ranges from about 1:1 to about 50:1, about 1:1 to about 25:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above ranges are also considered part of the invention.

[0404] Examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLenDMA), and 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLenDMA). DLin-C-DAP, 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP).Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio(propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or or its analogs, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or mixtures thereof. The cationic lipid may comprise from about 20 mol% to about 50 mol% or about 40 mol% of the total lipid present in the particle.

[0405] In another embodiment, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.The synthesis of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. Provisional Patent Application No. 61 / 107,998, filed October 23, 2008, which is incorporated herein by reference.

[0406] In one embodiment, the lipid-siRNA particles comprise 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane:10% DSPC:40% cholesterol:10% PEG-C-DOMG (molar percentages), with a particle size of 63.0±20 nm and an siRNA / lipid ratio of 0.027.

[0407] Ionic / non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine-4-( The lipid may be an anionic or neutral lipid, including, but not limited to, N-maleimidomethyl-cyclohexane-1-carboxylic acid (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), cholesterol, or mixtures thereof. When cholesterol is included, the non-cationic lipid may be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipid present in the particle.

[0408] The conjugated lipid that inhibits particle aggregation can be, for example, a PEG-lipid, including, but not limited to, polyethylene glycol (PEG)-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl (Ci2), PEG-dimyristyloxypropyl (Ci4), PEG-dipalmityloxypropyl (Ci6), or PEG-distearyloxypropyl (C]8). The conjugated lipid that inhibits particle aggregation can be 0 mol% to about 20 mol% or about 2 mol% of the total lipid present in the particle....

Claims

1. 1. A double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of angiotensinogen (AGT) in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2; substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides; and The sense strand is complexed with a ligand attached at the 3' end, the double stranded RNAi agent.

2. 1. A double-stranded ribonucleic acid (RNAi) agent for inhibiting angiotensinogen (AGT) expression in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region; The sense strand is nucleotides 2801-2101; 803-843; 834-859; 803-859; 803-875; 834-875; 847-875; 1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1624; 1584-1621; 2035-2144; 2070-2144; 2070-2103; 2201-2223; 2227-2360; 2227-2304; 2290-2318; 2304-2350; 2304-2326; 2320-2342; 2333 of the nucleotide sequence of SEQ ID NO:

1. -2360;2333-2358;485-503;517-535;560-578;635-653;803-821;814-832;822-840;825-843;834-852;837-855;841-859;855-873;967-985;1247- 1265;1248-1266;1249-1267;1251-1269;1253-1271;1566-1584;1570-1588;1572-1590;1574-1592;1584-1602;1587-1605;1591-1609;1592-1610; 1595-1613; 1601-1619; 1602-1620; 1605-1623; 1729-1747; 1738-1756; 1739-1757; 1741-1769; 1767-1785; 1810-1828; 1827-1845; 1880-1989; 1892- 1914;1894-1914;1894-2012;2035-2053;2046-2064;2057-2075;2070-2088;2072-2090;2078-2096;2078-2107;2078-2011;2080-2098;2081-2099; 2081-2104; 2081-2011; 2082-2100; 2084-2102; 2084-2011; 2090-2108; 2100-2118; 2111-2129; 2124-2142; 2125-2143; 2167-2185; 2179-2197; 2201- 2219;2202-2220;2203-2221;2204-2222;2227-2245;2230-2248;2234-2252;2244-2264;2255-2273;2266-2284;2268-2286;2270-2288;2279-2297;2281-2299; 2283-2301; 2284-2302; 2285-2303; 2286-2304; 2288-2306; 2290-2308; 2291-2309; 2291-2311; 2291-2318; 2291-2315; 2292-2310; 2294-2312; 22 96-2314; 2299-2317; 2304-2322; 2304-2329; 2306-2324; 2307-2325; 2309-2327; 2309-2329; 2309-2342; 2309-2350; 2309-2358; 2314-2332; 2316-2334; 2317- or 2341-2359, the antisense strand comprising at least 15 contiguous nucleotides which differ by no more than 3 nucleotides from said nucleotides at the corresponding positions in the nucleotide sequence of SEQ ID NO:2, and A double-stranded RNAi agent, wherein the antisense strand is substantially complementary to the sense strand.

3. The double stranded RNAi agent of claim 2, wherein substantially all of the nucleotides of the sense strand are modified nucleotides, substantially all of the nucleotides of the antisense strand are modified nucleotides, or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides.

4. The double-stranded RNAi agent of claim 2, wherein the sense strand is complexed with a ligand attached at the 3' end.

5. The double-stranded RNAi agent of any one of claims 1 to 4, wherein every nucleotide of the sense strand and every nucleotide of the antisense strand are modified nucleotides.

6. The double-stranded RNAi agent of any of claims 1-4, wherein the sense strand and the antisense strand comprise a complementary region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the sequences listed in any one of Tables 3, 4, 7, 8, 11, 13, and 15.

7. The double-stranded RNAi agent of any of claims 1 and 3-6, wherein at least one of the modified nucleotides is selected from the group consisting of a 3' terminal deoxythymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, a non-locked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base containing nucleotide, a nucleotide comprising a 5'-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl ester derivative or a dodecanoic acid bisdecylamide group.

8. The double-stranded RNAi agent of any one of claims 1 to 7, wherein at least one strand comprises a 3' overhang of at least one nucleotide.

9. The double-stranded RNAi agent of any one of claims 1 to 7, wherein at least one strand comprises a 3' overhang of at least two nucleotides.

10. A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting expression of angiotensinogen (AGT) in a cell, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region that is complementary to a portion of an mRNA encoding AGT, and each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent has the formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3' ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) [In the formula, i, j, k and l each independently represent 0 or 1; p, p', q and q' are each independently 0-6; Each N ａ and N ａ ' represents an oligonucleotide sequence containing 0-25 nucleotides that are either independently modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; Each N ｂ and N ｂ ' represents an oligonucleotide sequence containing 0-10 nucleotides, which are independently either modified or unmodified, or a combination thereof; each n ｐ , n ｐ ', n ｑ and n ｑ ', each of which may be present or absent, independently represent an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides; N ｂ The modification at Y is different from the modification at N. ｂ the modification at Y′ is different from the modification at Y′, and The sense strand is complexed with at least one ligand. A double-stranded RNAi drug represented by:

11. 11. The double-stranded RNAi agent of claim 10, wherein i is 0 and j is 0, or i is 1 and j is 1, or both i and j are 0, or both i and j are 1.

12. 11. The double-stranded RNAi agent of claim 10, wherein k is 0 and l is 0, or k is 1 and l is 1, or k and l are both 0, or k and l are both 1.

13. The double-stranded RNAi agent of claim 10, wherein XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'.

14. The double-stranded RNAi agent of claim 10, wherein the YYY motif occurs at or proximal to the cleavage site of the sense strand.

15. The double-stranded RNAi agent of claim 10, wherein the Y'Y'Y' motif occurs at positions 11, 12, and 13 from the 5' end of the antisense strand.

16. The double-stranded RNAi agent of claim 15, wherein Y' is 2'-O-methyl.

17. Formula (III) is represented by formula (IIIa): Sense: 5'n ｐ -N ａ -YYY-N ａ -n ｑ 3' Antisense: 3' ｐ '-N ａ’ -Y'Y'Y'-N ａ '-n ｑ The double-stranded RNAi agent of claim 10, represented by:

18. Formula (III) is a compound represented by formula (IIIb): Sense: 5'n ｐ -N ａ -YYY-N ｂ -ZZZ-N ａ -n ｑ 3' Antisense: 3' ｐ '-N ａ '-Y'Y'Y'-N ｂ '-Z'Z'Z'-N ａ '-n ｑ '5' (IIIb) [In the formula, each N ｂ and N ｂ ' independently represent an oligonucleotide sequence containing 1-5 modified nucleotides. The double-stranded RNAi agent of claim 10, wherein

19. Formula (III) is represented by formula (IIIc): Sense: 5'n ｐ -N ａ -XXX-N ｂ -YYY-N ａ -n ｑ 3' Antisense: 3' ｐ '-N ａ '-X'X'X'-N ｂ '-Y'Y'Y'-N ａ '-n ｑ '5' (IIIc) [In the formula, each N ｂ and N ｂ ' independently represent an oligonucleotide sequence containing 1-5 modified nucleotides. The double-stranded RNAi agent of claim 10, wherein

20. Formula (III) represents the formula (IIId): Sense: 5'n ｐ -N ａ -XXX-N ｂ -YYY-N ｂ -ZZZ-N ａ -n ｑ 3' Antisense: 3' ｐ '-N ａ '-X'X'X'-N ｂ '-Y'Y'Y'-N ｂ '-Z'Z'Z'-N ａ '-n ｑ '5' (IIId) [In the formula, each N ｂ and N ｂ Each N′ independently represents an oligonucleotide sequence containing 1-5 modified nucleotides; ａ and N ａ ' independently represent an oligonucleotide sequence containing 2-10 modified nucleotides. The double-stranded RNAi agent of claim 10, wherein

21. The double-stranded RNAi agent of any of claims 1 to 4 or claim 10, wherein the double-stranded region is 15-30 nucleotide pairs in length.

22. The double-stranded RNAi agent of claim 21, wherein the double-stranded region is 17-23 nucleotide pairs in length.

23. The double-stranded RNAi agent of claim 21, wherein the double-stranded region is 17-25 nucleotide pairs in length.

24. The double-stranded RNAi agent of claim 21, wherein the double-stranded region is 23-27 nucleotide pairs in length.

25. The double-stranded RNAi agent of claim 21, wherein the double-stranded region is 19-21 nucleotide pairs in length.

26. The double-stranded RNAi agent of claim 21, wherein the double-stranded region is 21-23 nucleotide pairs in length.

27. The double-stranded RNAi agent of any of claims 1 to 4 and 10, wherein each strand has 15-30 nucleotides.

28. The double-stranded RNAi agent of any of claims 1 to 4 and 10, wherein each strand has 19-30 nucleotides.

29. 11. The double stranded RNAi agent of claim 10, wherein the modification in the nucleotide is selected from the group consisting of LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.

30. The double-stranded RNAi agent of claim 29, wherein the modification in the nucleotide is a 2'-O-methyl modification or a 2'-fluoro modification.

31. The double-stranded RNAi agent of any of claims 1, 4, and 10, wherein the ligand is one or more GalNAc derivatives attached via a bivalent linker or a trivalent branched linker.

32. The ligand is 【Chemistry 1】 The double-stranded RNAi agent of any of claims 1, 4 and 10, wherein

33. The double-stranded RNAi agent of any one of claims 1, 4 and 10, wherein the ligand is attached to the 3' end of the sense strand.

34. 5. The RNAi agent comprising: 【Chemistry 2】 [In the formula, X is O or S.] The double-stranded RNAi agent of claim 33, wherein the double-stranded RNAi agent is complexed with a ligand represented by the formula:

35. The double-stranded RNAi agent of any of claims 1 to 4 and 10, wherein the RNAi agent further comprises at least one phosphorothioate or methyl nucleotide or methylphosphonate internucleotide linkage.

36. 36. The double-stranded RNAi agent of claim 35, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand.

37. 37. The double-stranded RNAi agent of claim 36, wherein the strand is an antisense strand.

38. 37. The double-stranded RNAi agent of claim 36, wherein the strand is the sense strand.

39. 36. The double stranded RNAi agent of claim 35, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand.

40. 40. The double-stranded RNAi agent of claim 39, wherein the strand is an antisense strand.

41. 40. The double stranded RNAi agent of claim 39, wherein the strand is the sense strand.

42. 36. The double-stranded RNAi agent of claim 35, wherein the phosphorothioate or methylphosphonate internucleotide linkages are at both the 5' and 3' ends of a strand.

43. 43. The double-stranded RNAi agent of claim 42, wherein the strand is an antisense strand.

44. The double-stranded RNAi agent of claim 35, wherein the RNAi agent comprises 6-8 phosphorothioate internucleotide linkages.

45. The double-stranded RNAi agent of claim 44, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

46. The double-stranded RNAi agent of any one of claims 1 to 4 and 10, wherein the base pair at one position at the 5' end of the antisense strand is an AU base pair.

47. The double-stranded RNAi agent of claim 10, wherein the Y nucleotide comprises a 2'-fluoro modification.

48. The double-stranded RNAi agent of claim 10, wherein the Y' nucleotide comprises a 2'-O-methyl modification.

49. The double-stranded RNAi agent of claim 10, wherein p'>0.

50. The double-stranded RNAi agent of claim 10, wherein p'=2.

51. 51. The double-stranded RNAi agent of claim 50, wherein q'=0, p=0, q=0, and the p' overhanging nucleotides are complementary to the target mRNA.

52. 51. The double-stranded RNAi agent of claim 50, wherein q'=0, p=0, q=0, and the p' overhanging nucleotide is not complementary to the target mRNA.

53. 51. The double-stranded RNAi agent of claim 50, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

54. At least one n ｐ 54. The double stranded RNAi agent of any of claims 49-53, wherein:

55. All n ｐ 55. The double stranded RNAi agent of claim 54, wherein:

56. The double-stranded RNAi agent of any of claims 1 to 4 and 10, wherein the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 3, 4, 7, 8, 11, 13 and 15.

57. A double-stranded ribonucleic acid (RNAi) agent for inhibiting expression of angiotensinogen (AGT), comprising: the double-stranded RNAi agent comprises a sense strand and an antisense strand that form a double-stranded region; wherein the sense strand comprises at least 15 contiguous nucleotides which differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides which differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2; substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end; substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and The double-stranded RNAi agent, wherein the sense strand is conjugated to one or more GalNAc derivatives attached to the 3' end via a bivalent linker or a trivalent branched linker.

58. The sense strand is selected from the group consisting of nucleotides 2801-2101; 803-843; 834-859; 803-859; 803-875; 834-875; 847-875; 1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1624; 1584-1621; 2035-2144; 2070-2144; 2070-2103; 2201-2223; 2227-2360; 2227-2304; 2290-2318; 2304-2350; 2304-2326; 2320-2342; 2333 of the nucleotide sequence of SEQ ID NO:

1. -2360;2333-2358;485-503;517-535;560-578;635-653;803-821;814-832;822-840;825-843;834-852;837-855;841-859;855-873;967-985;1247- 1265;1248-1266;1249-1267;1251-1269;1253-1271;1566-1584;1570-1588;1572-1590;1574-1592;1584-1602;1587-1605;1591-1609;1592-1610; 1595-1613; 1601-1619; 1602-1620; 1605-1623; 1729-1747; 1738-1756; 1739-1757; 1741-1769; 1767-1785; 1810-1828; 1827-1845; 1880-1989; 1892- 1914;1894-1914;1894-2012;2035-2053;2046-2064;2057-2075;2070-2088;2072-2090;2078-2096;2078-2107;2078-2011;2080-2098;2081-2099; 2081-2104; 2081-2011; 2082-2100; 2084-2102; 2084-2011; 2090-2108; 2100-2118; 2111-2129; 2124-2142; 2125-2143; 2167-2185; 2179-2197; 2201- 2219;2202-2220;2203-2221;2204-2222;2227-2245;2230-2248;2234-2252;2244-2264;2255-2273;2266-2284;2268-2286;2270-2288;2279-2297;2281-2299; 2283-2301; 2284-2302; 2285-2303; 2286-2304; 2288-2306; 2290-2308; 2291-2309; 2291-2311; 2291-2318; 2291-2315; 2292-2310; 2294-2312; 22 96-2314; 2299-2317; 2304-2322; 2304-2329; 2306-2324; 2307-2325; 2309-2327; 2309-2329; 2309-2342; 2309-2350; 2309-2358; 2314-2332; 2316-2334; 2317- or 2341-2359, the antisense strand comprising at least 15 contiguous nucleotides which differ by no more than 3 nucleotides from said nucleotides at the corresponding positions in the nucleotide sequence of SEQ ID NO:2, and 58. The double-stranded RNAi agent of claim 57, wherein the antisense strand is substantially complementary to the sense strand.

59. 59. The double-stranded RNAi agent of claim 57 or 58, wherein every nucleotide of the sense strand and every nucleotide of the antisense strand comprises a modification.

60. 1. A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting expression of angiotensinogen (AGT) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding AGT, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3' ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) [In the formula, i, j, k and l each independently represent 0 or 1; p, p', q and q' are each independently 0-6; Each N ａ and N ａ ' represents an oligonucleotide sequence containing 0-25 nucleotides that are either independently modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; Each N ｂ and N ｂ ' represents an oligonucleotide sequence containing 0-10 nucleotides, which are independently either modified or unmodified, or a combination thereof; each n ｐ , n ｐ ', n ｑ and n ｑ ', each of which may be present or absent, independently represent an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ The modification at Y is different from the modification at N. ｂ the modification at Y′ is different from the modification at Y′, and The sense strand is complexed with at least one ligand. A double-stranded RNAi drug represented by:

61. 1. A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting expression of angiotensinogen (AGT) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding AGT, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3' ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) [In the formula, i, j, k and l each independently represent 0 or 1; each n ｐ , n ｐ ', n ｑ and n ｑ ', each of which may be present or absent, independently represent an overhanging nucleotide; p, q and q′ are each independently 0-6; n ｐ '>0, and at least one n ｐ ' is linked to adjacent nucleotides via phosphorothioate bonds, Each N ａ and N ａ ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; Each N ｂ and N ｂ ' represents an oligonucleotide sequence containing 0-10 nucleotides, which are independently either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ The modification at Y is different from the modification at N. ｂ the modification at Y′ is different from the modification at Y′, and The sense strand is complexed with at least one ligand. A double-stranded RNAi drug represented by:

62. 1. A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting expression of angiotensinogen (AGT) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding AGT, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3' ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) [In the formula, i, j, k and l each independently represent 0 or 1; each n ｐ , n ｐ ', n ｑ and n ｑ ', each of which may be present or absent, independently represent an overhanging nucleotide; p, q and q′ are each independently 0-6; n ｐ '>0, and at least one n ｐ ' is linked to adjacent nucleotides via phosphorothioate bonds, Each N ａ and N ａ ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; Each N ｂ and N ｂ ' represents an oligonucleotide sequence containing 0-10 nucleotides, which are independently either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ The modification at Y is different from the modification at N. ｂ the modification at Y′ is different from the modification at Y′, and The sense strand is complexed with at least one ligand, where the ligand is one or more GalNAc derivatives attached via a bivalent linker or a trivalent branched linker. A double-stranded RNAi drug represented by:

63. 1. A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting expression of angiotensinogen (AGT) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding AGT, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ - (XXX) ｉ -N ｂ -YYY-N ｂ - (ZZZ) ｊ -N ａ -n ｑ 3' Antisense: 3' ｐ '-N ａ '-(X'X'X') ｋ -N ｂ '-Y'Y'Y'-N ｂ '-(Z'Z'Z') ｌ -N ａ '-n ｑ '5' (III) [In the formula, i, j, k and l each independently represent 0 or 1; each n ｐ , n ｐ ', n ｑ and n ｑ ', each of which may be present or absent, independently represent an overhanging nucleotide; p, q and q′ are each independently 0-6; n ｐ '>0, and at least one n ｐ ' is linked to adjacent nucleotides via phosphorothioate bonds, Each N ａ and N ａ ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; Each N ｂ and N ｂ ' represents an oligonucleotide sequence containing 0-10 nucleotides, which are independently either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y' and Z'Z'Z' each independently represent one motif of three identical modifications in three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; N ｂ The modification at Y is different from the modification at N. ｂ the modification at Y′ is different from the modification at Y′, and the sense strand comprises at least one phosphorothioate bond; The sense strand is complexed with at least one ligand, where the ligand is one or more GalNAc derivatives attached via a bivalent linker or a trivalent branched linker. A double-stranded RNAi drug represented by:

64. 1. A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting expression of angiotensinogen (AGT) in a cell, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of an mRNA encoding AGT, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent having a structure represented by formula (III): Sense: 5'n ｐ -N ａ -YYY-N ａ -n ｑ 3' Antisense: 3' ｐ '-N ａ '-Y'Y'Y'-N ａ '-n ｑ’ 5'(IIIa) [In the formula, each n ｐ , n ｐ ', n ｑ and n ｑ ', each of which may be present or absent, independently represent an overhanging nucleotide; p, q and q′ are each independently 0-6; n ｐ '>0, and at least one n ｐ ' is linked to adjacent nucleotides via phosphorothioate bonds, Each N ａ and N ａ ' represents an oligonucleotide sequence containing 0-25 nucleotides that are independently either modified or unmodified, or a combination thereof, each sequence containing at least two different modified nucleotides; YYY and Y'Y'Y' each independently represent a motif of three identical modifications in three consecutive nucleotides, the modifications being 2'-O-methyl or 2'-fluoro modifications; the sense strand comprises at least one phosphorothioate bond; The sense strand is complexed with at least one ligand, where the ligand is one or more GalNAc derivatives attached via a bivalent linker or a trivalent branched linker. A double-stranded RNAi drug represented by:

65. A double-stranded ribonucleic acid (RNAi) agent comprising a sense strand and an antisense strand, wherein the sense strand and the antisense strand comprise a sequence selected from the group consisting of any one of the sequences listed in any one of Tables 3, 4, 7, 8, 11, 13, and 15.

66. A composition comprising a modified antisense polynucleotide drug, the drug being capable of inhibiting expression of AGT in a cell and comprising a sequence complementary to a sense sequence selected from the group of sequences listed in any one of Tables 3, 4, 7, 8, 11, 13 and 15, the polynucleotide being about 14 to about 30 nucleotides in length.

67. A vector comprising the double-stranded RNAi agent of claim 2.

68. A cell comprising the double-stranded RNAi agent of any one of claims 1-4, 10, 57, 58, and 60-65.

69. A pharmaceutical composition comprising the double-stranded RNAi agent of any one of claims 1-4, 10, 57, 58 and 60-65, or a composition comprising the modified antisense polynucleotide agent of claim 66, or a vector of claim 67.

70. 70. The pharmaceutical composition of claim 69, wherein the double-stranded RNAi agent is administered in a non-buffered solution.

71. 71. The pharmaceutical composition of claim 70, wherein the non-buffered solution is saline or water.

72. 70. The pharmaceutical composition of claim 69, wherein the double-stranded RNAi agent is administered with a buffer.

73. 73. The pharmaceutical composition of claim 72, wherein the buffer comprises an acetate buffer, a citrate buffer, a prolamine, a carbonate buffer, or a phosphate buffer, or any combination thereof.

74. 74. The pharmaceutical composition of claim 73, wherein the buffer is phosphate buffered saline (PBS).

75. 1. A method for inhibiting angiotensinogen (AGT) expression in a cell, comprising: (a) contacting a cell with a composition comprising a double-stranded RNAi agent of any one of claims 1-4, 10, 57, 58, and 60-65, or a modified antisense polynucleotide agent of claim 66, a vector of claim 67, or a pharmaceutical composition of any one of claims 69-74; and (b) maintaining the cells produced in step (a) for a time sufficient to allow the mRNA transcripts of the AGT gene to be degraded, thereby inhibiting expression of the AGT gene in the cells. A method comprising:

76. 76. The method of claim 75, wherein the cell is present in a subject.

77. 77. The method of claim 76, wherein the subject is a human.

78. 78. The method of claim 77, wherein the subject is suffering from an angiotensinogen-related disorder.

79. 79. The method according to any one of claims 75 to 78, wherein AGT expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%.

80. A method of treating a subject having an angiotensinogen (AGT)-related disorder, comprising administering to the subject a therapeutically effective amount of a composition comprising the double-stranded RNAi agent of any one of claims 1-4, 10, 57, 58, and 60-65, or the modified antisense polynucleotide agent of claim 66, the vector of claim 67, or the pharmaceutical composition of any one of claims 69-74, thereby treating the subject.

81. 1. A method of treating a subject having an angiotensinogen (AGT)-related disorder, comprising subcutaneously administering to the subject a therapeutically effective amount of a double-stranded ribonucleic acid (RNAi) agent, thereby treating the subject, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region; the sense strand comprises at least 15 contiguous nucleotides which differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides which differ by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2; substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end; substantially all of the nucleotides of the sense strand comprise a modification selected from the group consisting of a 2'-O-methyl modification and a 2'-fluoro modification; the sense strand comprises two phosphorothioate internucleotide linkages at the 5' end, and The sense strand is conjugated to one or more GalNAc derivatives attached to the 3' end via a bivalent linker or a trivalent branched linker. method.

82. the sense strand is selected from the group consisting of nucleotides 2801-2101; 803-843; 834-859; 803-859; 803-875; 834-875; 847-875; 1247-1271; 1566-1624; 1570-1624; 1584-1624; 1584-1624; 1584-1621; 2035-2144; 2070-2144; 2070-2103; 2201-2223; 2227-2360; 2227-2304; 2290-2318; 2304-2350; 2304-2326; 2320-2342; 2333 of the nucleotide sequence of SEQ ID NO:1; -2360;2333-2358;485-503;517-535;560-578;635-653;803-821;814-832;822-840;825-843;834-852;837-855;841-859;855-873;967-985;1247- 1265;1248-1266;1249-1267;1251-1269;1253-1271;1566-1584;1570-1588;1572-1590;1574-1592;1584-1602;1587-1605;1591-1609;1592-1610; 1595-1613; 1601-1619; 1602-1620; 1605-1623; 1729-1747; 1738-1756; 1739-1757; 1741-1769; 1767-1785; 1810-1828; 1827-1845; 1880-1989; 1892- 1914;1894-1914;1894-2012;2035-2053;2046-2064;2057-2075;2070-2088;2072-2090;2078-2096;2078-2107;2078-2011;2080-2098;2081-2099; 2081-2104; 2081-2011; 2082-2100; 2084-2102; 2084-2011; 2090-2108; 2100-2118; 2111-2129; 2124-2142; 2125-2143; 2167-2185; 2179-2197; 2201- 2219;2202-2220;2203-2221;2204-2222;2227-2245;2230-2248;2234-2252;2244-2264;2255-2273;2266-2284;2268-2286;2270-2288;2279-2297;2281-2299; 2283-2301; 2284-2302; 2285-2303; 2286-2304; 2288-2306; 2290-2308; 2291-2309; 2291-2311; 2291-2318; 2291-2315; 2292-2310; 2294-2312; 229 6-2314; 2299-2317; 2304-2322; 2304-2329; 2306-2324; 2307-2325; 2309-2327; 2309-2329; 2309-2342; 2309-2350; 2309-2358; 2314-2332; 2316-2334; 2317-23 2339-2357; 2340-2358; or 2341-2359, and the antisense strand comprises at least 15 contiguous nucleotides which differ by no more than 3 nucleotides from the nucleotides at the corresponding positions in the nucleotide sequence of SEQ ID NO:2, and 82. The method of claim 81, wherein the antisense strand is substantially complementary to the sense strand.

83. 83. The method of claim 81 or 82, wherein every nucleotide of the sense strand and every nucleotide of the antisense strand comprises a modification.

84. The method of any one of claims 81-83, wherein the subject is a human.

85. Angiotensinogen-related disorders include hypertension, borderline hypertension, primary hypertension, secondary hypertension, hypertensive emergency, hypertensive urgency, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, labile hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetes 84. The method of any one of claims 81 to 83, wherein the condition is selected from the group consisting of chronic cardiomyopathy, glomerulosclerosis, aortic stenosis, aortic aneurysm, endocardial fibrosis, Cushing's syndrome and other glucocorticoid excess states including chronic steroid therapy, pheochromocytoma, reninoma, secondary aldosteronism and other mineralocorticoid excess states, sleep apnea, thyroid / parathyroid disease, heart failure, myocardial infarction, angina pectoris, stroke, diabetes mellitus, renal disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), and fetal growth restriction.

86. 84. The method of any one of claims 81 to 83, wherein the angiotensinogen-related disorder is pregnancy-associated hypertension.

87. 84. The method of any one of claims 81-83, wherein the double-stranded RNAi agent is administered at a dose of about 0.01 mg to about 10 mg or about 0.5 mg to about 50 mg per kg of body weight.

88. 88. The method of claim 87, wherein the double-stranded RNAi agent is administered at a dose of about 10 mg to about 30 mg per kg of body weight.

89. 88. The method of claim 87, wherein the double-stranded RNAi agent is administered at a dose of about 3 mg per kg of body weight.

90. 88. The method of claim 87, wherein the double-stranded RNAi agent is administered at a dose of about 10 mg per kg of body weight.

91. 88. The method of claim 87, wherein the double-stranded RNAi agent is administered at a dose of about 0.5 mg per kg of body weight twice weekly.

92. 88. The method of claim 87, wherein the double-stranded RNAi agent is administered at a dose of about 10 mg per kg of body weight every other week.

93. 88. The method of claim 87, wherein the double-stranded RNAi agent is administered once a week at a dosage of about 0.5-1 mg per kg of body weight.

94. 88. The method of claim 87, wherein the double-stranded RNAi agent is administered subcutaneously.

95. 88. The method of claim 87, wherein the double-stranded RNAi agent is administered intravenously.

96. 88. The method of claim 87, wherein the RNAi agent is administered in two or more doses.

97. 97. The method of claim 96, wherein the RNAi agent is administered at an interval selected from the group consisting of one administration about every 12 hours, about every 24 hours, about every 48 hours, about every 72 hours, and about every 96 hours.

98. 97. The method of claim 96, wherein the RNAi agent is administered twice a week.

99. 97. The method of claim 96, wherein the RNAi agent is administered every two weeks.

100. 84. The method of any one of claims 81 to 83, comprising administering to the subject a further therapeutic agent.

101. Further therapeutic agents include diuretics, angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta blockers, vasodilators, calcium channel blockers, aldosterone antagonists, alpha ２ The method of claim 100, wherein the antihypertensive agent is selected from the group consisting of a renin inhibitor, a renin blocker, a peripherally acting adrenergic agonist, a selective D1 receptor partial agonist, a non-selective alpha-adrenergic antagonist, a synthetic steroidal antimineralocorticoid drug, or any combination of the above, and a hypertension medication formulated as a combination agent.

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