CN118076361A

CN118076361A - Cell death-inducing DFFA-like effector B (CIDEB) iRNA compositions and methods of use thereof

Info

Publication number: CN118076361A
Application number: CN202280058994.5A
Authority: CN
Inventors: J·祖贝; J·D·麦金因克; M·K·施莱格尔; A·卡斯托雷诺; L·邦杜兰特; D·奥扎
Original assignee: Alnylam Pharmaceuticals Inc
Current assignee: Alnylam Pharmaceuticals Inc
Priority date: 2021-08-31
Filing date: 2022-08-31
Publication date: 2024-05-24

Abstract

The present invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting CIDEB genes, and methods of inhibiting CIDEB expression, and methods of using such dsRNA compositions to treat subjects that would benefit from reduced CIDEB expression, such as subjects having CIDEB-associated diseases, disorders, or conditions.

Description

Cell death-inducing DFFA-like effector B (CIDEB) iRNA compositions and methods of use thereof

Cross reference to related applications

The present application claims the benefit of priority from U.S. provisional application No. 63/239,271, filed on month 8, 2021, and claims the benefit of priority from U.S. provisional application No. 63/341,848, filed on month 5, 2022, 13. The entire contents of the foregoing application are hereby incorporated by reference. Sequence listing

The present application comprises a sequence listing that has been electronically submitted in XML format and is hereby incorporated by reference in its entirety. The XML copy created at month 8 and 30 of 2022 was named A108868_1300WO_SL.xml and was 3,873,182 bytes in size.

Background

Cell death induces DFFA-like effector B (CIDEB), a member of the CIDE protein family, to be expressed primarily in the liver and small intestine. CIDEB are Endoplasmic Reticulum (ER) and Lipid Droplet (LD) related proteins. Overexpression of CIDEB protein induces cell death, but the physiological function of CIDEB is more closely related to various lipid metabolic pathways, particularly the VLDL pathway.

Non-alcoholic fatty liver disease (NAFLD) is the most common form of liver disease in all modern industrialized economic body areas of the world, including korea and many other asian countries. Patients often exhibit no symptoms or no specificity in clinical features. In contrast, liver abnormalities are accidentally discovered by liver imaging, particularly by ultrasound examination, and/or the presence of elevated liver enzymes (alanine aminotransferase [ ALT ] and gamma-glutamyl transpeptidase). Diagnosis of NAFLD requires exclusion of other conditions, particularly viral hepatitis, heavy drinking and exposure to potentially hepatotoxic drugs. According to protocols such as the asia-pacific NAFLD guidelines, the term NAFLD now remains for cases of fatty liver associated with overnutrition metabolic complications, often with central obesity and overweight.

Nonalcoholic steatohepatitis (NASH) is considered to be a progressive form of nonalcoholic fatty liver disease (NAFLD) and is characterized by liver steatosis, inflammation, hepatocyte damage, and varying degrees of fibrosis. Adipose tissue dysfunction and liver inflammatory response play an important role during NASH development. Cell and molecular response mechanisms also promote liver inflammation by inducing a chronic inflammatory response that leads to hepatocyte damage in the absence of fatty liver.

Thus, there is a need for improved methods of treating chronic inflammatory diseases of the liver, such as NASH, comprising agents capable of selectively and effectively inhibiting the CIDEB gene.

Disclosure of Invention

There is a need for improved methods of treating chronic inflammatory diseases of the liver, such as NASH, including agents capable of selectively and effectively inhibiting the CIDEB gene. Current standard of care for subjects with chronic inflammatory diseases includes lifestyle changes (diet and exercise, smoking cessation, alcohol withdrawal, etc.), steroid and/or non-steroid anti-inflammatory drugs, management of related co-morbidities (e.g., hypertension, hyperlipidemia, diabetes), and the like. Once established, chronic inflammatory conditions can sustain inflammation, tissue damage, release of pro-inflammatory injury associated molecular patterns (DAMP) from damaged cells, and self-sustaining circulation of cytokine release, resulting in further inflammation. Elimination of liver inflammation can be achieved by using active, physiological pro-resolution mechanisms rather than classical passive blocking of pro-inflammatory mediators. (Schuster et al, nature review-gastroenterology and Hepatology (Nature Reviews Gastroenterology & Hepatology), vol.15, pp.349-364, 2018).

The present invention provides iRNA compositions that affect RNA-induced silencing complex (RISC) -mediated cleavage of an RNA transcript of a cell death-inducing DFFA-like effector b (CIDEB) gene. The CIDEB gene may be within a cell, for example, a cell in a subject (e.g., human). The invention also provides methods of using the iRNA compositions of the invention to inhibit CIDEB gene expression and/or treat a subject that would benefit from inhibiting or reducing CIDEB gene expression, e.g., a subject suffering from or susceptible to a CIDEB-related disease (e.g., a chronic inflammatory disease).

Accordingly, in one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell expression of cell death-induced DFFA-like effector b (CIDEB). The dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises a sequence that hybridizes to SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides differing by NO more than 1, 2 or 3 nucleotides from the nucleotide sequence of SEQ ID NO:2 by at least 15 consecutive nucleotides differing by no more than 1, 2 or 3 nucleotides. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises a sequence from SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence from SEQ ID NO:2, and at least 15 consecutive nucleotides of the nucleotide sequence of 2.

In another aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB). The dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to an mRNA encoding CIDEB comprising at least 15 contiguous nucleotides differing by no more than 1,2, or 3 nucleotides from any of the antisense sequences listed in tables 3-6. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to an mRNA encoding CIDEB comprising at least 15 contiguous nucleotides from any one of the antisense sequences listed in tables 3-6.

In one embodiment, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell death-induced expression of DFFA-like effector b (CIDEB), wherein the dsRNA agent comprises: (a) An antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700555; and a sense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700555; (b) An antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700821; and a sense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700821; (c) An antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700369; and a sense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700369; (d) An antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1699976; and a sense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1699976; (e) An antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700374; and a sense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700374; (f) An antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700314; and a sense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700314; (g) An antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700376; and a sense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700376; (h) An antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1699964; and a sense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1699964; or (i) an antisense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700556; and a sense strand comprising a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1, 2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700556.

In another embodiment, the complementing region comprises a sequence identical to SEQ ID NO:1, or at least 15 consecutive nucleotides 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2095-2117、2096-2118、2102-2124、2103-2125、2106-2128、2109-2131、2111-2133、2112-2134、2113-2135、2115-2137、2117-2139、2241-2263、2250-2272、2253-2275、2300-2322、2301-2323、2343-2365、2347-2369、2349-2371、1796-1818、1270-1292、1624-1646、1795-1817、1822-1844、1847-1869、1582-1604、1582-1604、1582-1604、1570-1592、2110-2132、1954-1976、2013-2035、1840-1862、2114-2136、1565-1587、1808-1830、2249-2271、1614-1636、1655-1677、1581-1603、1717-1739、1717-1739、1717-1739、1883-1905、1894-1916、1957-1979、1957-1979、1957-1979、1786-1808、1681-1703、1589-1611、2098-2120、1327-1349、2432-2454、1374-1396、1911-1933、1372-1394、1816-1838、2240-2262、1577-1599、2105-2127、2092-2114、1951-1973、2346-2368、1781-1803、2014-2036、1410-1432、1940-1962、1718-1740、1859-1881、1744-1766、1787-1809、1787-1809、1787-1809、1855-1877 or 2082-2104 that differ by no more than 1, 2, or 3 nucleotides. In some embodiments, the complementary region comprises a sequence from SEQ ID NO:1 or at least 15 consecutive nucleotides ：29-51、67-89、154-176、163-185、173-195、184-206、196-218、206-228、257-279、270-292、446-468、459-481、468-490、518-540、530-552、641-663、687-709、702-724、711-733、727-749、758-780、769-791、781-803、790-812、807-829、839-861、850-872、874-896、907-929、917-939、958-980、974-996、983-1005、999-1021、1009-1031、1018-1040、1027-1049、1036-1058、1045-1067、1054-1076、1080-1102、1089-1111、1098-1120、1108-1130、1140-1162、1156-1178、1174-1196、1183-1205、1192-1214、1205-1227、1214-1236、1225-1247、1254-1276、1263-1285、1273-1295、1282-1304、1292-1314、1316-1338、1337-1359、1350-1372、1364-1386、1375-1397、1408-1430、1417-1439、1429-1451、1454-147、1478-1500、1487-1509、1496-1518、1507-1529、1519-1541、1542-1564、1552-1574、1562-1584、1573-1595、1585-1607、1597-1619、1607-1629、1623-1645、1633-1655、1642-1664、1651-1673、1668-1690、1677-1699、1691-1713、1700-1722、1712-1734、1749-1771、1764-1786、1773-1795、1784-1806、1796-1818、1807-1829、1824-1846、1833-1855、1847-1869、1856-1878、1865-1887、1881-1903、1896-1918、1905-1927、1921-1943、1938-1960、1948-1970、1961-1983、1970-1992、1994-2016、2008-2030、2017-2039、2056-2078、2066-2088、2075-2097、2087-2109、2096-2118、2106-2128、2116-2138、2129-2151、2176-2198、2185-2207、2196-2218、2207-2229、2220-2242、2236-2258、2247-2269、2256-2278、2265-2287、2274-2296、2299-2321、2309-2331、2318-2340、2349-2371、2371-2393、2382-2404、2391-2413、2401-2423、1267-1289、1270-1292、1271-1293、1272-1294、1273-1295、1274-1296、1275-1297、1276-1298、1278-1300、1285-1307、1294-1316、1295-1317、1327-1349、1330-1352、1371-1393、1372-1394、1374-1396、1407-1429、1410-1432、1413-1435、1414-1436、1415-1437、1416-1438、1419-1441、1420-1442、1421-1443、1422-1444、1425-1447、1426-1448、1427-1449、1428-1450、1429-1451、1430-1452、1431-1453、1432-1454、1433-1455、1478-1500、1498-1520、1500-1522、1501-1523、1502-1524、1503-1525、1504-1526、1545-1567、1548-1570、1549-1571、1550-1572、1551-1573、1559-1581、1560-1582、1562-1584、1565-1587、1567-1589、1568-1590、1569-1591、1572-1594、1577-1599、1580-1602、1581-1603、1582-1604、1583-1605、1584-1606、1589-1611、1590-1612、1593-1615、1616-1638、1617-1639、1624-1646、1626-1648、1627-1649、1628-1650、1634-1656、1635-1657、1648-1670、1655-1677、1656-1678、1657-1679、1658-1680、1659-1681、1661-1683、1681-1703、1710-1732、1711-1733、1712-1734、1713-1735、1716-1738、1717-1739、1718-1740、1720-1742、1744-1766、1751-1773、1752-1774、1775-1797、1781-1803、1784-1806、1786-1808、1787-1809、1788-1810、1789-1811、1790-1812、1795-1817、1796-1818、1797-1819、1799-1821、1800-1822、1801-1823、1808-1830、1811-1833、1816-1838、1822-1844、1824-1846、1825-1847、1826-1848、1827-1849、1828-1850、1829-1851、1830-1852、1831-1853、1837-1859、1838-1860、1840-1862、1841-1863、1842-1864、1843-1865、1844-1866、1846-1868、1847-1869、1848-1870、1850-1872、1855-1877、1856-1878、1857-1879、1858-1880、1859-1881、1860-1882、1880-1902、1882-1904、1883-1905、1885-1907、1886-1908、1894-1916、1895-1917、1896-1918、1897-1919、1898-1920、1899-1921、1900-1922、1911-1933、1933-1955、1934-1956、1936-1958、1937-1959、1940-1962、1945-1967、1946-1968、1948-1970、1949-1971、1951-1973、1954-1976、1957-1979、1958-1980、1959-1981、1960-1982、1961-1983、1962-1984、2011-2033、2013-2035、2014-2036、2016-2038、2074-2096、2076-2098、2082-2104、2085-2107、2086-2108、2087-2109、2088-2110、2089-2111、2090-2112、2092-2114、2095-2117、2098-2120、2105-2127、2107-2129、2108-2130、2110-2132、2112-2134、2114-2136、2192-2214、2239-2261、2240-2262、2249-2271、2250-2272、2253-2275、2300-2322、2346-2368、2347-2369、2348-2370、2432-2454、2433-2455、2434-2456、1267-1289、1276-1298、1277-1299、1279-1301、1283-1305、1284-1306、1285-1307、1286-1308、1292-1314、1295-1317、1319-1341、1328-1350、1329-1351、1330-1352、1331-1353、1332-1354、1340-1362、1341-1363、1342-1364、1343-1365、1344-1366、1345-1367、1346-1368、1368-1390、1371-1393、1373-1395、1375-1397、1408-1430、1417-1439、1418-1440、1419-1441、1423-1445、1424-1446、1430-1452、1431-1453、1437-1459、1443-1465、1478-1500、1503-1525、1512-1534、1544-1566、1545-1567、1546-1568、1547-1569、1552-1574、1553-1575、1560-1582、1561-1583、1563-1585、1566-1588、1567-1589、1570-1592、1571-1593、1572-1594、1573-1595、1574-1596、1578-1600、1579-1601、1580-1602、1584-1606、1585-1607、1586-1608、1593-1615、1595-1617、1599-1621、1600-1622、1603-1625、1609-1631、1611-1633、1612-1634、1613-1635、1614-1636、1616-1638、1618-1640、1619-1641、1620-1642、1621-1643、1622-1644、1623-1645、1625-1647、1629-1651、1632-1654、1633-1655、1635-1657、1640-1662、1645-1667、1647-1669、1651-1673、1656-1678、1657-1679、1660-1682、1680-1702、1704-1726、1705-1727、1707-1729、1709-1731、1713-1735、1714-1736、1715-1737、1716-1738、1719-1741、1720-1742、1749-1771、1773-1795、1774-1796、1775-1797、1776-1798、1778-1800、1782-1804、1783-1805、1784-1806、1785-1807、1791-1813、1792-1814、1793-1815、1805-1827、1809-1831、1810-1832、1812-1834、1813-1835、1815-1837、1817-1839、1818-1840、1819-1841、1826-1848、1833-1855、1834-1856、1836-1858、1838-1860、1839-1861、1841-1863、1844-1866、1846-1868、1847-1869、1851-1873、1852-1874、1853-1875、1854-1876、1856-1878、1860-1882、1861-1883、1880-1902、1881-1903、1882-1904、1884-1906、1887-1909、1888-1910、1889-1911、1893-1915、1900-1922、1902-1924、1910-1932、1912-1934、1917-1939、1936-1958、1941-1963、1942-1964、1944-1966、1945-1967、1949-1971、1955-1977、1962-1984、2011-2033、2015-2037、2016-2038、2074-2096、2075-2097、2076-2098、2083-2105、2091-2113、2093-2115、2094-2116、2095-2117、2096-2118、2102-2124、2103-2125、2106-2128、2109-2131、2111-2133、2112-2134、2113-2135、2115-2137、2117-2139、2241-2263、2250-2272、2253-2275、2300-2322、2301-2323、2343-2365、2347-2369、2349-2371、1796-1818、1270-1292、1624-1646、1795-1817、1822-1844、1847-1869、1582-1604、1582-1604、1582-1604、1570-1592、2110-2132、1954-1976、2013-2035、1840-1862、2114-2136、1565-1587、1808-1830、2249-2271、1614-1636、1655-1677、1581-1603、1717-1739、1717-1739、1717-1739、1883-1905、1894-1916、1957-1979、1957-1979、1957-1979、1786-1808、1681-1703、1589-1611、2098-2120、1327-1349、2432-2454、1374-1396、1911-1933、1372-1394、1816-1838、2240-2262、1577-1599、2105-2127、2092-2114、1951-1973、2346-2368、1781-1803、2014-2036、1410-1432、1940-1962、1718-1740、1859-1881、1744-1766、1787-1809、1787-1809、1787-1809、1855-1877 or 2082-2104 of any of the following nucleotides of 1.

In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

In one embodiment, substantially all of the nucleotides of the sense strand comprise a modification. In another embodiment, substantially all of the nucleotides of the antisense strand comprise a modification. In yet another embodiment, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise modifications.

In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB). The dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a sequence identical to SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides differing by NO more than 1, 2 or 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3' end. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises a sequence from SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence from SEQ ID NO:2, wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and wherein the sense strand is conjugated to a ligand attached at the 3' terminus.

In one embodiment, all nucleotides of the sense strand comprise modifications. In another embodiment, all nucleotides of the antisense strand comprise a modification. In yet another embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand comprise modifications.

In some embodiments, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 3' -terminal deoxythymine (dT) nucleotides, 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxymodified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2' -amino modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-alkyl modified nucleotides, 2' -hydroxy modified nucleotides, 2' -methoxyethyl modified nucleotides, 2' -O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, tetrahydropyran modified nucleotides, 1, 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, phosphorothioate group containing nucleotides, methylphosphonate group containing nucleotides, 5' -phosphate mimetic containing nucleotides, ethylene glycol modified nucleotides and 2-O- (N-methylacetamide) modified nucleotides, and combinations thereof.

In one embodiment, the nucleotide modification is a2 '-O-methyl modification and/or a 2' -fluoro modification.

The complementary region may be at least 17 nucleotides in length; 19 to 30 nucleotides in length; 19 to 25 nucleotides in length; or 21 to 23 nucleotides in length.

Each strand may be no more than 30 nucleotides in length, e.g., each strand is independently 19 to 30 nucleotides in length; each strand is independently 19 to 25 nucleotides in length; each strand is independently 21 to 23 nucleotides in length.

The dsRNA may include at least one strand comprising a 3' overhang of at least 1 nucleotide; or at least one strand comprising a 3' overhang of at least 2 nucleotides.

In some embodiments, the dsRNA agent further comprises a ligand.

In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

In one embodiment, the ligand is

In one embodiment, the dsRNA agent is conjugated to the ligand, as shown in the following schematic

And wherein X is O or S.

In one embodiment, the X is O.

In one embodiment, the complementary region comprises any one of the antisense sequences in tables 3-6.

In one aspect, the invention provides a double strand for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB). The dsRNA 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 CIDEB, wherein each strand is about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (Ij):

Sense of meaning ：5′n_p-N_a-(XXX)_i-N_b-YYY-N_b-(ZZZ)_j-N_a-n_q3′

Antisense sense ：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′(Ij),

Wherein the method comprises the steps of

I. j, k and l are each independently 0 or 1;

p, p ', q and q' are each independently 0 to 6;

Each N _a and N _a' independently represents an oligonucleotide sequence comprising 0 to 25 modified or unmodified nucleotides or a combination thereof, each sequence comprising at least two different modified nucleotides;

Each N _b and N _b' independently represents an oligonucleotide sequence comprising 0 to 10 modified or unmodified nucleotides 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 overhang nucleotide;

XXX, YYY, ZZZ, X ' X ' X ', Y ' Y ' Y ' and Z ' Z ' Z ' each independently represent a motif of three identical modifications on three consecutive nucleotides;

The modification on N _b is different from the modification on Y, and the modification on N _b 'is different from the modification on Y'; and

Wherein the sense strand is conjugated to at least one ligand.

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

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

In one embodiment, the yyyy motif is present at or near the cleavage site of the sense strand, e.g., the Y 'motif is present at positions 11, 12 and 13 from the 5' end of the antisense strand.

In one embodiment, formula (Ij) is represented by formula (Ik):

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' (Ik).

In another embodiment, formula (Ij) is represented by formula (Il):

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' (Il)

Wherein each Nb and Nb' independently represents an oligonucleotide sequence comprising 1 to 5 modified nucleotides.

In yet another embodiment, formula (Ij) is represented by formula (Im):

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 '(Im) wherein each N _b and N _b' independently represents an oligonucleotide sequence comprising 1 to 5 modified nucleotides.

In another embodiment, formula (Ij) is represented by formula (In):

sense: 5'n _p-N_a-XXX-N_b-YYY-N_b-ZZZ-N_a-n_q 3'

Antisense sense ：3′n_p′-N_a′-X′X′X′-N_b′-Y′Y′Y′-N_b′-Z′Z′Z′-N_a′-n_q′5′(In)

Wherein each N _b and N _b 'independently represents an oligonucleotide sequence comprising 1 to 5 modified nucleotides, and each N _a and N _a' independently represents an oligonucleotide sequence comprising 2 to 10 modified nucleotides.

Each strand may be no more than 30 nucleotides in length, e.g., each strand is independently 19 to 30 nucleotides in length.

In one embodiment, the modification on the nucleotide is selected from the group consisting of: LNA, HNA, ceNA, 2 '-methoxyethyl, 2' -O-alkyl, 2 '-O-allyl, 2' -C-allyl, 2 '-fluoro, 2' -O-methyl, 2 '-deoxy, 2' -hydroxy, and combinations thereof.

In one embodiment, the modification on the nucleotide is a2 '-O-methyl modification or a 2' -fluoro modification.

In one embodiment, Y ' is a 2' -O-methyl or 2' -fluoro modified nucleotide.

In one embodiment, at least one strand of the dsRNA agent comprises a3 'overhang of at least 1 nucleotide or a 3' overhang of at least 2 nucleotides.

In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3' end of one strand. In one embodiment, the strand is an antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5' end of one strand. In one embodiment, the strand is an antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the strand is an antisense strand. In another embodiment, the strand is the sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkages are located at both the 5 'and 3' ends of one strand.

In one embodiment, the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

In one embodiment, p' > 0. In another embodiment, p' =2.

In one embodiment, q '=0, p=0, q=0, and the p' overhang nucleotide is complementary to the target mRNA. In another embodiment, q '=0, p=0, q=0, and the p' overhang nucleotide is not complementary to the target mRNA.

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

In one embodiment, at least one n _p' is linked to an adjacent nucleotide via a phosphorothioate linkage. In another embodiment, at least one n _p' is linked to an adjacent nucleotide by a phosphorothioate linkage.

In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand comprise modifications.

In one embodiment, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives linked by a monovalent, divalent or trivalent branched linker.

In one embodiment, the ligand is

And wherein X is O or S.

In one embodiment, the X is O.

In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB). The dsRNA 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 CIDEB, wherein each strand is about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (Ij):

Sense of meaning ：5′n_p-N_a-(XXX)_i-N_b-YYY-N_b-(ZZZ)_j-N_a-n_q3′

Antisense sense ：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′(Ij)

Wherein:

i. j, k and l are each independently 0 or 1;

p, p ', q and q' are each independently 0 to 6;

XXX, YYY, ZZZ, X 'X', Y 'and Z' each independently represent a motif of three identical modifications on three consecutive nucleotides, and wherein the modification is a2 '-O-methyl modification or a 2' -fluoro modification; on N _b

The modification is different from the modification on Y, and the modification on N _b 'is different from the modification on Y'; and

Wherein the sense strand is conjugated to at least one ligand.

Sense of meaning ：5′n_p-N_a-(XXX)_i-N_b-YYY-N_b-(ZZZ)_j-N_a-n_q3′

Wherein:

i. j, k and l are each independently 0 or 1;

each n _p、n_q and n _q', each of which may or may not be present, independently represents an overhang nucleotide;

p, q and q' are each independently 0 to 6;

n _p '> 0 and at least one n _p' is linked to adjacent nucleotides via phosphorothioate linkages;

XXX, YYY, ZZZ, X 'X', Y 'and Z' each independently represent a motif of three identical modifications on three consecutive nucleotides, and wherein the modification is a2 '-O-methyl modification or a 2' -fluoro modification;

Wherein the sense strand is conjugated to at least one ligand.

Sense of meaning ：5′n_p-N_a-(XXX)_i-N_b-YYY-N_b-(ZZZ)_j-N_a-n_q3′

Antisense sense ：3′n_p′-Na′-(X′X′X′)_k-N_b′-Y′Y′Y′-N_b′-(Z′Z′Z′)_l-N_a′-n_q′5′(Ij)

Wherein:

i. j, k and l are each independently 0 or 1;

p, q and q' are each independently 0 to 6;

Wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc derivatives linked by a monovalent, divalent or trivalent branched linker.

Sense of meaning ：5′n_p-N_a-(XXX)_i-N_b-YYY-N_b-(ZZZ)_j-N_a-n_q3′

Antisense sense 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′(Ij)

Wherein:

i. j, k and l are each independently 0 or 1;

p, q and q' are each independently 0 to 6;

The modification on N _b is different from the modification on Y, and the modification on N _b 'is different from the modification on Y';

Wherein the sense strand comprises at least one phosphorothioate linkage; and

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' (Ik)

Wherein:

p, q and q' are each independently 0 to 6;

YYY and Y ' each independently represent a motif of three identical modifications on three consecutive nucleotides, and wherein the modifications are 2' -O-methyl modifications and/or 2' -fluoro modifications;

Wherein the sense strand comprises at least one phosphorothioate linkage; and

In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB). The dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises a sequence identical to SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides differing by NO more than 1,2 or 3 nucleotides from the nucleotide sequence of SEQ ID NO:2, wherein substantially all nucleotides of the sense strand comprise modifications selected from the group consisting of 2 '-O-methyl modifications and 2' -fluoro modifications, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5 'end, wherein substantially all nucleotides of the antisense strand comprise modifications selected from the group consisting of 2' -O-methyl modifications and 2 '-fluoro modifications, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3 'end, and wherein the sense strand is conjugated with one or more GalNAc derivatives linked by a monovalent, divalent or trivalent branched linker at the 3' end. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises a sequence from SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence from SEQ ID NO:2, wherein substantially all nucleotides of the sense strand comprise modifications selected from the group consisting of 2 '-O-methyl modifications and 2' -fluoro modifications, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5 'terminus, wherein substantially all nucleotides of the antisense strand comprise modifications selected from the group consisting of 2' -O-methyl modifications 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, and wherein the sense strand is conjugated with one or more GalNAc derivatives conjugated through a monovalent, divalent, or trivalent branched linker at the 3' terminus.

In one embodiment, all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

In one embodiment, the complementary region comprises any one of the antisense sequences listed in tables 3-6.

In one embodiment, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of the nucleotide sequences of any of the agents listed in tables 3-6.

In various embodiments of the above dsRNA agents, the dsRNA agents target the hot spot region of mRNA encoding CIDEB.

In another aspect, the invention provides a dsRNA agent that targets a hot spot region of a cell death-inducing DFFA-like effector B (CIDEB) mRNA.

The invention also provides cells, vectors and pharmaceutical compositions comprising any of the dsRNA agents of the invention. The dsRNA agent may be formulated in an unbuffered solution, such as saline or water, or in a buffered solution, such as a solution comprising acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffered solution is Phosphate Buffered Saline (PBS).

In one aspect, the invention provides a method of inhibiting cell death-induced DFFA-like effector b (CIDEB) expression in a cell. The method comprises contacting the cell with a dsRNA agent or pharmaceutical composition of the invention, thereby inhibiting expression of CIDEB in the cell.

The cells may be in a subject (e.g., a human subject).

In one embodiment CIDEB expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or is inhibited to a level below the detection of CIDEB expression.

In one embodiment, the human subject has CIDEB-related diseases, disorders, or conditions. In one embodiment, the CIDEB-related disease, disorder, or condition is a chronic inflammatory disease, such as chronic inflammatory disease of the liver and other tissues. In one embodiment, the chronic inflammatory disease is chronic inflammatory liver disease. In one embodiment, the chronic inflammatory liver disease is selected from the group consisting of: accumulation of fat in the liver, inflammation of the liver, liver fibrosis, fatty liver disease (steatosis), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and cirrhosis.

In one aspect, the invention provides a method of inhibiting expression of CIDEB in a subject. The method comprises administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition of the invention, thereby inhibiting expression of CIDEB in the subject.

In another aspect, the invention provides a method of treating a subject having a CIDEB-related disease, disorder, or condition. The method comprises administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition of the invention, thereby treating the subject suffering from a CIDEB-related disease, disorder, or condition.

In another aspect, the invention provides a method of preventing at least one symptom in a subject suffering from a disease, disorder, or condition that would benefit from a decrease in expression of CIDEB genes. The method comprises administering to the subject a prophylactically effective amount of the agent dsRNA agent or pharmaceutical composition of the invention, thereby preventing at least one symptom in the subject suffering from a disease, disorder, or condition that would benefit from a reduction in expression of CIDEB genes.

In another aspect, the invention provides a method of reducing the risk of a subject suffering from steatosis to develop a chronic liver disease. The method comprises administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition of the invention, thereby reducing the risk of the subject suffering from steatosis developing chronic liver disease.

In one aspect, the invention provides a method of inhibiting lipid droplet accumulation in the liver of a subject suffering from a CIDEB-related disease, disorder, or condition. The method comprises administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition of the invention, and a dsRNA agent targeted to CIDEB gene or a pharmaceutical composition comprising a dsRNA agent targeted to CIDEB gene, thereby inhibiting accumulation of fat in the liver of the subject suffering from a CIDEB related disease, disorder or condition.

In another aspect, the invention provides a method of treating a subject having a CIDEB-related disease, disorder, or condition. The method comprises administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition of the invention, and a dsRNA agent targeted to CIDEB gene or a pharmaceutical composition comprising a dsRNA agent targeted to CIDEB gene, thereby treating the subject suffering from a CIDEB related disease, disorder or condition.

In another aspect, the invention provides a method of preventing at least one symptom in a subject suffering from a disease, disorder, or condition that would benefit from a decrease in expression of CIDEB genes. The method comprises administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition of the invention, and a dsRNA agent targeted to CIDEB gene or a pharmaceutical composition comprising a dsRNA agent targeted to CIDEB gene, thereby preventing at least one symptom of the subject suffering from a disease, disorder, or condition that would benefit from a reduction in expression of CIDEB gene.

In another aspect, the invention provides a method of reducing the risk of a subject suffering from steatosis to develop a chronic liver disease. The method comprises administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition of the invention, and a dsRNA agent targeted to CIDEB genes or a pharmaceutical composition comprising a dsRNA agent targeted to CIDEB genes, thereby reducing the risk of the subject suffering from steatosis developing chronic liver disease.

In another aspect, the invention provides a method of inhibiting the progression of steatosis to steatohepatitis in a subject suffering from steatosis. The method comprises administering to the subject a therapeutically effective amount of a dsRNA agent or pharmaceutical composition of the invention, and a dsRNA agent targeting CIDEB genes or a pharmaceutical composition comprising a dsRNA agent targeting CIDEB genes, thereby inhibiting the progression of steatosis to steatohepatitis in the subject.

In one embodiment, administration of a dsRNA agent or pharmaceutical composition to the subject results in a decrease in CIDEB protein activity, e.g., a decrease in CIDEB interaction with ApoB and/or a decrease in lipid maturation in the liver; CIDEB reduced protein accumulation, reduced CIDEB enzymatic activity, reduced CIDEB protein accumulation, and/or reduced fat accumulation and/or lipid droplet amplification in the liver of a subject.

In one embodiment, the CIDEB-related disease, disorder, or condition is a chronic inflammatory disease.

In one embodiment, the chronic inflammatory disease is chronic inflammatory liver disease.

In one embodiment, the chronic inflammatory liver disease is selected from the group consisting of: accumulation of fat in the liver, inflammation of the liver, liver fibrosis, fatty liver disease (steatosis), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and cirrhosis.

In one embodiment, the chronic inflammatory liver disease is non-alcoholic steatohepatitis (NASH).

In one embodiment, the subject is obese.

In one embodiment, the methods and uses of the invention further comprise administering an additional therapeutic agent to the subject.

In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01mg/kg to about 10mg/kg or about 0.5mg/kg to about 50 mg/kg.

The agent may be administered to the subject intravenously, intramuscularly or subcutaneously. In one embodiment, the agent is administered to the subject subcutaneously.

In one embodiment, the methods and uses of the invention further comprise determining the level of CIDEB in the subject.

In one aspect, the invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell expression of cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any one of the agents in tables 3-6, and the antisense strand comprises a nucleotide sequence of any one of the agents in tables 3-6, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand are modified nucleotides, and wherein the dsRNA agent is conjugated to a ligand.

Drawings

FIG. 1 depicts qPCR results of CIDEB mRNA in a single dose (3 mg/kg) study in mice using an exemplary human CIDEB dsRNA duplex. Results are presented as the percentage of remaining mRNA normalized to PBS.

FIG. 2 depicts qPCR results of CIDEB mRNA in a multi-dose (1.5 mg/kg and 0.75 mg/kg) study in mice using an exemplary human CIDEB dsRNA duplex. Results are presented as the percentage of remaining mRNA normalized to PBS.

Detailed Description

The present invention provides iRNA compositions that affect RNA-induced silencing complex (RISC) -mediated cleavage of RNA transcripts of CIDEB genes. The CIDEB gene may be within a cell, for example, a cell in a subject (e.g., human). The invention also provides methods of using the iRNA compositions of the invention to inhibit CIDEB gene expression, and to treat subjects who would benefit from inhibiting or reducing CIDEB gene expression, e.g., subjects who would benefit from reduced inflammation, e.g., subjects suffering from or susceptible to CIDEB-related diseases, disorders, or conditions, e.g., subjects suffering from or susceptible to chronic inflammatory diseases of the liver and other tissues, e.g., subjects suffering from chronic inflammatory liver diseases such as liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic steatohepatitis (NAFLD), alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), cirrhosis, HCV-related cirrhosis, drug-induced liver injury, hepatocyte necrosis, insulin insensitivity, and diabetes.

The targeting CIDEB iRNA of the invention can include an RNA strand (antisense strand) having a region of about 30 nucleotides or less in length, 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 nucleotides in length, that is substantially complementary to at least a portion of an mRNA transcript of the CIDEB gene.

In some embodiments, one or both strands of a double stranded RNAi agent of the invention are up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 consecutive nucleotides substantially complementary to at least a portion of the mRNA transcript of the CIDEB gene. In some embodiments, such iRNA agents having an antisense strand of longer length may comprise a second RNA strand (sense strand) of 20 to 60 nucleotides in length, wherein the sense strand and the antisense strand form a duplex of 18 to 30 consecutive nucleotides.

The use of iRNA agents described herein enables targeted degradation of the mRNA of CIDEB genes in mammals.

In particular, very low doses of iRNA can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of CIDEB gene expression. Thus, methods and compositions comprising these irnas are useful for treating subjects that would benefit from inhibiting or reducing expression of CIDEB genes, e.g., subjects that would benefit from reduced inflammation, e.g., subjects that have or are susceptible to CIDEB-related diseases, disorders or conditions, e.g., subjects that have or are susceptible to chronic inflammatory diseases of the liver and other tissues, e.g., subjects that have chronic inflammatory liver disease such as liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic steatohepatitis (NAFLD), alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), cirrhosis, HCV-related cirrhosis, drug-induced liver injury, hepatocyte necrosis, insulin insensitivity, and diabetes.

The following detailed description discloses how to make and use iRNA-containing compositions to inhibit CIDEB gene expression, as well as compositions and methods for treating subjects suffering from diseases and disorders that would benefit from inhibition and/or reduction of expression of such genes.

I. Definition of the definition

For easier understanding of the present invention, certain terms are first defined. In addition, it should be noted that whenever a value or range of values for a parameter is referred to, it is intended that values and ranges intermediate to the values recited are also intended to be part of the present invention.

The articles "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 article. For example, "an element" refers to one element or more than one element, e.g., a plurality of elements.

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

The term "or" is used herein to mean, and is used interchangeably with, the term "and/or" unless the context clearly indicates otherwise.

The term "about" is used herein to mean within typical tolerances in the art. For example, "about" may be understood as about 2 standard deviations of the mean. In certain embodiments, about ±10%. In certain embodiments, about ±5%. When "about" is present before a series of numbers or ranges, it is to be understood that "about" can modify each of the numbers in the series or ranges.

Unless otherwise indicated, the term "CIDEB", also referred to as "cell death inducing DFFA-like effector B", "cell death activator CIDE-B" or "cell death inducing DFF 45-like effector B", refers to a well known gene encoding CIDEB protein from any vertebrate or mammalian source including, but not limited to, humans, bovine, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys and guinea pigs.

The term also refers to fragments and variants of natural CIDEB that maintain at least one in vivo or in vitro activity of natural CIDEB.

CIDEB (a member of the CIDE family of proteins) are expressed predominantly in liver tissue and in the small intestine (e.g. jejunum and ileum sections of the small intestine) (Zhang et al, lipid research (Lipid Res.); 55 (7): 1279-87; 2014). CIDEB are Endoplasmic Reticulum (ER) and Lipid Droplet (LD) related proteins. Overexpression of CIDEB proteins as members of the CIDE family induces cell death, but the physiological function of CIDEB is more closely related to various lipid metabolic pathways, particularly the VLDL pathway. For example, CIDEB mediates VLDL lipidation and maturation by interaction with ApoB; CIDEB are also necessary for the biogenesis of VLDL transport vesicles and chylomicron lipidation in the small intestine. CIDEB mutant mice exhibit significantly increased insulin sensitivity and enhanced systemic metabolism and liver fatty acid oxidation rates (Li et al Diabetes 56 (10): 2523-32.2007). Thus CIDEB can represent a novel therapeutic target for the treatment of obesity, diabetes and hepatic steatosis (Li et al, diabetes 56 (10): 2523-32.2007). CIDEB is regulated by transcription of hepatocyte nuclear factor 4α (hnf4α), the most abundant transcription factor in the liver; hnf4α is critical for VLDL-mediated lipid transport and is involved in HCV assembly/release. Peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), a key transcriptional cofactor for HNF4 alpha, also regulates HCV production, and PGC-1 alpha stimulates VLDL assembly in a CIDEB-dependent manner (Cai et al, science report (SCIENTIFIC REPORTS), volume 6, article number 27778, 2016).

HCV entry into hepatocytes and HCV assembly requires CIDEB (Xu et al, journal of virology (J virol.)), 88, 8433-8444, 2014. CIDEB interacts with HCV NS5A protein and modulates association of HCV particles with ApoE. CIDEB also regulate the post-entry phase of the dengue virus (DENV) lifecycle (Cai et al, science report, volume 6, article No. 27778, 2016).

Exemplary nucleotide and amino acid sequences of CIDEB can be found, for example, in GenBank accession No. NM-001393338.1 of Chile (SEQ ID NO:1; reverse complement SEQ ID NO: 2).

Additional examples of CIDEB mRNA sequences can be readily obtained using publicly available databases (e.g., genBank, uniProt and OMIM).

Further information about CIDEB is provided, for example at http: in the NCBI gene database of// www.ncbi.nlm.nih.gov/gene/27141.

In some embodiments, the iRNA that is substantially complementary to the region of mouse or rat CIDEB mRNA cross-reacts with human CIDEB mRNA and represents a potential candidate for human targeting.

As used herein, the term "CIDEB" also refers to a particular polypeptide expressed in a cell by a naturally occurring DNA sequence variation of the CIDEB gene (e.g., a single nucleotide polymorphism in the CIDEB gene). Many Single Nucleotide Polymorphisms (SNPs) within CIDEB genes have been identified and can be found, for example, at NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/SNP).

As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the CIDEB gene, including mRNA that is the product of RNA processing of the primary transcript. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for directed cleavage of the iRNA at or near that portion of the nucleotide sequence of the mRNA molecule formed during transcription of the CIDEB gene.

The target sequence of the CIDEB gene may be about 9 to 36 nucleotides in length, for example, about 15 to 30 nucleotides in length. For example, the target sequence may be about 15 to 30 nucleotides 、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 nucleotides in length. Ranges and lengths intermediate to those described above are also considered part of the present invention.

As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by a sequence referred to using standard nucleotide nomenclature.

"G", "C", "A", "T" and "U" generally each represent nucleotides containing guanine, cytosine, adenine, thymine and uracil, respectively, as bases. However, it is understood that the term "ribonucleotide" or "nucleotide" may also refer to modified nucleotides, as described in further detail below, or alternative substitute parts (see e.g. table 2). It will be apparent to those skilled in the art that guanine, cytosine, adenine and uracil can be substituted with other moieties without substantially altering the base pairing properties of oligonucleotides including nucleotides containing such substituted moieties. For example, but not limited to, a nucleotide that includes inosine as its base may be base paired with a nucleotide containing adenine, cytosine, or uracil. Thus, in the nucleotide sequence of the dsRNA characteristic of the invention, the nucleotide containing uracil, guanine or adenine may be substituted with a nucleotide containing, for example, inosine. In another example, adenine and cytosine at any positions in the oligonucleotide may be substituted with guanine and uracil, respectively, to form a G-U wobble base pairing with the target mRNA. Sequences containing such substituted moieties are suitable for use in compositions and methods featuring the invention.

The terms "iRNA," "RNAi agent," "iRNA agent," and "RNA interfering agent" as used interchangeably herein refer to agents that contain RNA as defined herein, and which mediate targeted cleavage of RNA transcripts through an RNA-induced silencing complex (RISC) pathway. iRNA directs sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). iRNA regulates (e.g., inhibits) expression of CIDEB genes in cells, e.g., cells in a subject (e.g., a mammalian subject).

In one embodiment, the RNAi agents of the invention comprise single-stranded RNA that interacts with a target RNA sequence (e.g., CIDEB target mRNA sequence) to direct cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double stranded RNA introduced into the cell is cleaved into siRNA by a type III endonuclease called Dicer (Sharp et al, (2001) Genes and development (Genes Dev) 15:485). Dicer, a ribonuclease-III like enzyme, uses a characteristic two base 3' overhang to process dsRNA into 19 to 23 base pair short interfering RNA (Bernstein et al, (2001) Nature 409:363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), in which one or more helices cleave the siRNA duplex, thereby enabling the complementary antisense strand to direct 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 to induce silencing (Elbashir et al, (2001) Gene and development 15:188). Thus, in one aspect, the invention relates to single stranded RNA (sssiRNA) produced in a cell, and which facilitates the formation of RISC complexes to affect silencing of a target gene, i.e., CIDEB genes. Thus, the term "siRNA" is also used herein to refer to RNAi as described above.

In another embodiment, the RNAi agent can be a single stranded RNAi agent that is introduced into a cell or organism to inhibit a target mRNA. The single stranded RNAi agent (ssRNAi) binds to RISC endonuclease Argonaute 2, which then cleaves the target mRNA. Single stranded siRNA is typically 15 to 30 nucleotides and is chemically modified. Design and testing of single stranded RNAi agents is described in U.S. patent No. 8,101,348 and Lima et al, (2012) cell 150:883-894, the entire contents of each of which are hereby incorporated by reference. Any antisense nucleotide sequence described herein may be used as described herein or as described by Lima et al, (2012) cell 150; 883-894.

In another embodiment, the "iRNA" used in the compositions and methods of the invention is double stranded RNA, and is referred to herein as a "double stranded RNAi agent," double stranded RNA (dsRNA) molecule, "" dsRNA agent, "or" dsRNA. The term "dsRNA" refers to a complex of ribonucleic acid molecules having a duplex structure comprising two antiparallel and substantially complementary nucleic acid strands having "sense" and "antisense" orientations relative to a target RNA, i.e., CIDEB genes. In some embodiments of the invention, double-stranded RNA (dsRNA) triggers degradation of target RNA (e.g., mRNA) by a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

In general, the majority of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both strands may also include one or more non-ribonucleotides, such as deoxyribonucleotides and/or modified nucleotides. In addition, as used in this specification, an "RNAi agent" can comprise ribonucleotides with chemical modification; RNAi agents can comprise 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 encompasses substitution, addition or removal of internucleotide linkages, sugar moieties or nucleobases, such as functional groups or atoms. Modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. For the purposes of the present specification and claims, as used in siRNA-type molecules, any such modification is encompassed by "RNAi agents".

The duplex region may be any length that allows for specific degradation of the desired target RNA through the RISC pathway, and may be in the range of about 9 to 36 base pairs in length, for example 15 to 30 base pairs in length, for example 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, for example 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 base pairs in length. Ranges and lengths intermediate to those described above are also considered part of the present invention.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. If the two strands are part of one larger molecule and are thus connected by an uninterrupted nucleotide chain between the 3 'end of one strand and the 5' end of the corresponding other strand forming a duplex structure, the connected RNA strand is referred to as a "hairpin loop". The hairpin loop may include at least one unpaired nucleotide. In some embodiments, hairpin loops may include 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.

Where the two substantially complementary strands of the dsRNA comprise separate RNA molecules, those molecules need not be, but can be, covalently linked. In the case where the two strands are covalently linked by other means than an uninterrupted nucleotide chain between the 3 'end of one strand and the 5' end of the corresponding other strand forming the duplex structure, the linking structure is referred to as a "linker". The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to duplex structures, RNAi can include one or more nucleotide overhangs.

In one embodiment, the RNAi agents of the invention are dsRNA, each strand of which comprises less than 30 nucleotides, e.g., 17 to 27, 19 to 27, 17 to 25, 19 to 25, or 19 to 23 nucleotides, that interact with a target RNA sequence (e.g., CIDEB target mRNA sequence) to guide cleavage of the target RNA. In one embodiment, the RNAi agent of the invention is a dsRNA, each strand of which comprises 19 to 23 nucleotides, which interact with a target RNA sequence (e.g., CIDEB target mRNA sequence) to guide cleavage of the target RNA. In one embodiment, the sense strand is 21 nucleotides in length. In another embodiment, the antisense strand is 23 nucleotides in length.

As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide protruding from the duplex structure of an iRNA, e.g., dsRNA. For example, when the 3 'end of one strand of a dsRNA extends beyond the 5' end of the other strand, and vice versa, a nucleotide overhang is present. The dsRNA may include an overhang of at least one nucleotide; alternatively, the overhang may include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. Nucleotide overhangs may include or consist of: nucleotide/nucleoside analogs comprising deoxynucleotides/nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present on the 5 'end, 3' end or both ends of the antisense strand or sense strand of the dsRNA.

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, at the 3 'end and/or the 5' end of the overhang. In one embodiment, the overhang of the sense strand of the dsRNA at the 3 'end and/or 5' end has 1 to 10 nucleotides, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In another embodiment, one or more nucleotides in the overhang are replaced with a nucleoside thiophosphate.

In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can comprise an extension length longer than 10 nucleotides, e.g., 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides in length. In certain embodiments, the extended overhang is on the sense strand of the duplex. In certain embodiments, there is an extended overhang on the 3' end of the sense strand of the duplex. In certain embodiments, there is an extended overhang on the 5' end of the sense strand of the duplex. In certain embodiments, the extended overhang is on the antisense strand of the duplex. In certain embodiments, there is an extended overhang on the 3' end of the antisense strand of the duplex. In certain embodiments, there is an extended overhang on the 5' end of the antisense strand of the duplex. In certain embodiments, one or more nucleotides in the extended overhang are replaced with a nucleoside thiophosphate.

As used herein, the term "blunt end" or "blunt end" with respect to a dsRNA refers to the absence of unpaired nucleotides or nucleotide analogs, i.e., no nucleotide overhangs, at a given end of the dsRNA. One or both ends of the dsRNA may be blunt ended. When both ends of a dsRNA are blunt, the dsRNA is referred to as "blunt-ended". For clarity, "blunt-ended" dsRNA is a dsRNA that is blunt-ended at both ends, i.e., no nucleotide overhangs at either end of the molecule. The most common such molecules will be double stranded throughout their length.

The term "antisense strand" or "guide strand" refers to a strand of an iRNA, e.g., dsRNA, that includes a region that is substantially complementary to a target sequence (e.g., CIDEB mRNA).

As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence as defined herein (e.g., a target sequence, such as a CIDEB nucleotide sequence). In the case where the complementary region is not perfectly complementary to the target sequence, the mismatch may be in the internal or terminal region of the molecule. Typically, the most tolerable mismatches are in the terminal region, e.g., within 5,4, 3, or 2 nucleotides of the 5 'end and/or 3' end of the iRNA agent.

As used herein, the term "sense strand" or "follower strand" refers to an iRNA strand comprising a region that is substantially complementary to a region of the antisense strand of the term as defined herein.

As used herein, the term "cleavage region" refers to the region immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of the cleavage site and immediately adjacent to the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of the cleavage site and immediately adjacent to the cleavage site. In some embodiments, the cleavage site is specifically present at the site where nucleotides 10 and 11 of the antisense strand bind, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence relative to a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize under certain conditions to an oligonucleotide or polynucleotide comprising the second nucleotide sequence and form a duplex structure, as will be understood by the skilled artisan. For example, such conditions may be stringent conditions, where stringent conditions may comprise: 400mM NaCl,40mM PIPES,pH 6.4,1mM EDTA,50 ℃or 70℃for 12 to 16 hours, and then washed (see, for example, molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), sambrook et al (1989) Cold spring harbor laboratory Press (Cold Spring Harbor Laboratory Press)). Other conditions may be applied, such as physiologically relevant conditions that may be encountered inside an organism. The skilled person will be able to determine the set of conditions most suitable for the complementarity test of the two sequences depending on the end use of the hybridizing nucleotides.

Complementary sequences within an iRNA (e.g., within a dsRNA as described herein) comprise base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. However, where the first sequence is referred to herein as "substantially complementary" to the second sequence, the two sequences may be fully complementary, or they may form one or more, but typically no more than 5, 4, 3 or 2 post-hybridization mismatched base pairs for up to 30 base pairs of the duplex, while retaining the ability to hybridize under conditions most relevant to its end use, e.g., inhibiting gene expression via the RISC pathway. However, where two oligonucleotides are designed to form one or more single stranded overhangs upon hybridization, such overhangs should not be considered as a defined mismatch with respect to complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for the purposes described herein.

As used herein, a "complementary" sequence may also include, or be formed entirely of, non-Watson-Crick base pairs (non-Watson-Crick base pairs) and/or base pairs formed from non-natural and modified nucleotides, so long as the above requirements regarding its hybridization ability are met. Such non-Watson-Crick base pairs include, but are not limited to, G: u wobble base pairing or Holstein base pairing (Hoogstein base pairing).

The terms "complementary," "fully complementary," and "substantially complementary" herein may be used with respect to base matching between the sense strand and the antisense strand of a dsRNA or between the antisense strand of an RNAi agent and a target sequence, as will be understood from the context of its use.

As used herein, a polynucleotide that is substantially complementary to "at least a portion of a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA encoding CIDEB). For example, if the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding CIDEB, the polynucleotide is complementary to at least a portion of CIDEB mRNA.

Thus, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target CIDEB sequence. In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to a target CIDEB sequence and comprise a contiguous nucleotide sequence that is, over its entire length, identical to the sequence of SEQ ID NO:1 or SEQ ID NO:1, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In one embodiment, the RNAi agent of the invention comprises a sense strand that is substantially complementary to an antisense polynucleotide, which in turn is complementary to a target CIDEB sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence that is, over its entire length, identical to the sequence of SEQ ID NO:2 or SEQ ID NO:2, such as about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In some embodiments, an iRNA of the invention comprises an antisense strand that is substantially complementary to a target CIDEB sequence, and comprises a contiguous nucleotide sequence that is at least about 80% complementary, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementary or 100% complementary over its entire length to the nucleotide sequence of any one of the sense strands of tables 3-6 or the equivalent region of a fragment of any one of the sense strands of tables 3-6.

As used herein, the term "inhibit (inhibiting)" may be used interchangeably with "reduce," "silence," "down-regulate," "inhibit (suppressing)" and other similar terms, and includes any level of inhibition.

As used herein, the phrase "inhibiting expression of CIDEB gene" includes inhibiting expression of any CIDEB gene (e.g., mouse CIDEB gene, rat CIDEB gene, monkey CIDEB gene, or human CIDEB gene) as well as variants or mutants of CIDEB gene encoding CIDEB protein.

"Inhibiting the expression of CIDEB gene" includes any level of inhibition of CIDEB gene, e.g., at least partial inhibition of the expression of CIDEB gene, e.g., inhibition of at least about 20%. In certain embodiments, 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%.

The expression of CIDEB genes can be assessed based on the level of any variable associated with CIDEB gene expression (e.g., CIDEB mRNA level or CIDEB protein level). Expression of the CIDEB gene may also be assessed indirectly based on, for example, a decrease in CIDEB protein activity, e.g., a decrease in the interaction of CIDEB with ApoB and/or a decrease in lipid maturation in a tissue sample, such as a liver sample. Inhibition may be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level. The control level may be any type of control level used in the art, e.g., a pre-dosing baseline level, or a level determined in a similar subject, cell, or sample that has not been treated or treated with a control (e.g., a buffer-only control or an inactive agent control).

In one embodiment, at least partial inhibition of the expression of CIDEB gene is assessed by a decrease in the amount of CIDEB mRNA, the CIDEB mRNA can be isolated or detected from a first cell or group of cells that transcribes CIDEB gene, and that has been treated such that expression of CIDEB gene is inhibited, as compared to a second cell or group of cells (control cells) that is substantially the same as the first cell or group of cells but not so treated.

The extent of inhibition can be expressed in the following manner:

As used herein, the phrase "contacting a cell with an RNAi agent (e.g., dsRNA)" includes contacting a cell by any possible means. Contacting the cell with the iRNA agent includes contacting the cell with the iRNA in vitro or contacting the cell with the iRNA in vivo. The contacting may be performed directly or indirectly. Thus, for example, an RNAi agent can be brought into physical contact with a cell by an individual performing the method, or alternatively, an RNAi agent can be brought into a condition that allows or brings it into subsequent contact with a cell.

In vitro contacting of cells can be performed, for example, by incubating the cells with an RNAi agent. Contacting cells in vivo may be performed, for example, by injecting an RNAi agent into or near the tissue in which the cells are located, or by injecting an RNAi agent into another area, such as the blood stream or subcutaneous space, such that the agent will then reach the tissue in which the cells to be contacted are located. For example, the RNAi agent can contain or be conjugated to a ligand (e.g., galNAc 3) that directs the RNAi agent to a site of interest, such as the liver. Combinations of in vitro and in vivo contact methods are also possible. For example, the cells may also be contacted with an RNAi agent in vitro and subsequently transplanted into a subject.

In one embodiment, contacting the cell with the iRNA comprises "introducing" or "delivering" the iRNA into the cell by promoting or affecting uptake or uptake by the cell. The uptake or uptake of iRNA can occur by unassisted diffusion or active cellular processes, or by adjuvants or devices. The introduction of the iRNA into the cell may be in vitro and/or in vivo. For example, for in vivo introduction, the iRNA may be injected into a tissue site or administered systemically. In vivo delivery may also be performed by beta-glucan delivery systems, such as those described in U.S. patent nos. 5,032,401 and 5,607,677 and U.S. publication No. 2005/0281781, the entire contents of which are hereby incorporated by reference. In vitro introduction into cells comprises methods known in the art, such as electroporation and lipofection. Additional methods are described below and/or are known in the art.

The term "lipid nanoparticle" or "LNP" is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g. an iRNA or a plasmid from which an iRNA is transcribed. LNP is 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 hereby incorporated by reference.

As used herein, a "subject" is an animal, e.g., a mammal, including primates (e.g., humans, non-human primates, e.g., monkeys and chimpanzees), non-primates (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, mice, horses, and whales), or birds (e.g., ducks or geese).

In one embodiment, the subject is a human, such as a human being treated or evaluated for a disease, disorder, or condition that would benefit from a CIDEB expression reduction; a person at risk of a disease, disorder or condition that would benefit from CIDEB expression reduction; a human suffering from a disease, disorder or condition that would benefit from a CIDEB expression reduction; and/or treating a person who would benefit from a disease, disorder, or condition that has reduced CIDEB expression, as described herein.

As used herein, the term "treatment" or "treatment" refers to a beneficial or desired outcome, including, but not limited to, alleviation or amelioration of one or more symptoms associated with CIDEB gene expression and/or CIDEB protein production, such as CIDEB-associated diseases, e.g., chronic inflammatory diseases of the liver and other tissues. In one embodiment, the chronic inflammatory disease is a chronic inflammatory liver disease, such as inflammation of the liver, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic steatohepatitis (NAFLD), cirrhosis, alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), HCV-related cirrhosis, drug-induced liver injury, hepatocyte necrosis, hepatocellular carcinoma, insulin insensitivity, and/or diabetes. "treatment" may also mean an extension of survival compared to the expected survival without treatment.

The term "decrease" in the context of CIDEB-related diseases refers to a statistically significant decrease in such levels. The reduction may 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. In certain embodiments, the reduction is at least 20%. A "lower" in the context of a CIDEB level of a subject is an acceptable level that preferably falls within the normal range of individuals without such disorders.

As used herein, "prevent" or "prevention" when used in reference to a disease, disorder, or condition that would benefit from a decrease in expression of CIDEB genes, refers to a decreased likelihood that a subject will develop symptoms associated with such disease, disorder, or condition, e.g., symptoms of CIDEB gene expression, such as inflammation of the liver, liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), HCV-related cirrhosis, drug-induced liver injury, hepatocyte necrosis, hepatocellular carcinoma, insulin insensitivity, and/or diabetes. Failure to develop a disease, disorder or condition, or a reduction in the development of symptoms associated with such a disease, disorder or condition (e.g., at least about 10% reduction on a clinically accepted scale of the disease or disorder), or a delay in the reduction of manifestation of symptoms (e.g., reduction of inflammation, or lipid accumulation in the liver and/or reduction of lipid droplet amplification in the liver) (e.g., days, weeks, months or years) is considered effective prophylaxis.

As used herein, the term "CIDEB-related disease" is a disease or disorder caused by or associated with CIDEB gene expression or CIDEB protein production. The term "CIDEB-related diseases" includes diseases, disorders, or conditions that would benefit from reduced CIDEB gene expression or protein activity.

In one embodiment, the "CIDEB-related disease" is a chronic inflammatory disease. A "chronic inflammatory disease" is any disease, disorder or condition associated with chronic inflammation. Non-limiting examples of chronic inflammatory diseases include, for example, inflammation of the liver and other tissues. Non-limiting examples of chronic inflammatory liver disease include, for example, fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), HCV-associated cirrhosis, drug-induced liver damage, hepatocyte necrosis, hepatocellular carcinoma, insulin insensitivity, and/or diabetes.

As used herein, a "therapeutically effective amount" is intended to include an amount of an RNAi agent that is sufficient to effectively treat a disease (e.g., by reducing, ameliorating, or maintaining one or more symptoms of an existing disease or disorder) when administered to a subject having CIDEB-related diseases, disorders, or conditions. The "therapeutically effective amount" may vary depending on the RNAi agent, how the agent is administered, the disease and its severity as well as the medical history, age, weight, family history, genetic composition, type of previous or concomitant therapy (if any), and other individual characteristics of the subject to be treated.

As used herein, a "prophylactically effective amount" is intended to include an amount of iRNA sufficient to prevent or ameliorate a disease or one or more symptoms of a disease when administered to a subject having CIDEB-related diseases, disorders, or conditions. Improving a disease comprises slowing the progression of the disease or reducing the severity of the disease in later stages. The "prophylactically effective amount" may vary depending on the RNAi agent, how the agent is administered, the degree of risk of the disease, as well as the medical history, age, body weight, family history, genetic composition, type of previous or concomitant therapy (if any), and other individual characteristics of the patient to be treated.

"Therapeutically effective amount" or "prophylactically effective amount" also includes the amount of RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. The iRNA used in the methods of the invention can be administered in sufficient amounts to produce a reasonable benefit/risk ratio suitable for such treatment.

The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, or dosage forms which 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.

As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or stearic acid), or solvent encapsulation material that participates in carrying or transporting the subject compound from one organ or portion of the body to another organ or portion of the body. 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 may serve 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 carboxymethyl cellulose, 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 waxes; (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) Polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (19) ethanol; (20) a pH buffer solution; (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 employed in pharmaceutical formulations.

As used herein, the term "sample" includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, and tissues present in the subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. The tissue sample may comprise a sample from a tissue, organ or local area. For example, the sample may originate from a particular organ, a portion of an organ, or a fluid or cell within such an organ. In certain embodiments, the sample may be derived from the liver (e.g., whole liver or portions of the liver or certain types of cells in the liver, e.g., hepatocytes). In some embodiments, "subject-derived sample" refers to blood or plasma drawn from a subject.

II. IRNA of the invention

Described herein are irnas that inhibit expression of a target gene. In one embodiment, the iRNA inhibits CIDEB gene expression. In one embodiment, the iRNA agent includes a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting expression of a CIDEB gene in a cell, such as a hepatocyte, in a subject (e.g., a mammal, such as a human suffering from a chronic inflammatory disease, disorder, or condition).

The dsRNA includes an antisense strand having a complementary region that is complementary to at least a portion of an mRNA formed in expression of a CIDEB gene. 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 expressing a target gene, the iRNA inhibits expression of the target gene (e.g., a human, primate, non-primate, or rodent target gene) by at least about 10%, as determined by, for example, PCR or branched DNA (bDNA) based methods or by protein based methods (e.g., by immunofluorescence analysis using, for example, western blot or flow cytometry techniques).

The dsRNA comprises two RNA strands that are complementary and hybridize under conditions where the dsRNA will be used to form a duplex structure. One strand (the antisense strand) of the dsRNA comprises a region of complementarity that is substantially complementary and typically fully complementary to a target sequence. The target sequence may be derived from the sequence of mRNA formed during expression of the CIDEB gene. The other strand (the sense strand) contains a region complementary to the antisense strand such that the two strands hybridize and form a duplex structure when combined under appropriate conditions. As described elsewhere herein and as known in the art, the complementary sequence of a dsRNA can also be included as a self-complementary region of a single nucleic acid molecule relative to being located on a separate oligonucleotide.

Typically, duplex structures are 15 to 30 base pairs in length, for example 15 to 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 to 23 or 21 to 22 base pairs in length. Ranges and lengths intermediate to those described above are also considered part of the present invention.

Similarly, the region complementary to the target sequence is 15 to 30 nucleotides in length, for example 15 to 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 to 23 or 21 to 22 nucleotides in length. Ranges and lengths intermediate to those described above are also considered part of the present invention.

In some embodiments, the sense strand and the antisense strand of the dsRNA are each independently about 15 to about 30 nucleotides in length, or about 25 to about 30 nucleotides in length, e.g., each strand is independently 15 to 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 to 23 or 21 to 22 nucleotides in length. In some embodiments, the dsRNA is about 15 to about 23 nucleotides in length, or about 25 to about 30 nucleotides in length. In general, dsrnas are long enough to serve as substrates for Dicer enzymes. For example, dsRNA longer than about 21 to 23 nucleotides can be used as substrates for Dicer, as is well known in the art. As the skilled artisan will also recognize, the region targeted to the cleaved RNA will typically be part of a larger RNA molecule, typically an mRNA molecule. In related cases, a "portion" of an mRNA target is a contiguous sequence of the mRNA target that is sufficiently long to make it a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

Those of skill in the art will also recognize that the duplex region is a major functional portion of a dsRNA, such as a duplex region of about 9 to about 36 base pairs (e.g., about 10 to 36 、11-36,12-36,13-36,14-36,15-36,9-35,10-35,11-35,12-35,13-35,14-35,15-35,9-34,10-34,11-34,12-34,13-34,14-34,15-34,9-33,10-33,11-33,12-33,13-33,14-33,15-33,9-32,10-32,11-32,12-32,13-32,14-32,15-32,9-31,10-31,11-31,12-31,13-32,14-31,15-31,15-30,15-29,15-28,15-27,15-26,15-25,15-24,15-23,15-22,15-21,15-20,15-19,15-18,15-17,18-30,18-29,18-28,18-27,18-26,18-25,18-24,18-23,18-22,18-21,18-20,19-30,19-29,19-28,19-27,19-26,19-25,19-24,19-23,19-22,19-21,19-20,20-30,20-29,20-28,20-27,20-26,20-25,20-24,20-23,20-22,20-21,21-30,21-29,21-28,21-27,21-26,21-25,21-24,21 to 23 or 21 to 22 base pairs). Thus, in one embodiment, the RNA molecule or RNA molecule complex having a duplex region of greater than 30 base pairs is a dsRNA to the extent that it is processed into a functional duplex of, for example, 15 to 30 base pairs that targets the desired RNA for cleavage. Thus, one of ordinary skill will recognize that in one embodiment, the micrornas (mirnas) are dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, the iRNA agent useful for targeting CIDEB expression is not produced in the target cell by cleavage of a larger dsRNA.

The dsRNA as described herein may further include one or more single-stranded nucleotide overhangs, e.g., 1,2, 3, or 4 nucleotides. dsRNA with at least one nucleotide overhang has unexpectedly superior inhibition properties relative to its blunt-ended counterpart. Nucleotide overhangs may include or consist of: nucleotide/nucleoside analogs comprising deoxynucleotides/nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the overhanging nucleotides may be present on the 5 'end, 3' end or both ends of the antisense strand or sense strand of the dsRNA.

DsRNA can be synthesized by standard methods known in the art as discussed further below, for example, by using an automated DNA synthesizer, such as commercially available from, for example, biosearch (Applied Biosystems, inc.) of applied biosystems.

The iRNA compounds of the invention can be prepared using a two-step procedure. First, individual strands of a double-stranded RNA molecule are prepared separately. The component chains are then annealed. The individual strands of the siRNA compound may be prepared using either solution phase or solid phase organic synthesis or both. Organic synthesis offers the advantage that oligonucleotide chains comprising non-natural or modified nucleotides can be readily prepared. The single stranded oligonucleotides of the invention may be prepared using either solution phase or solid phase organic synthesis or both.

In one aspect, the dsRNA of the invention comprises at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequences are selected from the sequence sets provided in tables 3-6, and the corresponding nucleotide sequences of the antisense strand of the sense strand may be selected from the sequence sets of tables 3-6. In this regard, one of the two sequences is complementary to the other of the two sequences, wherein one of the sequences is substantially complementary to the mRNA sequence produced in the expression of the CIDEB gene. Thus, in this regard, a dsRNA will comprise two oligonucleotides, one of which is described in tables 3-6 as the sense strand (the follower strand) and the second oligonucleotide is described as the corresponding antisense strand (the guide strand) of the sense strand in tables 3-6. In one embodiment, the substantially complementary sequence of the dsRNA is contained on an isolated oligonucleotide. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

It should be appreciated that while the sequences in tables 3-6 are described as modified, unmodified, unconjugated, and/or conjugated sequences, the RNAs of the invention (e.g., the dsRNA of the invention) may include any of the unmodified, unconjugated, and/or differently modified and/or conjugated sequences described in tables 3-6.

It is well known to those skilled in the art that dsrnas having duplex structures of about 20 to 23 base pairs (e.g., 21 base pairs) have been known to be particularly effective in inducing RNA interference (Elbashir et al, (2001) journal of molecular biology (EMBO j.)), 20:6877-6888. However, others have found that shorter or longer RNA duplex structures may also be effective (Chu and Rana (2007) RNA 14:1714-1719; kim et al, (2005) Nature Biotechnology 23:222-226). In the above embodiments, due to the nature of the oligonucleotide sequences provided herein, the dsRNA described herein may comprise at least one strand of at least 21 nucleotides in length. It is reasonably expected that shorter duplexes, minus only few nucleotides at one or both ends, may be similarly effective compared to the dsRNA described above. Thus, dsRNA having a sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides derived from one of the sequences provided herein and whose ability to inhibit CIDEB gene expression differs from dsRNA comprising the complete sequence by no more than about 5%, 10%, 15%, 20%, 25% or 30% inhibition are considered to be within the scope of the invention.

In addition, the RNAs described in tables 3-6 identified sites in CIDEB transcripts that were susceptible to RISC-mediated cleavage. Thus, the invention further features targeting iRNA within the site. As used herein, an iRNA is said to be within a particular site of a targeted RNA transcript if it facilitates cleavage of the transcript at any position within the particular site. Such iRNA will typically comprise at least about 15 contiguous nucleotides from one of the sequences provided herein coupled to an additional nucleotide sequence taken from a region in the gene adjacent to the selected sequence.

Although the target sequence is typically about 15-30 nucleotides in length, within this range the suitability of a particular sequence for directing cleavage of any given target RNA varies greatly. The various software packages and guidelines shown herein provide guidance for the identification of the optimal target sequence for any given gene target, but empirical methods may also be employed in which a "window" or "mask" of a given size (21 nucleotides, as a non-limiting example) is placed literally or symbolically (including, for example, computer simulations) over the target RNA sequence to identify sequences within a range of sizes that can be used as target sequences. By moving the sequence "window" stepwise to one nucleotide upstream or downstream of the initial target sequence position, the next potential target sequence can be identified until a complete set of possible sequences for any given target size selected is identified. This process, combined with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those that perform best, can identify those RNA sequences that mediate optimal inhibition of target gene expression when targeted with iRNA agents. Thus, while the sequences identified herein represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively "shifting the window" by one nucleotide upstream or downstream of a given sequence to identify sequences having the same or better inhibition properties.

Furthermore, it is contemplated that for any sequence identified, further optimization can be achieved by systematically adding or removing nucleotides to produce longer or shorter sequences and testing the sequences produced by moving the target RNA up or down a longer or shorter size window from that point. Likewise, combining this method of generating new candidate targets with testing the effectiveness of iRNA based on these target sequences in inhibition assays as known in the art and/or as described herein can lead to further improvements in inhibition efficiency. Still further, such optimized sequences can be adjusted to further optimize the molecule (e.g., increase serum stability or circulation half-life, increase thermostability, enhance transmembrane delivery, target specific locations or cell types, increase interaction with silencing pathway enzymes, increase release from endosomes) as expression inhibitors by, for example, introducing modified nucleotides as described herein or known in the art, adding or altering overhangs, or other modifications as known in the art and/or discussed herein.

An iRNA agent as described herein may comprise one or more mismatches with a target sequence. In one embodiment, an iRNA as described herein comprises no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches with the target sequence, it is preferred that the mismatched region is not centered in the complementary region. If the antisense strand of the iRNA contains mismatches with the target sequence, it is preferred that the mismatches be limited to the last 5 nucleotides from the 5 'or 3' end of the complementary region. For example, for a 23 nucleotide iRNA agent, the strand complementary to the region of the CIDEB gene typically does not contain any mismatches within the center 13 nucleotides. The methods described herein or known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting CIDEB gene expression. Considering the efficacy of iRNA with mismatches in inhibiting the expression of CIDEB genes is important, especially if the specific complementary region in the CIDEB gene is known to have polymorphic sequence variations within the population.

The RNA target may have regions or spans of the target RNA nucleotide sequence that are relatively more susceptible or susceptible to RNA interference induced by binding of the RNAi agent to the region than other regions of the RNA target to mediate cleavage of the RNA target. Increased sensitivity to RNA interference within such "hot spot regions" (or simply "hot spots") means that an iRNA agent targeting that region may have higher efficacy in inducing iRNA interference than an iRNA agent targeting other regions of the target RNA. For example, without being bound by theory, the accessibility of a target RNA target region may affect the efficacy of an iRNA agent targeting that region, with some hot spots having higher accessibility. For example, secondary structures formed in an RNA target (e.g., within or near a hot spot region) may affect the ability of an iRNA agent to bind to the target region and induce RNA interference.

According to certain aspects of the invention, an iRNA agent can be designed to target a hot spot region of any of the target RNAs described herein, including any identified portion of the target RNA (e.g., a particular exon). As used herein, a hot spot region may refer to a region of about 19-200、19-150、19-100、19-75、19-50、21-200、21-150、21-100、21-75、21-50、50-200、50-150、50-100、50-75、75-200、75-150、75-100、100-200 or 100-150 nucleotides of a target RNA sequence for which targeting using RNAi agents provides a significantly higher probability of effective silencing relative to effective silencing of other regions targeted to the same target RNA. According to certain aspects of the invention, the hot spot region may comprise a restricted region of the target RNA, and in some cases, a substantially restricted region of the target, comprising, for example, less than half the length of the target RNA, such as about 5%, 10%, 15%, 20%, 25% or 30% of the length of the target RNA. Conversely, the other regions for which the hot spot is compared may cumulatively comprise at least a majority of the length of the target RNA. For example, the additional regions can cumulatively comprise at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the length of the target RNA.

The comparison region of the target RNA can be empirically assessed using efficacy data obtained from in vitro or in vivo screening assays to identify hot spots. For example, RNAi agents targeting individual regions across a target RNA can compare the frequency of effective iRNA agents binding to each region (e.g., the amount by which target gene expression is inhibited, e.g., as measured by mRNA expression or protein expression). In general, hotspots can be identified by observing clusters of multiple effective RNAi agents that bind to limited regions of RNA targets. By observing the efficacy of iRNA agents, the characteristics of the hot spots can be fully characterized by the accumulation of agents that cover at least about 60% of the region of interest identified as a hot spot, such as about 70%, about 80%, about 90% or about 95% or more of the length of the region, including both ends of the region (i.e., at least about 60%, 70%, 80%, 90% or 95% or more of the nucleotides within the region, including the nucleotides at each end of the region, targeted by the iRNA agent). According to some aspects of the invention, iRNA agents that exhibit at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% inhibition (e.g., no more than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% mrna remaining) of a region may be identified as effective.

Quantitative comparisons of inhibition measurements across different regions of defined sizes (e.g., 25nt, 30nt, 40nt, 50nt, 60nt, 70nt, 80nt, 90nt, or 100nt, 110nt, 120nt, 130nt, 140nt, 150nt, 160nt, 170nt, 180nt, 190nt, or 200 nt) can also be used to assess the convenience of targeting RNA regions. For example, the average inhibition level for each zone may be determined and the average value for each zone may be compared. The average inhibition level in the hot spot zone may be significantly higher than the average of all the evaluation zones. According to some aspects, the average level of inhibition in the hot spot zone may be at least about 10%, 20%, 30%, 40%, or 50% higher than the average value of the average zone. According to some aspects, the average level of inhibition in the hot spot zone may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviations higher than the average value of the average zone. The average inhibition level may be higher than the statistically significant (e.g., p < 0.05) amount. According to some aspects, each inhibition measurement within a hot spot region can be above a threshold amount (e.g., equal to or below a threshold amount of remaining mRNA). According to some aspects, each inhibition measurement within the region may be significantly higher than the average of all inhibition measurements over all measurement regions. For example, each inhibition measurement in a hot spot zone may be at least about 10%, 20%, 30%, 40%, or 50% higher than the average of all inhibition measurements. According to some aspects, each inhibition measurement may be at least about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 standard deviation higher than the average of all inhibition measurements. Each inhibition measurement may be higher than the average of all inhibition measurements by a statistically significant amount (e.g., p < 0.05). Criteria for assessing hot spots may include various combinations of the above criteria that are compatible (e.g., at least about an average inhibition level of a first amount and no inhibition measurement below a threshold level of a second amount, the second amount being less than the first amount).

Thus, it is expressly contemplated that any iRNA agent that can preferentially select for a hotspot region that targets a target RNA, including the specific exemplary iRNA agents described herein, is used to induce RNA interference of the target mRNA, as targeting such hotspot region may exhibit a strong inhibitory response relative to a region that targets a non-hotspot region. RNAi agents that target sequences that substantially overlap (e.g., at least about 70%, 75%, 80%, 85%, 90%, 95% of the length of the target sequence), or preferably lie entirely within the hotspot region, can be considered to target the hotspot region. The hot spot regions of the RNA targets of the invention may comprise any region where the data disclosed herein demonstrates that an effective RNAi agent has a higher targeting frequency, including by any criteria described elsewhere herein, whether or not such hot spot regions are explicitly specified.

In various embodiments, the dsRNA agents of the invention target the hot spot region of mRNA encoding CIDEB.

III modified iRNA of the invention

In one embodiment, the RNA (e.g., dsRNA) of the iRNA of the invention is unmodified and does not comprise chemical modifications and/or conjugation, e.g., as known in the art and described herein. In another embodiment, the RNA (e.g., dsRNA) of the iRNA of the invention is chemically modified to enhance stability or other beneficial properties. In certain embodiments of the invention, substantially all of the 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. The iRNA of the invention in which "substantially all nucleotides are modified" is largely but not completely modified and may include no more than 5, 4, 3, 2 or 1 unmodified nucleotides.

In some aspects of the invention, substantially all of the nucleotides of an iRNA of the invention are modified, and the iRNA agent comprises no more than 10 nucleotides that comprise 2' -fluoro modifications (e.g., no more than 92 ' -fluoro modifications, no more than 82 ' -fluoro modifications, no more than 72 ' -fluoro modifications, no more than 62 ' -fluoro modifications, no more than 52 ' -fluoro modifications, no more than 42 ' -fluoro modifications, no more than 32 ' -fluoro modifications, or no more than 2' -fluoro modifications). For example, in some embodiments, the sense strand comprises no more than 4 nucleotides that comprise 2' -fluoro modifications (e.g., no more than 32 ' -fluoro modifications, or no more than 2' -fluoro modifications). In other embodiments, the antisense strand comprises no more than 6 nucleotides that comprise 2 '-fluoro modifications (e.g., no more than 5 2' -fluoro modifications, no more than 42 '-fluoro modifications, or no more than 2' -fluoro modifications).

In other aspects of the invention, all nucleotides of an iRNA of the invention are modified, and an iRNA agent comprises no more than 10 nucleotides that comprise a 2' -fluoro modification (e.g., no more than 92 ' -fluoro modifications, no more than 82 ' -fluoro modifications, no more than 72 ' -fluoro modifications, no more than 62 ' -fluoro modifications, no more than 52 ' -fluoro modifications, no more than 42 ' -fluoro modifications, no more than 32 ' -fluoro modifications, or no more than 2' -fluoro modifications).

In one embodiment, the double stranded RNAi agents of the invention further comprise a 5' -phosphate or 5' -phosphate mimetic at the 5' nucleotide of the antisense strand. In another embodiment, the double stranded RNAi agent further comprises a 5 '-phosphate mimetic at the 5' nucleotide of the antisense strand. In a specific embodiment, the 5' -phosphate mimic is 5' -vinyl phosphonate (5 ' -VP). In one embodiment, the phosphate ester mimic is 5' -cyclopropyl phosphonate. In some embodiments, the 5 '-end of the antisense strand of the double-stranded iRNA agent does not contain a 5' -Vinyl Phosphonate (VP).

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 2 '-O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2 '-deoxymodified nucleotides, ethylene glycol modified nucleotides (GNAs), such as Ggn, cgn, tgn or Agn, nucleotides with 2' phosphates, such as G2p, C2p, A2p or U2p, and vinyl phosphonate nucleotides; and combinations thereof. In other embodiments, each of the duplex of tables 4 and 6 can be specifically modified to provide another double-stranded iRNA agent of the present disclosure. In one example, the 3' end of each sense duplex can be modified by removing the 3' end L96 ligand and exchanging two phosphodiester internucleotide linkages between the three 3' end nucleotides with phosphorothioate internucleotide linkages. That is, three 3' terminal nucleotides (N) of the sense sequence of the formula:

5′-N₁-...-N_n-2N_n-1N_nL96 3′

can be replaced by

5′-N₁-...-N_n-2sN_n-1sN_n 3′。

That is, for example, AD-1685156, sense sequence:

csusgcagAfaGfGfUfugacugcguuL96(SEQ ID NO：277)

can be replaced by

csusgcagAfaGfGfUfugacugcgsusu(SEQ ID NO：2922)

While the antisense sequence remains unchanged to provide another double stranded iRNA agent of the present disclosure.

The nucleic acids of the features of the invention may be synthesized and/or modified by art-recognized methods, such as those described in "Current protocols in nucleic acid chemistry (Current protocols in nucleic ACID CHEMISTRY)," Beaucage, S.L., et al (eds.), john Willi father, inc. (John Wiley & Sons, inc., new York, NY, USA), which is hereby incorporated by reference. Modifications include, for example, terminal modifications, such as 5 'terminal modifications (phosphorylation, conjugation, reverse ligation, etc.) or 3' terminal modifications (conjugation, DNA nucleotides, reverse ligation etc); base modification, e.g., substitution, removal (no base nucleotides) or conjugation of bases with stable bases, destabilizing bases or bases base pairing with extended partner pools; sugar modification (e.g., at the 2 'position or the 4' position) or sugar substitution; and/or backbone modifications, including modifications or substitutions of phosphodiester bonds. Specific examples of iRNA compounds that can be used in the embodiments described herein include, but are not limited to, RNAs that contain modified backbones or do not contain natural internucleoside linkages. In addition, RNAs having modified backbones include those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, modified RNAs that do not have phosphorus atoms in their internucleoside backbones can also be considered oligonucleotides. In some embodiments, the modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkylphosphonates (including 3 '-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3' -phosphoramidate and aminoalkyl amine phosphates), thiocarbonylphosphoramidates, thiocarbonylalkyl phosphonates, thiocarbonylalkyl phosphotriesters, and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these esters, and those with reversed polarity, wherein adjacent pairs of nucleoside units are linked in 3'-5' to 5'-3' or 2'-5' to 5 '-2'. Various salts, mixed salts and free acid forms are also included. In some embodiments of the invention, the dsRNA agent of the invention is in the free acid form. In other embodiments of the invention, the dsRNA agent of the invention is in salt form. In one embodiment, the dsRNA agent of the invention is in the form of a sodium salt. In certain embodiments, when the dsRNA agents of the invention are in the form of sodium salts, sodium ions are present in the agent as counter ions to substantially all of the phosphodiester and/or phosphorothioate groups present in the agent. Agents in which substantially all of the phosphodiester and/or phosphorothioate linkages have a sodium counterion comprise no more than 5, 4, 3, 2, or 1 phosphodiester and/or phosphorothioate linkages that do not have a sodium counterion. In some embodiments, when the dsRNA agents of the invention are in the form of sodium salts, sodium ions are present in the agent as counter ions to all phosphodiester and/or phosphorothioate groups present in the agent.

Representative U.S. patents teaching the preparation of the above-described phosphorus-containing bonds include, but are not limited to, U.S. patent No. 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;5,321,131;5,399,676;5,405,939;5,453,496;5,455,233;5,466,677;5,476,925;5,519,126;5,536,821;5,541,316;5,550,111;5,563,253;5,571,799;5,587,361;5,625,050;6,028,188;6,124,445;6,160,109;6,169,170;6,172,209;6,239,265;6,277,603;6,326,199;6,346,614;6,444,423;6,531,590;6,534,639;6,608,035;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, each of which is hereby incorporated by reference in its entirety.

Wherein the modified RNA backbone that does not comprise a phosphorus atom has a backbone formed by: short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatoms or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar moiety of the nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thioacetylacetyl backbones; methylene formylacetyl and thioformylacetyl backbones; an olefin-containing backbone; a sulfamate backbone; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; an amide backbone; and other backbones with mixed N, O, S and CH ₂ component moieties.

Representative U.S. patents that teach the preparation of the above-described oligonucleotides include, but are not limited to, U.S. patent 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;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 each of which are hereby incorporated by reference.

In other embodiments, suitable RNA mimics are contemplated for iRNA in which the sugar and internucleoside linkages of the nucleotide units, i.e., the backbone, are replaced with new groups. The base unit is maintained to hybridize to the appropriate nucleic acid target compound. One such oligomeric compound, i.e., an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a Peptide Nucleic Acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide containing backbone, especially an aminoethylglycine backbone. The nucleobases are retained and bound directly or indirectly to the aza nitrogen atoms of the amide moiety 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; no. 5,714,331; and U.S. Pat. No. 5,719,262, the entire contents of each of which are hereby incorporated by reference. Additional PNA compounds suitable for use in the iRNA of the present invention are described, for example, in Nielsen et al Science, 1991, 254, 1497-1500.

Some embodiments of features of the invention include RNAs with phosphorothioate backbones and oligonucleotides with heteroatom backbones, particularly- -CH ₂--NH--CH₂-、--CH₂--N(CH₃)--O--CH₂ - - [ known as methylene (methylimino) or MMI backbones ],--CH₂--O--N(CH₃)--CH₂--、--CH₂--N(CH₃)--N(CH₃)--CH₂-- and- -N (CH ₃)--CH₂--CH₂ - - [ wherein the natural phosphodiester backbone is represented as- -O- -P- -O- -CH ₂ - - ], as well as the amide backbone of U.S. Pat. No. 5,602,240 cited above.

The modified RNA may also include one or more substituted sugar moieties. An iRNA, e.g., dsRNA, as characterized herein may comprise one of the following at the 2' position: OH; f, performing the process; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl may be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_nO]_mCH₃、O(CH₂)._nOCH₃、O(CH₂)_nNH₂、O(CH₂)_nCH₃、O(CH₂)_nONH₂ and O (CH ₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. In other embodiments, the dsRNA comprises one of the following at the 2' position: c ₁ to C ₁₀ lower alkyl, substituted lower alkyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl 、SH、SCH₃、OCN、Cl、Br、CN、CF₃、OCF₃、SOCH₃、SO₂CH₃、ONO₂、NO₂、N₃、NH₂、 heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of iRNA, or groups for improving the pharmacodynamic properties of iRNA, and other substituents with similar properties. In some embodiments, the modification comprises a 2 '-methoxyethoxy (2' -O- -CH ₂CH₂OCH₃), also known as 2'-O- (2-methoxyethyl) or 2' -MOE (Martin et al, swiss chemical journal (Helv. Chim. Acta), 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2 '-dimethylaminooxyethoxy, i.e., the O (CH ₂)₂ON(CH₃) 2 group, also known as 2' -DMAOE, as described in the examples below, and 2 '-dimethylaminoethoxyethoxy (also known in the art as 2' -O-dimethylaminoethoxyethyl or 2 '-DMAEOE), i.e., 2' -o—ch ₂--O--CH₂--N(CH₂)₂. Additional exemplary modifications include: 5'-Me-2' -F nucleotide, 5'-Me-2' -OMe nucleotide, 5'-Me-2' -deoxynucleotide (both R and S isomers in these three families); 2' -alkoxyalkyl; and 2' -NMA (N-methylacetamide).

Other modifications include 2 '-methoxy (2' -OCH ₃), 2 '-aminopropoxy (2' -OCH ₂CH₂CH₂NH₂) and 2 '-fluoro (2' -F). Similar modifications can also be made at other positions on the RNA of the iRNA, particularly at the 3 'position of the sugar on the 3' terminal nucleotide or at the 5 'position of the 2' -5 'linked dsRNA and 5' terminal nucleotide. The iRNA may also have a glycomimetic, such as a cyclobutyl moiety in place of the pentose sugar. Representative U.S. patents teaching the preparation of such modified sugar structures include, but are not limited to, U.S. patent nos. 4,981,957, 5,118,800;5,319,080;5,359,044;5,393,878;5,446,137;5,466,786;5,514,785;5,519,134;5,567,811;5,576,427;5,591,722;5,597,909;5,610,300;5,627,053;5,639,873;5,646,265;5,658,873;5,670,633, and 5,700,920, some of which are co-owned by the present application. The entire contents of each of the foregoing are hereby incorporated by reference.

The iRNA of the invention may also comprise nucleobase (commonly referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases comprise the purine bases adenine (a) and guanine (G), as well as the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional nucleobases include those disclosed in U.S. Pat. No. 3,687,808, modified nucleosides in biochemistry, biotechnology and Medicine (Modified Nucleosides in Biochemistry, biotechnology and Medicine), herdewijn, P.edition Wiley-VCH Press (Wiley-VCH), those disclosed in 2008; those disclosed in polymer science and Engineering encyclopedia (The Concise Encyclopedia Of Polymer Science and Engineering), pages 858-859, kroschwitz, J.L. edited John wili parent, 1990, englisch et al (1991), international edition chemical applications (ANGEWANDTE CHEMIE, international Edition), 30:613, and a second set of the compounds disclosed in the text, and Sanghvi, Y s., chapter 15, dsRNA research and Applications (DSRNA RESEARCH and Applications), pages 289-302, crooke, s.t. and Lebleu, b. editions, CRC Press, 1993. Certain of these nucleobase pairs are particularly useful in increasing the binding affinity of oligomeric compounds that are characteristic of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. It has been shown that 5-methylcytosine substitution increases the stability of the nucleic acid duplex by 0.6 ℃ to 1.2 ℃ (Sanghvi, y.s., crooke, s.t. and Lebleu, b. Editions, dsRNA research and applications, CRC press, boca Raton, 1993, pages 276-278), and is an exemplary base substitution, even more particularly when combined with a 2' -O-methoxyethyl sugar modification.

Representative U.S. patents teaching the preparation of certain of the above-described modified nucleobases, as well as other modified nucleobases, include, but are not limited to, U.S. Pat. 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;5,594,121,5,596,091;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, each of which is hereby incorporated by reference in its entirety.

The iRNA of the invention may also be modified to include one or more Locked Nucleic Acids (LNAs). Locked nucleic acids are nucleotides having a modified ribose moiety, where the ribose moiety includes an additional bridge connecting the 2 'and 4' carbons. This structure effectively "locks" the ribose in the 3' -internal structure conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. Et al (2005) nucleic acids Ind 33 (1): 439-447; mook, OR. Et al (2007) molecular cancer therapeutics 6 (3): 833-843; grunwiller, A. Et al (2003) nucleic acids Ind 31 (12): 3185-3193).

The iRNA of the invention may also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by bridging of two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic system. In certain embodiments, the bridge connects the 4 'carbon and the 2' -carbon of the sugar ring. Thus, in some embodiments, the agents of the invention may comprise one or more Locked Nucleic Acids (LNAs). Locked nucleic acids are nucleotides having a modified ribose moiety, where the ribose moiety includes an additional bridge connecting the 2 'and 4' carbons. In other words, LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the 3' -internal structure conformation. The addition of locked nucleic acids to siRNA has been shown to increase siRNA stability in serum and reduce off-target effects (Elmen, J. Et al, (2005) nucleic acids Ind 33 (1): 439-447; mook, OR. Et al, (2007) molecular cancer therapeutics 6 (3): 833-843; grunwiller, A. Et al, (2003) nucleic acids Ind 31 (12): 3185-3193). Examples of bicyclic nucleosides for use in the present invention nucleotides include, but are not limited to nucleosides that include a bridge between the 4 'and 2' ribosyl ring atoms. In certain embodiments, antisense polynucleotide agents of the invention comprise one or more bicyclic nucleosides comprising a 4 'to 2' bridge. Examples of such 4 'to 2' bridged bicyclic nucleosides include, but are not limited to, 4'- (CH 2) -O-2' (LNA); 4'- (CH 2) -S-2';4'- (CH 2) 2-O-2' (ENA); 4'-CH (CH 3) -O-2' (also known as "constrained ethyl" or "cEt") and 4'-CH (CH 2OCH 3) -O-2' (and analogs thereof; see, for example, U.S. patent 7,399,845); 4'-C (CH 3) (CH 3) -O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH2-N (OCH 3) -2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-O-N (CH 3) -2' (see, e.g., U.S. patent publication No. 2004/0171570); 4'-CH2-N (R) -O-2', wherein R is H, C-C12 alkyl or a protecting group (see, e.g., U.S. patent No. 7,427,672); 4'-CH2-C (H) (CH 3) -2' (see, e.g., chattopadhyaya et al, journal of organic chemistry (j. Org. Chem.)), 2009, 74, 118-134; and 4'-CH2-C (=ch2) -2' (and analogs thereof; see, e.g., U.S. patent No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated by reference.

Additional representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. No. 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;US 2008/0039618; and U.S. Pat. No. 2009/0012281, the entire contents of each of which are hereby incorporated by reference.

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

The iRNA of the invention may also be modified to include one or more constrained ethyl nucleotides. As used herein, a "constrained ethyl nucleotide" or "cEt" is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4'-CH (CH 3) -0-2' bridge. In one embodiment, the constrained ethyl nucleotide is in an S conformation referred to herein as "S-cEt".

The iRNA of the invention may also comprise one or more "conformational restriction nucleotides" ("CRNs"). CRNs are nucleotide analogs having a linker linking the C2' and C4' carbons of ribose or the C3 and-C5 ' carbons of ribose. CRN locks the ribose ring in a stable conformation and increases the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in the optimal position for stability and affinity, resulting in less ribose ring wrinkling.

Representative publications teaching the preparation of some of the above CRNs include, but are not limited to, U.S. patent publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated by reference.

In some embodiments, an iRNA of the invention includes one or more monomers that are UNA (unlocking nucleic acid) nucleotides. UNA is an unlocked acyclic nucleic acid in which any bonds of the sugar have been removed, forming an unlocked "sugar" residue. In one example, UNA also encompasses monomers where the bond between C1'-C4' has been removed (i.e., covalent carbon-oxygen-carbon bonds between C1 'and C4' carbons). In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2 'and C3' carbons) has been removed (see nucleic acid seminar a series of books (nuc. Acids symp. Series), 52, 133-134 (2008) and Fluiter et al, molecular biological systems (mol. Biosystem.), 2009, 10, 1039, which are hereby incorporated by reference).

Representative U.S. disclosures teaching the preparation of UNA include, but are not limited to, U.S. patent No. 8,314,227; U.S. patent publication No. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated by reference.

The RNAi agents of the present disclosure can also comprise one or more "cyclohexene nucleic acids" or ("CeNA"). CeNA is a nucleotide analogue in which the furanose portion of DNA is replaced with a cyclohexene ring. Incorporation of cyclohexylnucleosides in the DNA strand increases the stability of the DNA/RNA hybrids. CeNA is stable in serum and does not degrade, and CeNA/RNA hybrids activate E.coli RNase H, resulting in RNA strand cleavage. (see Wang et al, journal of the American society of chemistry (am. Chem. Soc.) 2000, 122, 36, 8595-8602), which is hereby incorporated by reference.

Potentially stable modifications to the ends of the RNA molecule may include N- (acetamidohexanoyl) -4-hydroxyproline (Hyp-C6-NHAc), N- (hexanoyl-4-hydroxyproline) (Hyp-C6), N- (acetyl-4-hydroxyproline) (Hyp-NHAc), thymidine-2 '-0-deoxythymidine (ether), N- (aminohexanoyl) -4-hydroxyproline (Hyp-C6-amino), 2-behenoyl-uridine-3' -phosphate, inverted base dT (idT), and the like. The disclosure of this modification can be found in PCT publication No. WO 2011/005861.

Other modifications of the RNAi agents of the invention include 5' phosphates or 5' phosphate mimics, e.g., 5' terminal phosphates or phosphate mimics on the antisense strand of the RNAi agents. Suitable phosphate ester mimetics are disclosed, for example, in U.S. patent publication 2012/0157511, the entire contents of which are incorporated herein by reference.

In certain embodiments, the RNAi agents of the invention are agents that inhibit expression of CIDEB genes selected from the group consisting of the agents listed in tables 3-6. Any of these agents may further comprise a ligand.

A. Modified iRNA comprising the motif of the invention

In certain aspects of the invention, the double stranded RNAi agents of the invention comprise agents with chemical modifications, as disclosed in WO 2013/075035, for example, filed 11/16 in 2012, the entire contents of which are incorporated herein by reference.

Accordingly, the present invention provides double stranded RNAi agents capable of inhibiting expression of a target gene (i.e., CIDEB gene) in vivo. RNAi agents include a sense strand and an antisense strand. Each strand of the RNAi agent can be 12 to 30 nucleotides in length. For example, each strand may be 14 to 30 nucleotides in length, 17 to 30 nucleotides in length, 25 to 30 nucleotides in length, 27 to 30 nucleotides in length, 17 to 23 nucleotides in length, 17 to 21 nucleotides in length, 17 to 19 nucleotides in length, 19 to 25 nucleotides in length, 19 to 23 nucleotides in length, 19 to 21 nucleotides in length, 21 to 25 nucleotides in length, or 21 to 23 nucleotides in length. In one embodiment, the sense strand is 21 nucleotides in length. In one embodiment, the antisense strand is 23 nucleotides in length.

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

In one embodiment, the RNAi agent can comprise one or more overhanging regions and/or capping groups at the 3 'end, 5' end, or both of one or both strands. The length of the overhang may be 1 to 6 nucleotides, for example 2 to 6 nucleotides in length, 1 to 5 nucleotides in length, 2 to 5 nucleotides in length, 1 to 4 nucleotides in length, 2 to 4 nucleotides in length, 1 to 3 nucleotides in length, 2 to 3 nucleotides in length, or 1 to 2 nucleotides in length. An overhang may be the result of one strand being longer than the other, or the result of two strands of the same length being interleaved. The overhang may form a mismatch with the target mRNA, or it may be complementary to the gene sequence being targeted, or it may be another sequence. The first strand and the second strand may also be linked, for example by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhanging region of the dsRNAi agent can each independently be a modified or unmodified nucleotide, including but not limited to 2' -sugar modified, 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 (m 5 Ceo), and any combination thereof. For example, TT may be an overhang sequence at either end of either strand. The overhang may form a mismatch with the target mRNA, or it may be complementary to the gene sequence being targeted, or it may be another sequence.

The 5 'or 3' overhangs on the sense strand, antisense strand, or both strands of the RNAi agent can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with phosphorothioates between the two nucleotides, where 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, such a 3' overhang is present in the antisense strand. In one embodiment, such a 3' overhang is present in the sense strand.

RNAi agents may contain only a single overhang, which may enhance the interfering activity of RNAi without affecting its overall stability. For example, the single stranded overhang may be located at the 3 'end of the sense strand, or alternatively, at the 3' end of the antisense strand. RNAi may also have a blunt end located at the 5 'end of the antisense strand (or 3' end of the sense strand), and vice versa. Typically, the antisense strand of an RNAi has a nucleotide overhang at the 3 'end, and the 5' end is flat. While not wishing to be bound by theory, the asymmetric blunt end of the 5 'end of the antisense strand and the 3' overhang of the antisense strand facilitate the loading of the guide strand into the RISC process.

In one embodiment, the RNAi agent is a double-ended passivating agent 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, 9 starting from the 5' end. The antisense strand contains at least one motif of three 2 '-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 starting from the 5' end.

In another embodiment, the RNAi agent is a double-ended passivating agent 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, 10 starting from the 5' end. The antisense strand contains at least one motif of three 2 '-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 starting from the 5' end.

In yet another embodiment, the RNAi agent is a double-ended passivating agent 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, 11 starting from the 5' end. The antisense strand contains at least one motif of three 2 '-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 starting from the 5' end.

In one embodiment, the RNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5' end; the antisense strand contains at least one motif of three 2 '-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 starting from the 5' end, wherein one end of the RNAi agent is flat and the other end comprises a2 nucleotide overhang. Preferably, the 2 nucleotide overhang is located at the 3' end of the antisense strand.

When a2 nucleotide overhang is located at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are the overhang nucleotides and the third is the pairing nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent has two additional phosphorothioate internucleotide linkages between the terminal three nucleotides of both the 5 'end of the sense strand and the 5' end of the antisense strand. In one embodiment, each nucleotide in the sense and antisense strands of the RNAi agent, including a nucleotide that is part of a motif, is a modified nucleotide. In one embodiment, each residue is independently modified with 2 '-O-methyl or 3' -fluoro, e.g., in an alternating motif. Optionally, the RNAi agent further comprises a ligand (preferably GalNAc ₃).

In one embodiment, the RNAi agent comprises a sense strand and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, starting at position 1 to position 23 of the 5' terminal nucleotide (position 1) of the first strand, comprising at least 8 ribonucleotides; the antisense strand is 36 to 66 nucleotide residues in length and, starting from the 3' terminal nucleotide, comprises at least 8 ribonucleotides in the position that pairs with positions 1 to 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 at most 6 consecutive 3' terminal nucleotides are unpaired with the sense strand, thereby forming a1 to 6 nucleotide 3' single stranded overhang; wherein the 5 'end of the antisense strand comprises 10 to 30 consecutive nucleotides that are unpaired with the sense strand, thereby forming a 10 to 30 nucleotide single strand 5' overhang; wherein when the sense strand and the antisense strand are aligned for maximum complementarity, at least the 5 'end and 3' end nucleotides of the sense strand are paired with the nucleotide bases of the antisense strand, thereby forming a substantially double-stranded region between the sense strand and the antisense strand; and the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides of the antisense strand length to reduce target gene expression upon introduction of the double-stranded nucleic acid into a mammalian cell; and wherein the sense strand contains at least one motif modified by three 2' -F on three consecutive nucleotides, wherein at least one of the motifs is present at or near the cleavage site. The antisense strand contains at least one motif of three 2' -O-methyl modifications at or near the cleavage site at three consecutive nucleotides.

In one embodiment, an RNAi agent comprises a sense strand and an antisense strand, wherein the RNAi agent comprises a first strand of at least 25 and at most 29 nucleotides in length and a second strand of at most 30 nucleotides in length, having at least one motif with 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 to 4 nucleotides longer than the first strand at its 3 'end, wherein the duplex region is at least 25 nucleotides in length and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of the second strand length to reduce target gene expression upon introduction of the RNAi agent into a mammalian cell, and wherein dicer cleavage of the RNAi agent preferentially produces siRNA comprising the 3' end of the second strand, thereby reducing expression of the target gene in a mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at least one motif with three identical modifications on three consecutive nucleotides, one of which is present at a cleavage site in the sense strand.

In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, with one motif present at or near the cleavage site in the antisense strand.

For RNAi agents having duplex regions of 17 to 23 nucleotides in length, the cleavage site of the antisense strand is typically near the 10, 11, and 12 positions from the 5' end. Thus, three identical modified motifs may be present at positions 9, 10, 11 of the antisense strand; 10. 11, 12 positions; 11. 12, 13 positions; 12. 13, 14 positions; or positions 13, 14, 15, counting from nucleotide 1 of the 5 'end of the antisense strand, or counting from nucleotide 1 of the pairing within the 5' duplex region of the antisense strand. The cleavage site in the antisense strand may also vary depending on the length of the duplex region of the RNAi from the 5' end.

The sense strand of an RNAi agent can contain at least one motif of three identical modifications at three consecutive nucleotides at the cleavage site of the strand; and the antisense strand can have at least one motif of three identical modifications at or near three consecutive nucleotides of the strand at the cleavage site. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand may be arranged such that one motif of three nucleotides on the sense strand overlaps one motif of three nucleotides on the antisense strand by at least one nucleotide, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of an RNAi agent can contain more than one motif with three identical modifications on three consecutive nucleotides. The first motif may be present at or near the cleavage site of the strand, and the other motif may be a wing modification. The term "wing modification" herein refers to a motif present at another portion of a strand that is separated from the motif at or near the cleavage site of the same strand. The wing modifications are either adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are in close proximity to each other, then 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. There may be two or more wing modifications. For example, when two wing modifications are present, each wing modification may be present at one end relative to a first motif located at or near the cleavage site, or on either side of the leader motif.

As with the sense strand, the antisense strand of an RNAi agent can comprise more than one motif of three identical modifications on three consecutive nucleotides, with at least one motif present at or near the cleavage site of the strand. The antisense strand may also contain one or more wing modifications in an arrangement similar to the wing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense or antisense strand of the RNAi agent generally does not comprise the first or first two terminal nucleotides at the 3', 5', or both ends of the strand.

In another embodiment, the wing modification on the sense or antisense strand of the RNAi agent generally does not comprise the first or first two paired nucleotides within the duplex region at the 3', 5', or both ends of the strand.

When the sense and antisense strands of an RNAi agent each contain at least one flanking modification, the flanking modifications may fall on the same end of the duplex region and have an overlap of one, two, or three nucleotides.

When the sense strand and the antisense strand of the RNAi agent each comprise at least two winged modifications, the sense strand and the antisense strand can be arranged such that the two modifications, each from one strand, fall at one end of the duplex region, with an overlap of one, two, or three nucleotides; two modifications each from one strand fall at the other end of the duplex region, with an overlap of one, two, or three nucleotides; two modifications of one strand fall on each side of the leader motif, with one, two or three nucleotide overlaps in the duplex region.

In one embodiment, each nucleotide in the sense and antisense strands of the RNAi agent, including the nucleotide as part of the motif, can be modified. Each nucleotide may be modified with the same or different modifications, which may include one or more changes to one or two non-linked phosphate oxygens or one or more linked phosphate oxygens; a change in the composition of the ribose sugar, e.g., a change in the 2' hydroxyl group on the ribose sugar; large scale replacement of phosphate moieties with "dephosphorylation" linkers; modification or substitution of naturally occurring bases; substitution or modification of the ribose-phosphate backbone.

Since nucleic acids are polymers of subunits, many modifications occur at repeated positions within the nucleic acid, such as modifications of bases or phosphate moieties, or non-linking O of phosphate moieties. In some cases, the modification will occur at all subject positions in the nucleic acid, but in many cases will not. For example, the modification may occur only at the 3 'or 5' terminal position, may occur only in the terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. Modification may occur in the double-stranded region, the single-stranded region, or both. Modification may occur only in the double stranded region of the RNA, or may occur only in the single stranded region of the RNA. For example, phosphorothioate modifications at non-linked O positions may occur only at one or both ends, may occur only in the end regions, e.g., at positions on the end nucleotides or in the last 2, 3, 4, 5 or 10 nucleotides of the strand, or may occur in double-and single-stranded regions, particularly at the ends. One or more of the 5' ends may be phosphorylated.

For example, to enhance stability, a particular base may be included in the overhang, or a modified nucleotide or nucleotide substitute may be included in the single stranded overhang, such as in the 5 'or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhangs. In some embodiments, all or some of the bases in the 3 'or 5' overhangs may be modified, e.g., by modification described herein. Modifications may include, for example, modifications at the 2' position of ribose sugar using modifications known in the art, for example, ribose with deoxyribonucleotide, 2' -deoxy-2 ' -fluoro (2 ' -F), or 2' -O-methyl modifications instead of nucleobases, and modifications of phosphate groups, for example phosphorothioate modifications. The overhangs need not be homologous to the target sequence.

In one embodiment, each residue of the sense and antisense strands is independently modified with LNA, CRN, cET, UNA, HNA, ceNA, 2' -methoxyethyl, 2' -O-methyl, 2' -O-allyl, 2' -C-allyl, 2' -deoxy, 2' -hydroxy, or 2' -fluoro. The chain may contain more than one modification. In one embodiment, each residue of the sense and antisense strands is independently modified with 2 '-O-methyl or 2' -fluoro. The term "HNA" refers to hexitols or hexose nucleic acids.

There are typically at least two different modifications on the sense and antisense strands. These two modifications may be 2 '-O-methyl or 2' -fluoro modifications, or other modifications.

In one embodiment, N _a and/or N _b comprise an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. Alternate nucleotides may 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 of a nucleotide, the alternating motifs may be "ababababababab.", "aabbaabbaabbaabbab.", "aaabaabaaababaaab.", "aaabbbaabbabbb.", or "abccabcabc." or the like.

The types of modifications contained in the alternating motifs may be the same or different. For example, if A, B, C, D each represents one type of modification on a nucleotide, the alternating pattern, i.e., the modification on every other nucleotide, may be the same, but each sense strand or antisense strand may be selected from several modification possibilities within an alternating motif, such as "ababab.", "acacac.", "bdbd." or "cdcdcd.", etc.

In one embodiment, the RNAi agents of the invention comprise a pattern of modification of an alternating motif on the sense strand that is shifted relative to the pattern of modification of an alternating motif on the antisense strand. The offset may be such that the modified group of nucleotides of the sense strand corresponds to a different modified group of nucleotides of the antisense strand, and vice versa. For example, when the sense strand is paired with an antisense strand in a dsRNA duplex, the alternating motifs in the sense strand may begin with "ABABAB" from 5 'to 3' of the strand, and the alternating motifs in the antisense strand may begin with "BABABA" from 5 'to 3' of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with "AABBAABB" from 5 'to 3' of the strand, and the alternating motif in the antisense strand may start with "BBAABBAA" from 5 'to 3' of the strand within the duplex region, so there is a complete or partial transfer of modification pattern between the sense and antisense strands.

In one embodiment, the RNAi agent comprises a pattern of alternating motifs of 2 '-O-methyl modification and 2' -F modification on the sense strand initially offset from the pattern of alternating motifs of 2 '-O-methyl modification and 2' -F modification on the antisense strand, i.e., 2 '-O-methyl modified nucleotides on the bases of the sense strand pair with 2' -F modified nucleotides on the antisense strand, and vice versa. The 1 position of the sense strand may start with a 2'-F modification and the 1 position of the antisense strand may start with a 2' -O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand and/or the antisense strand interrupts the initial modification pattern present in the sense strand and/or the antisense strand. This pattern of modification of the sense strand and/or antisense strand by introducing three identical modifications of one or more motifs on three consecutive nucleotides into the sense strand and/or antisense strand surprisingly enhances the gene silencing activity against the target gene.

In one embodiment, when three identically modified motifs on three consecutive nucleotides are introduced into any strand, the modification of the nucleotide next to the motif is a modification that is different from the modification of the motif. For example, the sequence portion containing the motif is "..n _aYYYN_b.," where "Y" represents a modification of the motif for three identical modifications on three consecutive nucleotides, and "N _a" and "N _b" represent modifications to the modification of the nucleotide next to the motif "YYY" that are different from Y, and where N _a and N _b may be the same or different modifications. Alternatively, when wing modifications are present, N _a and/or N _b may or may not be present.

The RNAi agent can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. Phosphorothioate or methylphosphonate internucleotide linkage modifications may occur on any nucleotide of the sense or antisense strand or both strands in any position of the strand. For example, internucleotide linkage modifications may occur on each nucleotide on the sense and/or antisense strand; each internucleotide linkage modification may occur in alternating patterns on the sense strand and/or the antisense strand; or the sense strand or antisense strand may comprise two internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same as or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may be offset relative to the alternating pattern of internucleotide linkage modifications on the antisense strand. In one embodiment, the double stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In one 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 the 5 'end or the 3' end.

In one embodiment, the RNAi agent comprises phosphorothioate or methylphosphonate internucleotide linkage modifications in the overhanging region. For example, the overhang region may contain two nucleotides with phosphorothioate or methylphosphonate internucleotide linkages therebetween. Internucleotide linkage modifications may also be made to link the overhanging nucleotides to terminal pairing nucleotides within the duplex region. For example, at least 2,3, 4 or all of the overhang nucleotides can be linked by phosphorothioate or methylphosphonate internucleotide linkages, and optionally, additional phosphorothioate or methylphosphonate internucleotide linkages can be present, linking the overhang nucleotide to the paired nucleotide next to the overhang nucleotide. For example, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, two of which are the overhang nucleotides and the third is the pairing nucleotide adjacent to the overhang nucleotide. These terminal three nucleotides may be located 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 antisense strand.

In one embodiment, the 2-nucleotide overhang is located at the 3' end of the antisense strand and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides and the third nucleotide is the paired nucleotide next to the overhang nucleotide. Optionally, the RNAi agent can additionally have two phosphorothioate internucleotide linkages 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, the RNAi agent comprises a mismatch to the target, within the duplex, or a combination thereof. The mismatch may occur in the overhang region or the duplex region. Base pairs may be ordered based on their propensity to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairing on a single pairing basis, although the next nearest point or similar analysis may also be used). In promoting dissociation: a: u is better than G: c, performing operation; g: u is better than G: c, performing operation; and I: c is better than G: c (i=inosine). Mismatches, such as non-canonical or canonical pairs (as described elsewhere herein) are better than canonical (A: T, A: U, G: C) pairs; and pairing involving universal bases is preferred over canonical pairing.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within a duplex region starting from the 5' end of the antisense strand, said duplex region being independently selected from the group consisting of: a: u, G: u, I: c and mismatch pairs, e.g., non-canonical or canonical exopairs or pairs that contain universal bases, to promote dissociation of the antisense strand at the 5' end of the duplex.

In one embodiment, the nucleotide at position 1 within the duplex region starting from the 5' end in the 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 within the duplex region starting from the 5' end of the antisense strand is an AU base pair. For example, the first base pair in the duplex region from the 5' end of the antisense strand is an AU base pair.

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 deoxythymidines, e.g., two dT nucleotides on the 3' end of the sense and/or antisense strand.

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)

Wherein:

i and j are each independently 0 or 1;

p and q are each independently 0 to 6;

each N _a independently represents an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

Each N _b independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

Each of n _p and n _q independently represents an overhang nucleotide;

Wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represents a motif of three identical modifications on three consecutive nucleotides. Preferably, YYY are all 2' -F modified nucleotides.

In one embodiment, N _a and/or N _b comprise an alternating pattern of modifications.

In one embodiment, the YYY motif is present at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17 to 23 nucleotides in length, the YYY motif can be present at or near the cleavage site of the sense strand (e.g., can be present at positions 6, 7, 8;7, 8, 9;8, 9, 10;9, 10, 11;10, 11, 12 or 11, 12, 13), counting from nucleotide 1, starting from the 5' end; or optionally counting from the 5' end, starting from the 1 st paired nucleotide within the duplex region.

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 (b)

5′n_p-N_a-XXX-N_b-YYY-N_b-ZZZ-N_a-n_q 3′ (Id)。

When the sense strand is represented by formula (Ib), N _b represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N _a can independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the sense strand is represented by formula (Ic), N _b represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N _a can independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the sense strand is represented by formula (Id), each N _b independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Preferably, N _b is 0, 1,2, 3,4,5, or 6. Each N _a can independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

X, Y and Z may each be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand can be 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), each N _a may independently represent an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

In one embodiment, the antisense strand sequence of RNAi can be represented by formula (Ie):

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′ (Ie)

Wherein:

k and l are each independently 0 or 1;

p 'and q' are each independently 0 to 6;

Each N _a' independently represents an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

each N _b' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

Each of n _p 'and n _q' independently represents an overhang nucleotide;

wherein N _b 'and Y' do not have the same modification; and

X ' X ' X ', Y ' Y ' Y ' and Z ' Z ' Z ' each independently represent a motif of three identical modifications on three consecutive nucleotides.

In one embodiment, N _a 'and/or N _b' comprise an alternating pattern of modifications.

The Y ' Y ' Y ' motif is present at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17 to 23 nucleotides in length, the Y' motif can be present at positions 9, 10, 11 of the antisense strand; 10. 11, 12; 11. 12, 13; 12. 13, 14; or 13, 14, 15, counting from nucleotide 1, from the 5' end; or optionally counting from the 5' end, starting from the 1 st paired nucleotide within the duplex region. Preferably, the Y ' Y ' Y ' motif is present at positions 11, 12, 13.

In one embodiment, the Y 'Y' Y 'motifs are all 2' -OMe modified nucleotides.

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′ (Ig)；

5'n _q′-N_a′-Y′Y′Y′-N_b′-X′X′X′-n_p '3' (Ih); or (b)

5′n_q′-N_a′-Z′Z′Z′-N_b′-Y′Y′Y′-N_b′-X′X′X′-N_a′-n_p′3′ (Ii).

When the antisense strand is represented by formula (Ig), N _b' represents an oligonucleotide sequence comprising 0 to 10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N _a' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the antisense strand is represented by formula (Ih), N _b' represents an oligonucleotide sequence comprising 0 to 10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N _a' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the antisense strand is represented by formula (Ii), each N _b' independently represents an oligonucleotide sequence comprising 0 to 10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N _a' independently represents an oligonucleotide sequence comprising 2 to 20,2 to 15, or 2 to 10 modified nucleotides. Preferably, N _b is 0,1, 2,3, 4, 5, or 6.

In other embodiments, k is 0 and 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′ (If)。

When the antisense strand is represented by formula (If), each N _a' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

Each of X ', Y ', and Z ' may be the same or different from each other.

Each nucleotide of the sense and antisense strands may be independently modified with LNA, CRN, UNA, cEt, HNA, ceNA, 2 '-methoxyethyl, 2' -O-methyl, 2 '-O-allyl, 2' -C-allyl, 2 '-hydroxy, or 2' -fluoro. For example, each nucleotide of the sense strand and the antisense strand is independently modified with 2 '-O-methyl or 2' -fluoro. In particular, each X, Y, Z, X ', Y ', and Z ' may represent a 2' -O-methyl modification or a 2' -fluoro modification.

In one embodiment, when the duplex region is 21nt, the sense strand of the RNAi agent can contain YYY motifs present at positions 9, 10, and 11 of the strand, counting from nucleotide 1 of the 5 'end, or optionally counting from nucleotide 1 of the pairing within the duplex region of the 5' end; and Y represents a 2' -F modification. The sense strand may additionally contain an XXX motif or a ZZZ motif as a wing modification at the opposite end of the duplex region; and XXX and ZZZ each independently represent a 2'-OMe modification or a 2' -F modification.

In one embodiment, the antisense strand may comprise a Y ' motif present at positions 11, 12, 13 of the strand, counting from nucleotide 1 of the 5' end, or optionally, counting from nucleotide 1 of the pairing within the duplex region of the 5' end; and Y 'represents a 2' -O-methyl modification. The antisense strand may additionally contain an X 'motif or a Z' motif as a wing modification at opposite ends of the duplex region; and X 'X' X 'and Z' Z 'Z' each independently represent a 2'-OMe modification or a 2' -F modification.

The sense strand represented by any of the above formulas (Ia), (Ib), (Ic) and (Id) forms a duplex with the antisense strand represented by any of the formulas (If), (Ig), (Ih) and (Ii), respectively.

Thus, RNAi agents useful in the methods of the invention can comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex being represented by formula (Ij):

Sense: 5'np-Na- (XXX) i-Nb-YYY-Nb- (ZZZ) j-Na-nq 3'

Antisense: 3' np ' -Na ' - (X ' X ' X ') k-Nb ' -Y ' Y ' Y ' -Nb ' - (Z ' Z ' Z ') l-Na ' -nq '5'

(Ij)

Wherein:

i. j, k and l are each independently 0 or 1;

p, p ', q and q' are each independently 0 to 6;

Each Na and Na' independently represents an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two different modified nucleotides;

each Nb and Nb' independently represents an oligonucleotide sequence comprising 0 to 10 modified nucleotides;

each np ', np, nq' and nq, each of which may or may not be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X ' X ' X ', Y ' Y ' Y ' and Z ' Z ' Z ' each independently represent a motif of three identical modifications on three consecutive nucleotides.

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

An exemplary combination of sense and antisense strands forming an RNAi duplex comprises the formula:

5′np-Na-YYY-Na-nq 3′

3′np′-Na′-Y′Y′Y′-Na′nq′5′ (Ik)

5′np-Na-YYY-Nb-ZZZ-Na-nq 3′

3′np′-Na′-Y′Y′Y′-Nb′-Z′Z′Z′-Na′nq′5′ (Il)

5′np-Na-XXX-Nb-YYY-Na-nq 3′

3′np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Na′-nq′5′ (Im)

5′np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq 3′

3′np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Nb′-Z′Z′Z′-Na-nq′5′ (In)

when the RNAi agent is represented by formula (Ik), each Na independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the RNAi agent is represented by formula (Il), each Nb independently represents an oligonucleotide sequence comprising 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the RNAi agent is represented by formula (Im), each Nb, nb' independently represents an oligonucleotide sequence comprising 0 to 10, 0 to 7, 0 to 5,0 to 4, 0 to 2, or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

When the RNAi agent is represented by formula (In), each Nb, nb' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each Na, na' independently represents an oligonucleotide sequence comprising 2 to 20, 2 to 15 or 2 to 10 modified nucleotides. Each of Na, na ', nb, and Nb' independently includes an alternating pattern of modifications.

Each of X, Y and Z In formulas (Ij), (Ik), (Il), (Im) and (In) may be the same or different from each other.

When the RNAi agent is represented by formulas (Ij), (Ik), (Il), (Im), and (In), at least one Y nucleotide can form a base pair with one of the Y' nucleotides. Alternatively, at least two Y nucleotides form base pairs with corresponding Y' nucleotides; or all three Y nucleotides form base pairs with the corresponding Y nucleotide.

When the RNAi agent is represented by formula (Il) or (In), at least one Z nucleotide can form a base pair with one of the Z' nucleotides. Alternatively, at least two Z nucleotides form base pairs with corresponding Z' nucleotides; or all three Z nucleotides form base pairs with the corresponding Z' nucleotide.

When the RNAi agent is represented by formula (Im) or (In), at least one X nucleotide can form a base pair with one X' nucleotide. Alternatively, at least two X nucleotides form base pairs with corresponding X' nucleotides; or all three X nucleotides form base pairs with the corresponding X' nucleotide.

In one embodiment, the modification on the Y nucleotide is different from the modification on the Y ' nucleotide, the modification on the Z nucleotide is different from the modification on the Z ' nucleotide, and/or the modification on the X nucleotide is different from the modification on the X ' nucleotide.

In one embodiment, when the RNAi agent is represented by formula (In), the Na modification is a2 '-O-methyl modification or a 2' -mono-fluoro modification. In another embodiment, when the RNAi agent is represented by formula (In), the Na modification is a2 '-O-methyl modification or a 2' -fluoro modification, and np '> 0, and at least one np' is linked to an adjacent nucleotide a via a phosphorothioate linkage. In still other embodiments, when the RNAi agent is represented by formula (In), the Na modification is a2 '-O-methyl modification or a 2' -fluoro modification, np '> 0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked by a divalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (In), the Na modification is a2 '-O-methyl modification or a 2' -fluoro modification, np '> 0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked by a divalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (Ik), the Na modification is a2 '-O-methyl modification or a 2' -fluoro modification, np '> 0, and at least one np' is linked to an adjacent nucleotide via a phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives linked by a divalent or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer comprising at least two duplex represented by formulas (Ij), (Ik), (Il), (Im) and (In), wherein the duplex is connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes; or each duplex may target the same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer comprising three, four, five, six, or more duplex represented by formulas (Ij), (Ik), (Il), (Im), and (In), wherein the duplex is connected by a linker. The linker may be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each duplex may target the same gene or two different genes; or each duplex may target the same gene at two different target sites.

In one embodiment, the two RNAi agents represented by formulas (Ij), (Ik), (Il), (Im), and (In) are linked to each other at the 5 'end and one or both 3' ends, and optionally conjugated to a ligand. Each agent may target the same gene or two different genes; or each agent may target the same gene at two different target sites.

In certain embodiments, RNAi agents of the invention can contain a small amount of nucleotides containing a 2 '-fluoro modification, e.g., 10 or fewer nucleotides with a 2' -fluoro modification. For example, an RNAi agent can contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides with 2' -fluoro modifications. In particular embodiments, RNAi agents of the invention contain 10 nucleotides with 2' -fluoro modifications, e.g., 4 nucleotides with 2' -fluoro modifications in the sense strand and 6 nucleotides with 2' -fluoro modifications in the antisense strand. In another embodiment, the RNAi agents of the invention contain 6 nucleotides with 2' -fluoro modifications, e.g., 4 nucleotides with 2' -fluoro modifications in the sense strand and 2 nucleotides with 2' -fluoro modifications in the antisense strand.

In other embodiments, RNAi agents of the invention can comprise an ultra-low number of nucleotides containing a2 '-fluoro modification, e.g., 2 or fewer nucleotides containing a 2' -fluoro modification. For example, an RNAi agent can contain 2, 1 out of 0 nucleotides with 2' -fluoro modifications. In particular embodiments, an RNAi agent can contain 2 nucleotides with 2 '-fluoro modifications, e.g., 0 nucleotides with 2-fluoro modifications in the sense strand and 2 nucleotides with 2' -fluoro modifications in the antisense strand.

Various publications describe multimeric RNAi agents that can be used in the methods of the invention. Such publications include WO2007/091269, U.S. patent No. 7858769, WO2010/141511, WO2007/117686, WO2009/014887 and WO2011/031520, the entire contents of each of which are hereby incorporated by reference.

In certain embodiments, the compositions and methods of the present disclosure include Vinyl Phosphonate (VP) modification of RNAi agents as described herein. In an exemplary embodiment, the vinyl phosphonate of the present disclosure has the following structure:

for example, when the phosphate mimic is a5 '-Vinylphosphonate (VP), the 5' -terminal nucleotide may have the following structure,

Wherein indicates the position of the bond to the 5' position of the adjacent nucleotide;

R is hydrogen, hydroxy, methoxy, fluoro (e.g., hydroxy or methoxy), or another modification described herein; and

B is a nucleobase or modified nucleobase, optionally wherein B is adenine, guanine, cytosine, thymine or uracil.

The vinyl phosphonate of the present disclosure may be linked to the antisense strand or sense strand of the dsRNA of the present disclosure. In certain embodiments, a vinylphosphonate of the present disclosure is linked to the antisense strand of a dsRNA, optionally at the 5' end of the antisense strand of a dsRNA. The dsRNA agent may include a phosphorus-containing group at the 5' end of the sense strand or the antisense strand. The 5 'terminal phosphorus-containing group may be a 5' terminal phosphate (5 '-P), a 5' terminal phosphorothioate (5 '-PS), a 5' terminal phosphorothioate diester (5 '-PS 2), a 5' terminal vinylphosphonate (5 '-VP), a 5' terminal methylphosphonate (MePhos), or a5 '-deoxy-5' -C-malonyl. When the 5' terminal phosphorus-containing group is a 5' terminal vinyl phosphonate (5 ' -VP), the 5' -VP may be the 5' -E-VP isomer (i.e., trans-vinyl phosphonate,) The 5' -Z-VP isomer (i.e., cis-vinyl phosphate,/>)) Or a mixture thereof.

Vinyl phosphonate modifications are also contemplated for use in the compositions and methods of the present disclosure. Exemplary vinyl phosphate structures are:

for example, when the phosphate mimic is a 5 '-vinyl phosphate, the 5' -terminal nucleotide may have an immediate structure in which the phosphonate group is replaced with a phosphate.

As described in more detail below, RNAi agents containing conjugation of one or more carbohydrate moieties to the RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be linked to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of the d _s RNA agent may be replaced with another moiety, e.g., a non-carbohydrate (preferably, cyclic) carrier linked to a carbohydrate ligand. The 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 may 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 may be heteroatoms, such as nitrogen, oxygen, sulfur. The cyclic carrier may be a single ring system or may contain two or more rings, such as fused rings. The cyclic carrier may be a fully saturated ring system or it may contain one or more double bonds.

The ligand may be linked to the polynucleotide by a carrier. The carrier comprises (i) at least one "backbone attachment point", preferably two "backbone attachment points", and (ii) at least one "tether attachment point". As used herein, "backbone attachment point" refers to a functional group, such as a hydroxyl group, or a bond that is generally useful and suitable for incorporating a carrier into a backbone, such as a phosphate or modified phosphate (e.g., sulfur-containing) backbone of ribonucleic acid. In some embodiments, "tethered attachment point" (TAP) refers to a constituent ring atom of a cyclic carrier that attaches to a selected moiety, such as a carbon atom or a heteroatom (other than the atom providing the backbone attachment point). The moiety may be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, and polysaccharide. Optionally, the selected moiety is linked to the cyclic carrier via an intermediate tether. Thus, the cyclic carrier will typically include a functional group, such as an amino group, or typically provide a bond suitable for incorporation or tethering of another chemical entity (e.g., a ligand that makes up a ring).

The RNAi agent can be conjugated to the ligand via a carrier, wherein the carrier can be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3] dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl and decalinyl; preferably, the acyclic group is selected from a serinol backbone or a diethanolamine backbone.

In another embodiment of the invention, the iRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The RNAi agent can be represented by formula (L):

In formula (L), B1, B2, B3, B1', B2', B3', and B4' are each independently a modified nucleotide containing a nucleotide selected from the group consisting of: 2 '-O-alkyl, 2' -substituted alkoxy, 2 '-substituted alkyl, 2' -halo, ENA and BNA/LNA. In certain embodiments, B1, B2, B3, B1', B2', B3', and B4' each contain a 2' -OMe modification. In certain embodiments, B1, B2, B3, B1', B2', B3', and B4' each contain a 2'-OMe or 2' -F modification. In certain embodiments, at least one of B1, B2, B3, B1', B2', B3', and B4' contains a2 '-O-N-methylacetamido (2' -O-NMA) modification.

C1 is a thermally labile nucleotide located at a site opposite the seed region of the antisense strand (i.e., at positions 2 to 8 of the 5' end of the antisense strand). For example, C1 is located at a position of the sense strand that is paired with nucleotides at positions 2 to 8 of the 5' end of the antisense strand. In one example, C1 is located at position 15 from the 5' end of the sense strand. C1 nucleotides have a thermally labile modification, which may comprise an abasic modification; mismatches with the opposite nucleotide in the duplex; and sugar modifications, such as 2' -deoxy modifications or acyclic nucleotides, e.g., unlocking Nucleic Acids (UNA) or Glycerol Nucleic Acids (GNA). In certain embodiments, C1 has a heat labile modification selected from the group consisting of: i) Mismatches with the opposite nucleotide in the antisense strand; ii) an abasic modification selected from the group consisting of:

and iii) a sugar modification selected from the group consisting of:

Wherein B is a modified OR unmodified nucleobase, R ¹ and R ² are independently H, halogen, OR ₃ OR alkyl; and R ₃ is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. In certain embodiments, the heat labile modification in C1 is a mismatch selected from the group consisting of: g: G. g: A. g: u, G: t, A: A. a: C. c: C. c: u, C: t, U: u, T: t and U: t is a T; and optionally, at least one nucleobase in the mismatch pair is a 2' -deoxynucleobase. In one example, the thermally labile modification in C1 is GNA or/>

T1, T1', T2' and T3 'each independently represent a spatial volume comprising a modification of a nucleotide that provides the nucleotide with a spatial volume that is less than or equal to the spatial volume of the 2' -OMe modification. The spatial volume refers to the sum of the spatial effects of the modification. Methods for determining the spatial effect of nucleotide modifications are known to those skilled in the art. The modification may be at the 2' position of the ribose sugar of the nucleotide, or a modification to a non-ribonucleotide, an acyclic nucleotide, or the backbone of a nucleotide that is similar or equivalent to the 2' position of the ribose sugar, and provides the nucleotide with a steric bulk that is less than or equal to the steric bulk of the 2' -OMe modification. For example, T1', T2', and T3 'are each independently selected from DNA, RNA, LNA, 2' -F, and 2'-F-5' -methyl. In certain embodiments, T1 is DNA. In certain embodiments, T1' is DNA, RNA, or LNA. In certain embodiments, T2' is DNA or RNA. In certain embodiments, T3' is DNA or RNA.

N ¹、n³ and q ¹ are independently 4 to 15 nucleotides in length.

N ⁵、q³ and q ⁷ are independently 1 to 6 nucleotides in length.

N ⁴、q² and q ⁶ are independently 1 to 3 nucleotides in length; alternatively, n ⁴ is 0.

Q ⁵ is independently 0 to 10 nucleotides in length.

N ² and q ⁴ are independently 0 to 3 nucleotides in length.

Alternatively, n ⁴ is 0 to 3 nucleotides in length.

In certain embodiments, n ⁴ may be 0. In one example, n ⁴ is 0, and q ² and q ⁶ are 1. In another example, n ⁴ is 0, and q ² and q ⁶ are 1, with two phosphorothioate internucleotide linkage modifications in positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand), and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

In certain embodiments, n ⁴、q² and q ⁶ are each 1.

In certain embodiments, n ²、n⁴、q²、q⁴ and q ⁶ are each 1.

In certain embodiments, when the length of the sense strand is 19 to 22 nucleotides and n ⁴ is 1, C1 is located at positions 14 to 17 of the 5' end of the sense strand. In certain embodiments, C1 is located at position 15 of the 5' end of the sense strand.

In certain embodiments, T3 'begins at position 2 of the 5' end of the antisense strand. In one example, T3 'is located at position 2 from the 5' end of the antisense strand and q ⁶ is equal to 1.

In certain embodiments, T1 'begins at position 14 of the 5' end of the antisense strand. In one example, T1 'is located at position 14 from the 5' end of the antisense strand, and q ² is equal to 1.

In one exemplary embodiment, T3 'starts at position 2 in the 5' end of the antisense strand and T1 'starts at position 14 in the 5' end of the antisense strand. In one example, T3 'starts at position 2 in the 5' end of the antisense strand and q ⁶ is equal to 1, and T1 'starts at position 14 in the 5' end of the antisense strand and q ² is equal to 1.

In certain embodiments, T1 'and T3' are separated by 11 nucleotides in length (i.e., T1 'and T3' nucleotides are not counted).

In certain embodiments, T1 'is located at position 14 from the 5' end of the antisense strand. In one example, T1 'is located at position 14 from the 5' end of the antisense strand and q ² is equal to 1, and the modification is located at the 2 'position or at a position in the non-ribose, acyclic, or backbone that provides less steric bulk than the 2' -OMe ribose.

In certain embodiments, T3 'is located at position 2 from the 5' end of the antisense strand. In one example, T3 'is located at position 2 from the 5' end of the antisense strand and q ⁶ is equal to 1, and the modification is located at the 2 'position or at a position in the non-ribose, acyclic, or backbone that provides less or equal spatial volume than the 2' -OMe ribose.

In certain embodiments, T1 is located at the cleavage site of the sense strand. In one example, when the length of the sense strand is 19 to 22 nucleotides and n ² is 1, T1 is located at position 11 from the 5' end of the sense strand. In one exemplary embodiment, when the sense strand is 19 to 22 nucleotides in length and n ² is 1, T1 is located at the sense strand cleavage site at position 11 from the 5' end of the sense strand,

In certain embodiments, T2 'begins at position 6 of the 5' end of the antisense strand. In one example, T2 'is located at positions 6 to 10 from the 5' end of the antisense strand, and q ⁴ is 1.

In one exemplary embodiment, when the sense strand is 19 to 22 nucleotides in length and n ² is 1, T1 is located at the cleavage site of the sense strand, e.g., at position 11 from the 5' end of the sense strand; t1' is located at position 14 from the 5' end of the antisense strand and q ² is equal to 1, and the modification to T1' is located at the 2' position of the ribose sugar, or at a position in the non-ribose, acyclic, or backbone that provides less spatial volume than 2' -OMe ribose; t2 'is located at positions 6 to 10 from the 5' end of the antisense strand and q ⁴ is 1; and T3' is located at position 2 from the 5' end of the antisense strand and q ⁶ is equal to 1, and the modification to T3' is located at the 2' position or at a position in the non-ribose, acyclic, or backbone that provides less or equal spatial volume than the 2' -OMe ribose.

In certain embodiments, T2 'begins at position 8 of the 5' end of the antisense strand. In one example, T2 'starts at position 8 of the 5' end of the antisense strand and q ⁴ is 2.

In certain embodiments, T2 'begins at position 9 of the 5' end of the antisense strand. In one example, T2 'is located at position 9 from the 5' end of the antisense strand, and q ⁴ is 1.

In certain embodiments, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 1, B3' is 2' -OMe or 2' -F, q ⁵ is 6, T3' is 2' -F, q ⁶ is 1, B4' is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

In certain embodiments, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 1, B3' is 2'-OMe or 2' -F, q ⁵ is 6, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' _-F,q¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -OMe, and q ⁷ is 1.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 6, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 7, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -OMe, and q ⁷ is 1.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 6, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 7, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 1, B3' is 2'-OMe or 2' -F, q ⁵ is 6, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 1, B3' is 2'-OMe or 2' -F, q ⁵ is 6, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 5, T2' is 2'-F, q ⁴ is 1, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; optionally having at least 2 additional TTs at the 3' end of the antisense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 5, T2' is 2'-F, q ⁴ is 1, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; optionally having at least 2 additional TTs at the 3' end of the antisense strand; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -F, and q ⁷ is 1.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand).

RNAi agents may include a phosphorus-containing group at the 5' end of the sense strand or antisense strand. The phosphorus-containing group at the 5 'end can be a 5' phosphate (5 '-P), a 5' phosphorothioate (5 '-PS), a 5' -phosphorothioate diester (5 '-PS ₂), a 5' vinylphosphonate (5 '-VP), a 5' methylphosphonate (MePhos) or a 5 '-deoxy-5' -C-malonylWhen the 5' -terminal phosphorus-containing group is a 5' -terminal vinyl phosphonate (5 ' -VP), the 5' -VP may be the 5' -E-VP isomer (i.e., trans-vinyl phosphonate/>)) The 5' -Z-VP isomer (i.e., cis-vinyl phosphate) Or a mixture thereof.

In certain embodiments, the RNAi agent comprises a phosphorus-containing group at the 5' end of the sense strand. In certain embodiments, the RNAi agent comprises a phosphorus-containing group at the 5' end of the antisense strand.

In certain embodiments, the RNAi agent comprises 5' -P. In certain embodiments, the RNAi agent comprises 5' -P in the antisense strand.

In certain embodiments, the RNAi agent comprises 5' -PS. In certain embodiments, the RNAi agent comprises 5' -PS in the antisense strand.

In certain embodiments, the RNAi agent comprises 5' -VP. In certain embodiments, the RNAi agent comprises a 5' -VP in the antisense strand. In certain embodiments, the RNAi agent comprises 5' -E-VP in the antisense strand. In certain embodiments, the RNAi agent comprises a 5' -Z-VP in the antisense strand.

In certain embodiments, the RNAi agent comprises 5' -PS ₂. In certain embodiments, the RNAi agent comprises 5' -PS ₂ in the antisense strand.

In certain embodiments, the RNAi agent comprises 5' -PS ₂. In certain embodiments, the RNAi agent comprises 5 '-deoxy-5' -C-malonyl in the antisense strand.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -OMe, and q ⁷ is 1.RNAi agents also include 5' -PS.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -OMe, and q ⁷ is 1.RNAi agents also include 5' -P.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -OMe, and q ⁷ is 1.RNAi agents also include 5' -VP. The 5' -VP may be 5' -E-VP, 5' -Z-VP, or a combination thereof.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -OMe, and q ⁷ is 1.RNAi agents also include 5' -PS ₂.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -OMe, and q ⁷ is 1.RNAi agents also include 5 '-deoxy-5' -C-malonyl.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -P.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -VP. The 5' -VP may be 5' -E-VP, 5' -Z-VP, or a combination thereof.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS ₂.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5 '-deoxy-5' -C-malonyl.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1.RNAi agents also include 5' -P.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1.dsRNA agents also include 5' -PS.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1.RNAi agents also include 5' -VP. The 5' -VP may be 5' -E-VP, 5' -Z-VP, or a combination thereof.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1.RNAi agents also include 5' -PS ₂.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1.RNAi agents also include 5 '-deoxy-5' -C-malonyl.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5' -P.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5' -PS.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5' -VP. The 5' -VP may be 5' -E-VP, 5' -Z-VP, or a combination thereof.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5' -PS ₂.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5 '-deoxy-5' -C-malonyl.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -F, and q ⁷ is 1.RNAi agents also include 5' -P.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -F, and q ⁷ is 1.RNAi agents also include 5' -PS.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -F, and q ⁷ is 1.RNAi agents also include 5' -VP. The 5' -VP may be 5' -E-VP, 5' -Z-VP, or a combination thereof.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -F, and q ⁷ is 1.dsRNA RNA agents also include 5' -PS ₂.

In certain embodiments, B1 is 2' -OMe or 2' -F, n ¹ is 8, T1 is 2' F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2' OMe, n ⁵ is 3, B1' is 2' -OMe or 2' -F, q ¹ is 9, T1' is 2' -F, q ² is 1, B2' is 2' -OMe or 2' -F, q ³ is 4, T2' is 2' -F, q ⁴ is 2', B3' is 2' -OMe or 2' -F, q ⁵ is 5, T3' is 2' -F, q ⁶ is 1, B4' is 2' -F, and q ⁷ is 1.RNAi agents also include 5 '-deoxy-5' -C-malonyl.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -P.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -VP. The 5' -VP may be 5' -E-VP, 5' -Z-VP, or a combination thereof.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS ₂.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5 '-deoxy-5' -C-malonyl.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1.RNAi agents also include 5' -P.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1.RNAi agents also include 5' -PS.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1.RNAi agents also include 5' -VP. The 5' -VP may be 5' -E-VP, 5' -Z-VP, or a combination thereof.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1.RNAi agents also include 5' -PS ₂.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1.RNAi agents also include 5 '-deoxy-5' -C-malonyl.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -P.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -VP. The 5' -VP may be 5' -E-VP, 5' -Z-VP, or a combination thereof.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS ₂.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5 '-deoxy-5' -C-malonyl.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -P and targeting ligands. In certain embodiments, the 5' -P is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS and targeting ligands. In certain embodiments, the 5' -PS is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -VP (e.g., 5' -E-VP, 5' -Z-VP, or a combination thereof) and targeting ligand.

In certain embodiments, the 5' -VP is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS ₂ and targeting ligands. In certain embodiments, the 5' -PS ₂ is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5 '-deoxy-5' -C-malonyl and targeting ligands. In certain embodiments, the 5 '-deoxy-5' -C-malonyl is located at the 5 'end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5' -P and targeting ligands. In certain embodiments, the 5' -P is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5' -PS and targeting ligands. In certain embodiments, the 5' -PS is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5' -VP (e.g., 5' -E-VP, 5' -Z-VP, or a combination thereof) and targeting ligand. In certain embodiments, the 5' -VP is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5' -PS ₂ and targeting ligands. In certain embodiments, the 5' -PS ₂ is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -OMe, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications in positions 1 to 5 (counting from the 5 'end) of the sense strand, and two phosphorothioate internucleotide linkage modifications in positions 1 and 2, and two phosphorothioate internucleotide linkage modifications in positions 18 to 23 (counting from the 5' end) of the antisense strand. RNAi agents also include 5 '-deoxy-5' -C-malonyl and targeting ligands. In certain embodiments, the 5 '-deoxy-5' -C-malonyl is located at the 5 'end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -P and targeting ligands. In certain embodiments, the 5' -P is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS and targeting ligands. In certain embodiments, the 5' -PS is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -VP (e.g., 5' -E-VP, 5' -Z-VP, or a combination thereof) and targeting ligand. In certain embodiments, the 5' -VP is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS ₂ and targeting ligands. In certain embodiments, the 5' -PS ₂ is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, T2' is 2'-F, q ⁴ is 2, B3' is 2'-OMe or 2' -F, q ⁵ is 5, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5 '-deoxy-5' -C-malonyl and targeting ligands. In certain embodiments, the 5 '-deoxy-5' -C-malonyl is located at the 5 'end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -P and targeting ligands. In certain embodiments, the 5' -P is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS and targeting ligands. In certain embodiments, the 5' -PS is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -VP (e.g., 5' -E-VP, 5' -Z-VP, or a combination thereof) and targeting ligand. In certain embodiments, the 5' -VP is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is 1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is 1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5' -PS ₂ and targeting ligands. In certain embodiments, the 5' -PS ₂ is located at the 5' end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In certain embodiments, B1 is 2'-OMe or 2' -F, n ¹ is 8, T1 is 2'F, n ² is 3, B2 is 2' -OMe, n ³ is 7, n ⁴ is 0, B3 is 2'-OMe, n ⁵ is 3, B1' is 2'-OMe or 2' -F, q ¹ is 9, T1 'is 2' -F, q ² is1, B2 'is 2' -OMe or 2'-F, q ³ is 4, q ⁴ is 0, B3' is 2'-OMe or 2' -F, q ⁵ is 7, T3 'is 2' -F, q ⁶ is1, B4 'is 2' -F, and q ⁷ is 1; there are two phosphorothioate internucleotide linkage modifications within positions 1 to 5 of the sense strand (counting from the 5 'end of the sense strand) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2, and two phosphorothioate internucleotide linkage modifications within positions 18 to 23 of the antisense strand (counting from the 5' end of the antisense strand). RNAi agents also include 5 '-deoxy-5' -C-malonyl and targeting ligands. In certain embodiments, the 5 '-deoxy-5' -C-malonyl is located at the 5 'end of the antisense strand and the targeting ligand is located at the 3' end of the sense strand.

In a particular embodiment, the RNAi agent of the invention comprises:

(a) A sense strand having:

(i) A length of 21 nucleotides;

(ii) An ASGPR ligand linked to the 3' end, wherein the ASGPR ligand comprises three GalNAc derivatives linked by a trivalent branched linker; and

(Iii) 2' -F modifications at positions 1,3, 5, 7, 9 to 11, 13, 17, 19 and 21, and 2' -OMe modifications (counting from the 5' end) at positions 2,4, 6, 8, 12, 14 to 16, 18 and 20;

And

(B) An antisense strand, said antisense strand having:

(i) A length of 23 nucleotides;

(ii) 2' -OMe modifications at positions 1, 3, 5, 9, 11 to 13, 15, 17, 19, 21 and 23, and 2' f modifications at positions 2, 4, 6 to 8, 10, 14, 16, 18, 20 and 22 (counting from the 5' end); and

(Iii) Phosphorothioate internucleotide linkages (counting from the 5' end) between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23;

wherein the dsRNA agent has a two nucleotide overhang at the 3 'end of the antisense strand and a blunt end at the 5' end of the antisense strand.

In another particular embodiment, the RNAi agent of the invention comprises:

(a) A sense strand having:

(i) A length of 21 nucleotides;

(ii) An ASGPR ligand linked to the 3' end, wherein the ASGPR ligand comprises three GalNAc derivatives linked by a trivalent branched linker;

(iii) 2' -F modifications at positions 1,3, 5, 7, 9 to 11, 13, 15, 17, 19 and 21, and 2' -OMe modifications at positions 2,4, 6, 8, 12, 14, 16, 18 and 20 (counting from the 5' end); and

(Iv) Phosphorothioate internucleotide linkages (counting from the 5' end) between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3;

And

(B) An antisense strand, said antisense strand having:

(i) A length of 23 nucleotides;

(ii) 2' -OMe modifications at positions 1, 3, 5, 7, 9, 11 to 13, 15, 17, 19 and 21 to 23, and 2' f modifications at positions 2, 4, 6, 8, 10, 14, 16, 18 and 20 (counting from the 5' end); and

(Iii) Phosphorothioate internucleotide linkages (counting from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22 and between nucleotide positions 22 and 23;

Wherein the RNAi agent has two nucleotide overhangs at the 3 'end of the antisense strand and a blunt end at the 5' end of the antisense strand.

In another particular embodiment, the RNAi agent of the invention comprises:

(a) A sense strand having:

(i) A length of 21 nucleotides;

(iii) 2' -OMe modifications at positions 1 to 6, 8, 10 and 12 to 21, 2' -F modifications at positions 7 and 9, and deoxynucleotides (e.g., dT) at position 11 (counted from the 5' end); and

And

(B) An antisense strand, said antisense strand having:

(i) A length of 23 nucleotides;

(ii) 2' -OMe modifications at positions 1, 3, 7, 9, 11, 13, 15, 17 and 19 to 23, and 2' -F modifications at positions 2, 4 to 6, 8, 10, 12, 14, 16 and 18 (counting from the 5' end); and

In another particular embodiment, the RNAi agent of the invention comprises:

(a) A sense strand having:

(i) A length of 21 nucleotides;

(iii) 2'-OMe modifications at positions 1 to 6, 8, 10, 12, 14 and 16 to 21, and 2' -F modifications at positions 7, 9, 11, 13 and 15; and

And

(B) An antisense strand, said antisense strand having:

(i) A length of 23 nucleotides;

(ii) 2' -OMe modifications at positions 1,5, 7, 9, 11, 13, 15, 17, 19 and 21 to 23, and 2' -F modifications at positions 2 to 4, 6, 8, 10, 12, 14, 16, 18 and 20 (counting from the 5' end); and

In another particular embodiment, the RNAi agent of the invention comprises:

(a) A sense strand having:

(i) A length of 21 nucleotides;

(iii) 2'-OMe modifications at positions 1 to 9 and 12 to 21, and 2' -F modifications at positions 10 and 11; and

And

(B) An antisense strand, said antisense strand having:

(i) A length of 23 nucleotides;

(ii) 2' -OMe modifications at positions 1,3, 5, 7, 9, 11 to 13, 15, 17, 19 and 21 to 23, and 2' -F modifications at positions 2,4, 6, 8, 10, 14, 16, 18 and 20 (counting from the 5' end); and

In another particular embodiment, the RNAi agent of the invention comprises:

(a) A sense strand having:

(i) A length of 21 nucleotides;

(iii) 2'-F modifications at positions 1, 3, 5, 7, 9 to 11 and 13, and 2' -OMe modifications at positions 2,4,6, 8, 12 and 14 to 21; and

And

(B) An antisense strand, said antisense strand having:

(i) A length of 23 nucleotides;

(ii) 2' -OMe modifications at positions 1, 3, 5 to 7, 9, 11 to 13, 15, 17 to 19 and 21 to 23, and 2' -F modifications at positions 2, 4, 8, 10, 14, 16 and 20 (counting from the 5' end); and

In another particular embodiment, the RNAi agent of the invention comprises:

(a) A sense strand having:

(i) A length of 21 nucleotides;

(iii) 2'-OMe modifications at positions 1,2, 4, 6, 8, 12, 14, 15, 17 and 19 to 21, and 2' -F modifications at positions 3, 5,7, 9 to 11, 13, 16 and 18; and

And

(B) An antisense strand, said antisense strand having:

(i) A length of 25 nucleotides;

(ii) 2' -OMe modifications at positions 1, 4,6, 7, 9, 11 to 13, 15, 17 and 19 to 23, 2' -F modifications at positions 2,3, 5, 8, 10, 14, 16 and 18, and deoxynucleotides (e.g., dT) at positions 24 and 25 (counted from the 5' end); and

Wherein the RNAi agent has a four nucleotide overhang at the 3 'end of the antisense strand and a blunt end at the 5' end of the antisense strand.

In another particular embodiment, the RNAi agent of the invention comprises:

(a) A sense strand having:

(i) A length of 21 nucleotides;

(iii) 2'-OMe modifications at positions 1 to 6, 8 and 12 to 21, and 2' -F modifications at positions 7 and 9 to 11; and

And

(B) An antisense strand, said antisense strand having:

(i) A length of 23 nucleotides;

(ii) 2' -OMe modifications at positions 1,3 to 5, 7,8, 10 to 13, 15 and 17 to 23, and 2' -F modifications at positions 2,6, 9, 14 and 16 (counting from the 5' end); and

In another particular embodiment, the RNAi agent of the invention comprises:

(a) A sense strand having:

(i) A length of 21 nucleotides;

And

(B) An antisense strand, said antisense strand having:

(i) A length of 23 nucleotides;

(ii) 2' -OMe modifications at positions 1,3 to 5, 7, 10 to 13, 15 and 17 to 23, and 2' -F modifications at positions 2,6, 8,9, 14 and 16 (counting from the 5' end); and

In another particular embodiment, the RNAi agent of the invention comprises:

(a) A sense strand having:

(i) A length of 19 nucleotides;

(iii) 2'-OMe modifications at positions 1 to 4, 6 and 10 to 19, and 2' -F modifications at positions 5 and 7 to 9; and

And

(B) An antisense strand, said antisense strand having:

(i) A length of 21 nucleotides;

(ii) 2' -OMe modifications at positions 1,3 to 5, 7, 10 to 13, 15 and 17 to 21, and 2' -F modifications at positions 2,6, 8,9, 14 and 16 (counting from the 5' end); and

(Iii) Phosphorothioate internucleotide linkages (counting from the 5' end) between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 19 and 20, and between nucleotide positions 20 and 21;

In certain embodiments, the iRNA used in the methods of the invention is an agent selected from the agents listed in tables 3-6. These agents may further comprise a ligand.

IRNA conjugated to ligand

Another modification of the RNAs of the iRNAs of the present invention involves chemically linking one or more ligands, moieties, or conjugates to the iRNA that enhance the activity, cellular distribution, or cellular uptake of the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al, (1989) journal of the national academy of sciences, 86:6553-6556)), cholic acid (Manoharan et al, (1994) bioorganic and pharmaceutical chemistry rapid report (Biorg. Med. Chem. Let.)) (4): 1053-1060), thioethers, for example, andalusite-S-triphenylmethyl mercaptan (Manoharan et al, (1992) annual book of the new york academy of sciences (ann.n.y. Acad.sci.), 660:306-309; manoharan et al, (1993) fast report of bioorganic and pharmaceutical chemistry, 3: 2765-2770), thiocholesterol (Oberhauser et al, (1992) nucleic acids research, 20: 533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al, (1991) journal of molecular biology, european tissue, 10:1111-1118; kabanov et al, (1990) FEBS report (FEBS lett.), 259:327-330; svinarchuk et al, (1993) biochemistry (Biochimie), 75: 49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1, 2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al, (1995) Tetrahedron letters, 36:3651-3654; shea et al, (1990) nucleic acids research, 18: 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al, (1995) Nucleosides & Nucleotides (Nucleotides & Nucleotides), 14: 969-973) or adamantaneacetic acid (Manoharan et al, (1995) tetrahedral flash, 36: 3651-3654), palmityl moiety (Mishra et al, (1995) journal of biochemistry and biophysics (Biochim. Biophys. Acta), 1264: 229-237), or octadecylamine or hexylamino-carbonyloxy cholesterol moiety (Crooke et al, (1996) journal of pharmacology and experimental therapeutics (j.pharmacol.exp.ter.)), 277: 923-937).

In one embodiment, the ligand alters the distribution, targeting, or lifetime of the iRNA agent into which it is incorporated. In preferred embodiments, the ligand provides enhanced affinity for a selected target (e.g., a molecule, cell, or cell type), compartment (e.g., a cell or organ compartment, tissue, organ, or body region), for example, as compared to a species in which such ligand is not present. Preferred ligands will not participate in duplex pairing in the duplex nucleic acid.

The ligand may comprise naturally occurring substances, such as proteins (e.g., human Serum Albumin (HSA), low Density Lipoprotein (LDL), or globulin); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand may also be a recombinant molecule or a synthetic molecule, such as a synthetic polymer, e.g. a synthetic polyamino acid. Examples of polyamino acids include polyamino acids, i.e., polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymer, poly (L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N- (2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethacrylic acid), N-isopropylacrylamide polymer or polyphosphazine. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salts of polyamines, or alpha helical peptides.

The ligand may also comprise a targeting group, such as 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 (e.g. kidney cells). The targeting group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein a, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acid, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin a, biotin, or RGD peptide mimetic.

Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralene, mitomycin C (mitomycin C)), porphyrins (TPPC 4, texas porphyrin (texaphyrin), ring-extended porphyrins (SAPPHYRIN)), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1, 3-bis-O (hexadecyl) glycerol, geranyloxyhexyl, hexadecyl glycerol, borneol, menthol, 1, 3-propanediol, heptadecyl, palmitic acid, myristic acid, O3- (oleoyl) lithocholic acid, O3- (oleoyl) cholanic acid, dimethoxytrityl or phenazine), peptide conjugates (e.g., antennapedia mutant peptides, tat peptides), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [ MPEG ] ₂, polyamino groups, alkyl groups, substituted alkyl groups, radiolabelled markers, enzymes, haptens (e.g., biotin), transport/absorption enhancers (e.g., aspirin (aspirin), vitamin E, folic acid), synthetic ribonucleases (e.g., eu3+ complexes of imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, tetraazamacrocyclic compounds), dinitrophenyl, HRP, or AP.

The ligand may be a protein (e.g., glycoprotein) or peptide (e.g., a molecule having a specific affinity for the co-ligand) or an antibody (e.g., an antibody that binds to a particular cell type, such as a hepatocyte). The ligand may also comprise a hormone and a hormone receptor. They may also contain non-peptide substances such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose or multivalent fucose. The ligand may be, for example, lipopolysaccharide, an activator of p38MAP kinase or an activator of NF- κB.

The ligand may be a substance, such as a drug, which may increase uptake of the iRNA agent into the cell, e.g., by disrupting the cytoskeleton of the cell, e.g., by disrupting microtubules, microfilaments and/or intermediate filaments of the cell. The drug may be, for example, a taxonomic unit (taxon), vincristine (vincristine), vinblastine (vinblastine), cytochalasin (cytochalasin), nocodazole (nocodazole), jestilide (japlakinolide), lanchunkin A (latrunculin A), duponin (phalloidin), s Wen Heli A (swinholide A), yin Dannuo oct (indanocine), or mesitylene (myoservin).

In some embodiments, the ligand linked to an iRNA as 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, dialkyl glycerides, diacylglycerides, phospholipids, sphingolipids, naproxen (naproxen), ibuprofen (ibuprofen), vitamin E, biotin. Serum protein-binding oligonucleotides comprising a number of phosphorothioate linkages are also known, and thus short oligonucleotides comprising a number of phosphorothioate linkages in the backbone (e.g., oligonucleotides having about 5 bases, 10 bases, 15 bases, or 20 bases) are also suitable for use in the present invention as ligands (e.g., as PK modulating ligands). 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.

The ligand-conjugated oligonucleotides of the invention may be synthesized by using oligonucleotides with side chain reactive functionalities, such as derived from attaching a linker molecule to the oligonucleotide (as described below). Such reactive oligonucleotides may be reacted directly with commercially available ligands, synthetic ligands with any of a variety of protecting groups, or ligands having a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the invention may be conveniently and routinely prepared by well known solid phase synthesis techniques. Devices for such synthesis are sold by several suppliers, for example, application biosystems, inc. (Applied Biosystems (Foster City, calif.) containing Foster City, calif.). Any other means known in the art for such synthesis may additionally or alternatively be employed. Other oligonucleotides (e.g., phosphorothioates and alkylated derivatives) are also known to be prepared using similar techniques.

In the ligand-conjugated oligonucleotides and ligand molecules with sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleotides can be assembled on a suitable DNA synthesizer using standard nucleotides or nucleoside precursors or nucleotides or nucleoside conjugate precursors already bearing a linking moiety, ligand-nucleotide or nucleoside conjugate precursors already bearing a ligand molecule or non-nucleoside ligands bearing a building block.

When using nucleotide conjugate precursors that already carry a linking moiety, synthesis of the sequence-specific linked nucleoside is typically accomplished, and then the ligand molecule reacts with the linking moiety to form a ligand conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using a phosphoramidite derived from a ligand-nucleoside conjugate as well as standard and non-standard phosphoramidites commercially available and conventionally used for oligonucleotide synthesis.

A. Lipid conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such lipids or lipid-based molecules preferably bind to serum proteins, such as Human Serum Albumin (HSA). HSA binding ligands allow the conjugate to be distributed to target tissue, e.g., non-kidney target tissue of the body. For example, the target tissue may be the liver, including parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen (neproxin) or aspirin may be used. The lipid or lipid-based ligand may (a) increase resistance to conjugate degradation, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) may be used to modulate binding of HSA to a serum protein, such as HSA.

Lipid-based ligands can be used to inhibit (e.g., control) binding of the conjugate to a target tissue. For example, lipids or lipid-based ligands that bind more strongly to HSA will be less likely to be targeted to the kidneys 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.

In a preferred embodiment, the lipid based ligand binds to HSA. Preferably, it binds with sufficient affinity to HSA such that the conjugate will preferentially distribute to non-kidney tissue. However, it is preferred that the affinity is not so strong that HSA ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds to HSA weakly or not at all, so that the conjugate will preferentially distribute to the kidneys. Other moieties targeted to kidney cells may also be used instead of or in addition to lipid-based ligands.

On the other hand, the ligand is a moiety, such as a vitamin, that is taken up by the target cell (e.g., proliferating cell). These are particularly useful for treating diseases characterized by undesired cell proliferation, such as malignant or non-malignant types of diseases, such as cancer cells. Exemplary vitamins include vitamins A, E and K. Other exemplary vitamins that are included are B vitamins such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients that are taken up by target cells (e.g., hepatocytes). HSA and Low Density Lipoprotein (LDL) are also included.

B. Cell penetrating agent

In another aspect, the ligand is a cell penetrating agent, preferably a helical cell penetrating agent. Preferably, the agent is amphiphilic. Exemplary agents are peptides, such as tat or antennapedia mutant peptides. If the agent is a peptide, it may be modified, including peptidomimetics, inversion bodies, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an alpha helical agent having a lipophilic phase and a lipophobic phase.

The ligand may be a peptide or a peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptide mimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic profile of the iRNA, such as by enhancing cell recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, for example about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

The peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., consisting essentially of Tyr, trp, or Phe). The peptide moiety may be a dendrimer peptide, a constraint peptide or a cross-linked peptide. In another alternative, the peptide moiety may comprise a hydrophobic Membrane Translocation Sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 3). RFGF analogs (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 4)) containing a hydrophobic MTS may also be targeting moieties. The peptide moiety may be a "delivery" peptide that can carry a large polar molecule including peptides, oligonucleotides and proteins across the cell membrane. For example, it has been found that sequences from the HIV Tat protein (GRKKRRQRRRRRPPQ (SEQ ID NO: 5) and drosophila antennapedia mutein ((RQIKIWFQNRRMKWKK (SEQ ID NO: 6)) can act as delivery peptides the peptide or peptidomimetic can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead compound (OBOC) combinatorial libraries (Lam et al, nature 354:82-84, 1991).

RGD peptides for use in the compositions and methods of the invention may be linear or cyclic and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a particular tissue. RGD-containing peptides and peptide dimers may comprise D-amino acids and synthetic RGD mimics. In addition to RGD, other moieties that target integrin ligands can be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

"Cell penetrating peptide" is capable of penetrating a cell, such as a microbial cell (e.g., a bacterial or fungal cell) or a mammalian cell (e.g., a human cell). The microbial cell penetrating peptide may be, for example, an alpha-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide-containing peptide (e.g., an alpha-defensin, beta-defensin, or bacteriocin), or a peptide containing only one or two major amino acids (e.g., PR-39 or indomethacin). Cell penetrating peptides may also comprise Nuclear Localization Signals (NLS). For example, the cell penetrating peptide may be a bipartite amphiphilic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and NLS of the SV40 large T antigen (Simeoni et al, nucleic acids research 31:2717-2724, 2003).

C. Carbohydrate conjugates

In some embodiments of the compositions and methods of the invention, the iRNA oligonucleotide further comprises a carbohydrate. Carbohydrate conjugated iRNA is advantageous for in vivo delivery of nucleic acids and compositions suitable for in vivo therapeutic use as described herein. As used herein, "carbohydrate" refers to a compound that is a carbohydrate that itself is comprised of one or more monosaccharide units having at least 6 carbon atoms (which may be linear, branched, or cyclic), wherein an oxygen, nitrogen, or sulfur atom is bound to each carbon atom; or a compound having as part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched or cyclic), wherein an oxygen, nitrogen or sulfur atom is bonded to each carbon atom. Representative carbohydrates include sugars (mono-, di-, tri-and oligosaccharides containing about 4,5, 6, 7, 8 or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; disaccharides and trisaccharides include saccharides having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In one embodiment, the carbohydrate conjugates used in the compositions and methods of the invention are selected from the group consisting of:

Wherein Y is O or S and n is 3 to 6 (formula XXIV);

Wherein Y is O or S, and n is 3 to 6 (formula XXV);

Wherein X is O or S (formula XXVII); /(I)

In another embodiment, the carbohydrate conjugates used in the compositions and methods of the invention are monosaccharides. In one embodiment, the monosaccharide is N-acetylgalactosamine, e.g

Another representative carbohydrate conjugate for use in embodiments described herein includes, but is not limited to:

When one of X or Y is an oligonucleotide, the other is hydrogen.

In certain embodiments of the invention, galNAc or GalNAc derivative is linked to an iRNA agent of the invention by a monovalent linker. In some embodiments, galNAc or GalNAc derivative is linked to an iRNA agent of the invention through a divalent linker. In still other embodiments of the invention, galNAc or GalNAc derivative is linked to the iRNA agent of the invention through a trivalent linker.

In one embodiment, the double stranded RNAi agents of the invention comprise a GalNAc or GalNAc derivative linked to an iRNA agent, e.g., the 3 'or 5' end of the sense strand of a dsRNA agent as described herein. In another embodiment, the double stranded RNAi agent of the invention comprises a plurality (e.g., 2,3, 4, 5, or 6) galnacs or GalNAc derivatives, each of which is independently linked to a plurality of nucleotides of the double stranded RNAi agent by a plurality of monovalent linkers.

In some embodiments, for example, when two strands of an iRNA agent of the invention are part of one larger molecule, the larger molecule is joined by an uninterrupted nucleotide strand between the 3 'end of one strand and the 5' end of the corresponding other strand, forming a hairpin loop comprising a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop can independently comprise GalNAc or a GalNAc derivative joined by a monovalent linker.

In some 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.

Additional carbohydrate conjugates (and linkers) suitable for use in the present invention include those described in PCT publication nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Joint

In some embodiments, the conjugates or ligands described herein can be attached to an iRNA oligonucleotide through various linkers, which may be cleavable or non-cleavable.

The term "linker" or "linking group" means an organic moiety that connects two moieties of a compound, e.g., covalently connects two moieties of a compound. The linker typically includes a direct bond or atom, such as oxygen or sulfur; units such as NR8, C (O) NH, SO ₂、SO₂ NH or chains of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aralkyl, aralkenyl, aralkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl, heteroaryl, heterocycloalkenyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylakenyl, alkylarylalkenyl, alkenylaralkyl, alkenylarylalkenyl, alkynylarylalkyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkyl, alkynylheterocycloalkyl, alkylheterocycloalkenyl, alkenylheterocycloalkenyl, alkenylheterocycloalkynyl, alkynylheterocycloalkyl, alkynylheterocycloalkenyl, alkynylheterocycloalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, said one or more methylene groups may be blocked by: o, S, S (O), SO ₂, N (R8), C (O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl; wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

The cleavable linking group is one that is sufficiently stable outside the cell, but which cleaves after entry into the target cell to release the two parts of the linker that remain together. In preferred embodiments, cleavage of the cleavable linking group in a target cell or under a first reference condition (which may, for example, be selected to mimic or represent an intracellular condition) is 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 greater than cleavage rate in a subject's blood or under a second reference condition (which may, for example, be selected to mimic or represent a condition found in blood or serum).

Cleavable linking groups are susceptible to cleavage by a cleavage agent (e.g., pH, redox potential, or the presence of a degrading molecule). Generally, cleavage agents are more prevalent inside cells than in serum or blood, or are found at higher levels or activities. Examples of such degradation agents include: redox agents, selected for a particular substrate or not having substrate specificity, comprising, for example, an oxidation or reduction enzyme or reducing agent present in the cell, such as a thiol, which can cleave the redox cleavable linking group by reductive degradation; an esterase; endosomes or agents that can produce an acidic environment, such as those that produce a pH of five or less; enzymes that hydrolyze or degrade acid cleavable linkers can be used as broad acids, peptidases (which may be substrate specific), and phosphatases.

Cleavable linkage groups, such as disulfide linkages, may be pH sensitive. The pH of human serum was 7.4, while the average intracellular pH was slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH in the range of 5.5-6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers will have cleavable linking groups that cleave at a preferred pH, thereby releasing the cationic lipid from the ligand within the cell, or into a desired compartment of the cell.

The linker may comprise a cleavable linking group cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the cell targeted. For example, the liver targeting ligand may be linked to the cationic lipid through a linker comprising an ester group. Hepatocytes are rich in esterases and thus the linker will cleave more efficiently in hepatocytes than in non-esterase-rich cell types. Other esterase-enriched cell types include cells in the lung, kidney cortex and testes.

When targeting peptidase-rich cell types, such as hepatocytes and synovial cells, linkers containing peptide bonds may be used.

In general, the suitability of a candidate cleavable linking group can be assessed by testing the ability of the degrading agent (or condition) to cleave the candidate linking group. It would also be desirable to test candidate cleavable linking groups for their ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, a relative susceptibility to cleavage between a first condition and a second condition may be determined, wherein the first condition is selected to indicate cleavage in a target cell and the second condition is selected to indicate cleavage in other tissue or biological fluid, such as blood or serum. The evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. Initial evaluation was performed under cell-free or culture conditions and confirmed to be useful by further evaluation in whole animals. In preferred embodiments, the cleavage of a useful candidate compound in a cell (or under in vitro conditions selected to mimic intracellular conditions) is at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold greater than the cleavage rate in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

I. Redox cleavable linking groups

In one embodiment, the cleavable linking group is a redox cleavable linking group that cleaves upon reduction or oxidation. An example of a reducing cleavable linking group is a disulfide linking group (-S-). To determine whether a candidate cleavable linking group is a suitable "reducing cleavable linking group," or is suitable for use with a particular iRNA moiety and a particular targeting agent, for example, reference may be made to the methods described herein. For example, candidates can be evaluated by incubation with Dithiothreitol (DTT) or other reducing agent using reagents known in the art, which mimic the cleavage rate that would be observed in a cell (e.g., a target cell). Candidates may also be evaluated under conditions selected to mimic blood or serum conditions. In one, the candidate compound is cleaved in the blood up to about 10%. In other embodiments, the degradation of a useful candidate compound in a cell (or under in vitro conditions selected to mimic intracellular conditions) is at least about 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold greater than the cleavage rate in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzymatic kinetic assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

Phosphate-based cleavable linking groups

In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by an agent that degrades or hydrolyzes the phosphate group. Examples of agents in cells that cleave phosphate groups are enzymes in cells, such as phosphatases. An example of a phosphate-based linking group is -O-P(O)(ORk)-O-、-O-P(S)(ORk)-O-、-O-P(S)(SRk)-O-、-S-P(O)(ORk)-O-、-O-P(O)(ORk)-S-、-S-P(O)(ORk)-S-、-O-P(S)(ORk)-S-、-S-P(S)(ORk)-O-、-O-P(O)(Rk)-O-、-O-P(S)(Rk)-O-、-S-P(O)(Rk)-O-、-S-P(S)(Rk)-O-、-S-P(O)(Rk)-S-、-O-P(S)(Rk)-S-. a preferred embodiment is -O-P(O)(OH)-O-、-O-P(S)(OH)-O-、-O-P(S)(SH)-O-、-S-P(O)(OH)-O-、-O-P(O)(OH)-S-、-S-P(O)(OH)-S-、-O-P(S)(OH)-S-、-S-P(S)(OH)-O-、-O-P(O)(H)-O-、-O-P(S)(H)-O-、-S-P(O)(H)-O-、-S-P(S)(H)-O-、-S-P(O)(H)-S-、-O-P(S)(H)-S-. a preferred embodiment is-O-P (O) (OH) -O-. These candidates can be evaluated using methods similar to those described above.

Acid cleavable linking groups

In another embodiment, the cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments, the acid-cleavable linking group is cleaved in an acidic environment at 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 an agent (e.g., an enzyme) that can act as a generalized acid. In cells, specific low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid cleavable linkers. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. The acid cleavable group may have the general formula-c=nn-, C (O) O or-OC (O). Preferred embodiments are those wherein the carbon (alkoxy) attached to the oxygen of the ester is aryl, substituted alkyl or tertiary alkyl, such as dimethylpentyl or tertiary butyl. These candidates can be evaluated using methods similar to those described above.

Ester-based linking groups

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

V. peptide-based cleavage groups

In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes in the cell, such as peptidases and proteases. The peptide-based cleavable linking group is a peptide bond formed between amino acids to produce oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not comprise an amide group (-C (O) NH-). The amide groups may be formed between any alkylene, alkenylene or alkynylene groups. Peptide bonds are a special type of amide bond formed between amino acids to produce peptides and proteins. The peptide-based cleavage groups are typically limited to peptide bonds (i.e., amide bonds) formed between the amino acids that produce the peptide and protein and do not contain an entire amide functionality. The peptide-based cleavable linking group has the general formula-NHCHRAC (O) NHCHRBC (O) -, wherein RA and RB are R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

In one embodiment, the iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of linkers for iRNA carbohydrate conjugates with the compositions and methods of the invention include, but are not limited to:

When one of X or Y is an oligonucleotide, the other is hydrogen.

In certain embodiments of the compositions and methods of the present invention, the ligand is one or more "GalNAc" (N-acetylgalactosamine) derivatives linked by a divalent or trivalent branched linker.

In one embodiment, the dsRNA of the invention is conjugated to a divalent or trivalent branched linker selected from the group consisting of structures represented by any of formulas XLIV-xlviii:

Wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q B and q5C independently represent from 0 to 20 at each occurrence, and wherein the repeating units may be the same 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 Each occurrence is independently absent, CO, NH, O, S, OC (O), NHC (O), CH ₂、CH₂ NH, or CH ₂ O;

Q ^2A、Q^2B、Q^3A、Q^3B、Q^4A、Q^4B、Q^5A、Q^5B、Q^5C is independently at each occurrence absent, alkylene, substituted alkylene, wherein one or more methylene groups may be interrupted or capped by one or more of the following: o, S, S (O), SO ₂、N(R^N), C (R')=c (R "), c≡c, or C (O);

Each occurrence of R ^2A、R^2B、R^3A、R^3B、R^4A、R^4B、R^5A、R^5B、R^5C is independently absent, NH, O, S, CH ₂、C(O)O、C(O)NH、NHCH(R^a)C(O)、-C(O)-CH(R^a) -NH-, CO, ch=n-O, Or a heterocyclic group;

L ^2A、L^2B、L^3A、L^3B、L^4A、L^4B、L^5A、L^5B and L ^5C represent ligands; i.e., each occurrence is independently a monosaccharide (e.g., galNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R ^a is H or an amino acid side chain. Trivalent conjugated GalNAc derivatives are particularly suitable for use with RNAi agents for inhibiting expression of a target gene, such as those of formula XLVIII:

Wherein L ^5A、L^5B and L ^5C represent monosaccharides such as GalNAc derivatives.

Examples of suitable divalent and trivalent branched linker groups for conjugation to GalNAc derivatives include, but are not limited to, the structures of formula II, formula VII, formula XI, formula X and formula XIII mentioned above.

Representative U.S. patents teaching the preparation of RNA conjugates include, but are not limited to, 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;5,512,439;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;5,585,481;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;8,106,022, the entire contents of each of these patents are hereby incorporated by reference.

It is not necessary to modify all positions in a given compound uniformly, and in fact, more than one of the modifications described above may be incorporated into a single compound or even at a single nucleoside within an iRNA. The invention also encompasses iRNA compounds as chimeric compounds.

In the context of the present invention, a "chimeric" iRNA compound or "chimera" is an iRNA compound, preferably a dsRNA, which contains two or more chemically distinct regions, each region being composed of at least one monomer unit, i.e. a nucleotide in the case of a dsRNA compound. These irnas typically contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity to the target nucleic acid. Additional regions of iRNA may be used as a region capable of cleaving RNA: DNA or RNA: substrates for enzymes of RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves RNA: RNA strand of DNA duplex. Thus, activation of RNase H cleaves the RNA target, thereby greatly enhancing the efficiency of iRNA repressor gene expression. Thus, comparable results are generally obtained with shorter irnas when chimeric dsRNA is used, as compared to phosphorothioate deoxydsrna hybridized to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

In some cases, the RNA of the iRNA can be modified with a non-ligand group. Many non-ligand molecules have been conjugated to iRNA to enhance the activity, cellular distribution or cellular uptake of iRNA, and procedures for performing such conjugation are available in the scientific literature. Such non-ligand moieties have included lipid moieties such as cholesterol (Kubo, t et al, "biochem. Biophys. Res. Comm.)," biochem. Comm.), 2007, 365 (1): 54-61; letsinger et al, proc. Natl. Acad. Sci. U.S. 1989, 86:6553), cholic acid (Manoharan et al, J. Biol. Organic and pharmaceutical chemistry, 1994, 4:1053), thioethers, e.g., hexyl-S-triphenylmethyl mercaptan (Manoharan et al, J. New York, 1992, 660:306; manoharan et al, J. Bio Organic and pharmaceutical chemistry, 1993, 3:2765), thiocholesterol (Oberhauser et al, J. Nucleic acid research, 1992, 20:533), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al, J. European molecular biology, 1991, 10:111; kabanov et al, FEBS flash, 1990, 259:327; svinarchuk et al, biochemistry, 1993, 75:49), phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1, 2-di-O-hexadecyl-rac-glycerol-3-H-phosphonate (Marnoharan et al, tetrahedron flash, 1995, 36:3651; shea et al, nucleic acids research, 1990, 18:3777), polyamine or polyethylene glycol chains (Manoharan et al, nucleoside and nucleotide, 1995, 14:969) or adamantaneacetic acid (Manoharan et al, tetrahedron flash, 1995, 36:3651), palmityl moiety (Mishra et al, biochemistry and biophysics flash, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-carbonyloxy cholesterol moiety (Crooke et al, journal of pharmacology and experimental therapeutics, 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above. Typical conjugation protocols involve the synthesis of RNAs with amino linkers at one or more positions in the sequence. The amino group is then reacted with the conjugated molecule using an appropriate coupling or activating reagent. The conjugation reaction may be carried out with the RNA still bound to the solid support or in the solution phase after cleavage of the RNA. Purification of the RNA conjugate by HPLC generally yields the pure conjugate.

V. delivery of iRNA of the invention

For example, delivery can be accomplished by contacting the cells with an iRNA of the invention in vitro or in vivo.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) may be suitable for use with the iRNA of the present invention (see, e.g., akhtar S. And Julian RL. (1992) [ Trends in cell. Biol.) ] 2 (5): 139-144 and WO94/02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors that need to be considered for delivery of the iRNA molecule include, for example, biostability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The nonspecific effects of RNAi can be minimized by local administration, e.g., by direct injection or implantation into tissue or topical administration of the formulation. Local administration to the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may otherwise be injured by the agent or may degrade the agent, and allows administration of lower total doses of RNAi molecules. Several studies have shown successful knockdown of gene products when iRNA is administered locally. For example, intraocular delivery VEGF DSRNA by intravitreal injection (Tolentino, MJ. et al (2004) Retina (Retina) 24:132-138) in cynomolgus monkeys and by subretinal injection (Reich, SJ. et al (2003) molecular vision (mol. Vis.) 9:210-216) in mice has both been shown to prevent 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) molecular therapy (mol. Ther.) 11:267-274) and could extend survival of tumor-bearing mice (Kim, WJ. et al (2006) molecular therapy 14:343-350; li, S. Et al (2007) molecular therapy 15:515-523). RNA interference has also shown success in local delivery to the CNS by direct injection (Dorn, G. Et al, (2004) Nucleic Acids (Nucleic Acids) 32:e49; tan, PH. et al, (2005) Gene therapy 12:59-66; makimura, h. et al, (2002) BMC neuroscience (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) journal of neurophysiology (j. Neurophysiology.) 93: 594-602) and reaches the lungs by intranasal administration (Howard, ka. Et al, (2006) molecular therapy 14:476-484; zhang, x et al, (2004) journal of biochemistry (j.biol. Chem.)) (279: 10677-10684; bitko, v. et al, (2005) Nature medicine (Nat. Med.) 11: 50-55). To administer iRNA systemically to treat a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods are used to prevent rapid degradation of dsRNA by endo-and exonucleases in vivo. Modification of the RNA or drug carrier can also allow the iRNA composition to target the target tissue and avoid undesirable off-target effects. The iRNA molecule can be modified by chemical conjugation to a lipophilic group (e.g., cholesterol) to enhance cellular uptake and prevent degradation. For example, iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety is injected systemically into mice and results in knockdown of ApoB mRNA in both liver and jejunum (Soutschek, j. Et al (2004) Nature 432:173-178). In a mouse model of prostate cancer, conjugation of iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression (McNamara, jo et al ], (2006) natural biotechnology (nat. Biotechnol.)) (24:1005-1015). In alternative embodiments, the iRNA can be delivered using a drug delivery system (e.g., nanoparticle, dendrimer, polymer, liposome, or cationic delivery system). The positively charged cation delivery system promotes binding of (negatively charged) iRNA molecules and also enhances interactions at negatively charged cell membranes to allow efficient uptake of iRNA by cells. Cationic lipids, dendrimers, or polymers may bind to or be induced to form vesicles or micelles that encapsulate the iRNA (see, e.g., kim SH. et al (2008) journal of controlled release (Journal of Controlled Release) 129 (2): 107-116). When administered systemically, the formation of vesicles or micelles further prevents degradation of the iRNA. Methods for preparing and administering cationic iRNA complexes are well within the ability of those skilled in the art (see, e.g., sorensen, DR., et al, (2003) journal of molecular biology (J. Mol. Biol.) 327:761-766; verma, UN. Et al, (2003) clinical cancer research (Clin. Cancer Res.)) 9:1291-1300; arnold, AS et al, (2007) journal of hypertension (J. Hypertens.)) 25:197-205, which is incorporated herein by reference in its entirety). Some non-limiting examples of drug delivery systems that can be used for systemic delivery of iRNA include DOTAP (Sorensen, DR. et al (2003), supra; verma, UN. et al (2003), supra), oligofectamine, "solid nucleic acid lipid particles (solid nucleic ACID LIPID PARTICLES)" (Zimmermann, TS. et al, (2006) Nature 441:111-114), cardiolipin (Chien, PY. et al, (2005) cancer gene therapy 12:321-328; pal, a. Et al, (2005) journal of international oncology (Int j. Oncol.) 26: 1087-1091), polyethyleneimine (Bonnet ME. et al (2008) pharmaceutical research (pharm. Res.) 8 month 16 day electronic plate precedes the printing plate; aigner, A. (2006) [ journal of biomedical and biotechnology (J.biomed.Biotechnol.) ] 71659 ], arg-Gly-Asp (RGD) peptide (Liu, S. (2006) [ molecular medicine (mol.Pharm.) ] 3: 472-487) and polyamidoamines (Tomalia, DA. et al (2007) journal of the society of biochemistry (biochem. Soc. Trans.) 35:61-67; yoo, h. et al (1999) pharmaceutical research 16: 1799-1804). In some embodiments, the iRNA forms a complex with cyclodextrin for systemic administration. Methods of administration, as well as pharmaceutical compositions of iRNA and cyclodextrin, can be found in U.S. patent No. 7,427,605, incorporated herein by reference in its entirety.

A. vector-encoded iRNA of the invention

The iRNA targeting CIDEB genes can be expressed from transcriptional units inserted into DNA or RNA vectors (see, e.g., couture, A et al, TIG. (1996), 12:5-10; skillern, A et al, international PCT publication No. WO 00/22113, conrad, international PCT publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression may be transient (on the order of hours to weeks) or continuous (weeks to months or longer), depending on the particular construct and target tissue or cell type used. These transgenes may be introduced as linear constructs, circular plasmids, or viral vectors, which may be integrating or non-integrating vectors. Transgenes may also be constructed to allow them to be inherited as extrachromosomal plasmids (Gassmann et al, (1995) Proc. Natl. Acad. Sci. USA 92:1292).

One or more separate strands of iRNA may be transcribed from a promoter on the expression vector. Where two separate strands are to be expressed to produce, for example, dsRNA, the two separate expression vectors can be co-introduced (e.g., by transfection or infection) into the target cell. Alternatively, each individual strand of dsRNA can be transcribed by a promoter, both on the same expression plasmid. In one embodiment, the dsRNA is expressed as an inverted repeat polynucleotide linked by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

The iRNA expression vector is typically a DNA plasmid or viral vector. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for expression of an iRNA as described herein. Eukaryotic expression vectors are well known in the art and are available from a number of commercial sources. Generally, vectors are provided that contain restriction sites that facilitate insertion of the desired nucleic acid segment. Delivery of the iRNA expression vector can be systemic, such as by intravenous or intramuscular administration, by administration of target cells transplanted from a patient, followed by reintroduction into the patient, or by any other means that allows for the introduction of the desired target cells.

Viral vector systems that may be used with the methods and compositions described herein include, but are not limited to, (a) adenoviral vectors; (b) Retroviral vectors, including but not limited to lentiviral vectors, moronella leukemia virus (moloney murine leukemia virus), and the like; (c) an adeno-associated viral vector; (d) a herpes simplex virus vector; (e) SV 40 vector; (f) polyomavirus vectors; (g) papillomavirus vectors; (H) a picornaviral vector; (i) Poxvirus vectors such as orthopoxes (e.g., vaccinia virus vectors) or fowlpox (e.g., canary pox or fowlpox); and (i) helper-dependent or entero-free adenoviruses. Replication-defective viruses may also be advantageous. The different vectors will or will not be incorporated into the genome of the cell. If desired, the construct may comprise viral sequences for transfection. Alternatively, the construct may be incorporated into vectors capable of episomal replication, such as EPV and EBV vectors. Constructs for recombinant expression of iRNA will typically require regulatory elements, such as promoters, enhancers, and the like, to ensure expression of the iRNA in the target cell. Other aspects to be considered for vectors and constructs are known in the art.

VI pharmaceutical compositions of the invention

The invention also encompasses pharmaceutical compositions and formulations comprising the iRNA of the invention. Thus, in one embodiment, provided herein is a pharmaceutical composition comprising a double-stranded ribonucleic acid (dsRNA) agent that inhibits cell death-induced expression of DFFA-like effector b (CIDEB) in a cell, such as a hepatocyte, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises a sequence that hybridizes to SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides differing by NO more than 1, 2 or 3 nucleotides from the nucleotide sequence of SEQ ID NO:2 by at least 15 consecutive nucleotides differing by no more than 1, 2 or 3 nucleotides; and a pharmaceutically acceptable carrier. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises a sequence from SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides of the nucleotide sequence from SEQ ID NO:2, and at least 15 consecutive nucleotides of the nucleotide sequence of 2.

In another embodiment, provided herein is a pharmaceutical composition comprising a dsRNA agent that inhibits expression of CIDEB in a cell (e.g., a hepatocyte), wherein the dsRNA agent comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 1,2, or 3 nucleotides from any of the antisense sequences listed in tables 3-6; and a pharmaceutically acceptable carrier. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand comprising a complementary region comprising at least 15 contiguous nucleotides from any one of the antisense sequences listed in tables 3-6.

Pharmaceutical compositions containing the iRNA of the invention are useful for treating diseases or disorders associated with the expression or activity of CIDEB genes, for example, chronic inflammatory diseases.

Such pharmaceutical compositions are formulated based on the mode of delivery. One example is a composition formulated for systemic administration via parenteral delivery (e.g., by Intravenous (IV), intramuscular (IM), or for subcutaneous delivery). Another example is a composition formulated for direct delivery into the liver, for example by infusion into the liver, for example by continuous pump infusion. The pharmaceutical compositions of the invention may be administered in a dosage sufficient to inhibit expression of CIDEB genes. Typically, suitable dosages of the iRNA of the invention will range from about 0.001 to about 200.0 mg/kg of recipient weight/day, typically from about 1 to 50 mg/kg of body weight/day. Generally, suitable doses of the iRNA of the present invention will range from about 0.1mg/kg to about 5.0mg/kg, preferably about 0.3mg/kg and about 3.0 mg/kg.

Repeated dose regimens may include administration of therapeutic amounts of iRNA on a periodic basis, such as once every other day to once a year. In certain embodiments, the iRNA is administered about weekly, every 7-10 days, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, monthly, every 2 months, every 3 months (quarterly), every 4 months, every 5 months, or every 6 months.

After the initial treatment regimen, the treatment may be administered on a less frequent basis.

Those skilled in the art will appreciate that certain factors may affect the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatments, the overall health and/or age of the subject, and other diseases present. Furthermore, the treatment of a subject with a therapeutically effective amount of the composition may comprise a single treatment or a series of treatments. The estimation of effective doses and in vivo half-life of the individual irnas encompassed by the present invention can be performed using conventional methods or based on in vivo testing using a suitable animal model, as described elsewhere herein.

Advances in mouse genetics have resulted in a number of mouse models for studying various human diseases such as CIDEB-related diseases, disorders or conditions that would benefit from a reduction in CIDEB expression. Such models can be used for in vivo testing of iRNA, as well as for determining therapeutically effective doses. Such models can be used for in vivo testing of iRNA, as well as for determining therapeutically effective doses. Suitable mouse models are known in the art and include, for example, mice and rats fed a high fat diet (HFD; also known as western diet), methionine-choline deficiency (MCD) diet, or a high-fat (15%), high-cholesterol (1%) diet (HFHC); an obese (ob/ob) mouse containing a mutation in the obese (ob) gene (Wiegman et al, (2003) diabetes, 52:1081-1089); mice containing homozygous knockouts of LDL receptors (LDLR-/-mice; ishibashi et al, (1993) J.Clin.Ind.J CLIN INVEST, (2): 883-893); a diet-induced mouse model of atherosclerosis (Ishida et al, (1991) journal of lipid research (J lipid. Res.), 32:559-568); hybrid lipoprotein lipase knockout mouse models (Weistock et al, (1995) journal of clinical investigation 96 (6): 2555-2568); mice and rats fed a choline deficient L-amino acid defined high fat diet (CDAHFD) (Matsumoto et al, (2013) J.International Experimental pathology (int. J. Exp. Path) 94:93-103); mice and rats fed a high trans-fat cholesterol diet (HTF-C) (Clapper et al, (2013) [ J.Am. Physiol. Gastroidolite. Liver Physiol.) ] 305:G483-G495; mice and rats fed a high fat, high cholesterol, bile salt diet (HF/HC/BS) (Matsuzawa et al, (2007) Hepatology (Hepatology) 46:1392-1403); and mice and rats fed a high fat diet plus fructose (30%) water (Softic et al, (2018) journal of clinical investigation 128 (1) -85-96).

The pharmaceutical compositions of the present invention may be administered in a number of ways depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (e.g., by transdermal patch), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including by nebulizer); intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous (e.g., by implanted device); or intracranial (e.g., by intraparenchymal, intrathecal, or intraventricular administration).

IRNA can be delivered in a manner that targets a particular cell or tissue (e.g., liver hepatocytes of the liver)).

In some embodiments, the pharmaceutical compositions of the invention are suitable for intramuscular administration to a subject. In other embodiments, the pharmaceutical compositions of the invention are suitable for intravenous administration to a subject. In some embodiments of the invention, the pharmaceutical composition of the invention is suitable for subcutaneous administration to a subject, for example, using a 29g or 30g needle.

The pharmaceutical compositions of the invention may include the RNAi agents of the invention in an unbuffered solution (e.g., saline or water), or in a buffered solution, e.g., a buffered solution comprising acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

In one embodiment, a pharmaceutical composition of the invention, e.g., a composition suitable for subcutaneous administration, comprises an RNAi agent of the invention in Phosphate Buffered Saline (PBS). Suitable concentrations of PBS include, for example, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 6.5mM, 7mM, 7.5.mM, 9mM, 8.5mM, 9mM, 9.5mM, or about 10mM PBS. In one embodiment of the invention, the pharmaceutical composition of the invention comprises an RNAi agent of the invention dissolved in a solution of about 5mM PBS (e.g., 0.64mM NaH ₂PO₄,4.36mM Na₂HPO₄, 85mM NaCl). Values intermediate to the ranges and values noted above are also intended to be part of the invention. Further, it is intended to include ranges of values using any combination of the above values as upper and/or lower limits.

The pH of the pharmaceutical composition of the present invention may be from about 5.0 to about 8.0, from about 5.5 to about 8.0, from about 6.0 to about 8.0, from about 6.5 to about 8.0, from about 7.0 to about 8.0, from about 5.0 to about 7.5, from about 5.5 to about 7.5, from about 6.0 to about 7.5, from about 6.5 to about 7.5, from about 5.0 to about 7.2, from about 5.25 to about 7.2, from about 5.5 to about 7.2, from about 5.75 to about 7.2, from about 6.0 to about 7.2, from about 6.5 to about 7.2, or from about 6.8 to about 7.2. Ranges and values intermediate to those described above are also intended to be part of the present invention.

The osmolality (osmolality) of the pharmaceutical composition of the invention may be suitable for subcutaneous administration, for example not more than about 400mOsm/kg, such as 50 to 400mOsm/kg, 75 to 400mOsm/kg, 100 to 400mOsm/kg, 125 to 400mOsm/kg, 150 to 400mOsm/kg, 175 to 400mOsm/kg, 200 to 400mOsm/kg, 250 to 400mOsm/kg, 300 to 400mOsm/kg, 50 to 375mOsm/kg, 75 to 375mOsm/kg, 100 to 375mOsm/kg, 125 to 375mOsm/kg, 150 to 375mOsm/kg, 175 to 375mOsm/kg, 200 to 375mOsm/kg, 250 to 375mOsm/kg, 300 to 375mOsm/kg, 50 to 350mOsm/kg, 75 to 350mOsm/kg, 100 to 350mOsm/kg, 150 to 350mOsm/kg, 175 to 350mOsm/kg, 200 to 350mOsm/kg 250 to 350mOsm/kg, 50 to 325mOsm/kg, 75 to 325mOsm/kg, 100 to 325mOsm/kg, 125 to 325mOsm/kg, 150 to 325mOsm/kg, 175 to 325mOsm/kg, 200 to 325mOsm/kg, 250 to 325mOsm/kg, 300 to 350mOsm/kg, 50 to 300mOsm/kg, 75 to 300mOsm/kg, 100 to 300mOsm/kg, 125 to 300mOsm/kg, 150 to 300mOsm/kg, 175 to 300mOsm/kg, 200 to 300mOsm/kg, 250 to 300mOsm/kg, 50 to 250mOsm/kg, 75 to 250mOsm/kg, 100 to 250mOsm/kg, 125 to 250mOsm/kg, 150 to 250mOsm/kg, 175 to 350mOsm/kg, 200 to 250mOsm/kg, such as about 50、55,60,65,70,75,80,85,90,95,100,105,110,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250,255,260,265,270,275,280,285,295,300,305,310,320,325,330,335,340,345,350,355,360,365,370,375,380,385,390,395 or about 400mOsm/kg. Ranges and values intermediate to those described above are also intended to be part of the present invention.

The pharmaceutical composition of the invention comprising the RNAi agent of the invention can be present in a vial comprising about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0mL of the pharmaceutical composition. The concentration of the RNAi agent in the pharmaceutical composition of the invention can be about 10、15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115,130,125,130,135,140,145,150,175,180,185,190,195,200,205,210,215,230,225,230,235,240,245,250,275,280,285,290,295,300,305,310,315,330,325,330,335,340,345,350,375,380,385,390,395,400,405,410,415,430,425,430,435,440,445,450,475,480,485,490、495 or about 500mg/mL. In one embodiment, the concentration of the RNAi agent in the pharmaceutical composition of the present invention is about 100mg/mL. Values intermediate to the ranges and values noted above are also intended to be part of the invention.

The pharmaceutical compositions of the invention may comprise the dsRNA agents of the invention in free acid form. In other embodiments of the invention, the pharmaceutical compositions of the invention may comprise the dsRNA agents of the invention in salt form (e.g., sodium salt form). In certain embodiments, when the dsRNA agents of the invention are in the form of sodium salts, sodium ions are present in the agent as counter ions to substantially all of the phosphodiester and/or phosphorothioate groups present in the agent. Agents in which substantially all of the phosphodiester and/or phosphorothioate linkages have a sodium counterion comprise no more than 5, 4, 3, 2, or 1 phosphodiester and/or phosphorothioate linkages that do not have a sodium counterion. In some embodiments, when the dsRNA agents of the invention are in the form of sodium salts, sodium ions are present in the agent as counter ions to all phosphodiester and/or phosphorothioate groups present in the agent.

Pharmaceutical compositions and formulations for topical administration may comprise transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous bases, 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 characteristic of the invention is mixed with a topical delivery agent, such as a lipid, liposome, fatty acid ester, steroid, chelator, and surfactant. Suitable lipids and liposomes include neutral (e.g., dioleoyl phosphatidyl DOPE ethanolamine, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), negative (e.g., dimyristoyl phosphatidyl glycerol DMPG), and cationic (e.g., dioleoyl tetramethyl aminopropyl DOTAP and dioleoyl phosphatidyl ethanolamine DOTMA). The iRNA that is a feature of the invention may be encapsulated in a liposome or may form a complex therewith, in particular with a cationic liposome. Alternatively, the iRNA may be complexed with a lipid, in particular a cationic lipid. Suitable fatty acids and esters include, but are not limited to, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glycerol monooleate, glycerol dilaurate, glycerol 1-monocaprate, 1-dodecylazepan-2-one, acylcarnitines, acylcholines, or C _1-20 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, incorporated herein by reference.

A. iRNA formulations comprising membrane molecule assemblies

The iRNA used in the compositions and methods of the invention can be formulated for delivery in the form of a membrane molecule assembly (e.g., a liposome or micelle). In addition to microemulsions, there are a number of organized surfactant structures that have been studied and used in drug formulation. These include unilamellar, micellar, bilayer and vesicle. Vesicles (e.g., liposomes) are of great concern due to their specificity and duration of action provided from a drug delivery perspective. As used in this disclosure, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in one or more spherical bilayers.

Liposomes include unilamellar or multilamellar vesicles having a membrane formed of a lipophilic material and an aqueous interior. The aqueous portion comprises the composition (e.g., iRNA) to be delivered. The lipophilic material separates the aqueous interior from the aqueous exterior, which generally does not contain the iRNA composition, although in some examples it may. Cationic liposomes have the advantage of being able to fuse with the cell wall. Non-cationic liposomes, although not as effective as cell walls, are taken up by macrophages in the body.

In order to pass through intact mammalian skin, lipid vesicles must pass through a series of pores with a diameter of less than 50nm under the influence of a suitable transdermal gradient. Thus, it is desirable to use liposomes that are highly deformable and capable of passing through such pores.

Liposomes can be used to transfer and deliver active ingredients to the site of action. Because the liposome membrane is similar in structure to a biological membrane, when the liposome is applied to a tissue, the liposome begins to merge with the cell membrane, and as the liposome merges and the cell progresses, the liposome contents are emptied into the cell where the active agent can function.

Liposome formulations have been the focus of extensive research as a means of delivery for many drugs. There is increasing evidence that liposomes present several advantages over other formulations for topical administration. Such 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 ability to administer multiple drugs (hydrophilic and hydrophobic) into the skin.

Several reports detail the ability of liposomes to deliver agents comprising high molecular weight DNA into the skin. Compounds comprising analgesics, antibodies, hormones and high molecular weight DNA have been applied to the skin. Most applications result in targeting to the epidermis.

Liposomes containing iRNA agents can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent to form micelles with the lipid component. For example, the lipid component may be an amphiphilic cationic lipid or a lipid conjugate. The detergent may have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosinate. The iRNA agent formulation is then added to the micelle comprising the lipid component. Cationic groups on the lipids interact with the iRNA agent and condense around the iRNA agent to form liposomes. After condensation, the detergent is removed, e.g., by dialysis, to produce a liposomal formulation of the iRNA agent.

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

Methods for producing stable polynucleotide delivery vehicles incorporating polynucleotide/cationic lipid complexes as structural components of delivery vehicles are further described, for example, in WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation may also comprise one or more aspects of the exemplary methods described in the following: felgner, P.L. 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, molecular biology, 23:238, 1965; olson et al, "biochemistry. Acta)," 557:9,1979; szoka et al, journal of national academy of sciences 75:4194, 1978; mayhew et al, journal 775 of biochemistry and biophysics: 169, 1984; kim et al, "Proc. Biochemistry and BioPhysics Acta" 728:339, 1983; fukunaga et al endocrinology (endocrinol.) 115:757, 1984. Common techniques for preparing suitable sized lipid aggregates for use as delivery vehicles include sonication and freeze-thawing and extrusion (see, e.g., mayer et al, journal of biochemistry and biophysics 858:161, 1986). Microfluidization may be used when relatively uniform aggregates of small duration (50 nm to 200 nm) are desired (Mayhew et al, journal of biochemistry and biophysics 775:169, 1984). These methods are readily adaptable for packaging iRNA agent formulations into liposomes.

Liposomes fall into two broad categories. Cationic liposomes are positively charged liposomes that interact with negatively charged DNA molecules to form stable complexes. The positively charged DNA/liposome complex binds to the negatively charged cell surface and internalizes in endosomes. Due to the acidic pH in endosomes, liposomes burst, releasing their contents into the cytoplasm (Wang et al, biochemical and BioPhysics research Comm 1987, 147, 980-985).

Liposomes that are pH sensitive or negatively charged entrap DNA rather than complex with it. Since both DNA and lipids carry similar charges, rejection rather than complex formation occurs. However, some DNA is embedded within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al, J.controlled Release (Journal of Controlled Release), 1992, 19, 269-274).

One major type of liposome composition comprises phospholipids other than phosphatidylcholine of natural origin. Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are typically formed from dimyristoyl phosphatidylglycerol, whereas anionic fusogenic liposomes are formed predominantly from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from Phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed by a mixture of phospholipids and/or phosphatidylcholine and/or cholesterol.

Examples of other methods of introducing liposomes into cells in vitro and in vivo include U.S. patent No. 5,283,185 and U.S. patent No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; felgner, journal of biochemistry 269:2550, 1994; nabel, proc.Natl.Acad.Sci.90: 11307, 1993; nabel, human Gene therapy (Human Gene ter.)) 3:649, 1992; gershon, journal of biochemistry (biochem.) 32:7143, 1993; strauss journal of the european molecular biology (EMBO j.) 11:417, 1992.

Nonionic liposome systems have also been examined to determine their utility in delivering drugs to the skin, particularly systems that include nonionic surfactants and cholesterol. Cyclosporin a (cycloporin-a) was delivered into the dermis of the mouse skin using a non-ionic liposome formulation comprising Novasome ^TM I (glycerol dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome ^TM II (glycerol distearate/cholesterol/polyoxyethylene-10-stearyl ether). The results indicate that such a non-ionic liposome system is effective in promoting the deposition of cyclosporin a into different layers of the skin (Hu et al, s.t.p.pharmaceutical science (s.t.pharma.sci.)), 1994,4,6, 466.

Liposomes also include "sterically stabilized" liposomes, as used herein, the term refers to liposomes comprising one or more specific lipids which, when incorporated into a liposome, result in increased circulation life relative to liposomes lacking such specific lipids. Examples of sterically stabilized liposomes are those wherein a portion of the vesicle-forming lipid fraction of liposome (a) comprises one or more glycolipids, such as monosialoganglioside G _M1, or (B) liposomes derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. While not wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelins, or PEG-derived lipids, the enhanced circulation half-life of these sterically stabilized liposomes results from reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al, FEBS rapid report (FEBSLetters), 1987, 223, 42; wu et al, cancer research, 1993, 53, 3765).

Various liposomes including one or more glycolipids are known in the art. Papahadjoulos et al (New York science academy of years, 1987, 507, 64) report the ability of monosialoganglioside G _M1, galactosylsulfate and phosphatidylinositol to improve the blood half-life of liposomes. These findings are set forth in the following: gabizon et al (Proc. Natl. Acad. Sci. USA, 1988, 85, 6949). Liposomes are disclosed in both U.S. Pat. No. 4,837,028 to Allen et al and WO 88/04924, and include (1) sphingomyelin and (2) ganglioside G _M1 or galactosylsulfate. U.S. patent 5,543,152 (Webb et al) discloses liposomes comprising sphingomyelin. Liposomes comprising 1, 2-sn-dimyristoyl phosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In some embodiments, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with cell membranes. Non-cationic liposomes, while not effectively fused with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNA agents to macrophages.

Additional advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a variety of water-soluble and lipid-soluble drugs; liposomes can protect the iRNA encapsulated in their internal compartments from metabolism and degradation (Rosoff, pharmaceutical dosage form (Pharmaceutical Dosage Forms), lieberman, rieger and Banker (editions), 1988, volume 1, page 245). Important considerations for preparing liposome formulations are lipid surface charge, vesicle size, and aqueous volume of the liposome.

The positively charged synthetic cationic lipid N- [1- (2, 3-dioleoyloxy) 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 are capable of fusing with negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA agents (see, e.g., felgner, P.L. et al, proc. Natl. Acad. Sci. USA 8:7413-7417, 1987 and U.S. patent No. 4,897,355, description of DOTMA and its use with DNA).

DOTMA analogs, 1, 2-bis (oleoyloxy) -3- (trimethylammonio) propane (DOTAP) can be used in combination with phospholipids to form DNA complex vesicles. Lipofectin ^TM (Bethesda Research Laboratories, gaithersburg, md.) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells, including 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. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and effectively deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1, 2-bis (oleoyloxy) -3,3- (trimethylammonio) propane ("DOTAP") (Boehringer Mannheim, indianapolis, indiana) of Indianapolis, ind.) differs from DOTMA in that the oleoyl moiety is linked by an ester rather than an ether linkage.

Other reported cationic lipid compounds include those conjugated to a variety of moieties, including, for example, carboxy spermine conjugated to one of two types of lipids, and include compounds such as 5-carboxy spermino glycine octacosamide ("DOGS") (TRANSFECTAMINE ^TM, promega, madison, wisconsin, madison) and dipalmitoyl phosphatidylethanolamine 5-carboxy spermoyl-amide ("DPPES") (see, for example, U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate comprises derivatization of lipids with cholesterol ("DC-Chol") which has been formulated into liposomes in combination with DOPE (see Gao, x. And Huang, l., "communication of biochemistry and biophysics", 179:280, 1991). The lipopolylysine prepared by conjugation of polylysine with DOPE was reported to be effective for transfection in the presence of serum (Zhou, X. Et al, report of biochemistry and biophysics 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include dmriie and dmriie-HP (Vical company of lahoma, california (Vical, la Jolla, california)) and Lipofectamine (DOSPA) (Life technologies, inc. Of Gaithersburg, maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposome formulations are particularly suitable for topical administration; liposomes exhibit several advantages over other formulations. Such 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 ability to administer the iRNA agent into the skin. In some embodiments, the liposomes are used to deliver an iRNA agent to epidermal cells, and are also used to enhance penetration of the iRNA agent into dermal tissue, e.g., into the skin. For example, liposomes may be applied topically. Local delivery of drugs formulated as liposomes to the skin has been described (see, e.g., weiner et al J Targeted drug (Journal of Drug Targeting), 1992, volumes 405-410 and du Plessis et al J antiviral studies (ANTIVIRAL RESEARCH), 18, 1992, 259-265; mannino, R.J. and Fould-Fogerite, S., "biotechnology (Biotechniques)," 6:682-690, 1988; itani, T. Et al Gene (Gene) 56:267-276, 1987; nicola, C. Et al methods of enzymology (Enz.)) (149:157-176, 1987; straubinger, R.M. and Papahadiopoulos, D methods of enzymology 101:512-527, 1983; wang, C.Y. and Huang, L., "Proc.Sci.Natl.84:7851-7855, 1987).

Nonionic liposome systems have also been examined to determine their utility in delivering drugs to the skin, particularly systems that include nonionic surfactants and cholesterol. The drug was delivered into the dermis of the mouse skin using a non-ionic liposome formulation comprising Novasome ^TM I (glycerol dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome ^TM II (glycerol distearate/cholesterol/polyoxyethylene-10-stearyl ether). Such formulations with iRNA agents are useful for treating dermatological disorders.

Liposomes containing iRNA can be made highly deformable. Such deformability may allow the liposomes to penetrate through pores smaller than the average radius of the liposomes. For example, the delivery body is a class of deformable liposomes. The transfer body may be prepared by adding a surface edge activator (typically a surfactant) to a standard liposome composition. The transfer body comprising the iRNA may be delivered subcutaneously, e.g., by infection, in order to deliver the iRNA to keratinocytes in the skin. In order to pass through intact mammalian skin, lipid vesicles must pass through a series of pores with a diameter of less than 50nm under the influence of a suitable transdermal gradient. In addition, due to lipid properties, these transfer bodies can self-optimize (adapt to the shape of the pores, e.g. pores in the skin), repair themselves, and can often reach their targets without fragmentation, and often self-load.

Other formulations suitable for use in the present invention are described in WO 2008/042973.

The carrier is yet another type of liposome and is a highly deformable lipid aggregate that is an attractive candidate for drug delivery vehicles. The transfer body may be described as a lipid droplet, which is highly deformable such that it readily penetrates through pores smaller than the droplet. The delivery body is able to adapt to the environment in which it is used, e.g. it is self-optimizing (adapts to the shape of the pores in the skin), self-repairing, often reaches its target without fragmentation, and often self-loading. To prepare the transfer body, a surface edge activator, typically a surfactant, may be added to the standard liposome composition. Transfer bodies have been used to deliver serum albumin to the skin. The carrier-mediated delivery of serum albumin has been demonstrated to be as effective as subcutaneous injections of serum albumin-containing solutions.

Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the characteristics of many different types of surfactants, both natural and synthetic, is through the use of hydrophilic/lipophilic balance (HLB). The nature of the hydrophilic groups (also referred to as "heads") provides the most useful method for classifying the different surfactants used in the formulation (Rieger, pharmaceutical dosage form, majordson company, new york, 1988, page 285).

Surfactant molecules are classified as nonionic if they are not ionized. Nonionic surfactants find wide application in pharmaceuticals and cosmetics, and are useful in a variety of pH values. Typically, depending on its structure, its HLB value ranges from 2 to about 18. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glycerol esters, polyglycerol 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 category. Polyoxyethylene surfactants are the most popular members of the class of nonionic surfactants.

Surfactants are classified as anionic if they have a negative charge when dissolved or dispersed in water. Anionic surfactants include carboxylic acid esters such as soaps, acyl lactylates, acyl amides of amino acids, sulfates such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonate, acyl isothiooctoates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the class of anionic surfactants are alkyl sulfates and soaps.

Surfactants are classified as cationic if they have a positive charge when dissolved or dispersed in water. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are among the most commonly used members of such compounds.

Surfactants are classified as amphoteric if they have the ability to carry a positive or negative charge. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines and phospholipids.

The use of surfactants in pharmaceutical products, formulations and emulsions is reviewed (Rieger, pharmaceutical dosage form, makindel, new york, 1988, page 285).

IRNA used in the methods of the invention may also be provided as a micelle formulation. "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecule are inward, bringing hydrophilic portions into contact with surrounding water. If the environment is hydrophobic, the opposite arrangement exists.

Mixed micelle formulations suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of iRNA, an alkali metal C ₈ to C ₂₂ alkyl sulfate, and a micelle-forming compound. Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, glycerol monooleate, borage oil, evening primrose oil, menthol, trihydroxy oxo cholic acid glycine and pharmaceutically acceptable salts thereof, glycerol, polyglycerol, lysine, polylysine, glycerol trioleate, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate and mixtures thereof. The micelle-forming compound may be added simultaneously with or after the addition of the alkali metal alkyl sulfate. Essentially any type of ingredient mix will form mixed micelles, but mix vigorously in order to provide smaller size micelles.

In one method, a first micelle composition is prepared that contains RNAi 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 RNAi, an alkali metal alkyl sulfate, and at least one micelle-forming compound, and then adding the remaining micelle-forming compound with vigorous mixing.

Phenol or m-cresol may be added to the mixed micelle composition to stabilize the formulation and prevent bacterial growth. Alternatively, phenol or m-cresol may be added with the micelle-forming ingredients. After formation of the mixed micelle composition, an isotonic agent, such as glycerol, may also be added.

To deliver the micelle formulation as a spray, the formulation may be placed into an aerosol dispenser and the dispenser is filled with a propellant. The propellant under pressure is in liquid form in the dispenser. The ratio of the ingredients is adjusted so that the aqueous phase and the propellant phase are in one phase, i.e. there is one phase. If there are two phases, it may be necessary to shake the dispenser, for example through a metering valve, before dispensing a portion of the contents. The dispensed dose of medicament is advanced from the metering valve in the form of a fine spray.

The propellant may comprise a hydrochlorofluorocarbon, a hydrofluorocarbon, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1, 2 tetrafluoroethane) may be used.

The specific concentration of the essential components can be determined by relatively simple experimentation. For absorption by the oral cavity, it is often desirable to increase the dosage, for example at least two or three times, by injection or by gastrointestinal administration.

B. Lipid particles

The iRNA, e.g., dsRNA, agents of the invention may be fully encapsulated in a lipid formulation, e.g., LNP, e.g., other nucleic acid-lipid particles.

As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNP typically contains cationic lipids, non-cationic lipids, and lipids that prevent aggregation of particles (e.g., PEG-lipid conjugates). LNP is very useful for systemic applications because it exhibits an extended cycle life following intravenous (i.v.) injection and accumulates at distant sites (e.g., sites physically separated from the site of administration). As used herein, the term "SPLP" refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within lipid vesicles. LNPs include "pSPLP" comprising an encapsulated condensing agent-nucleic acid complex as shown in PCT publication No. WO 00/03683. The particles of the present invention typically have an average diameter of about 50nm to about 150nm, more typically about 60nm to about 130nm, more typically about 70nm to about 110nm, most typically about 70nm to about 90nm, and are substantially non-toxic. In addition, the nucleic acid when present in the nucleic acid-lipid particles of the invention resists degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods of making the same are disclosed, for example, in U.S. patent No. 5,976,567; 5,981,501 th sheet; 6,534,484 th sheet; 6,586,410 th sheet; 6,815,432 th sheet; in PCT publication No. WO 96/40964.

In certain embodiments, the ratio of lipid to drug (mass/mass ratio) (e.g., ratio of lipid to dsRNA) will be in the range of 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 present invention.

The cationic lipid may be, for example, N, N-dioleyl-N, N-dimethyl ammonium chloride (DODAC), N, N-distearyl-N, N-dimethyl ammonium bromide (DDAB), N- (I- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTAP), N- (I- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTMA), N, N-dimethyl-2, 3-dioleoyloxy) propylamine (DODMA), 1, 2-dioleoyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-Dihydrolinoleyloxy-N, N-dimethylaminopropane (DLenDMA), 1, 2-dialkylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1, 2-dialkylcarbamoyloxy-3- (dimethylamino) acetoxypropane (DLin-DAC), 1, 2-dialkylcarbamoyloxy-3-morpholinopropane (DLin-MA), 1, 2-dihydrooleoyl-3-dimethylaminopropane (DLinDAP), 1, 2-dihydrooleoyl thio-3-dimethylaminopropane (DLin-S-DMA), and, 1-linoleoyl-2-linoleoyl-3-dimethylaminopropane (DLin-2-DMAP), 1, 2-dioleoyloxy-3-trimethylaminopropane chloride (DLin-TMA. Cl), 1, 2-dioleoyloxy-3-trimethylaminopropane chloride (DLin-TAP. Cl), 1, 2-dioleoyloxy-3- (N-methylpiperazine) propane (DLin-MPZ) or 3- (N, N-dioleylamino) -1, 2-propanediol (DLinAP), 3- (N, N-dioleylamino) -1, 2-propanediol (DOAP), 1, 2-dioleyloxy-3- (2-N, n-dimethylamino) ethoxypropane (DLin-EG-DMA), 1, 2-dioleoyloxy-N, N-dimethylaminopropane (DLinDMA), 2-dioleyl-4-dimethylaminoethyl- [1,3] -dioxolane (DLin-K-DMA) or analogues thereof, (3 aR,5s,6 aS) -N, N-dimethyl-2, 2-di ((9Z, 12Z) -octadeca-9, 12-dienyl) tetrahydro-3 aH-cyclopenteno [ d ] [1,3] dioxolen-5-amine (ALN 100), (6Z, 9Z, 28z,31 z) -heptadecan-6, 9, 28, 31-tetraen-19-yl 4- (dimethylamino) butanoate (MC 3), 1' - (2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) (2-hydroxydodecylamino) ethyl) piperazin-1-yl) ethylazadiyl) docosan-2-ol (Tech G1) or a mixture thereof. The cationic lipid may comprise from about 20mol% to about 50mol% or about 40mol% of the total lipid present in the particle.

In certain embodiments, the compound 2, 2-dihydrooleoyl-4-dimethylaminoethyl- [1,3] -dioxolane may be used to prepare lipid siRNA nanoparticles. The synthesis of 2,2 dioleyl-4-dimethylaminoethyl- [1,3] -dioxolane is described in U.S. provisional patent application No. 61/107,998, filed on 10/23 of 2008, which is incorporated herein by reference.

In certain embodiments, the lipid siRNA particles comprise 40% 2, 2-dioleoyl-4-dimethylaminoethyl- [1,3] -dioxolane: 10% dspc:40% cholesterol: 10% PEG-C-DOMG (mole percent), wherein the particle size was 63.0.+ -. 20nm and the siRNA/lipid ratio was 0.027.

The non-cationic lipid may be a lipid comprising an anion or neutral lipid including, but not limited to, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl Oleoyl Phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid may comprise from about 5mol% to about 90mol%, about 10mol% or about 58mol% (if cholesterol is included) of the total lipid present in the particle.

Conjugated lipids that inhibit aggregation of particles may be, for example, polyethylene glycol (PEG) lipids, including but not limited to PEG-Diacylglycerol (DAG), PEG-Dialkoxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate may be, for example, PEG-dilauroxypropyl (Ci ₂), PEG-dimyristoxypropyl (Ci ₄), PEG-dipalmitoxypropyl (Ci ₆), or PEG-distearxypropyl (C ] ₈). The conjugated lipid that prevents aggregation of the particles may be from 0mol% to about 20mol% or about 2mol% of the total lipid present in the particles.

In some embodiments, the nucleic acid lipid particle further comprises cholesterol, which is, for example, about 10mol% to about 60mol% or about 48mol% of the total lipids present in the particle.

LNP01

In certain embodiments, lipid ND 98.4 HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed on 3/26 of 2008, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (E Wen Di polar lipid Co (Avanti Polar Lipids)) may be used to prepare lipid dsRNA nanoparticles (e.g., LNP01 particles). Stock solutions of each substance in ethanol can be prepared as follows: ND98, 133mg/ml; cholesterol, 25mg/ml, PEG-ceramide C16, 100mg/ml. The ND98, cholesterol, and PEG-ceramide C16 stock solutions may then be combined, for example, at a molar ratio of 42:48:10. The combined lipid solution may be mixed with an aqueous dsRNA solution (e.g., in sodium acetate at pH 5) such that the final ethanol concentration is about 35% to 45%, and the final sodium acetate concentration is about 100mM to 300mM. Lipid dsRNA nanoparticles typically spontaneously form upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a polycarbonate film (e.g., 100nm cutoff) using, for example, a hot barrel extruder, such as a Lipex extruder (Northern Lipids, inc.). In some cases, the extrusion step may be omitted. Ethanol removal and simultaneous buffer exchange can be achieved by, for example, dialysis or tangential flow filtration. The buffer may be exchanged with, for example, phosphate Buffered Saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, for example, in International application publication No. WO 2008/042973, which is incorporated herein by reference.

Additional exemplary lipid-dsRNA formulations are provided in table 1 below.

Table 1: exemplary lipid formulations

DSPC: distearoyl phosphatidylcholine

DPPC: dipalmitoyl phosphatidylcholine

PEG-DMG: PEG-dimyristoylglycerol (C14-PEG or PEG-C14) (PEG having an average molar weight of 2000)

PEG-DSG: PEG-Biphenylvinyl Glycerol (C18-PEG or PEG-C18) (PEG having an average molar weight of 2000)

PEG-cDMA: PEG-carbamoyl-1, 2-dimyristoyloxy propylamine (PEG with average molar weight of 2000)

Formulations comprising SNALP (1, 2-dioleoyloxy-N, N-dimethylaminopropane (DLinDMA)) are described in international publication No. WO2009/127060 filed 4/15 in 2009, which is hereby incorporated by reference.

Formulations comprising XTC are described, for example, in the following: U.S. provisional serial nos. 61/148,366 submitted on 1 month 29 of 2009; U.S. provisional serial nos. 61/156,851 submitted on 3/2 2009; U.S. temporary serial numbers 61/185,712 submitted on 6/10 2009; U.S. provisional serial No. 61/228,373 submitted 24 in 7 months 2009; U.S. provisional serial No. 61/239,686 filed on 9/3/2009 and international application PCT/US2010/022614 filed on 29/1/2010, which are hereby incorporated by reference.

Formulations comprising MC3 are described in, for example, U.S. provisional Serial No. 61/244,834, submitted at month 22 of 2009, U.S. provisional Serial No. 61/185,800, submitted at month 10 of 2009, and International application No. PCT/US10/28224, submitted at month 10 of 2010, which documents are hereby incorporated by reference.

Formulations comprising ALNY-100 are described, for example, international patent application PCT/US09/63933 filed on 11/10 2009, which is hereby incorporated by reference.

Formulations comprising C12-200 are described in the following: U.S. provisional serial No. 61/175,770, filed 5/2009, and international application No. PCT/US10/33777, filed 5/2010, which documents are hereby incorporated by reference.

Compositions and formulations for oral administration comprise powders or granules, microparticles, nanoparticles, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those wherein the dsRNA of the features of the invention is administered with one or more tonicity enhancing agent surfactants and chelating agents. Suitable surfactants comprise fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucuric acid, glycocholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, niu Huangji-24, 25-dihydro-sodium Fuxidate and sodium Ganod-hydrogen Fuxidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glyceryl monooleate, glyceryl dilaurate, glyceryl 1-monocaprate, 1-dodecylazepan-2-one, acylcarnitine, acylcholine or monoglyceride, diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, a combination of permeation enhancers is used, such as a combination of fatty acids/salts and bile acids/salts. An exemplary combination is the sodium salts of lauric acid, capric acid, and UDCA. Additional permeation enhancers include polyoxyethylene-9-dodecyl ether, polyoxyethylene-20-cetyl ether. The dsRNA characteristic of the invention may be delivered orally, in particulate form comprising spray-dried particles, or complexed to form micro-or nanoparticles. The dsRNA complexing agent comprises a polyamino acid; a polyimine; a polyacrylate; polyalkylacrylates, polyoxyethylenes, polyalkylcyanoacrylates; cationized gelatin, albumin, starch, acrylate, polyethylene glycol (PEG) and starch; polyalkylcyanoacrylates; DEAE-derived polyamines, pullulan, cellulose and starch. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyproteins, protamine, polyvinylpyridine, polythiodiethylaminomethyl ethylene P (TDAE), polyaminostyrene (e.g., para-amino), poly (methyl cyanoacrylate), poly (ethyl cyanoacrylate), poly (butyl cyanoacrylate), poly (isobutyl cyanoacrylate), poly (isohexide), DEAE-methacrylate, DEAE-hexyl acrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, Polymethacrylate, polyhexyl acrylate, poly (D, L-lactic acid), poly (DL-lactic-co-glycolic acid) (PLGA), alginate, and polyethylene glycol (PEG). Oral formulations of dsRNA and their preparation are described in detail in U.S. patent 6,887,906, U.S. publication No. 20030027780, and U.S. patent No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may comprise sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, tonicity enhancing agents, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be produced from a variety of components including, but not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Particularly preferred are formulations that target the liver in the treatment of liver disorders such as chronic inflammation of the liver.

The pharmaceutical formulations of the present invention may conveniently be presented in unit dosage form and may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of associating the active ingredient with a pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and fully associating the active ingredient with a liquid carrier or a fine solid carrier or both and then shaping the product if necessary.

The compositions of the present invention may be formulated into any of a number of possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. The aqueous suspension may further contain substances that increase the viscosity of the suspension, including for example sodium carboxymethyl cellulose, sorbitol, and/or dextran. The suspension may also contain stabilizers.

A number of liposomes including lipids derivatized with one or more hydrophilic polymers and methods of making the same are known in the art. Sunamoto et al (Japanese society of chemistry, inc. (Bull. Chem. Soc. Jpn.), 1980, 53, 2778) describe liposomes comprising a nonionic detergent 2C _1215G (containing a PEG moiety). Illum et al (FEBS flash, 1984, 167, 79) noted that hydrophilic coatings of polystyrene particles with polymeric glycols can significantly increase blood half-life. Carboxyl modified synthetic phospholipids by attachment of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. patent nos. 4,426,330 and 4,534,899). Klibanov et al (FEBS flash, 1990, 268, 235) describe experiments demonstrating that liposomes comprising Phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significantly increased blood circulation half-life. Blume et al (journal of biochemistry and biophysics (Biochimica et Biophysica Acta), 1990, 1029, 91) extend this observation to other PEG-derivatized phospholipids, such as DSPE-PEG formed from a combination of distearoyl phosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their outer surface are described in Fisher's European patent No. EP 0 445 131 B1 and WO 90/04384. The following describes liposome compositions containing 1 to 20 mole percent of PEG-derivatized PE and methods of use thereof: woodle et al (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al (U.S. Pat. No. 5,213,804 and European patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipopolymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both from Martin et al) and WO 94/20073 (Zalipsky et al). Liposomes comprising PEG modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. patent No. 5,540,935 (Miyazaki et al) and U.S. patent No. 5,556,948 (Tagawa et al) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surface.

A number of liposomes including nucleic acids are known in the art. WO 96/40062 (Thierry et al) discloses a method for encapsulating high molecular weight nucleic acids in liposomes. U.S. patent No. 5,264,221 (Tagawa et al) discloses protein-bound liposomes and states that the contents of such liposomes may contain dsRNA. U.S. patent No. 5,665,710 (Rahman et al) describes certain methods for encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 (from Love et al) discloses liposomes comprising dsRNA targeting the raf gene.

C. Additional formulations

I. Emulsion

The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems in which one liquid is dispersed in another liquid in the form of droplets, typically exceeding 0.1 μm in diameter (see, e.g., ansel's Pharmaceutical Dosage Forms and Drug DELIVERY SYSTEMS, allen, LV., popovich ng. And Ansel HC.,2004, litscott-willi Wilkins publishing company (Lippincott Williams & Wilkins, new York, NY) (8 th edition); idson, pharmaceutical dosage forms, lieberman, rieger and Banker (editions), 1988, majorddel, new York (MARCEL DEKKER, inc., new York, n.y.), volume 1, page 199; rosoff, pharmaceutical dosage forms, lieberman, rieger and Banker (eds.), 1988, marseidel, new York, N.Y., volume 1, page 245; block, pharmaceutical dosage form, lieberman, rieger and Banker (eds.), 1988, massel Dekker, N.Y., volume 2, page 335; higuchi et al, remington's Pharmaceutical Sciences, mark Publishing Co., iston, pa., 1985, page 301. Emulsions are typically two-phase systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. Typically, the emulsion may be of the water-in-oil (w/w) or oil-in-water (w/w) type. When the aqueous phase is finely divided into tiny droplets and dispersed into the bulk oil phase, the resulting composition is referred to as a water-in-oil (w/o) emulsion. Alternatively, when the oil phase is finely divided into tiny droplets and dispersed into the bulk aqueous phase, the resulting composition is referred to as an oil-in-water (o/w) emulsion. In addition to the dispersed phase and the active agent, the emulsion may contain additional components, which may be present in the aqueous phase, in the oil phase or as a separate phase itself, in the form of a solution. Pharmaceutical excipients, such as emulsifiers, stabilizers, dyes and antioxidants, may also be present in the emulsion as desired. The pharmaceutical emulsion may also be a variety of emulsions comprising more than two phases, for example in the case of oil-in-water (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations generally provide certain advantages over simple binary emulsions. Multiple emulsions in which the individual oil droplets of the o/w emulsion surround the water droplets constitute the w/o/w emulsion. Similarly, an oil droplet system surrounded by stable water droplets in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Typically, the dispersed or discontinuous phase of the emulsion disperses well into the external or continuous phase and this form is maintained by the viscosity of the emulsifier or formulation. Either phase of the emulsion may be semi-solid or solid, as is the case with emulsion ointment bases and creams. Other methods of stabilizing emulsions require the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can be broadly divided into four categories: synthetic surfactants, naturally occurring emulsifiers, absorbing bases and finely divided solids (see, e.g., ansair's pharmaceutical dosage form and drug delivery System, allen, LV., popovich NG. And Ansel HC.,2004, liPindect Williams Wills publishing company (8 th edition), idson, pharmaceutical dosage form, lieberman, rieger and Banker (editions), 1988, masaidel, new York, volume 1, page 199).

Synthetic surfactants (also known as surfactants) have found wide applicability in emulsion formulations and have been reviewed in the literature (see, e.g., ansell's pharmaceutical dosage form and drug delivery system, allen, LV., popovich ng. And Ansel HC.,2004, litscott willi wilkins publication company, n.y., 8 th edition); rieger, pharmaceutical dosage form, lieberman, rieger and Banker (editions), 1988, n.y., makerdel, volume 1, page 285; idson, pharmaceutical dosage form, lieberman, rieger and Banker (editions), 1988, makerdel, n.y., 1, page 199). Surfactants are generally amphiphilic and include hydrophilic and hydrophobic portions. The ratio of hydrophilicity to hydrophobicity of a surfactant is known as the hydrophilic/lipophilic balance (HLB) and is a valuable tool for classifying and selecting surfactants in the preparation of formulations. Surfactants can be classified into different categories based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see, e.g., ansels pharmaceutical dosage form and drug delivery System, allen, LV., popovich NG. And Ansel HC.,2004, liPing Kett Williams Wills publishing company (8 th edition), rieger, pharmaceutical dosage form, lieberman, rieger and Banker (editions), 1988, marseidel, volume 1, page 285, new York, N.Y.).

Naturally occurring emulsifiers used in the emulsion formulation include lanolin, beeswax, phospholipids, lecithins and acacia. The absorbing base has hydrophilic character such that it can absorb water to form a w/o emulsion, but still retain its semi-solid consistency, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids are also used as good emulsifiers, especially in combination with surfactants and viscous formulations. These include polar inorganic solids such as heavy metal hydroxides, non-swelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and non-polar solids such as carbon or glycerol tristearate.

A variety of non-emulsifying materials are also included in the emulsion formulation and contribute to the characteristics of the emulsion. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty acid esters, humectants, hydrocolloids, preservatives, and antioxidants (Block, pharmaceutical dosage form, lieberman, rieger and Banker (eds.), 1988, marseidel, new York, vol.1, page 335; idson, pharmaceutical dosage form, lieberman, rieger and Banker (eds.), 1988, marseidel, new York, vol.1, page 199).

Hydrocolloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (e.g., acacia, agar, alginic acid, carrageenan, guar gum, karaya gum and tragacanth), cellulose derivatives (e.g., carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (e.g., carbon polymers, cellulose ethers and carboxyvinyl polymers). These disperse or swell in water to form a colloidal solution that stabilizes the emulsion by forming a strong interfacial film around the dispersed phase droplets and by increasing the viscosity of the external phase.

Since emulsions typically contain many ingredients, such as carbohydrates, proteins, sterols, and phospholipids, which can readily support the growth of microorganisms, these formulations typically incorporate preservatives. Common preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, parabens and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. The antioxidants used may be free radical scavengers such as tocopherol, alkyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid and lecithin.

The use of emulsion formulations via the cutaneous, oral and parenteral routes and methods of their manufacture have been reviewed in the literature (see, e.g., ansel's pharmaceutical dosage form and drug delivery system, allen, LV., popovich ng. And Ansel HC.,2004, lipping willust publication company (8 th edition), idson, pharmaceutical dosage form, lieberman, rieger and Banker (editions), 1988, mazidel company, volume 1, page 199, new york). Emulsion formulations for oral delivery are very widely used due to ease of formulation and efficacy from the standpoint of absorption and bioavailability (see, e.g., pharmaceutical dosage forms and drug delivery systems of ansell, allen, LV., popovich ng. And Ansel HC.,2004, litscott-willi wilkins publishing company of new york, new york (8 th edition); rosoff, pharmaceutical dosage forms, lieberman, rieger and Banker (edit), 1988, majorddel company of new york, volume 1, page 245; idson, pharmaceutical dosage forms, lieberman, rieger and Banker (edit), 1988, majorddel company of new york, volume 1, page 199). Mineral oil-based laxatives, oil-soluble vitamins and high fat nutritional formulations are typically materials for oral administration as o/w emulsions.

Microemulsion(s)

In one embodiment of the invention, the composition of iRNA and nucleic acid is formulated as a microemulsion. Microemulsions may be defined as systems of water, oil and amphiphiles which are single optically isotropic and thermodynamically stable liquid solutions (see, e.g., ansell's pharmaceutical dosage form and drug delivery system, allen, LV., popovich ng. And Ansel HC.,2004, littermate willi wilkins publishing company (8 th edition), rosoff, pharmaceutical dosage form, lieberman, rieger and Banker (editions), 1988, mazidel company, volume 1, page 245, new york). Generally, microemulsions are systems prepared by first dispersing the oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component (typically a medium chain length alcohol) to form a transparent system. Microemulsions are therefore also described as thermodynamically stable, isotropically transparent dispersions of two immiscible liquids stabilized by interfacial films of surface active molecules (Leung and Shah, controlled release of drugs: polymers and aggregation systems (Controlled Release of Drugs: polymers and AGGREGATE SYSTEMS), rosoff, M. editors, 1989, VCH Publishers, new York, pages 185-215). Microemulsions are typically prepared by combining three to five components comprising oil, water, surfactant, cosurfactant and electrolyte. Whether a microemulsion is water-in-oil (w/o) or oil-in-water (o/w) type depends on the nature of the oil and surfactant used, as well as the structural and geometric accumulation of the polar head and hydrocarbon tail of the surfactant molecule (Schott, lemington pharmaceutical science, mark publication, iston, pa., 1985, page 271).

The phenomenological approach using phase diagrams has been widely studied and provides the skilled person with a comprehensive knowledge of how to formulate microemulsions (see, for example, ansell's pharmaceutical dosage form and drug delivery system, allen, LV., popovich ng and Ansel HC.,2004, the littermate willi wilkins publication company of new york, new york (8 th edition); rieger, pharmaceutical dosage form, lieberman, rieger and Banker (editions), 1988, the majorddel company of new york, volume 1, page 245; block, < pharmaceutical dosage form, lieberman, rieger and Banker (editions), 1988, the majorddel company of new york, volume 1, page 335). Microemulsions offer the advantage over traditional emulsions of dissolving water-insoluble drugs in spontaneously formed thermodynamically stable droplet formulations.

Surfactants for preparing the microemulsion include, but are not limited to, ionic surfactants, nonionic surfactants, brij 96, polyoxyethylene oleyl ether, polyglyceryl fatty acid esters, tetraglyceryl monolaurate (ML 310), tetraglyceryl monooleate (MO 310), hexaglyceryl monooleate (PO 310), hexaglyceryl pentaoleate (PO 500), decaglyceryl monocaprylate (MCA 750), decaglyceride monooleate (MO 750), decaglyceride continuous oleic acid (SO 750), decaglyceride decaoleate (DAO 750), alone or in combination with a co-surfactant. Cosurfactants, typically short chain alcohols such as ethanol, 1-propanol and 1-butanol, increase interfacial fluidity by penetrating into the surfactant film and creating disordered films due to the void spaces created between the surfactant molecules. However, microemulsions can be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, aqueous solutions of drugs, glycerol, PEG300, PEG400, polyglycerol, propylene glycol and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, captex 355, capmul MCM, fatty acid esters, medium chain (C8-C12) mono-, di-and tri-glycerides, polyoxyethylated glycerol fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils, and silicone oils.

Microemulsions are particularly interesting from the point of view of drug solubilization and enhanced drug absorption. Lipid-based microemulsions (both o/w and w/o) have been proposed to improve the oral bioavailability of drugs including peptides (see, e.g., U.S. Pat. No. 6,191,105; 7,063,860; 7,070,802; 7,157,099; constantinides et al, pharmaceutical research (Pharmaceutical Research), 1994, 11, 1385-1390; ritschel, methods and findings of experimental and clinical pharmacology (meth. Find. Clin. Pharmacol.), 1993, 13, 205). Microemulsions have the advantages of improved drug solubilization, protection of the drug from enzymatic hydrolysis, potential enhancement of drug absorption due to surfactant-induced changes in membrane fluidity and permeability, ease of preparation, ease of oral administration relative to solid dosage forms, improved clinical efficacy, and reduced toxicity (see, e.g., U.S. Pat. No. 6,191,105; 7,063,860; 7,070,802; 7,157,099; constantinides et al, drug Industy, 1994, 11, 1385; ho et al, J.Pharm. Sci.), 1996, 85, 138-143). Microemulsions can generally form spontaneously when the components of the microemulsion are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNA. Microemulsions are also effective for transdermal delivery of active ingredients in both cosmetic and pharmaceutical applications. The microemulsion compositions and formulations of the invention are expected to promote increased systemic absorption of iRNA and nucleic acids from the gastrointestinal tract, as well as improve local cellular uptake of iRNA and nucleic acids.

The microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), labrasol, and permeation enhancers to improve the characteristics of the formulations and enhance the absorption of the iRNA and nucleic acids of the present invention. The permeation enhancers used in the microemulsions of the present invention can be categorized as belonging to one of five broad classes-surfactants, fatty acids, bile salts, chelating agents and non-chelating non-surfactants (Lee et al, key reviews of Therapeutic Drug carrier systems (CRITICAL REVIEWS IN Therapeutic Drug CARRIER SYSTEMS), 1991, page 92). Each of these categories has been discussed above.

Micro-particles

The RNAi agents of the invention can be incorporated into particles (e.g., microparticles). Microparticles may be produced by spray drying, but may also be produced by other methods, including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

Penetration enhancer

In one embodiment, the present invention uses various permeation enhancers to achieve efficient delivery of nucleic acids, particularly iRNA, to the skin of an animal. Most drugs exist in solution in both ionized and non-ionized forms. However, only lipid-soluble or lipophilic drugs are generally easy to cross cell membranes. It has been found that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a permeation enhancer. In addition to facilitating diffusion of the non-lipophilic drug across the cell membrane, the permeation enhancer may also enhance the permeability of the lipophilic drug.

Permeation enhancers can be divided into one of five major classes, namely Surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-Surfactants (see, e.g., malmsten, m. Surfactants and polymers in drug delivery (Surfactants and polymers in drug delivery), informa medical health corporation of New York, NY), 2002; key reviews of therapeutic drug carrier systems by lee et al, 1991, page 92). Each of the above classes of penetration enhancers is described in more detail below.

A surfactant (or "surfactant-ACTIVE AGENT") is a chemical entity that, when dissolved in an aqueous solution, reduces the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that the absorption of iRNA through the mucosa is enhanced. These penetration enhancers include, for example, sodium dodecyl sulfate, polyoxyethylene-9-dodecyl ether, and polyoxyethylene-20-cetyl ether in addition to bile salts and fatty acids (see, for example, malmsten, m. surfactants and polymers in drug delivery, informa medical health company, new york, 2002; lee et al, key reviews of therapeutic drug delivery systems, 1991, page 92); and perfluorochemical emulsions, such as FC-43.Takahashi et al, J.Pharm.Pharmacol., 1988, 40, 252.

Various fatty acids and derivatives thereof as permeation enhancers include, for example, oleic acid, lauric acid, capric acid (n-capric acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, glycerol monooleate (1-monooleoyl-rac-glycerol), glycerol dilaurate, caprylic acid, arachidonic acid, glycerol 1-monodecanoate, 1-dodecylazepan-2-one, acyl carnitine, acyl choline, its C _1-20 alkyl esters (e.g., methyl, isopropyl and t-butyl) and its monoglycerides and diglycerides (i.e., oleic acid ester, lauric acid ester, capric acid ester, myristic acid ester, palmitic acid ester, stearic acid ester, linoleic acid ester, etc.) (see, for example, touitou, E. Et al, enhancement of Drug Delivery (ENHANCEMENT IN Drug Delivery), CRC publication (CRC Press, danves, mass.), 2006 et al, reviews of therapeutic Drug carrier systems, muir 92, harism, 33, U.S. 3, U.S. M. 3, U.S. 33, U.S. 3, 17, and so forth).

Physiological effects of bile include promoting the dispersion and absorption of lipids and fat-soluble vitamins (see, e.g., malmsten, M. "surfactants and polymers in drug delivery", informa medical health Inc., 2002; brunton, chapter 38: "Goodman & Gilman's The Pharmacological Basis of Therapeutics) of Goodman and Ji Erman therapeutics, 9 th edition, hardman et al, magla Hill, new York, 1996, pages 934-935). Various natural bile salts and synthetic derivatives thereof are used as permeation enhancers. Thus, the term "bile salt" encompasses any naturally occurring component of bile, as well as any synthetic derivative thereof. Suitable bile salts include, for example, cholic acid (or a pharmaceutically acceptable sodium salt thereof), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucurolactone (sodium glucuronate), glycocholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholate (sodium taurocholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), niu Huangji-24, 25-dihydro-sodium Fuxidate (STDHF), sodium dihydroFuxidate and polyoxyethylene-9-dodecyl ether (POE) (see, e.g., malmsten, m. surfactants and polymers in drug delivery, informa medical health corporation, 2002; critical reviews of therapeutic drug carrier systems by lee et al, 1991, page 92, new york; swinyard, chapter 39, lemington pharmaceutical science, 18 th edition, gennaro editions, mark publication, iston, pa., 1990, pages 782-783, muranishi, key reviews of therapeutic drug delivery systems, 1990,7,1-33, yamamoto et al J.Pharm. Exp. Ther.), 1992, 263, 25, yamamita et al J.pharmaceutical science, 1990, 79, 579-583.

Chelating agents as used in connection with the present invention may be defined as compounds that remove metal ions from solution by forming complexes with the solution, with the result that the absorption of iRNA through the mucosa is enhanced. With respect to its use as a permeation enhancer in the present invention, chelators also have the additional advantage of being DNase inhibitors, as most of the characterized DNA nucleases require divalent metal ions for catalysis and are therefore inhibited by chelators (Jarrett, J.chromatogrj., 1993, 618, 315-339). Suitable chelators include, but are not limited to, disodium ethylenediamine tetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate, and homovanillyl esters), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines) (see, e.g., katdare, A. Et al, "development of excipients for medicine, biotechnology, and drug delivery" (Excipient development for pharmaceutical, biotechnology, and drug delivery), CRC Press of Danfos, massachusetts, 2006; lee et al, "critical reviews of therapeutic drug vehicle systems", 1991, page 92; critical reviews of therapeutic drug vehicle systems ", 1990,7,1-33; buur et al," J controlled release ", 1990, 14, 43-51).

As used herein, a non-chelating non-surfactant penetration enhancing compound may be defined as a compound that exhibits insignificant activity as a chelating agent or surfactant, but still enhances the absorption of iRNA through the mucosa of the digestive tract (see, e.g., muranishi, critical review of therapeutic drug carrier systems, 1990,7,1-33). Such permeation enhancers include, for example, unsaturated cyclic ureas, 1-alkyl-and 1-alkenyl-aza-alkanone derivatives (Lee et al, key reviews of therapeutic drug delivery systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as sodium diclofenac, indomethacin, and phenylbutazone (Yamashita et al J.Pharmacology and pharmacology, 1987, 39, 621-626).

Agents that enhance the uptake of iRNA at the cellular level may also be added to the medicaments and other compositions of the invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al, PCT application WO 97/30731), are also known to enhance cellular uptake of dsRNA. Examples of commercially available transfection reagents include, for example, lipofectamine ^TM (Invitrogen, carlsberg, california; carlsbad, calif.), lipofectamine 2000 ^TM (Enjejun, calif.), 293 fectamine ^TM (Enjejun, calif.), cellfectamine ^TM (Enjejun, calif.), DMRIE-C ^TM (Enjejun, calif.), freeStyle ^TM MAX (Enjejun, calif.), cellfectamine, ten, calif.), cellfectamine, and combinations thereof, Lipofectamine ^TM 2000 CD (Enje corporation of Calsburgh, california), lipofectamine ^TM (Enje corporation of Calsburgh, california), RNAiMAX (Enje corporation of Calsburgh), oligofectamine ^TM (Enje corporation of Calsburgh, california), optifect ^TM (Enje corporation of Calsburgh, california), X-TREMEGENE Q2 transfection reagent (Roche corporation of Gherz, switzerland; grenzacherstrasse, switzerland)), DOTAP liposome transfection reagent (Switzerland), DOSPER liposome transfection reagent (Switzerland) or Fugene (Switzerland),Reagents (Promega, madison, wis.), transfast ^TM transfection reagent (Promega, madison, wis.), tfx ^TM -20 reagent (Promega, madison, wis.), tfx ^TM -50 reagent (Promega, madison, wis.), dreamFect ^TM (OZ Biosciences of Marseis, france; marseille, france)), ecoTransfect (OZ biosciences of Marseille, france), TRANSPASS ^a D1 transfection reagent (New England Biolabs of Yipusivelqi, massachusetts, USA (NEW ENGLAND Biolabs; ipswich, MA, USA), lyoVec ^TM/LipoGen^TM (invitrogen, san Diego, CA, USA), PERFECTIN transfection reagent (Genlantis, san Diego, CA; san Diego, CA, USA)), neuroPORTER transfection reagent (Genlantis company of San Diego, california), genePORTER transfection reagent (Genlantis company of San Diego, california), genePORTER transfection reagent (Genlantis company of San Diego, california), cytofectin transfection reagent (Genlantis company of San Diego, california), baculoPORTER transfection reagent (Genlantis company of San Diego, california), TroganPORTER ^TM transfection reagents (Genlantis, san diego, california), riboFect (Bioline, tao Du, ma, usa (Bioline; taunton, MA, USA)), plasFect (Bioline company of Tao Du, MA, USA), uniFECTOR (B-bridge international company of mountain view, ca, USA (B-Bridge International; mountain View, CA, USA)), sureFECTOR (B-bridge international company of Mountain View, california), hiFect ^TM (B-bridge international company of Mountain View, california), or the like.

Other agents may be used to enhance penetration of the administered nucleic acid, including glycols such as ethylene glycol and propylene glycol, pyrroles such as 2-pyrrole, azones and terpenes such as limonene and menthone.

V. vehicle

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, a "carrier compound" or "carrier" may refer to a nucleic acid or analog thereof that is inert (i.e., does not itself have biological activity), but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of the biologically active nucleic acid by, for example, degrading the biologically active nucleic acid or facilitating its removal from the circulation. Co-administration of the nucleic acid and the carrier compound (typically with an excess of the latter substance) can result in a significant reduction in the amount of nucleic acid recovered in the liver, kidney or other extra-circulatory reservoir, possibly due to competition for co-receptors between the carrier compound and the nucleic acid. For example, when a portion of phosphorothioate dsRNA is co-administered with polyinosinic acid, dextran sulfate, polycytidylic acid, or 4-acetamido-4 '-isothiocyanato-stilbene-2, 2' -disulfonic acid, recovery of a portion of phosphorothioate dsRNA in liver tissue can be reduced (Miyao et al, dsRNA research and development (DsRNA Res. Dev.), 1995,5, 115-121, takakura et al, dsRNA & nucleic acid Drug development (DsRNA & nucleic acid Drug Dev.)), 1996,6, 177-183.

Excipient(s)

In contrast to carrier compounds, "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. Excipients may be liquid or solid and are selected in consideration of the intended mode of administration so as to provide the desired volume, consistency, etc. when combined with the nucleic acid and other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethylcellulose, polyacrylates, dibasic calcium phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and a wetting agent (e.g., sodium lauryl sulfate, etc.).

Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not adversely react with nucleic acids may also be used to formulate the compositions of this invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, saline, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone and the like.

Formulations for topical application of nucleic acids may comprise sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents (such as alcohols), or solutions of nucleic acids in liquid or solid oil bases. The solution may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for parenteral administration that do not adversely react with nucleic acids may be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, saline solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone and the like.

Other components

The compositions of the present invention may additionally contain other accessory components conventionally present in pharmaceutical compositions at the level of use established in the art. Thus, for example, the compositions may contain additional compatible pharmaceutically active materials, e.g., antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain additional materials such as dyes, flavors, preservatives, antioxidants, opacifying agents, thickening agents, and stabilizers useful in physically formulating the various dosage forms of the compositions of the present invention. However, such materials, when added, should not unduly interfere with the biological activity of the components of the compositions of the present invention. The formulation may be sterilized and, if desired, mixed with adjuvants which do not deleteriously interact with the nucleic acids of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavoring and/or aromatic substances, and the like.

The aqueous suspension may contain substances that increase the viscosity of the suspension, including for example sodium carboxymethyl cellulose, sorbitol, and/or dextran. The suspension may also contain stabilizers.

In some embodiments, a pharmaceutical composition of the invention features (a) one or more iRNA compounds and (b) one or more agents that act through a non-RNAi mechanism and are useful in the treatment of CIDEB-related diseases, disorders, or conditions. Examples of such agents include, but are not limited to, pyridoxine, ACE inhibitors (angiotensin converting enzyme inhibitors), e.g., benazepril (Lotensin); angiotensin II receptor Antagonists (ARBs) (e.g. potassium chlorstudent, e.g. Merck & co)) For example, candesartan (Atacand); HMG-CoA reductase inhibitors (e.g., statins); calcium binding agents, such as sodium cellulose phosphate (Calcibind); diuretics, for example thiazide diuretics, such as hydrochlorothiazide (Microzide); insulin sensitizers, such as pparγ agonists pioglitazone, glp-lr agonists such as liraglutide, vitamin E, SGLT inhibitors, DPPIV inhibitors, and kidney/liver transplants; or a combination of any of the foregoing.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining LD ₅₀ (the dose lethal to 50% of the population) and ED ₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD ₅₀/ED₅₀. Compounds exhibiting high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of the compositions characterized herein is generally within the range of circulating concentrations comprising ED ₅₀ with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods of the invention, a therapeutically effective dose may be initially estimated from a cell culture assay. Dosages may be formulated in animal models to achieve a circulating plasma concentration range of the compound, or where appropriate, of the polypeptide product of the target sequence (e.g., to achieve a reduction in polypeptide concentration), which range includes IC ₅₀ (i.e., the concentration of the test compound that achieves half the maximum inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in the human body. The level in the plasma may be measured, for example, by high performance liquid chromatography.

In addition to their administration, as described above, the irnas featured in the invention can be administered in combination with other known agents that are effective in treating pathological processes mediated by CIDEB expression. In any event, the administering physician can adjust the amount and time of iRNA administration based on the results observed using standard measurements of efficacy known in the art or described herein.

Synthesis of cationic lipids:

Any of the compounds (e.g., cationic lipids, etc.) used in the nucleic acid lipid particles featuring the invention can be prepared by known organic synthesis techniques. Unless otherwise indicated, all substituents are defined below.

"Alkyl" means a straight or branched, acyclic or cyclic saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like, while saturated branched chain alkyl groups include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; and unsaturated cyclic alkyl groups include cyclopentenyl, cyclohexenyl, and the like.

"Alkenyl" means an alkyl group, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyl groups include both cis and trans isomers. Representative straight and branched alkenyl groups include ethenyl, propenyl, 1-butenyl, 2-butenyl, isobutenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2, 3-dimethyl-2-butenyl and the like.

"Alkynyl" means any alkyl or alkenyl group as defined above that additionally contains at least one triple bond between adjacent carbons. Representative straight and branched chain alkynyl groups include ethynyl, alkynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl, and the like.

"Acyl" means any alkyl, alkenyl or alkynyl group in which the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, -C (=o) alkyl, -C (=o) alkenyl, and-C (=o) alkynyl are acyl groups.

"Heterocycle" means a saturated, unsaturated or aromatic 5-to 7-membered monocyclic or 7-to 10-membered bicyclic, heterocyclic ring, and which contains 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatoms may optionally be quaternized, including bicyclic rings, wherein any of the above heterocyclic rings is fused to a benzene ring. The heterocycle may be attached through any heteroatom or carbon atom. The heterocyclic ring comprises heteroaryl as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinynyl (piperizynyl), hydantoin (hydantoinyl), valerolactamyl (valerolactamyl), oxiranyl (oxiranyl), oxetanyl (oxetanyl), tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms "optionally substituted alkyl", "optionally substituted alkenyl", "optionally substituted alkynyl", "optionally substituted acyl" and "optionally substituted heterocycle" mean that at least one hydrogen atom is replaced by a substituent when substituted. In the case of an oxo substituent ("=o"), two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocyclic ring 、-OR^x、-NR^xR^y、-NR^xC(＝O)R^y、-NR^xSO₂R^y、-C(＝O)R^x、-C(＝O)OR^x、-C(＝O)NR^xR^y、-SO_nR^x, and —so _nNR^xR^y, where n is 0, 1, or 2, R ^x and R ^y are the same or different and are independently hydrogen, alkyl, or heterocyclic, and each of the alkyl and heterocyclic substituents may be further substituted with one or more of: oxo, halogen, -OH, -CN, alkyl, -OR ^x, heterocycle 、-NR^xR^y、-NR^xC(＝O)R^y、-NR^xSO₂R^y、-C(＝O)R^x、-C(＝O)OR^x、-C(＝O)NR^xR^y、-SO_nR^x and-SO _nNR^xR^y.

"Halogen" means fluorine, chlorine, bromine or iodine.

In some embodiments, the methods of the inventive features may require the use of protecting groups. The protecting group methods are well known to those skilled in the art (see, e.g., protecting group in organic Synthesis (PROTECTIVE GROUPS IN ORGANIC SYNTHESIs), green, T.W., et al, wiley-Interscience, new York City, 1999). Briefly, in the context of the present invention, a protecting group is any group that reduces or eliminates the undesired reactivity of a functional group. Protecting groups may be added to the functional groups to mask their reactivity during certain reactions, and then removed to reveal the original functional groups. In some embodiments, an "alcohol protecting group" is used. An "alcohol protecting group" is any group that reduces or eliminates the undesirable reactivity of an alcohol functional group. The protecting groups may be added and removed using techniques well known in the art.

Synthesis of formula a:

In certain embodiments, the nucleic acid lipid particles of the features of the invention are formulated using a cationic lipid of formula a:

Wherein R1 and R2 are independently alkyl, alkenyl or alkynyl, each optionally substituted, and R3 and R4 are independently lower alkyl, or R3 and R4 may be taken together to form an optionally substituted heterocycle. In some embodiments, the cationic lipid is XTC (2, 2-dioleoyl-4-dimethylaminoethyl- [1,3] -dioxolane). In general, the lipids of formula a above can be prepared by the following reaction schemes 1 or 2, wherein all substituents are as defined above unless otherwise indicated.

Scheme 1

Lipid a, wherein R ₁ and R ₂ are independently alkyl, alkenyl or alkynyl, each of which may be optionally substituted, and R ₃ and R ₄ are independently lower alkyl, or R ₃ and R ₄ may together form an optionally substituted heterocycle, may be prepared according to scheme 1. Ketone 1 and bromide 2 may be purchased or prepared according to methods known to those of ordinary skill in the art. The reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 gives the lipid of formula a. The lipid of formula a may be converted to the corresponding ammonium salt using an organic salt of formula 5, wherein X is an anionic counterion selected from the group consisting of halogen, hydroxide, phosphate, sulfate, and the like.

Scheme 2

Alternatively, the ketone 1 starting material may be prepared according to scheme 2. Grignard reagent (GRIGNARD REAGENT) 6 and cyanide 7 may be purchased or prepared according to methods known to those of ordinary skill in the art. The reaction of 6 and 7 yields ketone 1. The conversion of ketone 1 to the corresponding lipid of formula a is described in scheme 1.

Synthesis of MC 3:

DLin-M-C3-DMA (i.e., (6Z, 9Z,28Z, 31Z) -heptadecane-6, 9, 28, 31-tetraen-19-yl 4- (dimethylamino) butyrate) was prepared as follows. A solution of (6Z, 9Z,28Z, 31Z) -heptadecan-6, 9, 28, 31-tetraen-19-ol (0.53 g), 4-N, N-dimethylaminobutyrate (0.51 g), 4-N, N-dimethylaminopyridine (0.61 g) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred overnight at room temperature. The solution was washed with dilute hydrochloric acid and then with dilute aqueous sodium bicarbonate. The organic fraction was dried over anhydrous magnesium sulfate, filtered and the solvent was removed on a rotary evaporator. The residue was passed down a silica gel column (20 g) using a 1% to 5% methanol/dichloromethane elution gradient. The fractions containing the purified product were combined and the solvent was removed to give a colorless oil (0.54 g).

ALNY-100 synthesis:

the synthesis of ketal 519[ ALNY-100] was performed using scheme 3 below:

515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09850 mol) in 200mL of double neck RBF (1L) of anhydrous THF was slowly added a solution of 514 (10 g,0.04926 mol) in 70mL of THF under nitrogen at 0deg.C. After complete addition, the reaction mixture was warmed to room temperature and then heated to reflux for 4 hours. The progress of the reaction was monitored by TLC. After the reaction was complete (determined by TLC), the mixture was cooled to 0 ℃ and quenched by careful addition of saturated Na2SO4 solution. The reaction mixture was stirred at room temperature for 4 hours and filtered off. The residue was washed thoroughly with THF. The filtrate and washings were mixed and diluted with 400mL dioxane and 26mL concentrated HCl and stirred at room temperature for 20 minutes. The volatiles were stripped under vacuum to provide 515 hydrochloride as a white solid. Yield: 7.12g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 250mL of two-necked RBF in 100mL of dry DCM was added NEt3 (37.2 mL,0.2669 mol) and cooled to 0deg.C under nitrogen. After slow addition of N- (benzyloxy-carbonyloxy) -succinimide (20 g,0.08007 mol) in 50mL dry DCM, the reaction mixture was warmed to room temperature. After the reaction was completed (2 to 3 hours by TLC), the mixture was washed with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL) in this order. The organic layer was then dried over anhydrous Na2SO4 and the solvent evaporated to give a crude material which was purified by silica gel column chromatography to give 516 as a viscous material. Yield rate ：11g(89％).1H-NMR(CDCl3,400MHz)：δ＝7.36-7.27(m,5H),5.69(s,2H),5.12(s,2H),4.96(br.,1H)2.74(s,3H),2.60(m,2H),2.30-2.25(m,2H).LC-MS[M+H]-232.3(96.94％).

Synthesis of 517A and 517B:

Cyclopentene 516 (5 g,0.02164 mol) was dissolved in a solution of 220mL of acetone and water (10:1) in a single neck 500mL RBF, and N-methylmorpholine-N-oxide (7.6 g,0.06492 mol) was added thereto followed by a solution of 4.2mL of 7.6% OsO4 (0.275 g,0.00108 mol) in t-butanol at room temperature. After the reaction was completed (about 3 hours), the mixture was quenched by addition of solid Na2SO3, and the resulting mixture was stirred at room temperature for 1.5 hours. The reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL), then with saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally brine (1×50 mL). The organic phase was dried over anhydrous Na ₂SO₄ and the solvent was removed in vacuo. Silica gel column chromatography purification of the crude material gave a mixture of diastereomers, which were separated by preparative HPLC. Yield: 6g of crude product 517A-Peak-1 (white solid ),5.13g(96％).1H-NMR(DMSO,400MHz)：δ＝7.39-7.31(m,5H),5.04(s,2H),4.78-4.73(m,1H),4.48-4.47(d,2H),3.94-3.93(m,2H),2.71(s,3H),1.72-1.67(m,4H).LC-MS-[M+H]-266.3,[M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry was confirmed by X-ray.

518 Synthesis:

Using a procedure analogous to that described for the synthesis of compound 505, the compound was obtained as a colourless oil 518(1.2g,41％).1H-NMR(CDCl3,400MHz)：δ＝7.35-7.33(m,4H),7.30-7.27(m,1H),5.37-5.27(m,8H),5.12(s,2H),4.75(m,1H),4.58-4.57(m,2H),2.78-2.74(m,7H),2.06-2.00(m,8H),1.96-1.91(m,2H),1.62(m,4H),1.48(m,2H),1.37-1.25(br m,36H),0.87(m,6H).HPLC-98.65％.

General procedure for the synthesis of compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added dropwise to an ice-cold solution of LAH in THF (1 m,2 eq). After complete addition, the mixture was heated at 40 ℃ for 0.5 hours and then cooled again in an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na ₂SO₄, then passed through Filtered and reduced to an oil. Column chromatography afforded pure 519 (1.3 g, 68%) as a colorless oil .13C NMR＝130.2,130.1(x2),127.9(x3),112.3,79.3,64.4,44.7,38.3,35.4,31.5,29.9(x2),29.7,29.6(x2),29.5(x3),29.3(x2),27.2(x3),25.6,24.5,23.3,226,14.1;Electrospray MS(+ve)：C44H80NO2(M+H)+Calc. having a molecular weight of 654.6, found 654.6.

Formulations prepared by standard methods or extrusion-free methods can be characterized in a similar manner. For example, the formulation is typically characterized by visual inspection. It should be a whitish translucent solution without aggregates or deposits. The particle size and particle size distribution of the lipid nanoparticles can be measured by using light scattering, for example Malvern Zetasizer Nano ZS (Malvern, USA, malvern, USA). The size of the particles should be about 20nm to 300nm, such as 40nm to 100nm. The particle size distribution should be unimodal. Total dsRNA concentration in the formulation was estimated using dye exclusion assay and the embedded fraction. Samples of formulated dsRNA can bind to the dye (e.g., RNA in the presence or absence of a surfactant (e.g., 0.5% Triton-X100) that disrupts the formulation(Molecular Probes) together. The total dsRNA in the formulation can be determined by signal from the surfactant-containing sample (relative to a standard curve). The embedded fraction was determined by subtracting the "free" dsRNA content (as measured by signal in the absence of surfactant) from the total dsRNA content. The percentage of embedded dsRNA is typically > 85%. For SNALP formulations, the particle size is at least 30nm, at least 40nm, at least 50nm, at least 60nm, at least 70nm, at least 80nm, at least 90nm, at least 100nm, at least 110nm, and at least 120nm. Suitable ranges are typically from about at least 50nm to about at least 110nm, from about at least 60nm to about at least 100nm, or from about at least 80nm to about at least 90nm.

The method of the invention

The invention also provides methods of using the iRNA of the invention and/or the composition of the invention to reduce and/or inhibit CIDEB expression in a cell (e.g., a cell of a subject, such as a hepatocyte). The method comprises contacting a cell with an RNAi agent of the invention or a pharmaceutical composition comprising an iRNA agent. In some embodiments, the cells are maintained for a time sufficient to obtain degradation of mRNA transcripts of CIDEB genes.

The invention also provides methods of reducing and/or inhibiting PNPLA3 expression in a cell (e.g., a cell of a subject, such as a hepatocyte) using an iRNA of the invention and/or a composition of the invention and a pharmaceutical composition that targets an iRNA agent comprising a Patatin-like phospholipase domain of 3 (PNPLA 3) genes and/or an iRNA agent comprising a target PNPLA 3.

In addition, the invention provides methods of inhibiting accumulation and/or expansion of lipid droplets in cells (e.g., cells of a subject, such as hepatocytes). The method comprises contacting the cell with an iRNA agent or a pharmaceutical composition comprising an iRNA agent of the invention and an iRNA agent that targets PNPLA genes and/or a pharmaceutical composition comprising an iRNA agent that targets PNPLA 3. In some embodiments, the cells are maintained for a time sufficient to obtain degradation of mRNA transcripts of CIDEB genes and PNPLA3 genes.

The reduction in gene expression may be assessed by any method known in the art. For example, a decrease in expression of CIDEB can be determined by determining the mRNA expression level of CIDEB using methods conventional to those of ordinary skill in the art (e.g., northern blot, qRT-PCR); by determining CIDEB protein levels using methods conventional to those of ordinary skill in the art (e.g., western blotting, immunological techniques). The reduction in CIDEB expression may also be assessed indirectly by measuring a reduction in biological activity of CIDEB, for example a reduction in the interaction of CIDEB with ApoB and/or a reduction in lipid maturation in the liver.

Suitable agents that target PNPLA genes are described, for example, in U.S. patent publication No. 2017/0340661, the entire contents of which are incorporated herein by reference.

In the methods of the invention, the cells may be contacted in vitro or in vivo, i.e., the cells may be in a subject.

The cells suitable for treatment using the methods of the invention can be any cells that express the CIDEB gene (and, in some embodiments, the PNPLA gene). The cells suitable for use in the methods of the invention can be mammalian cells, e.g., primate cells (e.g., human cells or non-human primate cells, e.g., monkey cells or chimpanzee cells), non-primate cells (e.g., cow cells, pig cells, camel cells, llama cells, horse cells, goat cells, rabbit cells, sheep cells, hamster cells, guinea pig cells, cat cells, dog cells, rat cells, mouse cells, lion cells, tiger cells, bear cells, or buffalo cells), bird cells (e.g., duck cells or goose cells), or whale cells. In one embodiment, the cell is a human cell, e.g., a human hepatocyte.

CIDEB expression is inhibited in the cell by at least about 5％、6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99％ or about 100%. In a preferred embodiment CIDEB expression is inhibited by at least 20%.

In some embodiments, PNPLA3 expression is also inhibited in the cell by at least about 5％、6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99％ or about 100%. In a preferred embodiment PNPLA expression is inhibited by at least 20%.

In one embodiment, the in vivo methods of the invention can comprise administering to a subject a composition comprising an iRNA, wherein the iRNA comprises a nucleotide sequence that is complementary to at least a portion of an RNA transcript of a CIDEB gene of a mammal to be treated.

In another embodiment, the in vivo methods of the invention can include administering to a subject a composition comprising a first iRNA agent and a second iRNA agent, wherein the first iRNA comprises a nucleotide sequence that is complementary to at least a portion of an RNA transcript of a CIDEB gene of a mammal to be treated and the second iRNA comprises a nucleotide sequence that is complementary to at least a portion of an RNA transcript of a PNPLA gene of a mammal to be treated.

When the organism to be treated is a mammal, such as a human, the composition may be administered by any means known in the art including, but not limited to, oral, intraperitoneal or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), intranasal, rectal and topical (including buccal and sublingual) administration. In certain embodiments, the composition is administered by intravenous infusion or injection. In certain embodiments, the composition is administered by subcutaneous injection.

In some embodiments, administration is by depot injection. Depot injections can release iRNA in a consistent manner over a long period of time. Thus, depot injections can reduce the frequency of administration required to obtain a desired effect (e.g., a desired inhibitory, or therapeutic or prophylactic effect of CIDEB). Depot injections may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In a preferred embodiment, the depot injection is subcutaneous injection.

In some embodiments, administration is by pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is an osmotic pump implanted subcutaneously. In other embodiments, the pump is an infusion pump. Infusion pumps may be used for intravenous, subcutaneous, arterial or epidural infusion. In a preferred embodiment, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

The iRNA of the invention may be present in a pharmaceutical composition, for example in a suitable buffer solution. The buffer solution may include acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is Phosphate Buffered Saline (PBS). The pH and osmolarity of the iRNA-containing buffer solution can be adjusted so that it is suitable for administration to a subject.

Alternatively, the iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposome formulation.

The mode of administration may be selected based on whether local or systemic treatment is desired or not, and based on the area to be treated. The route and site of administration may be selected to enhance targeting.

In one aspect, the invention also provides a method for inhibiting the expression of CIDEB genes in a mammal. The method comprises administering to the mammal a composition comprising dsRNA targeting a CIDEB gene in a cell of the mammal, thereby inhibiting expression of a CIDEB gene in the cell.

In some embodiments, the method comprises administering to the mammal a composition comprising dsRNA targeting a CIDEB gene in a cell of the mammal, thereby inhibiting expression of a CIDEB gene in the cell. In another embodiment, the method comprises administering to the mammal a pharmaceutical composition comprising a dsRNA agent that targets a CIDEB gene in a cell of the mammal.

In another aspect, the invention provides the use of an iRNA agent or pharmaceutical composition of the invention for inhibiting expression of CIDEB genes in a mammal.

In yet another aspect, the invention provides the use of an iRNA agent of the invention that targets CIDEB genes, or a pharmaceutical composition comprising such agent, in the manufacture of a medicament for inhibiting expression of a CIDEB gene in a mammal.

In one aspect, the invention also provides methods for inhibiting expression of CIDEB genes and PNPLA3 genes in a mammal. The method comprises administering to the mammal a composition comprising a dsRNA targeting a CIDEB gene in a cell of the mammal and a composition comprising a dsRNA targeting a PNPLA3 gene in a cell of the mammal, thereby inhibiting expression of a CIDEB gene and a PNPLA3 gene in the cell. In one embodiment, the method comprises administering to a mammal a pharmaceutical composition comprising a dsRNA agent that targets CIDEB gene and PNPLA3 gene in cells of the mammal.

In one aspect, the invention provides the use of an iRNA agent or pharmaceutical composition of the invention and dsRNA targeting PNPLA genes or a pharmaceutical composition comprising such agent for inhibiting expression of CIDEB genes and PNPLA3 genes in a mammal.

In a further aspect, the invention provides an iRNA agent of the invention that targets CIDEB genes, or a pharmaceutical composition comprising such agent, and the use of a dsRNA that targets PNPLA3 genes, or a pharmaceutical composition comprising such agent, in the manufacture of a medicament for inhibiting expression of CIDEB genes and PNPLA genes in a mammal.

The reduction in gene expression can be assessed by any method known in the art and by the methods described herein (e.g., qRT-PCR). The reduction in protein production can be assessed by any method known in the art and by the methods described herein (e.g., ELISA).

The invention also provides methods of treatment and prophylaxis comprising administering an iRNA agent, a pharmaceutical composition comprising an iRNA agent, or a vector comprising an iRNA of the invention to a subject suffering from or susceptible to developing a fatty liver-related disease, disorder, or condition.

In one aspect, the invention provides methods of treating a subject having a disorder that would benefit from reduced CIDEB expression (e.g., CIDEB-related disease).

The methods of treatment (and uses) of the invention comprise administering to a subject (e.g., a human) a therapeutically effective amount of a dsRNA agent that inhibits expression of CIDEB or a pharmaceutical composition that comprises a dsRNA that inhibits expression of CIDEB, thereby treating the subject.

In one aspect, the invention provides methods of preventing at least one symptom of a subject suffering from a disorder that would benefit from reduced CIDEB expression (e.g., a chronic inflammatory disease). The method comprises administering to the subject a prophylactically effective amount of a dsRNA agent or a pharmaceutical composition comprising dsRNA, thereby preventing at least one symptom of the subject.

In one embodiment, the CIDEB-related disease, disorder, or condition is a chronic inflammatory disease. Non-limiting examples of chronic inflammatory diseases include inflammation of the liver and other tissues. Non-limiting examples of chronic inflammatory liver disease include liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, hepatocyte necrosis, hepatocellular carcinoma, insulin sensitivity, and/or diabetes.

The invention also provides methods of treatment and prophylaxis comprising administering an iRNA agent, a pharmaceutical composition comprising an iRNA agent, or a vector comprising an iRNA of the invention and an iRNA agent targeted to PNPLA, a pharmaceutical composition comprising such an iRNA agent, or a vector comprising such an iRNA to a subject suffering from or susceptible to developing a fatty liver-related disease, disorder, or condition.

The invention also provides the use of a therapeutically effective amount of an iRNA agent of the invention or a pharmaceutical composition comprising dsRNA that inhibits CIDEB expression for treating a subject (e.g., a subject that would benefit from a reduction and/or inhibition of CIDEB expression), e.g., a CIDEB-associated disease, e.g., a chronic inflammatory disease. Non-limiting examples of chronic inflammatory liver disease include liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, hepatocyte necrosis, hepatocellular carcinoma, insulin sensitivity, and/or diabetes.

In another aspect, the invention provides the use of an iRNA agent (e.g., dsRNA) of the invention that targets the CIDEB gene, or a pharmaceutical composition comprising an iRNA agent that targets the CIDEB gene, for the manufacture of a medicament for treating a subject (e.g., a subject who would benefit from reduced and/or inhibited expression of CIDEB (e.g., CIDEB-related disease)).

The invention also provides the use of a prophylactically effective amount of an iRNA agent of the invention or a pharmaceutical composition comprising dsRNA that inhibits CIDEB expression for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced CIDEB expression (e.g., a chronic inflammatory disease). Non-limiting examples of chronic inflammatory liver disease include liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, hepatocyte necrosis, hepatocellular carcinoma, insulin sensitivity, and/or diabetes.

In another aspect, the invention provides the use of an iRNA agent (e.g., dsRNA) of the invention that targets the CIDEB gene or a pharmaceutical composition comprising an iRNA agent that targets the CIDEB gene in the manufacture of a medicament for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced CIDEB expression (e.g., a chronic inflammatory disease). Non-limiting examples of chronic inflammatory liver disease include liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, hepatocyte necrosis, hepatocellular carcinoma, insulin sensitivity, and/or diabetes.

In one aspect, the invention also provides the use of a therapeutically effective amount of an iRNA agent of the invention or a pharmaceutical composition comprising a dsRNA that inhibits CIDEB expression in combination with a dsRNA that targets the PNPLA3 gene, or a pharmaceutical composition comprising such an agent, for treating a subject, for example a subject that would benefit from a reduction and/or inhibition of CIDEB expression, such as a CIDEB-associated disease, such as a chronic inflammatory disease. Non-limiting examples of chronic inflammatory liver disease include liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, hepatocyte necrosis, hepatocellular carcinoma, insulin sensitivity, and/or diabetes.

In one aspect, the invention also provides the use of an iRNA agent (e.g., dsRNA) of the invention that targets the CIDEB gene, or a pharmaceutical composition comprising an iRNA agent that targets the CIDEB gene in combination with a dsRNA that targets the PNPLA3 gene, or a pharmaceutical composition comprising such an agent, for preventing at least one symptom in a subject suffering from a disorder that would benefit from reduced CIDEB expression (e.g., chronic inflammatory disease). Non-limiting examples of chronic inflammatory liver disease include liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), cirrhosis, alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, hepatocyte necrosis, hepatocellular carcinoma, insulin sensitivity, and/or diabetes.

Combination methods for treating subjects (e.g., human subjects) having CIDEB-related diseases, disorders, or conditions, such as chronic inflammatory diseases, e.g., chronic inflammatory liver diseases, e.g., NASH, are useful for treating such subjects because silencing of PPNPLA3 reduces steatosis (i.e., liver fat).

Thus, in one aspect, the invention provides methods of treating a subject suffering from a disorder that would benefit from a decrease in CIDEB expression, e.g., a CIDEB-related disease, such as a chronic inflammatory disease (e.g., inflammation of the liver and other tissues). In one embodiment, the chronic inflammatory disease is chronic inflammatory liver disease (e.g., liver fibrosis, non-alcoholic steatohepatitis (NASH), non-alcoholic steatohepatitis (NAFLD), cirrhosis, alcoholic Steatohepatitis (ASH), alcoholic Liver Disease (ALD), HCV-associated cirrhosis, drug-induced liver injury, hepatocyte necrosis, and/or hepatocellular carcinoma). In one embodiment, the chronic inflammatory liver disease is NASH.

The combination treatment methods (and uses) of the invention comprise administering to a subject (e.g., a human subject) a therapeutically effective amount of a dsRNA agent that inhibits expression of CIDEB or a pharmaceutical composition comprising a dsRNA that inhibits expression of CIDEB, and a dsRNA agent that inhibits expression of PNPLA3 or a pharmaceutical composition comprising a dsRNA that inhibits expression of PNPLA3, thereby treating the subject.

In one aspect, the invention provides methods of preventing at least one symptom of a subject suffering from a disorder that would benefit from reduced CIDEB expression (e.g., a chronic inflammatory disease). The method comprises administering to the subject a prophylactically effective amount of a dsRNA agent or a pharmaceutical composition comprising dsRNA that inhibits expression of CIDEB, and a dsRNA agent that inhibits expression of PNPLA3 or a pharmaceutical composition comprising dsRNA that inhibits expression of PNPLA3, thereby preventing at least one symptom in the subject.

In one embodiment, the subject is heterozygous for a gene encoding a patatin-like phospholipase domain containing 3 (PNPLA) I148M variants, that is, the subject may have an allele of the gene encoding the PPNPLA I148M variant and another allele encoding a different variant. In another embodiment, the subject is homozygous for the gene encoding the PPNPLA I148M variant, that is, both alleles of the gene encode the PPNPLA I148M variant. In one embodiment, the subject is heterozygous for a gene encoding a patatin-like phospholipase domain containing 3 (PNPLA) I144M variants, that is, the subject may have an allele of the gene encoding the PNPLA I144M variant and another allele encoding a different variant. In another embodiment, the subject is homozygous for the gene encoding the PNPLA I144M variant, that is, both alleles of the gene encode the PNPLA I143M variant.

In certain embodiments of the invention, the methods may include identifying a subject that would benefit from a reduction in CIDEB expression. The method generally includes determining whether a sample from a subject comprises a nucleic acid encoding PNPLA Ile148Met variant or PNPLA Ile144Met variant. The method may further comprise classifying the subject as a candidate for treating or inhibiting liver disease by inhibiting CIDEB expression, by determining whether a sample from the subject comprises a first nucleic acid encoding a PNPLA protein comprising an I148M variation and a second nucleic acid encoding a functional CIDEB protein, and/or PNPLA protein and a functional CIDEB protein comprising an I144M variation, and classifying the subject as a candidate for treating or inhibiting liver disease by inhibiting CIDEB when both the first nucleic acid and the second nucleic acid are detected and/or when both proteins are detected.

The variant PNPLA Ile148Met variant or PNPLA Ile144Met variant may be any of the PNPLA Ile148Met variant and PNPLA Ile144Met variants described herein. The PNPLA Ile148Met variant or PNPLA Ile144Met variant may be detected by any suitable means (e.g., ELISA assay, RT-PCR, sequencing).

In some embodiments, the method further comprises determining whether the subject is homozygous or heterozygous for the PNPLA Ile148Met variant or the PNPLA Ile144Met variant. In some embodiments, the subject is homozygous for PNPLA Ile148Met variant or PNPLA Ile144Met variant. A subject homozygous for the PNPLA Ile148Met variant has two alleles of the gene encoding the PNPLA Ile148Met variant; a subject homozygous for the PNPLA Ile144Met variant has both alleles of the gene encoding the PNPLA Ile144Met variant. In some embodiments, the subject is heterozygous for PNPLA Ile148Met variant or PNPLA Ile144Met variant. A subject heterozygous for the PNPLA Ile148Met variant may have an allele of the gene encoding the PNPLA Ile148Met variant and another allele encoding a different PNPLA variant; a subject heterozygous for the PNPLA Ile144Met variant may have an allele of the gene encoding the PNPLA Ile144Met variant and another allele encoding a different PNPLA variant. In some embodiments, the subject is homozygous for the PNPLA Ile148Met variant. In some embodiments, the subject is heterozygous for PNPLA Ile148Met variant. In some embodiments, the subject is homozygous for the PNPLA Ile144Met variant. In some embodiments, the subject is heterozygous for the PNPLA Ile144Met variant.

In some embodiments, the method further comprises determining whether the subject is obese. In some embodiments, the subject is obese if the subject's Body Mass Index (BMI) exceeds 30kg/m ². Obesity may be a characteristic of a subject suffering from or at risk of developing liver disease. In some embodiments, the method further comprises determining whether the subject has fatty liver. Fatty liver may be a characteristic of a subject suffering from or at risk of developing liver disease. In some embodiments, the method further comprises determining whether the subject is obese and has fatty liver.

As used herein, "non-alcoholic fatty liver disease" used interchangeably with the term "NAFLD" refers to a disease defined by the presence of macrovascular steatosis with less than 20 grams of alcohol ingested per day. NAFLD is the most common liver disease in the united states and is commonly associated with insulin resistance/type 2 diabetes and obesity. NAFLD is manifested by steatosis, steatohepatitis, cirrhosis, and sometimes hepatocellular carcinoma. For reviews of NAFLD, see Tolman and Dalpiaz (2007) [ therapist and clinical risk management (ter. Clin. Risk. Manag.) ], 3 (6): 1153-1163, the entire contents of which are incorporated herein by reference.

As used herein, the terms "steatosis," "liver steatosis," and "fatty liver disease" refer to the accumulation of triglycerides and other fats in hepatocytes.

As used herein, the term "NAFLD" refers to non-alcoholic fatty liver disease. NAFLD is the most common form of liver disease in all modern industrialized economic areas of the world, including korea and many other asian countries. Patients often exhibit no symptoms or no specificity in clinical features. In contrast, liver abnormalities are accidentally discovered by liver imaging, particularly by ultrasound examination, and/or the presence of elevated liver enzymes (alanine aminotransferase [ ALT ] and gamma-glutamyl transpeptidase). Diagnosis of NAFLD requires exclusion of other conditions, particularly viral hepatitis, heavy drinking and exposure to potentially hepatotoxic drugs. According to protocols such as the asia-pacific NAFLD guidelines, the term NAFLD now remains for cases of fatty Liver associated with overnutrition metabolic complications, often accompanied by central obesity and overweight (Farrell et al, intestinal and Liver (glut river) 6 (2): 149-171, 2012).

As used herein, the term "non-alcoholic steatohepatitis" or "NASH" refers to liver inflammation and damage caused by the accumulation of fat in the liver. NASH is considered a progressive form of nonalcoholic fatty liver disease (NAFLD) and is characterized by liver steatosis, inflammation, hepatocyte damage, and varying degrees of fibrosis. Adipose tissue dysfunction and liver inflammatory response play an important role during NASH development. Cell and molecular response mechanisms also promote liver inflammation by inducing a chronic inflammatory response that leads to hepatocyte damage in the absence of fatty liver.

NASH is similar to alcoholic liver disease but occurs in people who drink little or no alcohol. NASH is mainly characterized by fat in the liver, inflammation and injury. Most people with NASH feel well and are unaware that they have liver problems. NASH, however, can be severe and can lead to cirrhosis, where the liver is permanently damaged and scarred and no longer able to function properly. NASH is generally first suspected in humans found to be elevated in liver tests included in the conventional blood test set, such as alanine Aminotransferase (ALT) or aspartate Aminotransferase (AST). NASH is suspected when further evaluation does not show a clear cause of liver disease (e.g. drug, viral hepatitis or overdose of alcohol) and when x-ray or imaging studies of liver show fat. The only means to demonstrate diagnosis of NASH and isolate it from simple fatty liver is liver biopsy.

As used herein, the term "cirrhosis" is defined histologically as a diffuse liver process characterized by fibrosis and transformation of normal liver structures into structurally abnormal nodules.

As used herein, the term "serum lipid" refers to any major lipid present in blood. Serum lipids may be present in the blood in free form or as part of a protein complex (e.g., lipoprotein complex). Non-limiting examples of serum lipids may include triglycerides, cholesterol, such as total cholesterol (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), very low density lipoprotein cholesterol (VLDL-C), and medium density lipoprotein cholesterol (IDL-C).

In one embodiment, the subject who would benefit from reduced expression of CIDEB (and in some embodiments PNPLA 3) is a subject, for example, suffering from type 2 diabetes and pre-diabetes or obesity; subjects with high fat levels (e.g., cholesterol) in the blood or with hypertension; subjects with certain metabolic disorders (including metabolic syndrome); subjects with rapid weight loss; subjects with certain infections (e.g., hepatitis c infections), or subjects that have been exposed to some toxins. In one embodiment, the subject who would benefit from a decrease in CIDEB expression (and in some embodiments, PPNPLA 3) is a subject of middle-aged or older age, for example; spanish, african-spanish-whites or african-americans; subjects taking certain medications, such as corticosteroids and cancer medications.

In the methods (and uses) of the invention, which comprise administering to a subject a first dsRNA agent targeted to CIDEB and a second dsRNA agent targeted to PNPLA, the first dsRNA agent and the second dsRNA agent may be formulated in the same composition or in different compositions and may be administered to the subject in the same composition or in separate compositions.

In one embodiment, the "iRNA" used in the methods of the invention is a "dual targeting RNAi agent. The term "dual targeted RNAi agent" refers to a molecule comprising a first dsRNA agent comprising a complex of ribonucleic acid molecules having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to a first target RNA (i.e., CIDEB gene), covalently linked to a molecule comprising a second dsRNA agent comprising a complex of ribonucleic acid molecules having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having "sense" and "antisense" orientations relative to a second target RNA (i.e., PNPLA gene). In some embodiments of the invention, the dual targeted RNAi agent triggers degradation of the first and second target RNAs (e.g., mRNA) by a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

The dsRNA agent is administered to the subject at a dose of about 0.1mg/kg to about 50 mg/kg. Generally, a suitable dosage will be in the range of about 0.1mg/kg to about 5.0mg/kg, preferably about 0.3mg/kg and about 3.0 mg/kg.

The iRNA agent may be administered periodically by intravenous infusion for a period of time. In certain embodiments, the treatment may be administered on a less frequent basis after the initial treatment regimen.

Administration of iRNA can reduce CIDEB levels, for example, in a patient's cells, tissue, blood, urine, or other compartments by at least about 5％、6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,39,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98％ or at least about 99% or more. In preferred embodiments, administration of the iRNA can reduce CIDEB levels in, for example, cells, tissues, blood, CSF samples, or other compartments of a patient by at least 20%.

Administration of iRNA can reduce PNPLA levels, for example, in a patient's cells, tissue, blood, urine, or other compartments by at least about 5％、6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,39,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98％ or at least about 99% or more. In preferred embodiments, administration of the iRNA can reduce PNPLA levels in, for example, cells, tissue, blood, CSF samples, or other compartments of a patient by at least 20%.

A smaller dose, e.g., 5% infusion response, may be administered to the patient and adverse effects, e.g., allergic reactions, monitored prior to administration of the full dose of iRNA. In another example, a patient may be monitored for undesired immunostimulatory effects, such as increased cytokine (e.g., TNF- α or INF- α) levels.

Alternatively, the iRNA may be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver a daily dose of the desired iRNA to the subject. The injection may be repeated over a period of time. The administration may be repeated periodically. In certain embodiments, the treatment may be administered on a less frequent basis after the initial treatment regimen. Repeated dose regimens may include periodic administration of a therapeutic amount of the iRNA, such as once every other day or once a year. In certain embodiments, the iRNA is administered about weekly, every 7-10 days, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, monthly, every 2 months, every 3 months (quarterly), every 4 months, every 5 months, or every 6 months.

In one embodiment, the method comprises administering a composition as characterized herein such that expression of the target CIDEB gene is reduced, e.g., for about 1,2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, the reduced expression of the target CIDEB gene is for an extended duration, such as at least about two, three, four days, or more, such as about one week, two weeks, three weeks, or four weeks, or more.

In another embodiment, the method comprises administering a composition as characterized herein such that expression of the target PNPLA gene is reduced, e.g., for about 1, 2,3, 4, 5, 6,7,8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, the reduced expression of the target PNPLA gene is for an extended duration, such as at least about two, three, four days, or more, such as about one week, two weeks, three weeks, or four weeks, or more.

Preferably, the iRNA useful in the methods and compositions characterized herein specifically targets the RNA (primary or processed) of the target CIDEB gene (and in some embodiments, the PNPLA gene). Compositions and methods for inhibiting expression of these genes using iRNA can be prepared and performed as described herein.

Administration of dsRNA according to the methods of the invention can result in a reduction in the severity, sign, symptom, or marker of such diseases or disorders in patients suffering from disorders of lipid metabolism. In this context, "reduced" means that such levels are reduced statistically or clinically significantly. The decrease may be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

The therapeutic or prophylactic efficacy of a disease can be assessed, for example, by measuring disease progression, disease relief, symptom severity, pain relief, quality of life, the dosage of drug required to maintain therapeutic effect, disease marker levels, or any other measurable parameter suitable for the given disease being treated or targeted for prevention. It is well within the ability of those skilled in the art to monitor the efficacy of treatment or prophylaxis by measuring any one or any combination of such parameters. For example, the efficacy of treating a disorder of lipid metabolism may be assessed, for example, by periodically monitoring one or more serum lipid levels (e.g., triglyceride levels). The comparison of the later reading with the initial reading provides an indication to the physician as to whether the treatment is effective. It is well within the ability of those skilled in the art to monitor the efficacy of treatment or prophylaxis by measuring any one or any combination of such parameters. With respect to administration of iRNA or pharmaceutical compositions thereof, a condition that is "effective against" lipid metabolism indicates that administration of at least a statistically significant fraction of a patient in a clinically appropriate manner produces beneficial effects, such as symptomatic improvement, cure, disease alleviation, life prolongation, quality of life improvement, or other effects that are generally considered positive by doctors familiar with treating conditions and related causes of lipid metabolism.

The therapeutic or prophylactic effect is evident when there is a statistically significant improvement in one or more parameters of the disease state, or because there is no deterioration or the expected symptoms appear. As an example, an advantageous change of at least 10% of the measurable parameter of the disease, and preferably an advantageous change of at least 20%, 30%, 40%, 50% or more, may be indicative of an effective treatment. Experimental animal models of a given disease as known in the art may also be used to determine the efficacy of a given iRNA drug or formulation of the drug.

The invention further provides methods of using the iRNA agents or pharmaceutical compositions of the invention, e.g., for treating a subject (e.g., a subject having a CIDEB-related disease, disorder or condition) that would benefit from a reduction or inhibition of CIDEB expression or CIDEB, in combination with other drugs or other methods of treatment, e.g., in combination with known drugs and/or known methods of treatment (e.g., such as those currently used to treat such disorders). In some embodiments, the invention provides methods of using the iRNA agents or pharmaceutical compositions of the invention and the PNPLA-targeted iRNA agents, e.g., for treating subjects (e.g., subjects suffering from CIDEB-associated diseases, disorders, or conditions (e.g., chronic inflammatory diseases)) that would benefit from a reduction and/or inhibition of CIDEB expression and PNPLA3 expression, in combination with other drugs or other therapies, e.g., in combination with known drugs and/or known therapies (e.g., such as those currently used to treat such disorders). For example, in certain embodiments, an iRNA agent or pharmaceutical composition of the invention is administered in combination with: e.g. pyridoxine, ACE inhibitors (angiotensin converting enzyme inhibitors) for lowering blood pressure, e.g. benazepril Li Yaoji, e.g. diuretics, beta-blockers, ACE inhibitors, angiotensin II receptor blockers, calcium channel blockers, alpha-2 receptor antagonists, combined alpha-blockers and beta-blockers, central agonists, peripheral adrenergic inhibitors and vasodilators; or cholesterol lowering agents such as statins, selective cholesterol absorption inhibitors, resins; lipid lowering therapy; insulin sensitizers, such as the ppary agonist pioglitazone; glp-1r agonists, such as liraglutide; vitamin E; SGLT2 inhibitors; or a DPPIV inhibitor; or a combination of any of the foregoing. In one embodiment, the iRNA agent or pharmaceutical composition of the invention is administered in combination with an agent that inhibits the expression and/or activity of a transmembrane 6 superfamily member 2 (TM 6SF 2) gene, e.g., an RNAi agent that inhibits the expression of a TM6SF2 gene.

The iRNA agent and the additional therapeutic agent and/or treatment may be administered simultaneously and/or in the same combination, e.g., subcutaneously, or the additional therapeutic agent may be administered as part of a separate composition or at a separate time and/or by another method known in the art or described herein.

VIII pharmaceutical kit

The invention also provides a kit for performing any one of the methods of the invention. Such kits include one or more RNAi agents and instructions for use, e.g., instructions for inhibiting expression of CIDEB in a cell by contacting the cell with an RNAi agent or pharmaceutical composition of the invention in an amount effective to inhibit expression of CIDEB. The kit may optionally further comprise means for contacting the cells with an RNAi agent (e.g., an injection device), or means for measuring the inhibition of CIDEB (e.g., means for measuring the inhibition of CIDEB mRNA and/or CIDEB proteins). Such means for measuring the inhibition of CIDEB may include means for obtaining a sample (e.g., such as a plasma sample) from a subject. The kits of the invention may optionally further comprise a means for administering an RNAi agent to a subject or a means for determining a therapeutically effective amount or a prophylactically effective amount.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNA and methods of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Examples

Example 1: CIDEB iRNA design, synthesis and selection

The nucleic acid sequences provided herein are represented using standard nomenclature. See abbreviations of table 2. It will be appreciated that these monomers, when present in the oligonucleotide, are linked to each other by a 5'-3' -phosphodiester linkage. Abbreviations are understood to omit 3' -phosphates when placed at the 3' -terminal position of the oligonucleotides (i.e., they are 3' -OH).

Table 2: abbreviations for nucleotide monomers used for nucleic acid sequence representation.

¹ The chemical structure of L96 is as follows:

² The chemical structure of uL96 is as follows:

Experimental method

This example describes methods for the design, synthesis and selection of CIDEB iRNA medicaments.

Bioinformatics

Reagent source

Where the source of reagents is not specifically set forth herein, such reagents may be obtained from any molecular biology reagent provider, the quality/purity criteria of which are applicable to molecular biology.

Transcripts

A set of sirnas targeting human cell death-inducing DFFA-like effector B (CIDEB; human NCBI REFSEQID NM _001393338.1;NCBI GeneID:27141) were designed using custom R and Python scripts. Human NM-001393338.1 REFSEQ mRNA has a length of 2482 bases.

SiRNA synthesis

SiRNA is synthesized and annealed using conventional methods known in the art.

Briefly, siRNA sequences were synthesized on a Mermade 192 synthesizer (bioautomatic company (BioAutomation)) on a1 μmol scale using solid support-mediated phosphoramide chemistry. The solid support is a controlled pore glass (500A) loaded with custom GalNAc ligands or universal solid support (AM biochemistry). Auxiliary synthesis reagents 2'-F and 2' -O-methyl RNA and deoxyphosphoramides were obtained from sammer femto-Fisher company (Thermo-Fisher) (Milwaukee, WI) and megawatt technology development limited company (Hongene) (China)). The corresponding phosphoramidites were used to introduce 2' f 2' -O-methyl, GNA (ethylene glycol nucleic acid), 5' phosphate and other modifications. Synthesis of 3' GalNAc conjugated single strands was performed on GalNAc modified CPG vectors. Antisense single strands were synthesized using custom CPG universal solid supports. The coupling time for all phosphoramidites (100 mM in acetonitrile) was 5 minutes using 5-ethylthio-1H-tetrazole (ETT) as activator (0.6M in acetonitrile). Phosphorothioate linkages were generated using a 50mM solution of 3- ((dimethylamino-methylene) amino) -3H-1,2, 4-dithiazole-3-thione (DDTT, available from CHEMGENES company (Wilmington, mass.) in anhydrous acetonitrile/pyridine (1:1 v/v). The oxidation time was 3 minutes. All sequences were synthesized with the final removal of the DMT group ("DMT off").

After completion of the solid phase synthesis, the oligoribonucleotides were cleaved from the solid support and deprotected in a sealed 96-well plate using 200 μl aqueous methylamine reagent at 60 ℃ for 20 min. For sequences containing 2 'ribose residues (2' -OH) protected with t-butyldimethylsilyl (TBDMS) groups, the second step deprotection was performed using TEA.3HF (triethylamine trihydrofluoride) reagent. 200. Mu.L of dimethyl sulfoxide (DMSO) and 300. Mu.L of TEA.3HF reagent were added to the methylamine deprotection solution, and the solution was incubated at 60℃for an additional 20 minutes. At the end of the cleavage and deprotection steps, the synthetic plate was allowed to reach room temperature and was precipitated by adding 1mL of acetone/ethanol mixture (9:1). The plates were cooled at-80 ℃ for 2 hours and the supernatant carefully decanted with the aid of a multichannel pipette. The oligonucleotide particles were resuspended in 20mM NaOAc buffer and desalted using a 5mL HiTrap size exclusion column (general electric medical Co., ltd. (GE HEALTHCARE)) on an AKTA purification system equipped with an A905 autosampler and a Frac 950 fraction collector. Desalted samples were collected in 96-well plates. Samples of each sequence were analyzed by LC-MS to confirm identity, quantified by UV (260 nm), and a selected set of samples were analyzed by IEX chromatography to determine purity.

Annealing of the single strand was performed on a Tecan liquid handling robot. Equimolar mixtures of sense and antisense single strands were combined and annealed in 96-well plates. After combining the complementary single strands, the 96-well plates were tightly sealed and heated in an oven at 100 ℃ for 10 minutes and slowly brought to room temperature over a period of 2 to 3 hours. The concentration of each duplex was normalized to 10 μm in 1X PBS and then submitted for in vitro screening assay.

In some cases, the duplex (dsRNA) is synthesized more than once. Different batches were marked with different extensions. For example, AD-1686813.1 and AD-1686813.2 are identical duplex of different batches. Duplex having the same ID but no extension or having different extensions have the same nucleotide sequence of the sense and antisense strand, e.g., ADAD-1686813, AD-1686813.1, and AD-1686813.2 have the same nucleotide sequence of the sense and antisense strand.

A detailed list of unmodified nucleotide sequences of the sense and antisense strands is shown in tables 3 and 5.

A detailed list of modified nucleotide sequences of the sense and antisense strands is shown in tables 4 and 6.

TABLE 3 unmodified sense and antisense strand sequences of human CIDEB dsRNA Agents

TABLE 4 modified sense and antisense strand sequences of human CIDEB dsRNA Agents

TABLE 5 unmodified sense and antisense strand sequences of human CIDEB dsRNA Agents

TABLE 6 modified sense and antisense strand sequences of human CIDEB dsRNA Agents

Example 2: CIDEB siRNA in vitro screening

Experimental method

Cell culture and transfection:

The Hepa1c1c7 cells were grown to near confluence in minimal essential medium (Ji Boke company (Gibco)) supplemented with 10% fbs (ATCC) in an atmosphere of 5% co ₂ at 37 ℃ and then released from the plates by trypsin digestion. Transfection was performed by adding 14.8 μl of Opti-MEM to 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, carlsbad ca, catalog No. 13778-150) of each siRNA duplex to a single well in a 96-well plate. The mixture was then incubated at room temperature for 15 minutes. Eighty μl of antibiotic-free complete growth medium containing about 2x 10 ⁴ cells was then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at final duplex concentrations of 10nM, 1nM and/or 0.1 nM.

Panc-1 cells were grown to near confluence in minimal essential medium (Ji Boke company (Gibco)) supplemented with 10% FBS (ATCC) at 37℃in an atmosphere of 5% CO ₂ and then released from the plates by trypsin digestion. Transfection was performed by adding 14.6. Mu.l of Opti-MEM plus 0.4. Mu.l of Lipofectamine 2000 per well to 5. Mu.l of each siRNA duplex to a single well in a 96-well plate. The mixture was then incubated at room temperature for 15 minutes. Eighty μl of antibiotic-free complete growth medium containing about 1.5×10 ⁴ cells was then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Dose experiments were performed at final duplex concentrations of 10nM and 0.1 nM.

Hep3B cells were grown to near confluence in minimal essential medium (Ji Boke company (Gibco)) supplemented with 10% fbs (ATCC) in an atmosphere of 5% co ₂ at 37 ℃ and then released from the plates by trypsin digestion. Transfection was performed by adding 14.6. Mu.l of Opti-MEM plus 0.4. Mu.l Lipofectamine RNAiMax per well to 5. Mu.l of each siRNA duplex to a single well in a 96-well plate. The mixture was then incubated at room temperature for 15 minutes. Eighty μl of antibiotic-free complete growth medium containing about 1.5×10 ⁴ cells was then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Dose experiments were performed at final duplex concentrations of 10nM, 1nM and 0.1 nM.

Total RNA isolation using DYNABEADS mRNA isolation kit:

RNA was isolated using an automated protocol on the BioTek-EL406 platform using DYNABEADs (England, cat. No. 61012). Briefly, 70. Mu.l of lysis/binding buffer and 10. Mu.l of lysis buffer containing 3. Mu.l of magnetic beads were added to the plates with cells. Plates were incubated for 10 minutes on an electromagnetic shaker at room temperature, and then magnetic beads were captured and the supernatant removed. The bead-bound RNA was then washed 2 times with 150. Mu.l of wash buffer A and once with wash buffer B. The beads were then washed with 150 μl of elution buffer, recaptured and the supernatant removed.

Synthesis using the ABI high capacity cDNA reverse transcription kit (applied biosystems, foster City, california, (Applied Biosystems, foster City, CA), catalog No. 4368813):

To the isolated RNA, ten. Mu.l of a master mix containing 1. Mu.l of 10 Xbuffer, 0.4. Mu.l of 25 XdNTPs, 1. Mu.l of 10 Xrandom primer, 0.5. Mu.l of reverse transcriptase, 0.5. Mu.l of RNase inhibitor and 6.6. Mu.l of H ₂ O was added per reaction. The plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, then at 37 ℃ for 2 hours.

Real-time PCR:

Two μl of cDNA and 5 μl of Lightcycler480 probe master mix (Roche, cat. No. 04887301001) were added to 0.5 μl of human GAPDH TAQMAN probe and 0.5 μl of human CIDEB probe per well in 384 well plates (Roche, cat. No. 04887301001). Real-time PCR was performed in the LightCycler480 real-time PCR system (Roche). Each duplex was tested at least twice and the data were normalized to cells transfected with non-targeted control siRNA. To calculate the relative fold change, real-time data were analyzed using the ΔΔct method and normalized to the assay using cells transfected with non-targeted control siRNA.

The screening results of dsRNA agents listed in table 6 in Hep3B cells are shown in table 7. Additional single dose (10 nM) screens of the dsRNA agents listed in Table 4 were performed in Hep3B cells. The CIDEB signal was normalized to GAPDH. The results are shown in table 8.

TABLE 7 CIDEB Single dose Screen in Hep3B cells

TABLE 8 CIDEB Single dose Screen in Hep3B cells

Example 3 in vitro screening methods

Subsets of the duplex were also assessed by transfection and free uptake in primary human hepatocytes and primary cynomolgus monkey hepatocytes.

Cell culture and transfection:

transfection and free uptake assays were performed in primary human hepatocytes (PHH, bioIVT) and primary cynomolgus monkey hepatocytes (PCH, bioIVT). Transfection was performed by adding 0.1 μl of Lipofectamine RNAiMax per well of Opti-MEM (Enjetty, calif. cat. No. 13778-150) to 5 μl of siRNA duplex per well in 384 well plates and incubating for 15 minutes at room temperature. Then 40 μl of Invitrogro CP medium (BioIVT, catalog number Z99029) containing about 10×10 ³ cells was added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Experiments were performed at 10nM, 1nM, 0.1nM and 0.01 nM. The free uptake assay was performed similarly to the transfection assay without Liporectamine RNAimax and cells were incubated for 48 hours prior to RNA purification. Experiments were performed at 250nM, 100nM, 10nM, 1 nM.

Total RNA isolation Using DYNABEADS mRNA isolation kit

RNA was isolated using the Dynabeads ^TM mRNA DIRECT^TM purification kit (Invitrogen ^TM, catalog No. 61012) using Highres Biosolution integration system. Briefly, 70. Mu.L of lysis/binding buffer and 10. Mu.L of lysis buffer containing 3. Mu.L of magnetic beads were added to the plates with cells. Plates were incubated for 10 minutes on an electromagnetic shaker at room temperature, and then magnetic beads were captured and the supernatant removed. The bead-bound RNA was then washed 2 times with 90. Mu.L of wash buffer A and once with 90. Mu.L of wash buffer B. The beads were then washed with 90 μl of elution buffer, recaptured and the supernatant removed. Complementary DNA (cDNA) was synthesized using a high capacity cDNA reverse transcription kit with RNAse inhibitor (Applied Biosystems ^TM, cat. 4374967) according to the manufacturer's recommendations. To the isolated RNA was added a master mix containing 1. Mu.L of 10 Xbuffer, 0.4. Mu.L of 25 Xdeoxyribonucleotide triphosphate, 1. Mu.L of 10 Xrandom primer, 0.5. Mu.L of reverse transcriptase, 0.5. Mu.L of RNase inhibitor and 6.6. Mu.L of H ₂ O per reaction. The plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room temperature, then at 37 ℃ for 2 hours.

Levels of CIDEB mRNA were quantified by performing RT-qPCR analysis. Mu.l of cDNA was added to a master mix containing 0.5. Mu.l of human or cyno GAPDH TAQMAN probe, 0.5. Mu.l of human or cyno CIDEB probe and 5. Mu.l of Lightcycler480 probe master mix (Roche, cat. No. 04887301001) in each well of a 384 well plate. Real-time PCR was performed in the LightCycler480 real-time PCR system (Roche). To calculate the relative fold change, real-time data were analyzed using the delta-delta threshold cycle (relative quantification) (delta C _q [ RQ ]) method [ SCHMITTGEN and Livak 2008], and normalized to control assays performed with PBS transfected cells. For all samples, CIDEB mRNA levels were first normalized to GAPDH as a reference gene. Data are expressed as a percentage of the remaining CIDEB mRNA relative to the mean PBS control, and error is expressed as Standard Deviation (SD), with gas derived from 4 transfection replicates.

The results of single dose transfection screening and free uptake screening of dsRNA agents in PCH are shown in table 9 and PHH cells are shown in table 10.

TABLE 9 transfection and free uptake in primary cynomolgus monkey hepatocytes

Table 10: transfection and free uptake in primary human hepatocytes

EXAMPLE 4 in vivo screening of dsRNA duplex

(1) Single dose study (3 mg/kg)

The duplex of interest identified from the in vitro studies described above was evaluated in vivo. Specifically, wild-type mice (C57 BL/6) were transduced intravenously with 2x 10 ¹⁰ viral particles encoding human CIDEB adeno-associated virus 8 (AAV 8) vector via retroorbital delivery on day 14 prior to dosing. Specifically, AAV8 encoding a portion of human CIDEB mRNA encoding the open reading frame and the 3' utr of human CIDEB mRNA, referred to as nm_001393338.1, referred to as VCAV-07736-AAV8.Hscideb-FL-trd, is administered to mice.

On day 0, a single 3mg/kg dose of duplex or Phosphate Buffered Saline (PBS) of interest was subcutaneously administered in groups of three mice. Table 11 provides the treatment design and provides the duplex of interest. On day 7 post-dosing, animals were sacrificed and liver samples were collected and flash frozen in liquid nitrogen. Liver mRNA was extracted and analyzed by RT-QPCR.

For all samples, human CIDEB Cq values were first normalized to Gapdh Cq values as reference genes to calculate liver CIDEB mRNA levels for each animal. For each group, liver CIDEB mRNA levels relative to Gapdh from individual treated animals were normalized to the group mean (±standard deviation [ SD ]) of relative CIDEB mRNA levels from the PBS group.

Data are expressed as a percentage of baseline values and as mean plus standard deviation. The results set forth in table 12 and shown in fig. 1 demonstrate that the exemplary duplex agents tested effectively reduced the levels of human CIDEB messenger RNAs in vivo.

TABLE 11 study design of in vivo single dose study

TABLE 12 qPCR results of in vivo single dose studies

(2) Multi-dose study (0.75 mg/kg and 1.5 mg/kg)

The duplex of interest identified from the in vitro studies described above was evaluated in vivo. Specifically, wild-type mice (C57 BL/6) were transduced with 2x 10 ¹⁰ viral particles encoding human CIDEB adeno-associated virus 8 (AAV 8) vector by intravenous via retroorbital delivery on day 14 prior to dosing. Specifically, AAV8 encoding a portion of human CIDEB mRNA encoding the open reading frame and the 3' utr of human CIDEB mRNA, referred to as nm_001393338.1, referred to as VCAV-07736-AAV8.Hscideb-FL-trd, is administered to mice.

On day 0, a single 0.75 or 1.5mg/kg dose of duplex or Phosphate Buffered Saline (PBS) of interest was subcutaneously administered in groups of three mice. Table 13 provides the study design and provides the duplex of interest. On day 10 post-dosing, animals were sacrificed and liver samples were collected and flash frozen in liquid nitrogen. Liver mRNA was extracted and analyzed by RT-QPCR.

Data are expressed as a percentage of baseline values and as mean plus standard deviation. The results set forth in table 14 and shown in fig. 2 demonstrate that the exemplary duplex agents tested effectively and dose dependently reduced the levels of human CIDEB messenger RNAs in vivo.

TABLE 13 study design of in vivo Multi-dose study

TABLE 14 qPCR results of in vivo multi-dose studies

CIDEB sequence

SEQ ID NO：1

NM-001393338.1 homo sapiens cell death-inducing DFFA-like effector b (CIDEB), transcript variant 7, mRNA

SEQ ID NO：2

SEQ ID NO:1 reverse complement

Claims

1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence that hybridizes to SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides differing by NO more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2 by at least 15 consecutive nucleotides differing by no more than 3 nucleotides.

2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the antisense strand comprises a region that is complementary to mRNA encoding CIDEB, said region comprising at least 15 contiguous nucleotides that differ by no more than 3 nucleotides from any one of the antisense sequences listed in tables 3-6.

3. The dsRNA agent of claim 1 or 2, wherein

(A) The nucleotide sequence contained in the antisense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700555, and the nucleotide sequence contained in the sense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700555;

(b) The nucleotide sequence contained in the antisense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700821, and the nucleotide sequence contained in the sense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700821;

(c) The nucleotide sequence contained in the antisense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700369, and the nucleotide sequence contained in the sense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700369;

(d) The nucleotide sequence contained in the antisense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1699976, and the nucleotide sequence contained in the sense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1699976;

(e) The nucleotide sequence contained in the antisense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700374, and the nucleotide sequence contained in the sense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700374;

(f) The nucleotide sequence contained in the antisense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700314, and the nucleotide sequence contained in the sense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700314;

(g) The antisense strand comprises a nucleotide sequence comprising at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700376; and the nucleotide sequence contained in the sense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the nucleotide sequence of the sense strand of duplex AD-1700376;

(h) The nucleotide sequence contained in the antisense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1699964, and the nucleotide sequence contained in the sense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1699964; or (b)

(I) The nucleotide sequence contained in the antisense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the antisense strand nucleotide sequence of duplex AD-1700556, and the nucleotide sequence contained in the sense strand comprises at least 15 consecutive nucleotides with 0, 1,2 or 3 mismatches from the sense strand nucleotide sequence of duplex AD-1700556.

4. The dsRNA agent of any one of claims 1 to 3, wherein the dsRNA agent comprises at least one modified nucleotide.

5. The dsRNA agent of any one of claims 1 to 4, wherein substantially all nucleotides of the sense strand comprise a modification.

6. The dsRNA agent of any one of claims 1 to 4, wherein substantially all nucleotides of the antisense strand comprise a modification.

7. The dsRNA agent of any one of claims 1 to 4, wherein substantially all nucleotides of the sense strand and substantially all nucleotides of the antisense strand comprise a modification.

8. A double-stranded RNA (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the double-stranded RNA agent comprises a sense strand and an antisense strand forming a double-stranded region,

Wherein the sense strand comprises a sequence identical to SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides differing by NO more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:2 by at least 15 consecutive nucleotides differing by no more than 3 nucleotides,

Wherein substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand are modified nucleotides, and

Wherein the sense strand is conjugated to a ligand attached at the 3' end.

9. The dsRNA agent of claim 8, wherein all nucleotides of the sense strand comprise a modification.

10. The dsRNA agent of claim 8, wherein all nucleotides of the antisense strand comprise a modification.

11. The dsRNA agent of claim 8, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand comprise modifications.

12. The dsRNA agent of any one of claims 4 to 11, wherein at least one of the modified nucleotides is selected from the group consisting of: deoxynucleotides, 3' -terminal deoxythymine (dT) nucleotides, 2' -O-methyl modified nucleotides, 2' -fluoro modified nucleotides, 2' -deoxymodified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2' -amino modified nucleotides, 2' -O-allyl modified nucleotides, 2' -C-alkyl modified nucleotides, 2' -hydroxy modified nucleotides, 2' -methoxyethyl modified nucleotides, 2' -O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, tetrahydropyran modified nucleotides, 1, 5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, phosphorothioate group containing nucleotides, methylphosphonate group containing nucleotides, 5' -phosphate mimetic containing nucleotides, ethylene glycol modified nucleotides and 2-O- (N-methylacetamide) modified nucleotides, and combinations thereof.

13. The dsRNA agent of claim 12, wherein the nucleotide modification is a 2 '-O-methyl modification and/or a 2' -fluoro modification.

14. The dsRNA agent of any one of claims 1 to 13, wherein the length of the complementary region is at least 17 nucleotides.

15. The dsRNA agent of any one of claims 1 to 14, wherein the complementary region is 19 to 30 nucleotides in length.

16. The dsRNA agent of claim 15, wherein the complementary region is 19 to 25 nucleotides in length.

17. The dsRNA agent of claim 16, wherein the complementary region is 21 to 23 nucleotides in length.

18. The dsRNA agent of any one of claims 1 to 17, wherein each strand is no more than 30 nucleotides in length.

19. The dsRNA agent of any one of claims 1 to 18, wherein each strand is independently 19 to 30 nucleotides in length.

20. The dsRNA agent of claim 19, wherein each strand is independently 19 to 25 nucleotides in length.

21. The dsRNA agent of claim 19, wherein each strand is independently 21 to 23 nucleotides in length.

22. The dsRNA agent of any one of claims 1 to 21, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

23. The dsRNA agent of any one of claims 22, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

24. The dsRNA agent of any one of claims 1 to 7 and 12 to 23, further comprising a ligand.

25. The dsRNA agent of claim 24, wherein the ligand is conjugated to the 3' end of the sense strand of the dsRNA agent.

26. The dsRNA agent of claim 8 or 25, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.

27. The dsRNA agent of claim 26, wherein the ligand is

28. The dsRNA agent of claim 27, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic diagram

And wherein X is O or S.

29. The dsRNA agent of claim 28, wherein the X is O.

30. The dsRNA agent of claim 2, wherein the complementary region comprises any one of the antisense sequences in tables 3-6.

31. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA 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 mRNA encoding CIDEB, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (Ij):

sense of meaning ：5′n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

Antisense sense ：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′ (Ij)

Wherein:

i. j, k and l are each independently 0 or 1;

p, p ', q and q' are each independently 0 to 6;

Wherein the sense strand is conjugated to at least one ligand.

32. The dsRNA agent of claim 31, wherein i is 0; j is 0; i is 1; j is 1; i and j are both 0; or i and j are both 1.

33. The dsRNA agent of claim 31, wherein k is 0; l is 0; k is 1; l is 1; k and l are both 0; or k and l are both 1.

34. The dsRNA agent of claim 31, wherein XXX is complementary to X ' X ' X ', YYY is complementary to Y ' and ZZZ is complementary to Z '.

35. The dsRNA agent of claim 31, wherein the YYY motif is present at or near a cleavage site of the sense strand.

36. The dsRNA agent of claim 31, wherein the Y 'motif is present at positions 11, 12 and 13 of the antisense strand from the 5' end.

37. The dsRNA agent of claim 31, wherein formula (Ij) is represented by formula (Ik):

sense: 5'n _p-N_a-Y Y Y-N_a-n_q 3'

Antisense: 3' n _p′-N_a′-Y′Y′Y′-N_a′-n_q '5' (Ik).

38. The dsRNA agent of claim 31, wherein formula (Ij) is represented by formula (Il):

Sense: 5'n _p-N_a-Y Y Y-N_b-Z Z Z-N_a-n_q 3'

Wherein each of N _b and N _b' independently represents an oligonucleotide sequence comprising 1 to 5 modified nucleotides.

39. The dsRNA agent of claim 31, wherein formula (Ij) is represented by formula (Im):

Sense: 5'n _p-N_a-X X X-N_b-Y Y Y-N_a-n_q 3'

Antisense: 3' n _p′-N_a′-X′X′X′-N_b′-Y′Y′Y′-N_a′-n_q '5' (Im)

40. The dsRNA agent of claim 31, wherein formula (Ij) is represented by formula (In):

sense: 5'n _p-N_a-X X X-N_b-Y Y Y-N_b-Z Z Z-N_a-n_q 3'

Antisense sense ：3′n_p′-N_a′-X′X′X′-N_b′-Y′Y′Y′-N_b′-Z′Z′Z′-N_a′-n_q′5′ (In)

41. The dsRNA agent of any one of claims 31 to 40, wherein the length of the complementary region is at least 17 nucleotides.

42. The dsRNA agent of any one of claims 31 to 40, wherein the length of the complementary region is 19 to 30 nucleotides.

43. The dsRNA agent of claim 42, wherein the complementary region is 19 to 25 nucleotides in length.

44. The dsRNA agent of claim 43, wherein the complementary region is 21 to 23 nucleotides in length.

45. The dsRNA agent of any one of claims 31 to 44, wherein each strand is no more than 30 nucleotides in length.

46. The dsRNA agent of any one of claims 31 to 44, wherein each strand is independently 19 to 30 nucleotides in length.

47. The dsRNA agent of any one of claims 31 to 46, wherein said modification on said nucleotide is selected from the group consisting of: LNA, HNA, ceNA, 2 '-methoxyethyl, 2' -O-alkyl, 2 '-O-allyl, 2' -C-allyl, 2 '-fluoro, 2' -O-methyl, 2 '-deoxy, 2' -hydroxy, and combinations thereof.

48. The dsRNA agent of claim 47, wherein the modification on the nucleotide is a2 '-O-methyl modification and/or a 2' -fluoro modification.

49. The dsRNA agent of any one of claims 31 to 47, wherein the Y ' is a 2' -O-methyl or 2' -fluoro modified nucleotide.

50. The dsRNA agent of any one of claims 31 to 49, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide.

51. The dsRNA agent of any one of claims 31 to 50, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides.

52. The dsRNA agent of any one of claims 31 to 51, wherein the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

53. The dsRNA agent of claim 52, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 3-terminal end of one strand.

54. The dsRNA agent of claim 53, wherein the strand is the antisense strand.

55. The dsRNA agent of claim 53, wherein the strand is the sense strand.

56. The dsRNA agent of claim 52, wherein the phosphorothioate or methylphosphonate internucleotide linkage is located at the 5' end of one strand.

57. The dsRNA agent of claim 56, wherein the strand is the antisense strand.

58. The dsRNA agent of claim 56, wherein the strand is the sense strand.

59. The dsRNA agent of claim 52, wherein the phosphorothioate or methylphosphonate internucleotide linkages are located at both the 5 'and 3' ends of one strand.

60. The dsRNA agent of claim 31, wherein the base pair at position 1 of the 5' end of the antisense strand of the duplex is an AU base pair.

61. The dsRNA agent of claim 31, wherein p' > 0.

62. The dsRNA agent of claim 31, wherein p' =2.

63. The dsRNA agent of claim 62, wherein q '=0, p=0, q=0, and the p' overhang nucleotide is complementary to the target mRNA.

64. The dsRNA agent of claim 62, wherein q '=0, p=0, q=0, and the p' overhang nucleotide is not complementary to the target mRNA.

65. The dsRNA agent of claim 31, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

66. The dsRNA agent of claim 31, wherein at least one n _p' is linked to an adjacent nucleotide via a phosphorothioate linkage.

67. The dsRNA agent of claim 66, wherein all n _p' are linked to adjacent nucleotides via phosphorothioate linkages.

68. The dsRNA agent of claim 31, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand comprise modifications.

69. The dsRNA agent of any one of claims 31 to 68, wherein said ligand is conjugated to the 3' end of the sense strand of said dsRNA agent.

70. The dsRNA agent of claim 69, wherein the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives linked by a monovalent, divalent or trivalent branched linker.

71. The dsRNA agent of claim 70, wherein the ligand is

72. The dsRNA agent of claim 71, wherein said dsRNA agent is conjugated to said ligand as shown in the following schematic

And wherein X is O or S.

73. The dsRNA agent of claim 72, wherein the X is O.

74. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA 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 mRNA encoding CIDEB, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (Ij):

sense of meaning ：5′n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

Antisense sense ：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′ (Ij)

Wherein:

i. j, k and l are each independently 0 or 1;

p, p ', q and q' are each independently 0 to 6;

Wherein the sense strand is conjugated to at least one ligand.

75. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA 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 mRNA encoding CIDEB, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (Ij):

sense of meaning ：5′n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

Wherein:

i. j, k and l are each independently 0 or 1;

p, q and q' are each independently 0 to 6;

Wherein the sense strand is conjugated to at least one ligand.

76. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA 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 mRNA encoding CIDEB, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (Ij):

Sense of meaning ：5′n_p-N_a-(X X X)_i-N_b-YYY-N_b-(Z Z Z)_j-N_a-n_q 3′

Wherein:

i. j, k and l are each independently 0 or 1;

p, q and q' are each independently 0 to 6;

77. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA 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 mRNA encoding CIDEB, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (Ij):

sense of meaning ：5′n_p-N_a-(X X X)_i-N_b-Y Y Y-N_b-(Z Z Z)_j-N_a-n_q 3′

Wherein:

i. j, k and l are each independently 0 or 1;

p, q and q' are each independently 0 to 6;

Wherein the sense strand comprises at least one phosphorothioate linkage; and

78. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA 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 mRNA encoding CIDEB, wherein each strand is from about 14 to about 30 nucleotides in length, wherein the dsRNA agent is represented by formula (Ij):

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' (Ik)

Wherein:

p, q and q' are each independently 0 to 6;

YYY and Y ' each independently represent a motif of three identical modifications on three consecutive nucleotides, and wherein the modification is a 2' -O-methyl modification or a 2' -fluoro modification;

Wherein the sense strand comprises at least one phosphorothioate linkage; and

79. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region,

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,

Wherein substantially all of the nucleotides of the antisense strand comprise a modification selected from the group consisting of a2 '-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 linked by a monovalent, divalent or trivalent branched linker at the 3' terminus.

80. The dsRNA agent of claim 79, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand are modified nucleotides.

81. The dsRNA agent of any one of claims 3, 31 and 74 to 80, wherein the complementary region comprises any one of the antisense sequences listed in tables 3 to 6.

82. The dsRNA agent of any one of claims 1 to 81, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of the nucleotide sequences of any one of the agents listed in tables 3 to 6.

83. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting cell-expressed cell death-induced DFFA-like effector b (CIDEB), wherein the dsRNA agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises a nucleotide sequence of any one of the agents of tables 3-6, and the antisense strand comprises a nucleotide sequence of any one of the agents of tables 3-6,

Wherein the dsRNA agent is conjugated to a ligand.

84. The dsRNA agent of any one of claims 1 to 83, wherein said dsRNA agent targets a hot spot region of mRNA encoding CIDEB.

85. A dsRNA agent that targets a hot spot region of a cell death-inducing DFFA-like effector b (CIDEB) mRNA.

86. A cell containing the dsRNA agent of any one of claims 1 to 85.

87. A vector encoding at least one strand of the dsRNA agent of any one of claims 1 to 85.

88. A pharmaceutical composition for inhibiting cell death-inducing expression of a DFFA-like effector b (CIDEB) gene comprising the dsRNA agent of any one of claims 1 to 85.

89. The pharmaceutical composition of claim 88, wherein the agent is formulated in an unbuffered solution.

90. The pharmaceutical composition of claim 89, wherein the unbuffered solution is saline or water.

91. The pharmaceutical composition of claim 88, wherein the agent is formulated in a buffer solution.

92. The pharmaceutical composition of claim 91, wherein the buffer solution comprises acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof.

93. The pharmaceutical composition of claim 91, wherein the buffer solution is Phosphate Buffered Saline (PBS).

94. A method of inhibiting cell-expressed cell death from inducing DFFA-like effector b (CIDEB), the method comprising contacting the cell with the agent of any one of claims 1 to 84 or the pharmaceutical composition of any one of claims 88 to 93, thereby inhibiting expression CIDEB by the cell.

95. The method of claim 94, wherein the cell is in a subject.

96. The method of claim 95, wherein the subject is a human.

97. The method of any one of claims 94-96, wherein the CIDEB expression is inhibited by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or is inhibited to a level below the detection of CIDEB expression.

98. The method of claim 97, wherein the human subject has a CIDEB-related disease, disorder, or condition.

99. The method of claim 98, wherein the CIDEB-related disease, disorder, or condition is a chronic inflammatory disease.

100. The method of claim 99, wherein the chronic inflammatory disease is chronic inflammatory liver disease.

101. The method of claim 100, wherein the chronic inflammatory liver disease is associated with accumulation and/or amplification of lipid droplets in the liver.

102. The method of claim 100, wherein the chronic inflammatory liver disease is selected from the group consisting of: accumulation of fat in the liver, inflammation of the liver, liver fibrosis, fatty liver disease (steatosis), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and cirrhosis.

103. The method of claim 102, wherein the chronic inflammatory liver disease is non-alcoholic steatohepatitis (NASH).

104. A method of inhibiting expression of CIDEB in a subject, the method comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1 to 85 or the pharmaceutical composition of any one of claims 88 to 93, thereby inhibiting expression of CIDEB in the subject.

105. A method of treating a subject having a CIDEB-related disease, disorder, or condition, comprising administering to the subject a therapeutically effective amount of the agent of any one of claims 1-85 or the pharmaceutical composition of any one of claims 88-93, thereby treating the subject having a CIDEB-related disease, disorder, or condition.

106. A method of preventing at least one symptom of a subject suffering from a disease, disorder, or condition that would benefit from a reduction in CIDEB gene expression, the method comprising administering to the subject a prophylactically effective amount of the agent of any one of claims 1-85 or the pharmaceutical composition of any one of claims 88-93, thereby preventing at least one symptom of a subject suffering from a disease, disorder, or condition that would benefit from a reduction in CIDEB gene expression.

107. A method of reducing the risk of a subject suffering from non-alcoholic steatohepatitis (NASH) to develop a chronic liver disease, the method comprising administering to the subject a therapeutically effective amount of the dsRNA agent of any one of claims 1 to 85 or the pharmaceutical composition of any one of claims 88 to 93, thereby reducing the risk of the subject suffering from NASH to develop a chronic liver disease.

108. The method of any one of claims 104-107, wherein the CIDEB-related disease, disorder, or condition is a chronic inflammatory disease.

109. The method of claim 108, wherein the chronic inflammatory disease is chronic inflammatory liver disease.

110. The method of claim 109, wherein the chronic inflammatory liver disease is associated with accumulation and/or amplification of lipid droplets in the liver.

111. The method of claim 109, wherein the chronic inflammatory liver disease is selected from the group consisting of: accumulation of fat in the liver, inflammation of the liver, liver fibrosis, fatty liver disease (steatosis), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), and cirrhosis.

112. The method of claim 111, wherein the chronic inflammatory liver disease is non-alcoholic steatohepatitis (NASH).

113. The method of any one of claims 95-112, wherein said subject is obese.

114. The method of any one of claims 95-113, further comprising administering to the subject an additional therapeutic agent.

115. The method of any one of claims 95-114, wherein the dsRNA agent is administered to the subject at a dose of about 0.01mg/kg to about 10mg/kg or about 0.5mg/kg to about 50 mg/kg.

116. The method of any one of claims 95-115, wherein the agent is administered to the subject intravenously, intramuscularly, or subcutaneously.

117. The method of any one of claims 95-116, further comprising determining a level of CIDEB in the subject.