PROGRAMMED CELL DEATH 1 LIGAND 1 (PD-L1) iRNA COMPOSITIONS AND METHODS OF USE THEREOF

Abstract

To provide RNAi compositions which affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a PD-L1 gene.SOLUTION: A double stranded ribonucleic acid (RNAi) agent for inhibiting expression of programmed cell death 1 ligand 1 (PD-L1) comprises a sense strand and an antisense strand. The sense strand comprises at least 15 contiguous nucleotides differing by 3 or less nucleotides from any one selected from a group of partial sequences of a sequence consisting of specific nucleotide sequences. The antisense strand comprises at least 15 contiguous nucleotides differing by 3 or less nucleotides from the complementary portion of another specific nucleotide sequence. The RNAi agent comprises at least one modified nucleotide.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 213,224, filed on September 2, 2015, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on July 29, 2016 has the name 121301-04220_SL.txt and a size of 108,003 bytes.

Background Art

[0003] Background of the Invention Programmed cell death 1 ligand 1 (PD-L1) is a 290-amino acid type I transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. The expression of PD-L1 is involved in the avoidance of immune responses associated with chronic infections, such as chronic viral infections (including, for example, HIV, HBV, HCV, and HTLV), chronic bacterial infections (including, for example, Helicobacter pylori), and chronic parasitic infections (including, for example, Schistosoma mansoni). The expression of PD-L1 has been detected in a number of tissues and cell types, including T cells, B cells, macrophages, dendritic cells, and non-hematopoietic cells including endothelial cells, hepatocytes, muscle cells, and placenta.

[0004] The expression of PD-L1 is also involved in the suppression of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but immune elimination of tumors is rare. Part of this failure is due to immunosuppression by the tumor microenvironment. The expression of PD-L1 on many tumors is a component of this suppressive environment and acts in concert with other immunosuppressive signals. The expression of PD-L1 has been shown in situ on a wide variety of solid tumors, including breast, lung, colon, ovary, melanoma, bladder, liver, salivary, stomach, glioma, thyroid, thymic epithelial, head, and neck (Brown JA et al., 2003. J. Immunol. 170:1257-66; Dong H et al. 2002. Nat. Med. 8:793-800; Hamanishi J, et al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65; Strome SE et al. 2003. Cancer Res. 63:6501-5; Inman BA et al. 2007. Cancer 109:1499-505; Konishi J et al. 2004. Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007. Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007. Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004. Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N. 2006. Acta Histochem. 108:19-24). In addition, the expression of programmed cell death protein 1 (also known as PD-1 and CD279), the receptor for PD-L1, is upregulated on tumor-infiltrating lymphocytes, which also contributes to tumor immunosuppression (Blank C et al. 2003. J. Immunol. 171:4574-81).Most importantly, studies correlating the expression of PD-L1 on tumors with disease outcome have shown that PD-L1 expression strongly correlates with poor prognosis in renal cancer, ovarian cancer, bladder cancer, breast cancer, gastric cancer, and pancreatic cancer (Hamanishi J et al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA et al. 2007. Cancer 109:1499-505; Konishi J et al. 2004. Clin. Cancer Res. 10:5094-100; Nakanishi J et al. 2007. Cancer Immunol. Immunother. 56:1173-82; Nomi T et al. 2007. Clin. Cancer Res. 13:2151-57; Thompson RH et al. 2004. Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N. 2006. Acta Histochem. 108:19-24). In addition, these studies suggest that higher levels of PD-L1 expression on tumors may promote tumor stage progression and invasion into deeper tissue architecture.

[0005] The PD-1 pathway can also play a role in hematological malignancies. PD-L1 is expressed on multiple myeloma cells rather than on normal plasma cells (Liu J et al. 2007. Blood 110:296-304). PD-L1 is expressed on some primary T cell lymphomas, particularly anaplastic large cell T cell lymphoma (Brown JA et al., 2003. J. Immunol. 170:1257-66). PD-1 is highly expressed on T cells in angioimmunoblastic lymphoma, and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman DM et al. 2006. Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin lymphoma, T cells associated with lymphocyte or histiocyte (L&H) cells express PD-1. Microarray analysis using readouts of genes induced by PD-1 ligation suggests that tumor-associated T cells are in situ responsive to PD-1 signals in Hodgkin lymphoma (Chemnitz JM et al. 2007. Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T cells in HTLV-1-mediated adult T cell leukemia and lymphoma (Shimauchi T et al. 2007. Int. J. Cancer 121: 2585-90). These tumor cells are hyporesponsive to TCR signals.

[0006] Studies in animal models have demonstrated that PD-L1 on tumors inhibits T cell activation and tumor cell lysis and, in some cases, leads to increased tumor-specific T cell death (Dong H et al. 2002. Nat. Med. 8:793-800; Hirano F et al. 2005. Cancer Res. 65:1089-96). Tumor-associated APCs can also utilize the PD-1:PD-L pathway to control the anti-tumor T cell response. The expression of PD-L1 on tumor-associated myeloid DC populations is upregulated by tumor environmental factors (Curiel TJ et al. 2003. Nat. Med. 9:562-67). Plasmacytoid dendritic cells (DCs) in the tumor-draining lymph nodes of B16 melanoma express IDO, which potently activates the suppressive activity of regulatory T cells. The suppressive activity of regulatory T cells treated with IDO required cell contact with IDO-expressing DCs (Sharma MD et al. 2007. J. Clin. Invest. 117:2570-82).

[0007] Accordingly, there is a need in the art for effective treatments for PD-L1-related diseases such as infectious diseases, such as chronic intracellular infectious diseases, such as viral diseases, such as hepatitis infection, or bacterial infections, such as tuberculosis infection; and cancers, such as liver cancer, such as hepatocellular carcinoma. SUMMARY OF THE INVENTION

[0008] The present invention provides an iRNA composition that affects RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the PD-L1 gene. The PD-L1 gene can be inside cells, such as cells in a subject such as a human.

[0009] Accordingly, in one aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of programmed cell death 1 ligand 1 (PD-L1), wherein the RNAi comprises a sense strand and an antisense strand, the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides.

[0010] In certain embodiments, the sense strand and the antisense strand comprise a sequence selected from any of the sequences in Table 3. In other embodiments, the sense strand and the antisense strand comprise a sequence selected from any of the sequences in Table 5.

[0011] In one aspect, the present invention provides a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of programmed cell death 1 ligand 1 (PD-L1), wherein the RNAi comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides that differ from any one of the antisense sequences listed in Table 3 by no more than 3 nucleotides. In certain embodiments, the sense strand and the antisense strand comprise a sequence selected from any of the sequences in Table 5.

[0012] In certain embodiments, the RNAi comprises at least one modified nucleotide. In some embodiments, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise modifications. In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise modifications.

[0013] In one aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of programmed cell death 1 ligand 1 (PD-L1), wherein the RNAi agent comprises a sense strand and an antisense strand, the sense strand comprises at least 15 consecutive nucleotides which differ from any one of nucleotides 3221-3243, 351-372, 618-641, 618-639, 619-640, 620-641, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 1518-1539, 2103-2124, 2220-2261, 2220-2241, 2240-2261, 2648-2680, 2648-2669, 2658-2679, 2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3222-3243 of the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, the antisense strand comprises at least 15 consecutive nucleotides which differ from the complementary portion of the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides, and the RNAi agent comprises at least one modified nucleotide.

[0014] In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of programmed cell death 1 ligand 1 (PD-L1), wherein the RNAi agent comprises a sense strand and an antisense strand, and the antisense strand comprises a complementary region comprising at least 15 consecutive nucleotides which differ from any one of the antisense sequences in any one of double-stranded AD-67635, AD-67637, AD-67658, AD-67632, AD-67629, AD-67631, AD-67633, AD-67643, AD-67653, AD-67640, AD-67650, AD-67676, AD-67661, AD-67667, AD-67655, AD-67672, AD-67659, AD-67673, AD-67664, AD-67662, AD-67660, AD-67656, AD-67628, AD-67647, AD-67626, or AD-67645 by no more than 3 nucleotides.

[0015] In one aspect, the sense and antisense strands comprise a nucleotide sequence selected from the group consisting of any one of the nucleotide sequences in any one of the double-stranded AD-67635, AD-67637, AD-67658, AD-67632, AD-67629, AD-67631, AD-67633, AD-67643, AD-67653, AD-67640, AD-67650, AD-67676, AD-67661, AD-67667, AD-67655, AD-67672, AD-67659, AD-67673, AD-67664, AD-67662, AD-67660, AD-67656, AD-67628, AD-67647, AD-67626, or AD-67645.

[0016] In one aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of programmed cell death 1 ligand 1 (PD-L1), the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, the antisense strand comprising at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand being modified nucleotides, and the sense strand being conjugated to a ligand attached to the 3' end.

[0017] In one aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of PD-L1, which comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from nucleotides 3221-3243, 351-372, 618-641, 618-639, 619-640, 620-641, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 1518-1539, 2103-2124, 2220-2261, 2220-2241, 2240-2261, 2648-2680, 2648-2669, 2658-2679, 2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3222-3243 of the nucleotide sequence of SEQ ID NO:1 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the corresponding complementary positions of the nucleotide sequence of SEQ ID NO:2 by three or fewer nucleotides, whereby the antisense strand is complementary to at least 15 consecutive nucleotides of the sense strand that differ by three or fewer nucleotides.

[0018] In certain embodiments, the sense strand comprises at least 15 consecutive nucleotides that differ from nucleotides 3221-3243, 351-372, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 1518-1539, 2103-2124, 2220-2261, 2220-2241, 2240-2261, 2648-2680, 2648-2669, 2658-2679, 2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3222-3243 of the nucleotide sequence of SEQ ID NO:1 by three or fewer nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the corresponding complementary positions of the nucleotide sequence of SEQ ID NO:2 by three or fewer nucleotides, whereby the antisense strand is complementary to at least 15 consecutive nucleotides of the sense strand that differ by three or fewer nucleotides.

[0019] In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides that differ by three or fewer nucleotides from nucleotides 3221-3243, 1093-1115, 1093-1114, 1094-1115, 3221-3242, or 3222-3243 of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides that differ by three or fewer nucleotides from the corresponding complementary positions of the nucleotide sequence of SEQ ID NO:2, whereby the antisense strand is complementary to at least 15 contiguous nucleotides of the sense strand that differ by three or fewer nucleotides.

[0020] In another aspect, the invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of programmed cell death 1 ligand 1 (PD-L1), the double-stranded RNAi agent comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by three or fewer nucleotides from any one of nucleotides 3221-3243, 351-372, 618-641, 618-639, 619-640, 620-641, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 1518-1539, 2103-2124, 2220-2261, 2220-2241, 2240-2261, 2648-2680, 2648-2669, 2658-2679, 2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3222-3243 of the nucleotide sequence of SEQ ID NO:1, the antisense strand comprises at least 15 contiguous nucleotides that differ by three or fewer nucleotides from the complementary portion of the nucleotide sequence of SEQ ID NO:2, substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise nucleotide modifications, and the sense strand is conjugated to a ligand attached to the 3' end.

[0021] In certain embodiments, substantially all of the nucleotides of the sense strand or substantially all of the nucleotides of the antisense strand are modified nucleotides, or substantially all of the nucleotides of both strands are modified; wherein the sense strand is conjugated to a ligand attached to the 3' end.

[0022] In one aspect, the present invention provides a double-stranded RNAi agent for inhibiting the expression of PD-L1, comprising a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides from nucleotides 3221-3243, 351-372, 618-641, 618-639, 619-640, 620-641, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 1518-1539, 2103-2124, 2220-2261, 2220-2241, 2240-2261, 2648-2680, 2648-2669, 2658-2679, 2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3222-3243 of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides from the corresponding complementary positions of the nucleotide sequence of SEQ ID NO:2, whereby the antisense strand is complementary to at least 15 consecutive nucleotides of the sense strand.

[0023] In certain embodiments, the agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides from nucleotides 3221-3243, 351-372, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 1518-1539, 2103-2124, 2220-2261, 2220-2241, 2240-2261, 2648-2680, 2648-2669, 2658-2679, 2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3222-3243 of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides from the corresponding complementary positions of the nucleotide sequence of SEQ ID NO:2, whereby the antisense strand is complementary to at least 15 consecutive nucleotides of the sense strand.

[0024] In certain embodiments, the agent comprises a sense strand and an antisense strand that form a double-stranded region, wherein the sense strand comprises at least 15 consecutive nucleotides from nucleotides 3222-3243, 1093-1115, 1093-1114, 1094-1115, 3221-3243, or 3221-3242 of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises at least 15 consecutive nucleotides from the corresponding complementary positions of the nucleotide sequence of SEQ ID NO:2, whereby the antisense strand is complementary to at least 15 consecutive nucleotides of the sense strand. In certain embodiments, substantially all of the nucleotides of the sense strand are modified nucleotides. In certain embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides. In certain embodiments, substantially all of the nucleotides of both strands are modified. In a preferred embodiment, the sense strand is conjugated to a ligand attached to the 3' end.

[0025] In certain embodiments, the sense strand and the antisense strand comprise a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences listed in any one of Tables 3 and 5. For example, in certain embodiments, the sense strand and the antisense strand comprise a complementary region comprising at least 15 consecutive nucleotides that differ by three or fewer nucleotides from any one of the antisense sequences of duplexes AD-67635, AD-67637, AD-67658, AD-67632, AD-67629, AD-67631, AD-67633, AD-67643, AD-67653, AD-67640, AD-67650, AD-67676, AD-67661, AD-67667, AD-67655, AD-67672, AD-67659, AD-67673, AD-67664, AD-67662, AD-67660, AD-67656, AD-67628, AD-67647, AD-67626, or AD-67645. In certain embodiments, the sense strand and the antisense strand comprise a complementary region comprising at least 15 consecutive nucleotides of any one of the antisense sequences of the aforementioned duplexes.

[0026] In some embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand include modifications. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of deoxy-nucleotide, 3'-terminal deoxy-thymine (dT) nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide, unlocked nucleotide, conformationally restricted nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino modified nucleotide, 2'-O-allyl modified nucleotide, 2'-C-alkyl modified nucleotide, 2'-hydroxyl modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing phosphorothioate group, nucleotide containing methylphosphonate group, nucleotide containing 5'-phosphate, and nucleotide containing 5'-phosphate mimetic. In another embodiment, the modified nucleotide includes a short sequence of 3'-terminal deoxy-thymine nucleotides (dT).

[0027] In certain embodiments, substantially all of the nucleotides of the sense strand are modified. In certain embodiments, substantially all of the nucleotides of the antisense strand are modified. In certain embodiments, substantially all of the nucleotides of both the sense strand and the antisense strand are modified.

[0028] In certain embodiments, the double-strand includes the modified antisense strand provided in Table 5. In certain embodiments, the double-strand includes the modified sense strand provided in Table 5. In certain embodiments, the double-strand includes the modified double-strand provided in Table 5.

[0029] In certain embodiments, the complementary region between the antisense strand and the target is at least 17 nucleotides in length. For example, the complementary region between the antisense strand and the target is 19 to 21 nucleotides in length, and for example, the complementary region is 21 nucleotides in length. In preferred embodiments, each strand is 30 nucleotides in length or less.

[0030] In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide, for example, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0031] In many embodiments, the RNAi agent further comprises a ligand. The ligand can be conjugated to the 3' end of the sense strand of the RNAi agent. The ligand is, without limitation a N-acetylgalactosamine (GalNAc) derivative including TIFF2025106281000001.tif56128.

[0032] Exemplary RNAi agents conjugated to a ligand as shown in the following schematic: TIFF2025106281000002.tif58128 wherein X is O or S. In one embodiment, X is O.

[0033] In certain embodiments, the ligand can be a cholesterol moiety.

[0034] In certain embodiments, the complementary region comprises one of the antisense sequences of either Table 3 or Table 5. In another embodiment, the complementary region consists of one of the antisense sequences of either Table 3 or Table 5.

[0035] In another aspect, the present invention is a double-stranded RNAi agent capable of inhibiting the expression of PD-L1, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding PD-L1, each strand being from about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent being represented by formula (III): TIFF2025106281000003.tif11129wherein i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently from 0 to 6; each N a and N a ' represents an oligonucleotide sequence comprising from 0 to 25 nucleotides, either modified or unmodified or a combination thereof, each sequence comprising at least two differently modified nucleotides; each N b and N b ' represents an oligonucleotide sequence comprising from 0 to 10 nucleotides, either modified or unmodified or a combination thereof; each n p , n p ', n q , and n q ' each independently represents an overhang nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif having 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, providing a double-stranded RNAi agent.

[0036] In certain embodiments, i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1. In another embodiment, k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1. In another embodiment, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'. In another embodiment, the YYY motif appears at or near the cleavage site of the sense strand. In another embodiment, the Y'Y'Y' motif appears at positions 11, 12, and 13 from the 5'-end of the antisense strand. In one embodiment, Y' is 2'-O-methyl.

[0037] For example, formula (III) can be represented by formula (IIIa): TIFF2025106281000004.tif11128.

[0038] In another embodiment, formula (III) is represented by formula (IIIb): TIFF2025106281000005.tif11128 wherein each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides.

[0039] Alternatively, formula (III) can be represented by formula (IIIc): TIFF2025106281000006.tif11128 wherein each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides.

[0040] Furthermore, formula (III) can be represented by formula (IIId): TIFF2025106281000007.tif11128 wherein each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and each N a and Na ' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides.

[0041] In certain embodiments, the double-stranded region is 15 to 30 nucleotide pairs in length. For example, the double-stranded region can be 17 to 23 nucleotide pairs in length. The double-stranded region can be 17 to 25 nucleotide pairs in length. The double-stranded region can be 23 to 27 nucleotide pairs in length. The double-stranded region can be 19 to 21 nucleotide pairs in length. The double-stranded region can be 21 to 23 nucleotide pairs in length.

[0042] In certain embodiments, each strand has 15 to 30 nucleotides. In other embodiments, each strand has 19 to 30 nucleotides.

[0043] Modifications on the nucleotide can be selected from the group consisting of, without limitation, LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. In another embodiment, the modification on the nucleotide is a 2'-O-methyl or 2'-fluoro modification.

[0044] In certain embodiments, the ligand is one or more GalNAc derivatives linked via a monovalent linker or a divalent or trivalent branched linker. In one embodiment, the ligand is as follows: TIFF2025106281000008.tif56128.

[0045] The ligand can be attached to the 3'-end of the sense strand.

[0046] An exemplary structure of the RNAi agent conjugated with the ligand is shown in the following schematic diagram: TIFF2025106281000009.tif47128.

[0047] In certain embodiments, the ligand can be a cholesterol moiety.

[0048] In certain embodiments, the RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. For example, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3' end of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, i.e., the sense strand and the antisense strand.

[0049] In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5' end of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, i.e., the sense strand and the antisense strand.

[0050] In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at both the 5' and 3' ends of one strand, i.e., the sense strand or the antisense strand; or at the ends of both strands, i.e., the sense strand and the antisense strand.

[0051] In certain embodiments, the base pair at position 1 at the 5' end of the double-stranded antisense strand is an AU base pair.

[0052] In certain embodiments, the Y nucleotide contains a 2'-fluoro modification. In another embodiment, the Y' nucleotide contains a 2'-O-methyl modification. In another embodiment, p'>0. In some embodiments, p' = 2. In some embodiments, q' = 0, p = 0, q = 0, and the p' overhang nucleotide is complementary to the target mRNA. In some embodiments, q' = 0, p = 0, q = 0, and the p' overhang nucleotide is non-complementary to the target mRNA.

[0053] In certain embodiments, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

[0054] In certain embodiments, at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate linkage. In other embodiments, all n p ' are linked to an adjacent nucleotide via a phosphorothioate linkage.

[0055] In certain embodiments, the RNAi agent is selected from the group of RNAi agents listed in any one of Tables 3 and 5. In certain embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand include modifications.

[0056] In one aspect, the present invention is a double-stranded RNAi agent capable of inhibiting the expression of PD-L1 in cells, the double-stranded RNAi agent comprising a sense strand complementary to the antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding PD-L1, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent being represented by formula (III): TIFF2025106281000010.tif11129wherein i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently from 0 to 6; each N a and N a ' independently represent an oligonucleotide sequence comprising from 0 to 25 nucleotides, either modified or unmodified or a combination thereof, each sequence comprising at least two differently modified nucleotides; each N b and N b ' independently represent an oligonucleotide sequence comprising from 0 to 10 nucleotides, either modified or unmodified or a combination thereof; each n p n p ' n q and n q' independently represents an overhanging nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif having three identical modifications on three consecutive nucleotides, where the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modification above is different from the modification on Y and N b The modification on'is different from the modification on Y' and where the sense strand is conjugated with at least one ligand, A double-stranded RNAi agent is provided.

[0057] In one aspect, the present invention is a double-stranded RNAi agent capable of inhibiting the expression of PD-L1 in cells, the double-stranded RNAi agent comprising a sense strand complementary to the antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding PD-L1, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent being represented by formula (III): TIFF2025106281000011.tif11129wherein i, j, k, and l are each independently 0 or 1; each n that may or may not be present p n q and n q ' independently represents an overhanging nucleotide; p, q, and q' are each independently 0 to 6; n p '> 0 and at least one n p ' is linked to the adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represents an oligonucleotide sequence comprising 0 to 25 nucleotides that are either modified or unmodified or a combination thereof, each sequence comprising at least two differently modified nucleotides; each N b and N b' represents an oligonucleotide sequence containing from 0 to 10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif having three identical modifications on three consecutive nucleotides, where the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modification above is different from the modification on Y, and N b The modification on' is different from the modification on Y', and wherein the sense strand is conjugated with at least one ligand, A double-stranded RNAi agent is provided.

[0058] In one aspect, the present invention is a double-stranded RNAi agent capable of inhibiting the expression of PD-L1 in cells, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, the antisense strand comprises a region complementary to a part of the mRNA encoding PD-L1, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent is represented by formula (III): TIFF2025106281000012.tif11129Wherein i, j, k, and l are each independently 0 or 1; each n that may or may not be present p , n q , and n q ' 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 the adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represents an oligonucleotide sequence containing from 0 to 25 nucleotides, either modified or unmodified, or a combination thereof, each sequence comprising at least two differently modified nucleotides; each N b and N b' represents an oligonucleotide sequence containing 0 to 10 nucleotides, either modified or unmodified, or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif having three identical modifications on three consecutive nucleotides, where the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modification above is different from the modification on Y, and N b The modification on'is different from the modification on Y', and Here, the sense strand is conjugated with at least one ligand, and the ligand is one or more GalNAc derivatives linked via a monovalent linker or a divalent or trivalent branched linker, A double-stranded RNAi agent is provided.

[0059] In one aspect, the present invention is a double-stranded RNAi agent capable of inhibiting the expression of PD-L1 in cells, the double-stranded RNAi agent comprising a sense strand complementary to the antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding PD-L1, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent being represented by formula (III): TIFF2025106281000013.tif11129Wherein i, j, k, and l are each independently 0 or 1; each n that may or may not be present p , n q , and n q ' 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 an adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, either modified or unmodified, or a combination thereof, each sequence containing at least two differently modified nucleotides; each N b and Nb ' represents an oligonucleotide sequence containing 0 to 10 nucleotides, which may be either modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif having three identical modifications on three consecutive nucleotides, where the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modification above is different from the modification on Y, and N b The modification on'is different from the modification on Y', and wherein the sense strand contains at least one phosphorothioate bond; the sense strand is conjugated to at least one ligand, and the ligand is one or more GalNAc derivatives linked via a monovalent linker or a divalent or trivalent branched linker, A double-stranded RNAi agent is provided.

[0060] In one aspect, the present invention is a double-stranded RNAi agent capable of inhibiting the expression of PD-L1 in cells, the double-stranded RNAi agent comprising a sense strand complementary to the antisense strand, the antisense strand comprising a region complementary to a part of the mRNA encoding PD-L1, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent being represented by formula (III): TIFF2025106281000014.tif11128Wherein each n, which may or may not be present when each is present p , n q , and n q ' 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 an adjacent nucleotide via a phosphorothioate bond; each N a and N a' represents an oligonucleotide sequence containing 0 to 25 nucleotides, either independently modified or unmodified or a combination thereof, each sequence containing at least two differently modified nucleotides; YYY and Y'Y'Y' each independently represent one motif having three identical modifications on three consecutive nucleotides, where the modification is a 2'-O-methyl or 2'-fluoro modification, and wherein the sense strand contains at least one phosphorothioate bond; the sense strand is conjugated to at least one ligand, and the ligand is one or more GalNAc derivatives conjugated via a monovalent linker or a divalent or trivalent branched linker, A double-stranded RNAi agent is provided.

[0061] In one aspect, the present invention is a double-stranded RNAi agent for inhibiting the expression of PD-L1, the double-stranded RNAi agent comprising a sense strand and an antisense strand forming a double-stranded region, the sense strand containing at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO:1 by three or fewer nucleotides, the antisense strand containing at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO:2 by three or fewer nucleotides, substantially all of the nucleotides of the sense strand containing a modification selected from 2'-O-methyl modification and 2'-fluoro modification, the sense strand containing two phosphorothioate internucleotide linkages at the 5' end, substantially all of the nucleotides of the antisense strand containing a modification selected from 2'-O-methyl modification and 2'-fluoro modification, the antisense strand containing two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand being conjugated to one or more GalNAc derivatives conjugated via a monovalent linker or a divalent or trivalent branched linker at the 3' end. A double-stranded RNAi agent is provided.

[0062] In another aspect, the present invention provides a double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PD-L1, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, the sense strand comprising at least 15 consecutive nucleotides that differ from nucleotides 3221-3243, 351-372, 618-641, 618-639, 619-640, 620-641, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 1518-1539, 2103-2124, 2220-2261, 2220-2241, 2240-2261, 2648-2680, 2648-2669, 2658-2679, 2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3222-3243 of the nucleotide sequence of SEQ ID NO:1 by three or fewer nucleotides, the antisense strand comprising at least 15 consecutive nucleotides that differ from the complementary portion of the nucleotide sequence of SEQ ID NO:2 by three or fewer nucleotides, substantially all of the nucleotides of the sense strand comprising a nucleotide modification selected from the group consisting of 2'-O-methyl modification and 2'-fluoro modification, the sense strand comprising two phosphorothioate internucleotide linkages at the 5' end, substantially all of the nucleotides of the antisense strand comprising a nucleotide modification selected from the group consisting of 2'-O-methyl modification and 2'-fluoro modification, the antisense strand comprising two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand being conjugated to one or more GalNAc derivatives bound via a divalent or trivalent branched linker at the 3' end.

[0063] In certain embodiments, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides. In certain embodiments, each strand has 19-30 nucleotides.

[0064] In certain embodiments, substantially all of the nucleotides of the sense strand are modified. In certain embodiments, substantially all of the nucleotides of the antisense strand are modified. In certain embodiments, substantially all of the nucleotides of both the sense strand and the antisense strand are modified.

[0065] In one aspect, the present invention provides a cell containing an RNAi agent described herein.

[0066] In one aspect, the present invention provides a vector encoding at least one strand of an RNAi agent, wherein the RNAi agent comprises a region complementary to at least a portion of the mRNA encoding PD-L1, the RNAi is 30 base pairs or less in length, and the RNAi agent targets the mRNA for cleavage. In certain embodiments, the complementary region is at least 15 nucleotides in length. In certain embodiments, the complementary region is 19 to 23 nucleotides in length.

[0067] In one aspect, the present invention provides a cell containing a vector described herein.

[0068] In one aspect, the present invention provides a pharmaceutical composition for inhibiting the expression of the PD-L1 gene, comprising the RNAi agent of the present invention. In one embodiment, the RNAi agent is administered in an unbuffered solution. In certain embodiments, the unbuffered solution is saline or water. In other embodiments, the RNAi agent is administered in a buffered solution. In such embodiments, the buffered solution can comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. For example, the buffered solution can be phosphate buffered saline (PBS).

[0069] In one aspect, the present invention provides a pharmaceutical composition comprising the double-stranded RNAi agent of the present invention and a lipid formulation. In certain embodiments, the lipid formulation comprises an LNP. In certain embodiments, the lipid formulation comprises an MC3.

[0070] In one aspect, the present invention provides a method for inhibiting PD-L1 expression in a cell, comprising: (a) contacting the cell with the double-stranded RNAi agent of the present invention or the pharmaceutical composition of the present invention; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the PD-L1 gene, thereby inhibiting the expression of the PD-L1 gene in the cell. In certain embodiments, the cell is inside a subject, such as a human subject, such as a female or male human. In a preferred embodiment, the expression of PD-L1 is inhibited by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or below the detection threshold of the assay method used.

[0071] In one aspect, the present invention provides a method for treating a subject having a disease or disorder that would benefit from a reduction in the expression of PD-L1, comprising administering to the subject a therapeutically effective amount of the RNAi agent of the present invention or the pharmaceutical composition of the present invention, thereby treating the subject.

[0072] In one aspect, the present invention provides a method for preventing at least one symptom in a subject having a disease or disorder that would benefit from a reduction in the expression of PD-L1, comprising administering to the subject a prophylactically effective amount of the RNAi agent of the present invention or the pharmaceutical composition of the present invention, thereby preventing at least one symptom in a subject having a disorder that would benefit from a reduction in the expression of PD-L1.

[0073] In certain embodiments, administration of the RNAi to the subject causes a decrease in the PD-L1 signaling pathway. In certain embodiments, administration of the RNAi causes a decrease in the level of PD-L1 in the subject, such as the serum level of PD-L1 in the subject.

[0074] In certain embodiments, the PD-L1-related disease is an infectious disease such as a chronic intracellular infectious disease, such as a viral disease, such as hepatitis infection, or a bacterial infection, such as tuberculosis infection.

[0075] In certain embodiments, the PD-L1 related disease is cancer, such as liver cancer, such as hepatocellular carcinoma.

[0076] In certain embodiments, the invention further comprises the step of administering an antiviral agent to a subject having a PD-L1 related disease. In certain embodiments, the antiviral agent is a nucleotide or nucleoside analog. In certain embodiments, the antiviral agent is for the treatment of hepatitis virus infection, such as HBV infection, HDV infection. In certain embodiments, the antiviral agent is not an immune stimulant.

[0077] In certain embodiments, the invention further comprises the step of administering a chemotherapeutic agent to a subject having a PD-L1 related disease.

[0078] In certain embodiments where the PD-L1 related disease is cancer, the subject is further treated for the cancer. In certain embodiments, the treatment for the cancer includes surgery. In certain embodiments, the treatment for the cancer includes irradiation. In certain embodiments, the treatment for the cancer includes administration of a chemotherapeutic agent.

[0079] In various embodiments, the RNAi agent is administered at a dose of about 0.01 mg / kg to about 10 mg / kg or about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the RNAi agent is administered at a dose of about 10 mg / kg to about 30 mg / kg. In certain embodiments, the RNAi agent is administered at a dose selected from about 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, 3 mg / kg, 5 mg / kg, 10 mg / kg, and 30 mg / kg. In certain embodiments, the RNAi agent is administered about once a week, once a month, once every two months, or once a quarter (i.e., once every three months) at a dose of about 0.1 mg / kg to about 5.0 mg / kg.

[0080] In certain embodiments, the RNAi agent is administered to the subject once a week. In certain embodiments, the RNAi agent is administered to the subject once a month. In certain embodiments, the RNAi agent is administered once a quarter (i.e., once every three months).

[0081] In one aspect, the RNAi agent is administered subcutaneously to a subject.

[0082] In various aspects, the methods of the invention further comprise measuring the PD-L1 level in a subject. In certain aspects, a decrease in the expression level or activity level of the PD-L1 signaling pathway indicates that a PD-L1-related disease is being treated.

[0083] In various aspects, a surrogate marker of PD-L1 expression is measured. For example, in the treatment of an infectious disease, the presence of a pathogen, e.g., a protein or nucleic acid from the pathogen, e.g., HBsAg, HBeAg, HB cccDNA, is detected. In certain aspects, an indicator of the immune response to the pathogen, e.g., an anti-HBs antibody, is detected. In certain aspects, a change indicating effective treatment of the infection, preferably a clinically meaningful change in the surrogate marker, is detected. In the treatment of cancer, demonstration of stabilization or reduction of tumor burden using the RECIST criteria can be used as a surrogate marker for reduction in the expression or activity of PD-L1. [Inventive Concept 1001] A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of programmed cell death 1 ligand 1 (PD-L1), wherein the RNAi agent comprises a sense strand and an antisense strand, the sense strand comprising at least 15 consecutive nucleotides that differ from any one of nucleotides 3221-3243, 351-372, 618-641, 618-639, 619-640, 620-641, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 1518-1539, 2103-2124, 2220-2261, 2220-2241, 2240-2261, 2648-2680, 2648-2669, 2658-2679, 2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3222-3243 of the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, the antisense strand comprising at least 15 consecutive nucleotides that differ from the complementary portion of the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides, and the RNAi agent comprising at least one modified nucleotide, a double-stranded RNAi agent. [Inventive item 1002] A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of programmed cell death 1 ligand 1 (PD-L1), wherein the RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a complementary region comprising at least 15 consecutive nucleotides that differ from any one of the antisense sequences in any one of double-stranded AD-67635, AD-67637, AD-67658, AD-67632, AD-67629, AD-67631, AD-67633, AD-67643, AD-67653, AD-67640, AD-67650, AD-67676, AD-67661, AD-67667, AD-67655, AD-67672, AD-67659, AD-67673, AD-67664, AD-67662, AD-67660, AD-67656, AD-67628, AD-67647, AD-67626, or AD-67645 by no more than 3 nucleotides, a double-stranded RNAi agent. [Inventive item 1003] The dsRNA agent of the present invention 1001 or 1002, wherein the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of any one of the nucleotide sequences in any one of the double-stranded AD-67635, AD-67637, AD-67658, AD-67632, AD-67629, AD-67631, AD-67633, AD-67643, AD-67653, AD-67640, AD-67650, AD-67676, AD-67661, AD-67667, AD-67655, AD-67672, AD-67659, AD-67673, AD-67664, AD-67662, AD-67660, AD-67656, AD-67628, AD-67647, AD-67626, or AD-67645. [The present invention 1004] The dsRNA agent according to any one of the present inventions 1001 to 1003, wherein substantially all of the nucleotides of the sense strand or substantially all of the nucleotides of the antisense strand comprise a nucleotide modification. [The present invention 1005] The dsRNA agent according to any one of the present inventions 1001 to 1003, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification. [The present invention 1006] A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of programmed cell death 1 ligand 1 (PD-L1), wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region. The sense strand contains at least 15 consecutive nucleotides that differ from any one of nucleotides 3221 - 3243, 351 - 372, 618 - 641, 618 - 639, 619 - 640, 620 - 641, 1093 - 1115, 1093 - 1114, 1094 - 1115, 1167 - 1188, 1293 - 1314, 1518 - 1539, 2103 - 2124, 2220 - 2261, 2220 - 2241, 2240 - 2261, 2648 - 2680, 2648 - 2669, 2658 - 2679, 2659 - 2680, 3143 - 3164, 3198 - 3219, 3221 - 3242, or 3222 - 3243 of the nucleotide sequence of SEQ ID NO:1 by no more than 3 nucleotides, and the antisense strand contains at least 15 consecutive nucleotides that differ from the complementary part of the nucleotide sequence of SEQ ID NO:2 by no more than 3 nucleotides. Substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand contain nucleotide modifications, and the sense strand is conjugated to a ligand attached to the 3'-end. A double-stranded RNAi agent. [Invention 1007] The double-stranded RNAi agent of Invention 1006, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand contain modifications. [Invention 1008] At least one of the nucleotide modifications is selected from the group consisting of deoxy-nucleotide, 3'-terminal deoxy-thymine (dT) nucleotide, 2'-O-methyl modified nucleotide, 2'-fluoro modified nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide, unlocked nucleotide, conformationally fixed nucleotide, constrained ethyl nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2'-hydroxyl-modified nucleotide, 2'-methoxyethyl modified nucleotide, 2'-O-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate, nucleotide containing unnatural base, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing phosphorothioate group, nucleotide containing methylphosphonate group, nucleotide containing 5'-phosphate, and nucleotide containing 5'-phosphate mimetic, and is an RNAi agent of any one of the present inventions 1001 to 1006. [The present invention 1009] The RNAi agent of the present invention 1008, wherein the nucleotide modification comprises a short sequence of 3'-terminal deoxy-thymine nucleotide (dT). [The present invention 1010] The RNAi agent of the present invention 1002, wherein the complementary region is at least 17 nucleotides in length. [The present invention 1011] The RNAi agent of the present invention 1002, wherein the complementary region is 19 to 21 nucleotides in length. [The present invention 1012] The RNAi agent of the present invention 1011, wherein the complementary region is 19 nucleotides in length. [The present invention 1013] The RNAi agent of any one of the present inventions 1001, 1002, and 1006, wherein each strand is 30 nucleotides or less in length. [The present invention 1014] The RNAi agent of any one of the present inventions 1001, 1002, and 1006, wherein at least one strand comprises a 3'-overhang of at least 1 nucleotide. [The present invention 1015] An RNAi agent according to any one of the present inventions 1001, 1002, and 1006, wherein at least one strand contains a 3'-overhang of at least 2 nucleotides. [The present invention 1016] An RNAi agent according to any one of the present inventions 1001 to 1005, further comprising a ligand. [The present invention 1017] An RNAi agent according to the present invention 1016, wherein the ligand is conjugated to the 3'-end of the sense strand of the dsRNA agent. [The present invention 1018] An RNAi agent according to the present invention 1006 or 1016, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative. [The present invention 1019] An RNAi agent according to the present invention 1018, wherein the ligand is as follows: TIFF2025106281000015.tif54128. [The present invention 1020] An RNAi agent according to the present invention 1018, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic diagram: TIFF2025106281000016.tif56128 wherein X is O or S. [The present invention 1021] An RNAi agent according to the present invention 1020, wherein X is O. [The present invention 1022] An RNAi agent according to the present invention 1002, wherein the complementary region contains any one of the antisense sequences in Tables 3 and 5. [The present invention 1023] An RNAi agent according to the present invention 1002, wherein the complementary region consists of any one of the antisense sequences in Table 3 or Table 5. [The present invention 1024] A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-L1, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding PD-L1, each strand being from about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent being represented by formula (III): TIFF2025106281000017.tif10128Wherein i, j, k, and l are each independently 0 or 1; p, p', q, and q' are each independently from 0 to 6; Each N a and N a ' represents an oligonucleotide sequence containing from 0 to 25 nucleotides, either modified or unmodified or a combination thereof, each sequence containing at least two differently modified nucleotides; Each N b and N b ' represents an oligonucleotide sequence containing from 0 to 10 nucleotides, either modified or unmodified or a combination thereof; Each n p , n p ', n q , and n q ' independently represents an overhang nucleotide; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent a motif having 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, Double-stranded RNAi agent. [Invention 1025] i is 0; j is 0; i is 1; j is 1; both i and j are 0; or both i and j are 1, the double-stranded RNAi agent of the present invention 1024. [The present invention 1026] k is 0; l is 0; k is 1; l is 1; both k and l are 0; or both k and l are 1, the double-stranded RNAi agent of the present invention 1024. [The present invention 1027] XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z', the double-stranded RNAi agent of the present invention 1024. [The present invention 1028] The YYY motif appears at or near the cleavage site of the sense strand, the double-stranded RNAi agent of the present invention 1024. [The present invention 1029] The Y'Y'Y' motif appears at positions 11, 12, and 13 from the 5'-end of the antisense strand, the double-stranded RNAi agent of the present invention 1024. [The present invention 1030] Y' is 2'-O-methyl, the double-stranded RNAi agent of the present invention 1029. [The present invention 1031] The double-stranded RNAi agent of the present invention 1024, wherein formula (III) is represented by formula (IIIa): TIFF2025106281000018.tif10128. [The present invention 1032] The double-stranded RNAi agent of the present invention 1024, wherein formula (III) is represented by formula (IIIb): TIFF2025106281000019.tif10128 wherein each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides. [The present invention 1033] The double-stranded RNAi agent of the present invention 1024, wherein formula (III) is represented by formula (IIIc): TIFF2025106281000020.tif10128 wherein each N b and Nb ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides. [Inventive Item 1034] The double-stranded RNAi agent of Inventive Item 1024, wherein formula (III) is represented by (IIId): TIFF2025106281000021.tif10128 wherein each N b and N b ' independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, and each N a and N a ' independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides. [Inventive Item 1035] The double-stranded RNAi agent of Inventive Item 1006 or 1024, wherein the double-stranded region has a length of 15 to 30 nucleotide pairs. [Inventive Item 1036] The double-stranded RNAi agent of Inventive Item 1035, wherein the double-stranded region has a length of 17 to 23 nucleotide pairs. [Inventive Item 1037] The double-stranded RNAi agent of Inventive Item 1035, wherein the double-stranded region has a length of 17 to 25 nucleotide pairs. [Inventive Item 1038] The double-stranded RNAi agent of Inventive Item 1035, wherein the double-stranded region has a length of 23 to 27 nucleotide pairs. [Inventive Item 1039] The double-stranded RNAi agent of Inventive Item 1035, wherein the double-stranded region has a length of 19 to 21 nucleotide pairs. [Inventive Item 1040] The double-stranded RNAi agent of Inventive Item 1006 or 1024, wherein the double-stranded region has a length of 21 to 23 nucleotide pairs. [Inventive Item 1041] The double-stranded RNAi agent of Inventive Item 1006 or 1024, wherein each strand has 15 to 30 nucleotides. [Inventive Item 1042] The double-stranded RNAi agent of any one of Inventive Items 1006, 1024, and 1034, wherein each strand has 19 to 30 nucleotides. [Inventive Item 1043] The double-stranded RNAi agent of the present invention 1006 or 1024, wherein the nucleotide modification is selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof. [The present invention 1044] The double-stranded RNAi agent of the present invention 1043, wherein the nucleotide modification is a 2'-O-methyl or 2'-fluoro modification. [The present invention 1045] The double-stranded RNAi agent of the present invention 1006 or 1024, wherein the ligand is one or more GalNAc derivatives linked via a divalent or trivalent branched linker; or cholesterol. [The present invention 1046] The double-stranded RNAi agent of the present invention 1024, wherein the ligand is as follows: TIFF2025106281000022.tif54128. [The present invention 1047] The double-stranded RNAi agent of the present invention 1024, wherein the ligand is attached to the 3' end of the sense strand. [The present invention 1048] The double-stranded RNAi agent of the present invention 1047, conjugated to a ligand as shown in the following schematic diagram: TIFF2025106281000023.tif46128. [The present invention 1049] The double-stranded RNAi agent of the present invention 1006 or 1024, further comprising at least one phosphorothioate or methylphosphonate nucleotide internucleoside linkage. [The present invention 1050] The double-stranded RNAi agent of the present invention 1049, wherein the phosphorothioate or methylphosphonate nucleotide internucleoside linkage is at the 3' end of one strand. [The present invention 1051] The double-stranded RNAi agent of the present invention 1050, wherein the strand is the antisense strand. [The present invention 1052] The double-stranded RNAi agent of the present invention 1050, wherein the strand is the sense strand. [The present invention 1053] A double-stranded RNAi agent of the present invention 1049, wherein the phosphorothioate or methylphosphonate nucleotide linkage is at the 5'-end of one strand. [The present invention 1054] A double-stranded RNAi agent of the present invention 1053, wherein the strand is an antisense strand. [The present invention 1055] A double-stranded RNAi agent of the present invention 1053, wherein the strand is a sense strand. [The present invention 1056] A double-stranded RNAi agent of the present invention 1049, wherein the phosphorothioate or methylphosphonate nucleotide linkage is at both the 5'-end and the 3'-end of one strand. [The present invention 1057] A double-stranded RNAi agent of the present invention 1056, wherein the strand is an antisense strand. [The present invention 1058] A double-stranded RNAi agent of the present invention 1006 or 1024, wherein the base pair at the 1st position at the 5'-end of the double-stranded antisense strand is an AU base pair. [The present invention 1059] A double-stranded RNAi agent of the present invention 1024, wherein the Y nucleotide contains a 2'-fluoro modification. [The present invention 1060] A double-stranded RNAi agent of the present invention 1024, wherein the Y' nucleotide contains a 2'-O-methyl modification. [The present invention 1061] A double-stranded RNAi agent of the present invention 1024, wherein p'>0. [The present invention 1062] A double-stranded RNAi agent of the present invention 1024, wherein p'=2. [The present invention 1063] A double-stranded RNAi agent of the present invention 1062, wherein q'=0, p=0, q=0, and the p' overhang nucleotide is complementary to the target mRNA. [The present invention 1064] A double-stranded RNAi agent of the present invention 1062, wherein q'=0, p=0, q=0, and the p' overhang nucleotide is non-complementary to the target mRNA. [The present invention 1065] The double-stranded RNAi agent of the present invention 1056, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides. [The present invention 1066] At least one n p ' of any of the double-stranded RNAi agents of the present inventions 1061 to 1065, which is linked to an adjacent nucleotide via a phosphorothioate bond. [The present invention 1067] All n p ' of the double-stranded RNAi agent of the present invention 1066, which is linked to an adjacent nucleotide via a phosphorothioate bond. [The present invention 1068] The double-stranded RNAi agent of the present invention 1024, which is selected from the group of RNAi agents listed in Table 3 and Table 5. [The present invention 1069] The double-stranded RNAi agent of the present invention 1024, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand contain modifications. [The present invention 1070] A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-L1 in cells, wherein the double-stranded RNAi agent comprises a sense strand complementary to the antisense strand, the antisense strand comprises a region complementary to a part of the mRNA encoding PD-L1, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent is represented by formula (III): TIFF2025106281000024.tif10128 wherein, 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 containing 0 to 25 nucleotides, either modified or unmodified or a combination thereof, and each sequence contains at least two differently modified nucleotides; Each N b and N b' represents an oligonucleotide sequence containing 0 to 10 nucleotides, either modified or unmodified, or a combination thereof; each n, which may or may not be present p , n p ', n q , and n q ' represents an overhang nucleotide, independently; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif having three identical modifications on three consecutive nucleotides, where the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modification on N is different from the modification on Y, and the modification on N b ' is different from the modification on Y', and where the sense strand is conjugated to at least one ligand, double-stranded RNAi agent. [Inventive Concept 1071] A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-L1 in cells, wherein the double-stranded RNAi agent comprises a sense strand complementary to an antisense strand, the antisense strand comprises a region complementary to a part of the mRNA encoding PD-L1, each strand is about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent is represented by formula (III): TIFF2025106281000025.tif10128In the formula, i, j, k, and l are each independently 0 or 1; each n, which may or may not be present p , n q , and n q ' represents an overhang nucleotide, independently; p, q, and q' are each independently 0 to 6; n p '>0, and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond; each Na and N a ' each independently represents an oligonucleotide sequence comprising from 0 to 25 nucleotides, either modified or unmodified or a combination thereof, each sequence comprising at least two differently modified nucleotides; each N b and N b ' each independently represents an oligonucleotide sequence comprising from 0 to 10 nucleotides, either modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif having three identical modifications on three consecutive nucleotides, where the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modification above 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, double-stranded RNAi agent. [Inventive concept 1072] A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-L1 in cells, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand comprising a region complementary to a portion of the mRNA encoding PD-L1, each strand being about 14 to about 30 nucleotides in length, the double-stranded RNAi agent being represented by formula (III): TIFF2025106281000026.tif10128 wherein, i, j, k, and l are each independently 0 or 1; each n, which may or may not be present in each case, p , n q , and n q ' each 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 the adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, either modified or unmodified or a combination thereof, and each sequence contains at least two differently modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, either modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif having three identical modifications on three consecutive nucleotides, where the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modification above is different from the modification on Y, and the modification on N b ' is different from the modification on Y', and where the sense strand is conjugated to at least one ligand, and the ligand is one or more GalNAc derivatives linked via a divalent or trivalent branched linker, Double-stranded RNAi agent. [Inventive concept 1073] A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-L1 in cells, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand containing a region complementary to a part of the mRNA encoding PD-L1, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent being represented by formula (III): TIFF2025106281000027.tif10128 wherein, i, j, k, and l are each independently 0 or 1; each n that may or may not be present p , n q , and n q ' 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 an adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represents an oligonucleotide sequence containing 0 to 25 nucleotides, either modified or unmodified or a combination thereof, and each sequence contains at least two differently modified nucleotides; each N b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides, either modified or unmodified or a combination thereof; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif having three identical modifications on three consecutive nucleotides, where the modification is a 2'-O-methyl or 2'-fluoro modification; N b The modification above is different from the modification on Y, and the modification on N b ' is different from the modification on Y', where the sense strand contains at least one phosphorothioate bond and where the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a divalent or trivalent branched linker, Double-stranded RNAi agent. [Inventive item 1074] A double-stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-L1 in cells, the double-stranded RNAi agent comprising a sense strand complementary to an antisense strand, the antisense strand containing a region complementary to a part of the mRNA encoding PD-L1, each strand being about 14 to about 30 nucleotides in length, and the double-stranded RNAi agent being represented by formula (III): TIFF2025106281000028.tif10128 wherein, each n that may or may not be present p , n q , and n q ' independently represent overhang nucleotides; p, q, and q' are each independently 0 to 6; n p '>0 and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond; each N a and N a ' independently represent oligonucleotide sequences containing 0 to 25 nucleotides, either modified or unmodified or a combination thereof, and each sequence contains at least two differently modified nucleotides; YYY and Y'Y'Y' each independently represent a motif having three identical modifications on three consecutive nucleotides, where the modification is a 2'-O-methyl or 2'-fluoro modification, wherein the sense strand contains at least one phosphorothioate bond and wherein the sense strand is conjugated to at least one ligand, the ligand being one or more GalNAc derivatives linked via a divalent or trivalent branched linker, double-stranded RNAi agent. [Invention 1075] A double-stranded ribonucleic acid (RNAi) agent for inhibiting the expression of PD-L1, wherein the double-stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, The sense strand contains at least 15 consecutive nucleotides that differ by three or fewer nucleotides from nucleotides 3221 to 3243, 351 to 372, 618 to 641, 618 to 639, 619 to 640, 620 to 641, 1093 to 1115, 1093 to 1114, 1094 to 1115, 1167 to 1188, 1293 to 1314, 1518 to 1539, 2103 to 2124, 2220 to 2261, 2220 to 2241, 2240 to 2261, 2648 to 2680, 2648 to 2669, 2658 to 2679, 2659 to 2680, 3143 to 3164, 3198 to 3219, 3221 to 3242, or 3222 to 3243 of the nucleotide sequence of SEQ ID NO:1, and the antisense strand contains at least 15 consecutive nucleotides that differ by three or fewer nucleotides from the complementary portion of the nucleotide sequence of SEQ ID NO:2. Substantially all of the nucleotides of the sense strand contain a nucleotide modification selected from the group consisting of 2'-O-methyl modification and 2'-fluoro modification. The sense strand contains two phosphorothioate internucleotide linkages at the 5' end. Substantially all of the nucleotides of the antisense strand contain a nucleotide modification selected from the group consisting of 2'-O-methyl modification and 2'-fluoro modification. The antisense strand contains two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and The sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker at the 3' end. Double-stranded RNAi agent. [Inventive Item 1076] The double-stranded RNAi agent of Inventive Item 1075, wherein all nucleotides of the sense strand and all nucleotides of the antisense strand contain a nucleotide modification. [Inventive Item 1077] The double-stranded RNAi agent of Inventive Item 1075, wherein each strand has 19 to 30 nucleotides. [Inventive Item 1078] A cell containing any one of the RNAi agents of the present invention 1001, 1002, 1006, 1024, and 1070 to 1075. [The present invention 1079] A pharmaceutical composition for inhibiting the expression of the PD-L1 gene, comprising any one of the RNAi agents of the present invention 1001 to 1077. [The present invention 1080] The pharmaceutical composition of the present invention 1079, wherein the RNAi agent is administered in an unbuffered solution. [The present invention 1081] The pharmaceutical composition of the present invention 1080, wherein the unbuffered solution is physiological saline or water. [The present invention 1082] The pharmaceutical composition of the present invention 1079, wherein the RNAi agent is administered together with a buffer solution. [The present invention 1083] The pharmaceutical composition of the present invention 1082, wherein the buffer solution contains acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof. [The present invention 1084] The pharmaceutical composition of the present invention 1082, wherein the buffer solution is phosphate-buffered saline (PBS). [The present invention 1085] A pharmaceutical composition comprising any one of the double-stranded RNAi agents of the present invention 1001 to 1005 and a lipid preparation. [The present invention 1086] The pharmaceutical composition of the present invention 1085, wherein the lipid preparation contains LNP. [The present invention 1087] The pharmaceutical composition of the present invention 1085, wherein the lipid preparation contains MC3. [The present invention 1088] A step of contacting a cell with any one of the double-stranded RNAi agents of the present invention 1001 to 1077 or any one of the pharmaceutical compositions of the present invention 1079 to 1087, thereby inhibiting the expression of the PD-L1 gene in the cell A method for inhibiting the expression of PD-L1 in a cell, comprising the above step. [The present invention 1089] The method of the present invention 1088, wherein the cell is in a subject. [The present invention 1090] The method of the present invention 1089, wherein the subject is a human. [The present invention 1091] The method according to any one of the present inventions 1088 to 1090, wherein the expression of PD-L1 is inhibited by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%; or below the detection level of the assay. [The present invention 1092] Administering to a subject a therapeutically effective amount of any RNAi agent of the present inventions 1001 to 1077 or any pharmaceutical composition of the present inventions 1079 to 1087, thereby treating the subject A method of treating a subject having a disease or disorder that would benefit from a reduction in the expression of PD-L1, comprising [The present invention 1093] The method of the present invention 1092, wherein administration of RNAi to the subject causes a decrease in the PD-L1 signaling pathway. [The present invention 1094] The method of the present invention 1092, wherein the disorder is a PD-L1-related disease. [The present invention 1095] The method of the present invention 1094, wherein the PD-L1-related disease is an infection. [The present invention 1096] The method of the present invention 1095, wherein the infection is a chronic intracellular infection. [The present invention 1097] The method according to any one of the present inventions 1094 to 1096, wherein the PD-L1-related disease is a viral infection. [The present invention 1098] The method according to any one of the present inventions 1094 to 1097, wherein the PD-L1-related disease is a hepatitis virus infection. [The present invention 1099] The method according to any one of the present inventions 1095 to 1098, further comprising detecting at least one sign or symptom of the infection in the subject. [The present invention 1100] The method of the present invention 1092, wherein the PD-L1-related disease is cancer. [The present invention 1101] The method according to any one of the present inventions 1092 to 1100, wherein the subject is a human. [The present invention 1102] Any of the methods of the present invention 1092 - 1101, further comprising the step of administering an agent for treating infection or cancer. [The present invention 1103] Any of the methods of the present invention 1092 - 1102, wherein the dsRNA agent is administered at a dose of about 0.01 mg / kg to about 50 mg / kg. [The present invention 1104] Any of the methods of the present invention 1092 - 1103, wherein the dsRNA agent is administered subcutaneously to the subject. [The present invention 1105] Any of the methods of the present invention 1092 - 1104, further comprising the step of measuring the level of the PD - L1 signaling pathway in the subject. [The present invention 1106] Any of the methods of the present invention 1092 - 1104, further comprising the step of measuring the level of PD - L1 in the subject.

Brief Description of the Drawings

[0084]

Figure 1

Modes for Carrying Out the Invention

[0085] Detailed Description of the Invention The present invention provides an iRNA composition that causes RNA - induced silencing complex (RISC) - mediated cleavage of RNA transcripts of the programmed cell death 1 ligand 1 (PD - L1) gene. This gene can be intracellular, for example, within cells in a subject such as a human. The use of these iRNAs enables targeted degradation of the mRNA of the corresponding gene (PD - L1 gene) in mammals.

[0086] The iRNAs of the present invention are designed to target the human PD-L1 gene, including gene portions conserved in PD-L1 orthologs of other mammalian species. Without intending to be bound by theory, it is believed that the combination or sub-combination of the aforementioned properties and specific target sites or specific modifications in these iRNA agents confers improved efficacy, stability, potency, durability, and safety to the iRNAs of the present invention.

[0087] Accordingly, the present invention will benefit from using an iRNA composition that causes RNA-induced silencing complex (RISC)-mediated cleavage of the RNA transcript of the PD-L1 gene to inhibit or reduce the expression of the PD-L1 gene, such as disorders that would benefit from this, such as PD-L1-related diseases such as infections, such as viral infections, such as hepatitis virus infection, or cancers such as liver cancer, such as hepatocellular carcinoma, to treat a subject having the same.

[0088] In particular, very low dosages of the iRNAs of the present invention can result in significant inhibition of the expression of the corresponding gene (PD-L1 gene) as a result of specifically and efficiently mediating RNA interference (RNAi).

[0089] The iRNA of the present invention has a length of about 30 nucleotides or less, 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 nucleotides in length, and includes an RNA strand (antisense strand) having a region that is substantially complementary to at least a part of the mRNA transcript of the PD-L1 gene. In certain embodiments, the iRNA of the present invention has a region of at least 19 consecutive nucleotides that is substantially complementary to at least a part of the mRNA transcript of the PD-L1 gene, and can include a longer length, for example, a maximum length of 66 nucleotides, for example, 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length. The iRNA having an antisense strand of longer length preferably includes a second RNA strand (sense strand) having a length of 20-60 nucleotides, wherein the sense strand and the antisense strand form a double strand of 18-30 consecutive nucleotides. The use of these iRNAs enables targeted degradation of the mRNA of the corresponding gene (PD-L1 gene) in mammals. In particular, very low dosages of the iRNA of the present invention can result in significant inhibition of the expression of the corresponding gene (PD-L1 gene) as a result of specifically and efficiently mediating RNA interference (RNAi).Using in vitro and in vivo assays, the inventors have demonstrated that iRNAs targeting the PD-L1 gene can not only result in a significant inhibition of PD-L1 expression mediated by RNAi, but also reduce signal transduction via the PD-L1 pathway, which will reduce one or more of the symptoms associated with PD-L1-related diseases such as infectious diseases, e.g., viral diseases or chronic intracellular infections; or cancers. Accordingly, methods and compositions containing these iRNAs are useful for treating subjects having PD-L1-related diseases such as infectious diseases, e.g., viral diseases or chronic intracellular infections, or cancers. The methods and compositions herein are useful for reducing the level of PD-L1 in a subject, e.g., particularly in a subject having a chronic intracellular infection, particularly a chronic liver infection, or a tumor, particularly liver PD-L1 in a subject having a tumor.

[0090] The following detailed description discloses compositions, uses, and methods for making and using compositions containing iRNAs to inhibit the expression of the PD-L1 gene and to treat subjects having a disease or disorder that would benefit from a reduction in the expression of the PD-L1 gene.

[0091] I. Definitions To facilitate a better understanding of the present invention, certain terms are first defined. In addition, it should be noted that when a value or range of values of a parameter is recited, intermediate values and ranges between the recited values are also intended to be part of the present invention.

[0092] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element, or more than one element, e.g., a plurality of elements.

[0093] The term "comprising" is used herein to mean "including, but not limited to" and is used interchangeably therewith.

[0094] As used herein, the term "or" is used to mean the term "and / or" and is used interchangeably therewith, unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood to mean "sense strand or antisense strand, or sense strand and antisense strand".

[0095] The term "about" is used herein to mean within a typical tolerance range in the art. For example, "about" can be understood to be about two standard deviations from the average. In certain embodiments, about means ±10%. In certain embodiments, about means ±5%. When "about" is present before a series of numbers or a range, it is understood that "about" can modify each of the numbers in that series or range.

[0096] The term "at least" before a number or a series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that can also be logically included as apparent from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21 - nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the specified property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in that series or range.

[0097] As used herein, "below" or "less than" is understood as a logical lower value or integer from zero up to and including the value adjacent to that phrase and as logical in the context. For example, a duplex having an overhang of "two nucleotides or less" has an overhang of two, one, or zero nucleotides. When "below" is present before a series of numbers or a range, it is understood that "below" can modify each of the numbers in that series or range.

[0098] If there is a conflict between the array and the site it indicates on the transcript or another array, the nucleotide sequence listed in the specification shall take precedence.

[0099] The various aspects of the present invention can be combined as determined appropriate by those skilled in the art.

[0100] "Programmed death 1 ligand 1", also known as B7-H, B7H1, PDL1, PD-L1, PDCD1L1, PDCD1LG1, B7 homolog 1, PDCD1 ligand 1, and programmed cell death ligand 1, has been shown to be constitutively expressed on mouse T and B cells, DCs, macrophages, mesenchymal stem cells, and bone marrow-derived mast cells. Expression of PD-L1 has also been found on a wide range of non-hematopoietic cells and is upregulated on some cell types after activation. When stimulated with IFN-γ, PD-L1 is expressed on T cells, NK cells, macrophages, bone marrow DCs, B cells, epithelial cells, and vascular endothelial cells (Flies DB and Chen L (2007) J Immunother. 30 (3): 251-60). PD-L1 is significantly expressed on macrophages. Further information regarding PD-L1 is provided, for example, in the NCBI Gene database at www.ncbi.nlm.nih.gov / gene / 29126 (incorporated herein by reference as of the filing date of the present application).

[0101] As used herein, "programmed death 1 ligand 1," which is used interchangeably with the term "PD-L1" (and optionally, any of the other recognized names listed above), refers to the native gene encoding the programmed death 1 ligand 1 protein. The amino acid sequences and full coding sequences of the reference sequences of the human PDL-1 gene can be found, for example, in GenBank accession numbers GI:390979638 (RefSeq accession number NM_001267706.1; SEQ ID NO:1; SEQ ID NO:2) and GenBank accession number GI:292658763 (RefSeq accession number NM_014143.3; SEQ ID NO:9 and 10). Additional splice variants are provided, for example, in Grzywnowicz et al., PLoS One. 2012;7:e35178, which is incorporated herein by reference. Mammalian orthologs of the human PD-L1 gene can be found, for example, in GI:755563510 (RefSeq accession number XM_006527249.2, mouse; SEQ ID NO:3 and SEQ ID NO:4); GI:672040129 (RefSeq accession number XM_006231248.2, rat; SEQ ID NO:5 and SEQ ID NO:6); GenBank accession number GI:544494555 (RefSeq accession number XM_005581779.1, cynomolgus monkey; SEQ ID NO:7 and SEQ ID NO:8).

[0102] Several natural SNPs are known and can be found, for example, in the NCBI SNP database at www.ncbi.nlm.nih.gov / SNP / snp_ref.cgi?locusId=29126, which lists SNPs in human PD-L1 (incorporated herein by reference as of the filing date of this application). In preferred embodiments, such natural variants are included within the scope of the PD-L1 gene sequence.

[0103] Additional examples of the mRNA sequence of PD-L1 are readily available using publicly available databases such as GenBank, UniProt, and OMIM.

[0104] As used herein, the term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the PD-L1 gene, including the mRNA that is the RNA processing product of the primary transcript. The target portion of the sequence is long enough to at least serve as a substrate for iRNA-directed cleavage in or near a portion of the nucleotide sequence of the mRNA molecule formed during the transcription of the PD-L1 gene. In one aspect, the target sequence is within the protein-coding region of PD-L1.

[0105] The target sequence can be about 9 to 36 nucleotides in length, for example, about 15 to 30 nucleotides in length. For example, the target sequence can be 15 to 30 nucleotides in length, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides in length. Ranges and lengths intermediate to those listed above are also intended to be part of the present invention.

[0106] As used herein, the term "strand comprising a sequence" refers to an oligonucleotide that includes the nucleotide strand described by the sequence referred to, using standard nucleotide nomenclature.

[0107] "G", "C", "A", "T", and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymidine, and uracil as bases, respectively. However, the terms "ribonucleotide" or "nucleotide" can also refer to modified nucleotides or alternative substitution moieties as further detailed below (see, for example, Table 2). One of ordinary skill in the art is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of oligonucleotides containing nucleotides with such substitution moieties. For example, without limitation, a nucleotide containing inosine as a base can form base pairs with nucleotides containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced, for example, by a nucleotide containing inosine in the nucleotide sequence of the dsRNA taken up in the present invention. In another example, adenine and cytosine at any position of an oligonucleotide can be replaced by guanine and uracil, respectively, to form G-U wobble base pairs with a target mRNA. Sequences containing such substitution moieties are suitable for the compositions and methods taken up in the present invention.

[0108] The terms "iRNA", "RNAi agent", "iRNA agent", and "RNA interference agent", which are used interchangeably herein, refer to an agent containing an RNA as defined herein that mediates the targeted cleavage of an RNA transcript via the RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA via a process known as RNA interference (RNAi). iRNA regulates, for example, inhibits the expression of the PD-L1 gene in cells, for example, in cells within a subject such as a mammalian subject.

[0109] In one aspect, the RNAi agent of the present invention comprises a single-stranded RNA that interacts with a target RNA sequence, for example, a PD-L1 target mRNA sequence, and directs cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are thought to be degraded into siRNAs by type III endonucleases known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). The ribonuclease III-like enzyme Dicer processes these dsRNAs into small interfering RNAs of 19-23 base pairs with characteristic 2-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex and enable the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect, the present invention relates to a single-stranded RNA (siRNA) generated intracellularly that promotes the formation of the RISC complex and causes silencing of the target gene, i.e., the PD-L1 gene. Thus, the term "siRNA" is also used herein to refer to the above-described iRNA.

[0110] In certain embodiments, the RNAi agent can be a single-stranded siRNA (ssRNAi) that is introduced into a cell or organism to inhibit a target mRNA. The single-stranded RNAi agent binds to Argonaute 2, a RISC endonuclease, which then cleaves the target mRNA. Single-stranded siRNAs are generally 15 to 30 nucleotides in length and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Patent No. 8,101,348 and Lima et al., (2012) Cell 150:883-894, each of which is incorporated herein by reference in its entirety. Any of the antisense nucleotide sequences described herein can be used as a single-stranded siRNA described herein or as one that has been chemically modified by the method described in Lima et al., (2012) Cell 150:883-894.

[0111] In certain embodiments, the "iRNA" used in the compositions, uses, and methods of the invention is double-stranded RNA and is referred to herein as "double-stranded RNAi agent", "double-stranded RNA (dsRNA) molecule", "dsRNA agent", or "dsRNA". The term "dsRNA" refers to a ribonucleic acid molecular complex having a double-stranded structure that includes two antiparallel and substantially complementary nucleic acid strands that are said to have "sense" and "antisense" orientations with respect to a target RNA, i.e., the PD-L1 gene. In some embodiments of the invention, the double-stranded RNA (dsRNA) induces degradation of a target RNA, such as mRNA, by a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi.

[0112] Generally, the majority of the nucleotides of each strand of a dsRNA molecule are ribonucleotides, but as detailed herein, each or both strands can also include one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Additionally, as used herein, "iRNA" may include ribonucleotides with chemical modifications; the iRNA may include substantial modifications to multiple nucleotides. The term "modified nucleotide" as used herein independently refers to a nucleotide having a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase, or any combination thereof. Thus, the term modified nucleotide encompasses substitutions, additions, or removals of, for example, functional groups or atoms to the internucleoside linkage, the sugar moiety, or the nucleobase. Modifications suitable for use in the agents of the present invention include all types of modifications disclosed herein or known in the art. Any such modifications used with siRNA-type molecules are encompassed by "iRNA" or "RNAi agent" for the purposes of this specification and the claims.

[0113] The double-stranded region can be of any length that enables the desired target RNA-specific degradation via the RISC pathway, and is in the range of about 9 to 36 base pairs in length, for example, about 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, in the range of about 15 to 30, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs in length. Ranges and lengths intermediate to those listed above are also intended to be part of the present invention.

[0114] The two strands that form the double-stranded structure can be different parts of one larger RNA molecule, or they can be separate RNA molecules. If the two strands are parts of one larger molecule and are thus connected by a nucleotide strand that is not interrupted between the 3' end of one strand that forms the double-stranded structure and the 5' end of each other strand, the connecting RNA strand is called a "hairpin loop". A hairpin loop can contain at least one unpaired nucleotide. In some embodiments, a hairpin loop can contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, a hairpin loop can be 10 nucleotides or less. In some embodiments, a hairpin loop can be 8 unpaired nucleotides or less. In some embodiments, a hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, a hairpin loop can be 4-8 nucleotides.

[0115] If the two substantially complementary strands of dsRNA are composed of separate RNA molecules, these molecules need not be covalently linked, but they can be linked. If the two strands are covalently linked by means other than a nucleotide strand that is not interrupted between the 3' end of one strand and the 5' end of each other strand and form a double-stranded structure, the connecting structure is called a "linker". The RNA strands can have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of dsRNA minus any overhangs present in the double strand. In addition to the double-stranded structure, RNAi can include one or more nucleotide overhangs.

[0116] In certain embodiments, the iRNA agents of the invention are dsRNAs in which each strand comprises 19 to 23 nucleotides and interact with a target RNA sequence, for example, the PD-L1 gene. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are broken down into siRNAs by type III endonucleases known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes the dsRNA into small interfering RNAs of 19 to 23 base pairs with characteristic 2-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex and allow the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0117] In one aspect, the iRNA of the present invention is a 24 - 30 nucleotide dsRNA that interacts with a target RNA sequence, such as a PD-L1 target mRNA sequence, and directs cleavage of the target RNA, or could potentially be even longer, such as 25 - 35, 27 - 53, or 27 - 49 nucleotides. Without wishing to be bound by theory, long double-stranded RNAs introduced into cells are broken down into siRNAs by type III endonucleases known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes dsRNA into small interfering RNAs with a characteristic 2-base 3' overhang of 19 - 23 base pairs (Bernstein, et al., (2001) Nature 409:363). Next, the siRNA is incorporated into the RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

[0118] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double-stranded iRNA. For example, there is a nucleotide overhang when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand, or vice versa. The dsRNA can include an overhang of at least one nucleotide; alternatively, the overhang can include at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang can comprise, consist essentially of, or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Further, the nucleotide(s) of the overhang can be present on either the 5' end, 3' end, or both ends of either the antisense or sense strand of the dsRNA.

[0119] In certain embodiments, the antisense strand of the dsRNA has an overhang of 1 to 10 nucleotides, such as 0 to 3, 1 to 3, 2 to 4, 2 to 5, 4 to 10, 5 to 10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at the 3' or 5' end. In certain embodiments, the overhangs on the sense strand or antisense strand, or both, can include an extended length that is longer than 10 nucleotides in length, such as 1 to 30 nucleotides, 2 to 30 nucleotides, 10 to 30 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, the extended overhang is present at the 3' end of the sense strand of the duplex. In certain embodiments, the extended overhang is present at 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, the extended overhang is at the 3' end of the antisense strand of the duplex. In certain embodiments, the extended overhang is present at the 5' end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang are replaced with nucleoside thiophosphates.

[0120] "Blunt" or "blunt end" means that there are no unpaired nucleotides at the respective end of the double-stranded RNAi agent, i.e., no nucleotide overhang. A "blunt-ended" double-stranded RNAi agent is double-stranded over its entire length, i.e., there are no nucleotide overhangs at either end of the molecule. The RNAi agents of the invention include agents that do not have a nucleotide overhang at one end (i.e., agents having one overhang and one blunt end), or do not have nucleotide overhangs at either end.

[0121] The term "antisense strand" or "guide strand" refers to a strand of an iRNA, e.g., a dsRNA, that contains a region substantially complementary to a target sequence, e.g., a PD-L1 mRNA. As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence, e.g., a target sequence, e.g., a PD-L1 nucleotide sequence that is synonymous herein. If the complementary region is not completely complementary to the target sequence, there may be mismatches in the internal or terminal regions of the molecule. Generally, the most tolerant mismatches are in the terminal regions, e.g., within 5, 4, 3, 2, or 1 nucleotide at the 5' or 3' end of the iRNA. In some embodiments, the double-stranded RNAi agents of the invention contain nucleotide mismatches in the antisense strand. In some embodiments, the double-stranded RNAi agents of the invention contain nucleotide mismatches in the sense strand. In some embodiments, the nucleotide mismatches are within 5, 4, 3, 2, or 1 nucleotide from the 3' end of the iRNA, e.g., in another embodiment, the nucleotide mismatch is at the 3' terminal nucleotide of the iRNA.

[0122] As used herein, the term "sense strand" or "passenger strand" refers to an iRNA strand that contains a region substantially complementary to a region of the antisense strand that is defined as such herein.

[0123] As used herein, the term "cleavage region" refers to the region located immediately adjacent to the cleavage site. The cleavage site is the site on the target where cleavage occurs. In some embodiments, the cleavage region contains the 3 bases immediately adjacent to either side of the cleavage site. In some embodiments, the cleavage region contains the 2 bases immediately adjacent to one side of 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 contains nucleotides 11, 12, and 13.

[0124] Unless otherwise indicated, as used herein, the term "complementary," when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to an oligonucleotide or polynucleotide containing the first nucleotide sequence that can hybridize to an oligonucleotide or polynucleotide containing the second nucleotide sequence to form a double-stranded structure under certain conditions, as understood by those of ordinary skill in the art. Such conditions can be, for example, stringent conditions, where stringent conditions can include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, 12 - 16 hours at 50 °C or 70 °C, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press"). Other conditions can be applied, such as physiologically relevant conditions that can be encountered within an organism. Those of ordinary skill in the art can determine the optimal set of conditions for checking the complementarity of two sequences according to the ultimate application of the hybridized nucleotides.

[0125] In an iRNA, for example, the complementary sequences within the dsRNAs described herein include base pairing between an oligonucleotide or polynucleotide containing a first nucleotide sequence and an oligonucleotide or polynucleotide containing a second nucleotide sequence over all or part of the length of one or both nucleotide sequences. Such sequences can be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences can be fully complementary or they can form one or more, but generally 5, 4, 3, or 2 or fewer, mismatched base pairs while maintaining the ability to hybridize under conditions optimal for their ultimate application, such as inhibition of gene expression via the RISC pathway, during hybridization of a duplex of up to 30 base pairs. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches with respect to the determination of complementarity. For example, a dsRNA comprising one 21-nucleotide-long oligonucleotide and another 23-nucleotide-long oligonucleotide, wherein the longer oligonucleotide contains a 21-nucleotide sequence that is fully complementary to the shorter oligonucleotide, shall still be referred to as "fully complementary" for the purposes described herein.

[0126] As used herein, "complementary" sequences include, or can be fully formed from, base pairs formed from non-Watson-Crick base pairs or non-natural and modified nucleotides, so long as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogsteen-type base pairing.

[0127] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" can be used with respect to base pairing between the sense and antisense strands of a dsRNA or between the antisense strand of a double-stranded RNAi agent and a target sequence, as understood from the context in which they are used.

[0128] 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 the mRNA of interest (e.g., the mRNA encoding the PD-L1 gene). For example, a polynucleotide is complementary to at least a portion of the PD-L1 mRNA if its sequence is substantially complementary to an uninterrupted portion of the mRNA encoding the PD-L1 gene.

[0129] Thus, in one aspect, the sense strand polynucleotides and antisense polynucleotides disclosed herein are fully complementary to the target PD-L1 sequence.

[0130] In other aspects, the antisense polynucleotides disclosed herein are substantially complementary to the target PD-L1 sequence and are at least about 80% complementary, e.g., at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary or 100% complementary over the entire length of a contiguous nucleotide sequence that is equivalent to any one of the nucleotide sequences of SEQ ID NO:1 or a fragment of any one of SEQ ID NO:1.

[0131] In some embodiments, the iRNA of the present invention comprises a sense strand that is substantially complementary to an antisense polynucleotide, the antisense polynucleotide is then complementary to the target PD-L1 sequence, and the sense strand is at least about 80% complementary over the entire length to the nucleotide sequence of any one of the antisense strands of Table 3 or Table 5, or an equivalent region of a fragment of any one of the antisense strands of Table 3 and Table 5, for example about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary, continuous nucleotide sequence.

[0132] In some embodiments, the iRNA of the present invention comprises an antisense strand that is substantially complementary to the target PD-L1 sequence and comprises a continuous nucleotide sequence that is at least 80% complementary over the entire length to the nucleotide sequence of any one of the sense strands of Table 3 or Table 5, or an equivalent region of a fragment of any one of the sense strands of Table 3 and Table 5, for example about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.

[0133] In aspects of the invention, the agent for use in the methods and compositions of the invention is a single-stranded antisense oligonucleotide molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotide can inhibit translation stoichiometrically by base pairing with the mRNA and physically interfering with the translation machinery. See Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense oligonucleotide molecule can be from about 14 to about 30 nucleotides in length and can have a sequence complementary to the target sequence. For example, the single-stranded antisense oligonucleotide molecule can comprise a sequence that is at least 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

[0134] As used herein, the phrase "contacting a cell with an iRNA," such as dsRNA, includes contacting the cell by any possible means. Contacting a cell with an iRNA includes contacting the cell with the iRNA in vitro or contacting the cell with the iRNA in vivo. Contacting can be done directly or indirectly. Thus, for example, the iRNA can be placed in physical contact with the cell by an individual performing the method, or alternatively, the iRNA can be placed in a situation that allows or causes it to subsequently contact the cell.

[0135] Contacting the cells in vitro can be effected, for example, by incubating the cells with the iRNA. Contacting the cells in vivo can be effected, for example, by injecting the iRNA into or in the vicinity of the tissue in which the cells are located, or by injecting the iRNA into another region, such as the bloodstream or subcutaneous cavity, such that, for example subsequently, the agent reaches the tissue in which the cells to be contacted are located. For example, the iRNA may contain or be conjugated to a ligand that directs the iRNA to the site of interest, such as the liver, for example, GalNAc, for example, GalNAc3. Combinations of in vitro and in vivo contacting methods are also possible. For example, the cells may be contacted with the iRNA in vitro and subsequently transplanted into a subject.

[0136] In certain embodiments, contacting the cells with the iRNA includes "introducing" or "delivering" the iRNA into the cells by facilitating or causing uptake or absorption into the cells. Absorption or uptake of the iRNA can occur through passive diffusion or active cellular processes, or by means of an auxiliary agent or device. Introducing the iRNA into the cells can be in vitro or in vivo. For example, for in vivo introduction, the iRNA can be injected into a tissue site or systemically administered. In vivo delivery can also be effected by a beta-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677, and U.S. Patent Application Publication No. 2005 / 0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into the cells includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein or are known in the art.

[0137] The term "lipid nanoparticle" or "LNP" refers to a vesicle containing a lipid layer that encapsulates a nucleic acid molecule, such as a pharmaceutically active molecule like an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0138] As used herein, "subject" refers to a mammal, including a primate (human, non-human primate such as a monkey and a chimpanzee), a non-primate (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamster, guinea pig, cat, dog, rat, mouse, horse, and whale), or a bird (e.g., duck or goose), etc., that expresses a target gene either endogenously or heterologously, provided that the target gene sequence has sufficient complementarity with an iRNA agent to facilitate knockdown of the target. In certain embodiments, the subject is a human being who is being treated or evaluated for a disease, disorder, or condition that would benefit from a reduction in the expression or replication of the PD-L1 gene; a human being at risk of a disease, disorder, or condition that would benefit from a reduction in the expression of the PD-L1 gene; a human being having a disease, disorder, or condition that would benefit from a reduction in the expression of the PD-L1 gene; or a human being who is being treated for a disease, disorder, or condition that would benefit from a reduction in the expression of the PD-L1 gene. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human.

[0139] As used herein, the term "treating" or "treatment" refers, without limitation, to beneficial or desirable results, including alleviation or amelioration of one or more symptoms associated with the expression of the PD-L1 gene or the production of the PD-L1 protein, such as infection, particularly chronic intracellular infection, such as chronic viral infection, or reduction or improvement of cancer. "Treatment" can also mean extending the survival period as compared to the predicted survival period in the absence of treatment. Treatment can include prevention of the occurrence of co-morbidities, such as reduced liver damage in a subject with liver infection.

[0140] The term "reduce" in the context of the level of expression of the PD-L1 gene or production of the PD-L1 protein, or in relation to a disease marker or symptom in a subject, refers to a statistically significant decrease in such level. The decrease can be, for example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% less compared to an appropriate control, or less than the detection level for the detection method. In certain embodiments, the expression of the target is normalized, i.e., reduced to a level that is accepted as within the normal range for individuals without such disorder. In certain embodiments, the method includes a clinically meaningful inhibition of the expression of PD-L1, as demonstrated by a clinically meaningful outcome, for example, after treating a subject with an agent for reducing the expression of PD-L1.

[0141] As used herein, the terms "programmed cell death 1 ligand 1 related disease" or "PD-L1 related disease" refer to a disease or disorder caused by or associated with the expression of the PD-L1 gene or production of the PD-L1 protein. The term "PD-L1 related disease" includes diseases, disorders or conditions that would benefit from a decrease in the expression, replication, or protein activity of the PD-L1 gene. Non-limiting examples of PD-L1 related diseases include, for example, infections, particularly chronic intracellular infections, such as viral infections, such as hepatitis infections, or cancer.

[0142] In certain embodiments, the PD-L1 related disease is an infection, particularly a chronic, intracellular infection, such as a viral infection, such as a hepatitis virus infection, such as hepatitis B infection or hepatitis D infection. In certain embodiments, the infection is a chronic bacterial infection, such as tuberculosis. In certain embodiments, the PD-L1 related disease is cancer, particularly liver cancer, such as hepatocellular carcinoma (HCC).

[0143] As used herein, a "therapeutically effective amount" is intended to include an amount of iRNA sufficient to effect the treatment (e.g., by reducing, ameliorating or maintaining an existing disease or one or more symptoms of a disease or its associated co - morbidities) of a subject having an infection, particularly a chronic intracellular infection, or cancer, or other PD - L1 related diseases, when administered to a patient. A "therapeutically effective amount" can vary depending on the iRNA, how it is administered, the disease and its severity and history, age, weight, family history, genetic makeup, stage of the pathological process mediated by the expression of the PD - L1 gene, if any, the type of prior or concomitant treatment, and other individual characteristics of the patient being treated.

[0144] A "therapeutically effective amount" also includes an amount of iRNA that produces a desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The iRNA employed in the methods of the present invention can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment. A therapeutically effective amount includes, as appropriate, an amount that results in a clinically meaningful change or stabilization of an indicator of the disease or condition.

[0145] The term "pharmaceutically acceptable" as used herein is employed to refer to compounds, materials, compositions, or dosage forms suitable for use in contact with the tissues of human and animal subjects within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response, or other problems or complications, and at a reasonable benefit / risk ratio.

[0146] As used herein, the phrase "pharmaceutically acceptable carrier" means a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricants, talc, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), or solvent encapsulating material, etc., a pharmaceutically acceptable material, composition or vehicle that is involved in transporting or delivering the subject compound from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials that can 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) tragacanth powder; (5) malt; (6) gelatin; (7) lubricants such as magnesium state, sodium lauryl sulfate and talc; (8) excipients such as cocoa butter and suppository wax; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, 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) ethyl alcohol; (20) pH buffering solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents such as polypeptides and amino acids; (23) serum components such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations are included.

[0147] As used herein, the term "sample" includes not only similar liquids, cells, or tissues isolated from a subject, but also populations of liquids, cells, or tissues present within the subject. Examples of biological liquids include blood, serum and serous fluid, plasma, cerebrospinal fluid, ocular fluid, lymph, urine, saliva, and the like. Tissue samples can include samples from tissue, organs, or local regions. For example, a sample can be derived from a particular organ, a part of an organ, or the liquid or cells within those organs. In certain embodiments, the sample can be derived from the liver (e.g., the whole liver or a particular region of the liver, or a particular cell type in the liver such as hepatocytes). "A sample derived from a subject" can refer to blood drawn from the subject or plasma obtained from the subject. In certain embodiments when detecting the level of PD-L1, the "sample" preferably refers to a tissue or body fluid from the subject in which PD-L1 is detectable prior to administration of the agent of the invention, e.g., a liver biopsy or tumor biopsy from a subject with a liver infection. In certain subjects, e.g., healthy subjects, the level of PD-L1 may be undetectable from some body fluids, cell types, and tissues.

[0148] I. iRNA of the Invention The present invention provides an iRNA that inhibits the expression of the PD-L1 gene. In a preferred embodiment, the iRNA is a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of the PD-L1 gene in cells, such as cells inside a mammal, for example, a human having a PD-L1 related disease, such as a chronic infection. The dsRNA agent contains an antisense strand having a complementary region that is at least partially complementary to the mRNA formed in the expression of the PD-L1 gene. The complementary region is about 30 nucleotides or less in length (for example, about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). When contacting cells expressing the PD-L1 gene, the iRNA inhibits the expression of the PD-L1 gene (for example, human, primate, non-primate, or avian PD-L1 gene) by at least about 20%, preferably at least 30%, as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or a protein-based method such as immunofluorescence analysis using Western blotting or flow cytometry techniques. In a preferred embodiment, the inhibition of expression is determined by the qPCR method provided in the examples, for example, at a double-stranded concentration of 10 nM. The level of reduction can be compared, for example, to an appropriate historical control or a pooled population sample control.

[0149] The dsRNA contains two RNA strands that are complementary and hybridize under the conditions in which the dsRNA is used to form a double-stranded structure. One strand of the dsRNA (antisense strand) is substantially complementary to the target sequence and generally contains a completely complementary complementary region. The target sequence can be obtained from the mRNA sequence formed during the expression of the PD-L1 gene. The other strand (sense strand) contains a region complementary to the antisense strand, such that the two strands hybridize and form a double-stranded structure when combined under appropriate conditions. As described elsewhere herein and known in the art, the complementary sequences of the dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

[0150] Generally, the double-stranded structure is about 15 to 30 base pairs in length, for example, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 base pairs in length. Ranges and lengths intermediate to those listed above are also intended to be part of the present invention.

[0151] Similarly, the region complementary to the target sequence is about 15 to 30 nucleotides in length, for example, 15 to 29, 15 to 28, 15 to 27, 15 to 26, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 15 to 20, 15 to 19, 15 to 18, 15 to 17, 18 to 30, 18 to 29, 18 to 28, 18 to 27, 18 to 26, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 18 to 20, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides in length. Ranges and lengths intermediate to those listed above are also intended to be part of the present invention.

[0152] In some embodiments, the dsRNA is about 15 to 23 nucleotides in length, or about 25 to 30 nucleotides in length. Generally, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNA longer than about 21 to 23 nucleotides in length may serve as a substrate for Dicer. As those skilled in the art will also recognize, the RNA region targeted for cleavage is often part of a larger RNA molecule, often an mRNA molecule. Where relevant, a "portion" of the mRNA target is a continuous sequence of the mRNA target that is long enough to be capable of serving as a substrate for RNAi-directed cleavage (i.e., cleavage via the RISC pathway).

[0153] Those skilled in the art also recognize that a double-stranded region, for example, about 9 to about 36 base pairs, such as 10 - 36, 11 - 36, 12 - 36, 13 - 36, 14 - 36, 15 - 36, 9 - 35, 10 - 35, 11 - 35, 12 - 35, 13 - 35, 14 - 35, 15 - 35, 9 - 34, 10 - 34, 11 - 34, 12 - 34, 13 - 34, 14 - 34, 15 - 34, 9 - 33, 10 - 33, 11 - 33, 12 - 33, 13 - 33, 14 - 33, 15 - 33, 9 - 32, 10 - 32, 11 - 32, 12 - 32, 13 - 32, 14 - 32, 15 - 32, 9 - 31, 10 - 31, 11 - 31, 12 - 31, 13 - 32, 14 - 31, 15 - 31, 15 - 30, 15 - 29, 15 - 28, 15 - 27, 15 - 26, 15 - 25, 15 - 24, 15 - 23, 15 - 22, 15 - 21, 15 - 20, 15 - 19, 15 - 18, 15 - 17, 18 - 30, 18 - 29, 18 - 28, 18 - 27, 18 - 26, 18 - 25, 18 - 24, 18 - 23, 18 - 22, 18 - 21, 18 - 20, 19 - 30, 19 - 29, 19 - 28, 19 - 27, 19 - 26, 19 - 25, 19 - 24, 19 - 23, 19 - 22, 19 - 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, is the main functional part of the dsRNA. Thus, in one aspect, an RNA molecule or a complex of RNA molecules having a double-stranded region larger than 30 base pairs is dsRNA in the range that is processed into a functional double strand (e.g., 15 - 30 base pairs) targeting the desired RNA for cleavage. Thus, those skilled in the art recognize that in one aspect, miRNA is dsRNA. In another aspect, the dsRNA is not a naturally occurring miRNA. In another aspect, an iRNA agent useful for targeting the expression of the PD-L1 gene is not generated in target cells by cleavage of larger dsRNA.

[0154] The dsRNAs described herein can further comprise one or more single-stranded nucleotide overhangs, e.g., overhangs of 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have superior inhibitory properties compared to their blunt-ended counterparts. The nucleotide overhang can comprise or consist of nucleotide / nucleoside analogs including deoxynucleotides / nucleosides. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Further, the nucleotide(s) of the overhang can be present on the 5′ end, 3′ end, or both ends of the antisense or sense strand of the dsRNA.

[0155] For example, dsRNAs can be synthesized by use of an automated DNA synthesizer (e.g., commercially available from Biosearch, Applied Biosystems, Inc.) by standard methods known in the art, as further described below.

[0156] The double-stranded RNAi compounds of the invention can be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Next, the constituent strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase and / or solid-phase organic synthesis or both. Organic synthesis offers the advantage of allowing easy preparation of oligonucleotide strands containing unnatural or modified nucleotides. Similarly, the single-stranded oligonucleotides of the invention can be prepared using solution-phase and / or solid-phase organic synthesis or both.

[0157] In one aspect, the dsRNA of the present invention comprises at least two nucleotide sequences, namely a sense sequence and an antisense sequence. The sense strand is selected from the group of sequences provided in Tables 3 and 5, and the corresponding antisense strand of the sense strand is selected from the group of sequences in Tables 3 and 5. In this aspect, one of the two sequences is complementary to the other of the two sequences, and one of the sequences is substantially complementary to the sequence of the mRNA produced during the expression of the PD-L1 gene. Thus, in this aspect, the dsRNA comprises two oligonucleotides, where one oligonucleotide is described as the sense strand in Table 3 or 5, and the second oligonucleotide is described as the antisense strand corresponding to the sense strand in Table 3 or 5. In certain embodiments, the substantially complementary sequences of the dsRNA are contained in separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained in one oligonucleotide.

[0158] Although the sequences in Table 3 are not described as modified or conjugated sequences, it is understood that the RNA of the iRNA of the present invention, such as the dsRNA of the present invention, may comprise any one of the sequences shown in Table 3, or the modified sequences of Table 5, or the conjugated sequences of Table 5. In other words, the present invention encompasses the dsRNAs of Tables 3 and 5 that are unmodified, unconjugated, modified, or conjugated as described herein.

[0159] Those skilled in the art are well aware that dsRNAs having a double-stranded structure of about 20 to 23 base pairs, for example 21 base pairs, are particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA double-stranded structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the above aspect, depending on the nature of the oligonucleotide sequences provided in either Table 3 or 5, the dsRNAs described herein can include at least one strand that is at least 21 nucleotides in length. It can be reasonably expected that shorter double-stranded ones having sequences with a few nucleotides subtracted from one or both ends of one of the sequences in Tables 3 and 5 can be equally effective compared to the above dsRNAs. Thus, dsRNAs having sequences of at least 15, 16, 17, 18, 19, 20, or more consecutive nucleotides obtained from one of the sequences in Tables 3 and 5, and whose ability to inhibit the expression of the PD-L1 gene differs from that of the dsRNA containing the complete sequence by only about 5, 10, 15, 20, 25, or 30% or less of inhibition, are considered to be within the scope of the present invention.

[0160] In addition, the RNAs provided in Tables 3 and 5 identify sites in the PD-L1 transcript that are sensitive to RISC-mediated cleavage. Thus, the present invention further encompasses iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if it promotes cleavage of the RNA transcript somewhere within that particular site. Such iRNAs generally include at least about 15 consecutive nucleotides from one of the sequences provided in Tables 3 and 5, linked to additional nucleotide sequences taken from regions adjacent to the selected sequences in the PD-L1 gene.

[0161] Target arrays generally are about 15 to 30 nucleotides in length, while there is wide variation in the suitability of specific sequences within this range to direct cleavage of any given target RNA. The various software packages and guidelines described herein give guidance for identification of target sequences optimal for any given gene target, but a "window" or "mask" of a given size (e.g., 21 nucleotides, as a non-limiting example) can be placed literally or metaphorically (including in silico) on the target RNA sequence to identify sequences within the size range that can serve as target sequences. Potential target sequences can be identified by gradually moving the sequence "window" one nucleotide upstream or downstream of the first target sequence position until a complete set of possible sequences is identified for any given target size selected. This process, combined with the systematic synthesis and testing of identified sequences (using assays described herein or known in the art or provided herein) to identify sequences that mediate the best inhibition of target gene expression when targeted by an iRNA agent, can identify RNA sequences that, for example, the sequences identified in Tables 3 and 5 represent effective target sequences, while it is contemplated that further optimization of inhibition efficiency can be achieved by gradually "moving the window" one nucleotide upstream or downstream of a given sequence to identify sequences having equivalent or better inhibition characteristics.

[0162] Furthermore, for any of the sequences identified, for example, in Tables 3 and 5, additional optimization can also be achieved, for instance, by systematically adding or removing nucleotides to generate longer or shorter sequences, and by examining those sequences generated by moving a window of longer or shorter size either upstream or downstream of the target RNA from that point. Also, in the inhibition assays known in the art or described herein, coupling this approach for generating new candidate targets with the examination of the efficacy of iRNAs based on their target sequences can lead to further improvement in inhibition efficiency. Additionally, such optimized sequences can be further optimized as expression inhibitors, for example, by introducing modified nucleotides as described herein or known in the art, adding or changing in the overhangs, or by other modifications known in the art or described herein (e.g., increasing serum stability or circulation half-life, increasing thermal stability, enhancing transmembrane delivery, targeting specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes).

[0163] The iRNAs described herein can contain one or more mismatches with the target sequence. In one aspect, the iRNAs described herein contain three or fewer mismatches. If the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferred that the region of the mismatch is not located at the center of the complementary region. If the antisense strand of the iRNA contains a mismatch with the target sequence, it is preferred that the mismatch is limited to within the last five nucleotides from either the 5'-end or the 3'-end of the complementary region. For example, for a 23-nucleotide iRNA agent, the strand complementary to the region of the PD-L1 gene generally contains no mismatches within the central 13 nucleotides. It is possible to determine whether an iRNA containing a mismatch with the target sequence is effective in inhibiting the expression of the PD-L1 gene using the methods described herein or methods known in the art. Considering the effectiveness of an iRNA having a mismatch in inhibiting the expression of the PD-L1 gene is particularly important when it is known that a specific complementary region in the PD-L1 gene has polymorphic sequence diversity within a population.

[0164] II. Modified iRNAs of the Invention In certain aspects, the iRNAs of the invention, e.g., the RNA of the dsRNA, are unmodified, e.g., do not include chemical modifications or conjugations known in the art and described herein. In other aspects, the iRNAs of the invention, e.g., the RNA of the dsRNA, are chemically modified to enhance stability or other beneficial characteristics. In certain aspects of the invention, substantially all of the nucleotides of the iRNAs of the invention are modified. In other aspects of the invention, all or substantially all of the nucleotides of the iRNA are modified, i.e., five or fewer, four or fewer, three or fewer, two or fewer, or one or fewer unmodified nucleotides are present within the strand of the iRNA.

[0165] The nucleic acids employed in the present invention can be synthesized or modified by well-established methods in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated by reference. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, inverted linkage) or 3'-end modifications (conjugation, DNA nucleotides, inverted linkage, etc.); base modifications, such as substitution of a base with a stabilizing base, a labile base, or a base that base pairs with a wide repertoire of partners, removal of a base (abasic nucleotide), or conjugated bases; sugar modifications (e.g., at the 2'- or 4'-position) or substitution of the sugar; or backbone modifications, including modifications or substitutions of the phosphodiester bond. Specific examples of iRNA compounds useful in the aspects described herein include, without limitation, RNAs that contain a modified backbone or do not contain native internucleoside linkages. RNAs with modified backbones include, inter alia, those that do not have phosphorus atoms 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 the internucleoside backbone can also be considered oligonucleosides. In some aspects, the modified iRNA has a phosphorus atom in its internucleoside backbone.

[0166] Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates having the normal 3'-5' linkage, 2'-5' linkage analogs thereof, and those having an inverted polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts and free acid forms.

[0167] Representative U.S. patents teaching the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 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. Reissue Patent No. 39464, the entire contents of each of which are incorporated herein by reference.

[0168] Modified RNA backbones that do not contain phosphorus atoms have backbones formed by short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatom and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatom or heterocyclic nucleoside linkages. These include those having a morpholino linkage (partially formed from the sugar moiety of the nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and methylenethioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others in which N, O, S, and CH2 constituent moieties are mixed.

[0169] Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 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 incorporated herein by reference.

[0170] Both the sugar of the nucleotide unit and the internucleoside linkage, i.e., the backbone, are replaced with novel groups in suitable RNA mimics intended for use in the iRNAs provided herein. The base units are maintained for hybridization with suitable nucleic acid target compounds. One such oligomeric compound, which is an RNA mimic shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, specifically an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to the azanitrogen atoms of the amide portions of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0171] Some aspects taken up in the present invention include RNAs having a phosphorothioate backbone, as well as oligonucleosides having a heteroatom backbone, particularly --CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- [known as the methylene(methylimino) or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-- of U.S. Patent No. 5,489,677 described above [wherein the natural phosphodiester backbone is represented as --O--P--O--CH2--] and the amide backbone of U.S. Patent No. 5,602,240 described above. In some aspects, the RNAs taken up herein have the morpholino backbone structure of U.S. Patent No. 5,034,506 described above.

[0172] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs taken up herein, for example, dsRNAs can contain at the 2'-position one of the following: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C 10 alkyl or C2-C 10 and can contain one of alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2) n O] m CH3, O(CH2). n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2, and O(CH2) n ON[(CH2) n CH3)]2 are included, where n and m are from 1 to about 10. In other embodiments, the dsRNA is at the 2'-position the following: C1-C 10It includes one of lower alkyl, substituted lower alkyl, aralkyl, aralkyl, O - aralkyl or O - aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, a group for improving the pharmacokinetic properties of iRNA, or a group for improving the pharmacodynamic properties of iRNA, and other substituents having similar properties. In some embodiments, the modification comprises 2'-methoxyethoxy (2'-O--CH2CH2OCH3), also known as 2'-O-(2 - methoxyethyl) or 2'-MOE (Martin et al., Helv. Chim. Acta, 1995, 78:486 - 504), i.e., an alkoxy - alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, described in the examples below herein, i.e., the O(CH2)2ON(CH3)2 group, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O - dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2. Further exemplary modifications include: 5'-Me - 2'-F nucleotides, 5'-Me - 2'-OMe nucleotides, 5'-Me - 2'-deoxynucleotides (both R and S isomers of these three families); 2'-alkoxyalkyl; and 2'-NMA (N - methylacetamide).

[0173] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the iRNA, particularly at the 3'-terminal nucleotide or at the 3'-position of the sugar in a 2'-5' linked dsRNA and the 5'-position of the 5'-terminal nucleotide. The iRNA can also have a sugar mimetic such as a cyclobutyl moiety instead of a pentofuranosyl sugar. Representative U.S. patents that teach 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 owned by the same owner as the present application. The entire content of each of the above is incorporated herein by reference.

[0174] iRNA can also include modifications or substitutions of nucleobases (often simply referred to as "bases" in the art). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include deoxy-thymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, 2-propyl derivatives and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine and 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and 8-substituted guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and 5-substituted cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine (daazaadenine) and other synthetic and natural nucleobases such as 3-deazaguanine and 3-deazaadenine.Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of oligomeric compounds taken up by the present 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. 5-Methylcytosine substitution has been shown to increase the stability of nucleic acid duplexes by 0.6 to 1.2 °C, more particularly when combined with 2'-O-methoxyethyl sugar modification (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and is an exemplary base substitution.

[0175] Representative U.S. patents that teach the preparation of some of these other modified nucleobases, in addition to the modified nucleobases described above, include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 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, the entire contents of each of which are incorporated herein by reference.

[0176] The RNA of the iRNA can also be modified to include one or more locked nucleic acids (LNAs). A locked nucleic acid is a nucleotide having a modified ribose moiety, wherein the ribose moiety is a nucleotide that includes an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose into a 3'-end conformational arrangement. The addition of locked nucleic acids to siRNA has been shown to increase the stability of siRNA in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0177] In one aspect, the iRNA of the present invention comprises one or more monomers that are unlocked nucleic acid (UNA) nucleotides. UNA is an unlocked acyclic nucleic acid in which one of the sugar linkages has been removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' carbon and the C4' carbon) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' carbon and the C3' carbon) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0178] The RNA of the iRNA can also be modified to include one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a bridge of two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that forms a bicyclic ring system by connecting two carbon atoms of the sugar ring. In certain embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in some embodiments, the agent of the present invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2'-carbon and the 4'-carbon. In other words, LNA is a nucleotide that includes a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose into a 3'-end conformational arrangement. Adding 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 Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present invention include, but are not limited to, nucleosides that include a bridge between the 4'-ribosyl ring atom and the 2'-ribosyl ring atom. In certain embodiments, the antisense polynucleotide agent of the present invention includes one or more bicyclic nucleosides that include a 4' to 2' bridge.Examples of such 4'-to-2' bridged bicyclic nucleosides include, but are not limited to, 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and its analogs; see, e.g., U.S. Patent No. 7,399,845); 4'-C(CH3)(CH3)-O-2' (and its analogs; see, e.g., U.S. Patent No. 8,278,283); 4'-CH2-N(OCH3)-2' (and its analogs; see, e.g., U.S. Patent No. 8,278,425); 4'-CH2-O-N(CH3)-2' (see, e.g., U.S. Patent Application Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Patent No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and its analogs; see, e.g., U.S. Patent No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0179] Additional representative U.S. patents and U.S. patent application publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US2008 / 0039618; and US2009 / 0012281, the entire contents of each of which are incorporated herein by reference.

[0180] For example, any of the aforementioned bicyclic nucleosides having one or more stereochemical sugar configurations including α-L-ribofuranose and β-D-ribofuranose can be prepared (see International Publication No. 99 / 14226).

[0181] The RNA of the iRNA can also be modified to include one or more locked ethyl nucleotides. As used herein, "locked ethyl nucleotide" or "cEt" is a locked nucleic acid containing a bicyclic sugar moiety including a 4'-CH(CH3)-O-2' bridge. In one embodiment, the locked ethyl nucleotide is in the S conformation, referred to herein as "S-cEt".

[0182] The iRNA of the present invention may also include one or more "conformationally restricted nucleotides" ("CRNs"). A CRN is a nucleotide analog having a linker connecting the C2' and C4' carbons of ribose, or the C3 and C5' carbons of ribose. The CRN locks the ribose ring into a stable conformation and increases the hybridization affinity for mRNA. The linker is long enough to place oxygen in an optimal position for stability and affinity and reduces puckering of the ribose ring.

[0183] Exemplary publications that teach some of the above preparations of CRNs include, but are not limited to, US Patent Application Publication No. 2013 / 0190383; and PCT Publication WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0184] In one aspect, the iRNA of the present invention comprises one or more monomers that are unlocked nucleic acid (UNA) nucleotides. UNA is an unlocked acyclic nucleic acid in which one of the sugar linkages has been removed to form an unlocked "sugar" residue. In one example, UNA also includes monomers in which the bond between C1'-C4' (i.e., the covalent carbon-oxygen-carbon bond between the C1' carbon and the C4' carbon) has been removed. In another example, the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' carbon and the C3' carbon) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0185] Exemplary U.S. patent publications that teach the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Application Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0186] Potential stabilizing modifications to the ends of the RNA molecule can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and others. The disclosure of this modification can be found in PCT Publication WO2011 / 005861.

[0187] Other modifications of the nucleotides of the iRNA of the present invention include 5'-phosphate or 5'-phosphate mimics, such as 5'-terminal phosphate or phosphate mimics on the antisense strand of the iRNA. Suitable phosphate mimics are disclosed, for example, in US Patent Application Publication No. 2012 / 0157511, the entire content of which is incorporated herein by reference.

[0188] A. Modified iRNA Containing the Motif of the Present Invention In certain aspects of the present invention, the double-stranded RNAi agents of the present invention include agents having chemical modifications disclosed, for example, in International Publication No. WO 2013 / 075035, the entire content of each of which is incorporated herein by reference. International Publication No. WO 2013 / 075035 provides motifs having three identical modifications on three consecutive nucleotides in the sense or antisense strand of the dsRNAi agent, particularly at or near the cleavage site. In some embodiments, the sense and antisense strands of the dsRNAi agent may be fully modified in another manner. The introduction of these motifs interrupts the modification pattern of the sense or antisense strand, if it exists. The dsRNAi agent may optionally be conjugated to a GalNAc derivative ligand, for example, on the sense strand.

[0189] More specifically, gene silencing activity of the dsRNAi agent was observed when the sense and antisense strands of the double-stranded RNAi agent were fully modified to have one or more motifs having three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of the dsRNAi agent.

[0190] Accordingly, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene (i.e., the PD-L1 gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent can independently be 12 to 30 nucleotides in length. For example, each strand can independently 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.

[0191] The sense strand and the antisense strand typically form a double-stranded double-stranded RNA (``dsRNA''), also referred to herein as a ``dsRNAi agent''. The double-stranded region of the dsRNAi agent can be 12 to 30 nucleotide pairs in length. For example, the double-stranded 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 to 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 double-stranded region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

[0192] In certain embodiments, the sense strand and the antisense strand may be even longer. For example, in certain embodiments, the sense strand and the antisense strand are independently 25 to 35 nucleotides in length. In certain embodiments, each of the sense strand and the antisense strand is independently 27 to 53 nucleotides in length, such as 27 to 49, 31 to 49, 33 to 49, 35 to 49, 37 to 49, and 39 to 49 nucleotides in length.

[0193] In certain embodiments, the dsRNAi agent may contain one or more overhang regions or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhangs can independently be 1 to 6 nucleotides in length, 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. In certain embodiments, the overhang region can include an extended overhang region as provided above. The overhang can be the result of one strand being longer than the other or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the targeted gene sequence, or it can be another sequence. The first and second strands can also be joined, for example, by additional bases to form a hairpin, or joined by other non-base linkers.

[0194] In certain embodiments, the nucleotides in the overhang region of the dsRNAi agent can each independently be modified or unmodified nucleotides including, without limitation, 2′-sugar modifications such as 2′-F, 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combination thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA, or the overhang can be complementary to the targeted gene sequence, or it can be another sequence.

[0195] The 5'- or 3'-overhang in the sense strand, antisense strand, or both strands of a dsRNAi agent can be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3'-end of the sense strand, antisense strand, or both strands. In some embodiments, this 3'-overhang is present in the antisense strand. In some embodiments, this 3'-overhang is present in the sense strand.

[0196] A dsRNAi agent can contain only one overhang that can enhance the interference activity of RNAi without affecting its overall stability. For example, a single-stranded overhang can be located at the 3'-end of the sense strand or, alternatively, at the 3'-end of the antisense strand. RNAi can also have blunt ends located at the 5'-end of the antisense strand (or the 3'-end of the sense strand) or vice versa. Generally, the antisense strand of a dsRNAi agent has a nucleotide overhang at the 3'-end and a blunt 5'-end. Without wishing to be bound by theory, the asymmetric blunt ends at the 5'-end and the 3'-end overhang of the antisense strand are favorable for guide strand loading into the RISC process.

[0197] In certain embodiments, the dsRNAi agent is a 19-nucleotide long double-ended bluntmer, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 7, 8, and 9 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, and 13 from the 5'-end.

[0198] In other embodiments, the dsRNAi agent is a blunt-ended double-strand of 20 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8, 9, and 10 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, and 13 from the 5'-end.

[0199] In yet other embodiments, the dsRNAi agent is a blunt-ended double-strand of 21 nucleotides in length, and the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 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, and 13 from the 5'-end.

[0200] In certain embodiments, the dsRNAi agent comprises a 21-nucleotide sense strand and a 23-nucleotide antisense strand, the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9, 10, and 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, and 13 from the 5'-end, one end of the RNAi agent is blunt while the other end contains an overhang of two nucleotides. Preferably, the overhang of two nucleotides is at the 3'-end of the antisense strand.

[0201] When two nucleotide overhangs are at the 3'-end of the antisense strand, there can be two phosphorothioate internucleotide linkages between the terminal three nucleotides, where two of the three nucleotides are the overhang nucleotides and the third nucleotide is the base-pairing nucleotide adjacent to the overhang nucleotide. In one aspect, the RNAi agent additionally has 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 certain aspects, each nucleotide in the sense and antisense strands of the dsRNAi agent, including the nucleotides that are part of the motif, is a modified nucleotide. In certain aspects, each residue is independently modified, for example, as an alternating motif, with 2'-O-methyl or 3'-fluoro. The dsRNAi agent optionally further comprises a ligand (preferably, GalNAc3).

[0202] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand. The sense strand is 25-30 nucleotide residues in length, and starting from the 5'-terminal nucleotide (position 1), positions 1-23 of the first strand contain at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length, and starting from the 3'-terminal nucleotide, it contains at least 8 ribonucleotides at positions paired with positions 1-23 of the sense strand to form a double strand; at least the 3'-terminal nucleotides of the antisense strand do not pair with the sense strand, and up to 6 consecutive 3'-terminal nucleotides do not pair with the sense strand, thereby forming a 3'-single-stranded overhang of 1-6 nucleotides; the 5'-terminal of the antisense strand contains 10-30 consecutive nucleotides that do not pair with the sense strand, thereby forming a single-stranded 5'-overhang of 10-30 nucleotides; at least the 5'-terminal and 3'-terminal nucleotides of the sense strand form base pairs with the nucleotides of the antisense strand when the sense strand and the antisense strand are aligned for maximum complementarity, thereby forming a substantially double-stranded region between the sense strand and the antisense strand; the antisense strand is sufficiently complementary to the target RNA along at least 19 ribonucleotides in length of the antisense strand so as to reduce the expression of the target gene when the double-stranded nucleic acid is introduced into mammalian cells; the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, where at least one of the motifs appears at or near the cleavage site. The antisense strand contains at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

[0203] In certain embodiments, the dsRNAi agent comprises a sense strand and an antisense strand, the dsRNAi agent comprising a first strand having a length of at least 25 and at most 29 nucleotides and a second strand having a length of at most 30 nucleotides having at least one motif of three 2'-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5' end; the 3' end of the first strand and the 5' end of the second strand form blunt ends, the second strand is 1 to 4 nucleotides longer than the first strand at its 3' end, the double-stranded region is at least 25 nucleotides long, the second strand is sufficiently complementary to the target mRNA along at least 19 nucleotides of the second strand length such that when the RNAi agent is introduced into mammalian cells, it reduces the expression of the target gene, and Dicer cleavage of the dsRNAi agent preferentially yields siRNAs comprising the 3' end of the second strand, thereby reducing the expression of the target gene in a mammal. The dsRNAi agent optionally further comprises a ligand.

[0204] In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, wherein one of the motifs is present at the cleavage site of the sense strand.

[0205] In certain embodiments, the antisense strand of the dsRNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, wherein one of the motifs appears at or near the cleavage site of the antisense strand.

[0206] For a dsRNAi agent having a double-stranded region 17 to 23 nucleotides in length, the cleavage site of the antisense strand is typically near the 10th, 11th, and 12th positions from the 5' end. Thus, three identical modified motifs can start counting from the first nucleotide from the 5' end of the antisense strand, or from the first base-pairing nucleotide within the double-stranded region from the 5' end of the antisense strand, and can be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand. The cleavage site in the antisense strand can also vary depending on the length of the double-stranded region of the dsRNAi agent from the 5' end.

[0207] The sense strand of the dsRNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be aligned such that one motif of three nucleotides on the sense strand and one motif of three nucleotides on the antisense strand have an overlap of at least one nucleotide, that is, 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, there may be an overlap of at least two nucleotides, or all three nucleotides may overlap.

[0208] In some embodiments, the sense strand of the dsRNAi agent may contain a motif with more than one of the three same modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand, while the other motif may be a wing modification. As used herein, the term "wing modification" refers to a motif that occurs in another part of the same strand away from the motif at or near the cleavage site of the strand. The wing modification is either adjacent to the first motif or separated by at least one or more nucleotides. When the motifs are directly adjacent to each other, the chemical structures of the motifs are different from each other. When the motifs are separated by one or more nucleotides, the chemical structures can be the same or different. There may be two or more wing modifications. As an example, when there are two wing modifications, each wing modification may occur at one end relative to the first motif at or near the cleavage site or on both sides of the leading motif.

[0209] Similar to the sense strand, the antisense strand of the dsRNAi agent may contain a motif with more than one of the three same modifications on three consecutive nucleotides, and at least one of the motifs is present at or near the cleavage site of the strand. Similar to the wing modification that may be present on the sense strand, this antisense strand may also contain one or more wing modifications in the alignment.

[0210] In some embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not include the first one or two terminal nucleotides at the 3' end, 5' end, or both ends of the strand.

[0211] In other embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not include the first one or two base-pairing nucleotides within the double-stranded region at the 3' end, 5' end, or both ends of the strand.

[0212] When the sense and antisense strands of a dsRNAi agent each contain at least one wing modification, the wing modifications may be located at the same end of the double-stranded region and may have an overlap of one, two, or three nucleotides.

[0213] When the sense and antisense strands of a dsRNAi agent each contain at least two wing modifications, two modifications, one from each strand, are located at one end of the double-stranded region and have an overlap of one, two, or three nucleotides; two modifications, one from each strand, are located at the other end of the double-stranded region and have an overlap of one, two, or three nucleotides; and the sense and antisense strands can be aligned such that two modifications from one strand are on either side of a leader motif and have an overlap of one, two, or three nucleotides within the double-stranded region.

[0214] In some embodiments, each nucleotide in the sense and antisense strands of a dsRNAi agent, including nucleotides that are part of a motif, can be modified. Each nucleotide can be modified with the same or different modifications, which can include one or more alterations of one or both of non-bridging phosphate oxygens or bridging phosphate oxygens; alterations of components of the ribose sugar, such as the 2'-hydroxyl of the ribose sugar; large-scale substitution of the phosphate moiety with a "dephospho" linker; modification or substitution of natural bases; and substitution or modification of the ribose-phosphate backbone.

[0215] Since nucleic acids are polymers of subunits, many modifications, such as modifications of bases, or phosphate moieties, or non-bridging O of phosphate moieties, are present at repetitive positions within the nucleic acid. In some cases, the modification is present at all target positions in the nucleic acid, but often it is not. As an example, the modification may be present only at the 3' or 5' terminal positions, or may be present only in the terminal region, for example, at positions on the terminal nucleotide or only in the last 2, 3, 4, 5, or 10 nucleotides of the strand. The modification can be present in double-stranded regions, single-stranded regions, or both. The modification may be present only in the double-stranded region of the dsRNAi agent, or may be present only in the single-stranded region of the dsRNAi agent. For example, phosphorothioate modification at the non-bridging O position may be present only at one or both ends, may be present only in the terminal region, for example, at positions on the terminal nucleotide or only in the last 2, 3, 4, 5, or 10 nucleotides of the strand, or may be present particularly at the ends of double-stranded and single-stranded regions. One or more 5' ends can be phosphorylated.

[0216] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide surrogates in single-stranded overhangs, such as 5' or 3' overhangs, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3' or 5' overhang can be modified, for example, by the modifications described herein. Modifications can include, for example, the use of modifications at the 2' position of ribose sugars by modifications known in the art, such as the use of deoxyribonucleotides in place of ribonucleotides of nucleic acid bases, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications, and modifications of phosphate groups, such as phosphorothioate modifications. The overhang need not be homologous to the target sequence.

[0217] In some embodiments, 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'-hydroxyl, or 2'-fluoro. The strands can 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.

[0218] Typically, at least two different modifications are present on the sense and antisense strands. Those two modifications can be 2'-O-methyl or 2'-fluoro modifications, or others.

[0219] In certain embodiments, N a or N b includes an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, where each modification is present on alternating nucleotides of one strand. Alternating nucleotides can refer to every other nucleotide, every third nucleotide, or a similar pattern. For example, if A, B, and C each represent one type of modification to a nucleotide, the alternating motif can be "ABABABABABAB…", "AABBAABBAABB…", "AABAABAABAAB…", "AAABAAABAAAB…", "AAABBBAAABBB…", or "ABCABCABCABC…", etc.

[0220] The types of modifications contained in the alternating motif can be the same or different. For example, if A, B, C, D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modification on every other nucleotide, can be the same, but each of the sense and antisense strands can be selected from several possibilities of modifications within the alternating motif such as "ABABAB…", "ACACAC…", "BDBDBD…", or "CDCDCD…".

[0221] In some embodiments, the dsRNAi agent of the present invention comprises a modification pattern for alternating motifs on the sense strand that is shifted relative to the modification pattern for alternating motifs on the antisense strand. This shift can be such that the modified groups of the nucleotides of the sense strand correspond to the differently modified groups of the nucleotides of the antisense strand and vice versa. For example, when the sense strand forms base pairs in the antisense strand and the dsRNA duplex, the alternating motif in the sense strand may start with "ABABAB" from the 5' to 3' of the strand, and the alternating motif in the antisense strand may start with "BABABA" from the 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 the 5' to 3' of the strand such that there is a complete or partial shift in the modification pattern between the sense and antisense strands, and the alternating motif in the antisense strand may start with "BBAABBAA" from the 5' to 3' of the strand within the duplex region.

[0222] In some embodiments, the dsRNAi agent initially comprises a pattern of alternating motifs of 2'-O-methyl modifications and 2'-F modifications on the sense strand, which pattern has a shift relative to the pattern of alternating motifs of 2'-O-methyl modifications and 2'-F modifications on the antisense strand, i.e., the 2'-O-methyl modified nucleotides on the sense strand form base pairs with the 2'-F modified nucleotides on the antisense strand and vice versa. The first position of the sense strand may start with a 2'-F modification, and the first position of the antisense strand may start with a 2'-O-methyl modification.

[0223] Introduction of one or more motifs having three identical modifications on three consecutive nucleotides into the sense or antisense strand disrupts the first modification pattern present in the sense or antisense strand. This disruption of the modification pattern of the sense or antisense strand by introduction of one or more motifs having three identical modifications on three consecutive nucleotides into the sense or antisense strand can enhance the gene silencing activity against the target gene.

[0224] In some embodiments, when a motif having three identical modifications on three consecutive nucleotides is introduced into either strand, the modification of the nucleotides adjacent to the motif is a modification different from the modification of the motif. For example, the portion of the sequence containing the motif is “…N a YYYN b …”, where “Y” represents the modification of the motif having three identical modifications on three consecutive nucleotides, “N a ” and “N b ” represent the modifications of the nucleotides adjacent to the motif “YYY” that are different from the modification of Y, and N a and N b can be the same or different modifications. Alternatively, N a or N b can be present, absent or present when a wing modification is present.

[0225] The iRNA can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. Modifications of the phosphorothioate or methylphosphonate internucleotide linkages can be present at any nucleotide on the sense strand, the antisense strand, or both strands at any position of the strand. By way of example, the modification of the internucleotide linkage can be present on any nucleotide in the sense or antisense strand; the modification of each internucleotide linkage can be present in an alternating pattern on the sense or antisense strand; or the sense or antisense strand can contain the modification of both internucleotide linkages in an alternating pattern. The alternating pattern of the modification of the internucleotide linkages on the sense strand can be the same as or different from that on the antisense strand, and the alternating pattern of the modification of the internucleotide linkages on the sense strand can have a shift relative to the alternating pattern of the modification of the internucleotide linkages on the antisense strand. In one embodiment, the double-stranded RNAi agent comprises 6 to 8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises 2 phosphorothioate internucleotide linkages at the 5' end and 2 phosphorothioate internucleotide linkages at the 3' end, and the sense strand comprises at least 2 phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0226] In some embodiments, the dsRNAi agent comprises modifications of phosphorothioate or methylphosphonate nucleotide linkages in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate nucleotide linkage between the two nucleotides. Modification of the nucleotide linkages may also be done to link the overhang nucleotides to the terminal pairing nucleotides within the double-stranded region. For example, at least two, three, four, or all of the overhang nucleotides may be linked by phosphorothioate or methylphosphonate nucleotide linkages, and optionally, there may be additional phosphorothioate or methylphosphonate nucleotide linkages that link the overhang nucleotides to the pairing nucleotides adjacent to the overhang nucleotides. By way of example, there may be at least two phosphorothioate nucleotide linkages between the three terminal nucleotides, where two of these three nucleotides are overhang nucleotides and the third nucleotide is a pairing nucleotide adjacent to the overhang nucleotide. These three terminal nucleotides may be at the 3'-end of the antisense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, or the 5'-end of the antisense strand.

[0227] In some embodiments, the two-nucleotide overhangs are at the 3'-end of the antisense strand, there are two phosphorothioate nucleotide linkages between the three terminal nucleotides, two of the three nucleotides are overhang nucleotides, and the third nucleotide is a pairing nucleotide adjacent to the overhang nucleotide. Optionally, the dsRNAi agent may additionally have two phosphorothioate nucleotide linkages between the three terminal nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand.

[0228] In one aspect, the dsRNAi agent includes mismatches with the target, mismatches within the double strand, or combinations thereof. Mismatches can occur in the overhang region or the double strand region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., for the free energy of association or dissociation of specific pair formations, the simplest approach is to examine the pairs on an individual pair basis, but next neighbor analysis or similar analysis can also be used). With respect to promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; I:C is preferred over G:C (I = inosine). Mismatches, e.g., non-classical pair formation or pair formation other than classical (described elsewhere herein), are preferred over classical (A:T, A:U, G:C) pair formation; pair formation including universal bases is preferred over classical pair formation.

[0229] In certain aspects, the dsRNAi agent includes at least one of the first 1, 2, 3, 4, or 5 base pairs within the double strand region from the 5' end of the antisense strand, independently selected from the group of A:U, G:U, I:C, and a mismatch pair for promoting dissociation of the antisense strand at the 5' end of the double strand, e.g., non-classical pair formation or pair formation other than classical or pair formation including universal bases.

[0230] In certain aspects, the nucleotide at position 1 within the double strand region from the 5' end of the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs within the double strand region from the 5' end of the antisense strand is an AU base pair. For example, the first base pair within the double strand region from the 5' end of the antisense strand is an AU base pair.

[0231] In other embodiments, the nucleotide at the 3' end of the sense strand is deoxy-thymine (dT), or the nucleotide at the 3' end of the antisense strand is deoxy-thymine (dT). For example, there is a short sequence of deoxy-thymine nucleotides, such as two dT nucleotides, on the 3' end of the sense strand, the antisense strand, or both strands.

[0232] In certain embodiments, the sense strand sequence can be represented by formula (I): TIFF2025106281000029.tif4128 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 containing 0 to 25 modified nucleotides, and each sequence contains at least two differently modified nucleotides; each N b independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p and n q independently represent overhang nucleotides; Nb and Y do not have the same modification; XXX, YYY, and ZZZ each independently represent one motif having three identical modifications on three consecutive nucleotides. Preferably, YYY are all 2'-F modified nucleotides.

[0233] In some embodiments, N a or N b contains an alternating pattern of modifications.

[0234] In one aspect, the YYY motif is present at or near the cleavage site of the sense strand. For example, when the dsRNAi agent has a double-stranded region 17 to 23 nucleotides in length, the YYY motif can be present at or near the cleavage site of the sense strand starting from the first nucleotide counted from the 5'-end; or optionally, starting from the first base-pairing nucleotide within the double-stranded region counted from the 5'-end (e.g., it can be present at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11, 12; or 11, 12, 13).

[0235] In one aspect, 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 following formula: TIFF2025106281000030.tif17128.

[0236] When the sense strand is represented by formula (Ib), N b represents an oligonucleotide sequence containing 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 containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0237] When the sense strand is represented as formula (Ic), N b represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 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 containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0238] When the sense strand is represented by formula (Id), each N b independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Preferably, N bis 0, 1, 2, 3, 4, 5, or 6. Each N a independently can represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0239] Each of X, Y, and Z can be the same as or different from each other.

[0240] In another aspect, i is 0, j is 0, and the sense strand can be represented by the following formula: 5' n p -N a -YYY-N a -n q 3' (Ia).

[0241] When the sense strand is represented by formula (Ia), each N a independently can represent an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0242] In one aspect, the antisense strand sequence of RNAi can be represented by formula (II): TIFF2025106281000031.tif4128 where 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 containing 0 to 25 modified nucleotides, and each sequence contains at least two differently modified nucleotides; each N b ' independently represents an oligonucleotide sequence containing 0 to 10 modified nucleotides; each n p ' and n q ' independently represent overhang nucleotides; N b ' and Y' do not have the same modification; X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif having three identical modifications on three consecutive nucleotides.

[0243] In some embodiments, N a ' or N b ' includes an alternating pattern of modifications.

[0244] The Y'Y'Y' motif is present at or near the cleavage site of the antisense strand. For example, when the dsRNAi agent has a double-stranded region 17-23 nucleotides in length, the Y'Y'Y' motif can be present at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, starting from the 5' end and counting from the first nucleotide; or optionally, starting from the 5' end and counting from the first base-pairing nucleotide within the double-stranded region. Preferably, the Y'Y'Y' motif is present at positions 11, 12, 13.

[0245] In certain embodiments, the Y'Y'Y' motif consists entirely of 2'-OMe modified nucleotides.

[0246] In certain embodiments, k is 1 and l is 0, or k is 0 and l is 1, or both k and l are 1.

[0247] Thus, the antisense strand can be represented by the following formula: TIFF2025106281000032.tif17128.

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

[0249] When the antisense strand is represented by formula (IIc), N b ' represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0250] When the antisense strand is represented by formula (IId), each N b ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2, or 0 modified nucleotides. Each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5, or 6.

[0251] In other embodiments, k is 0, l is 0, and the antisense strand can be represented by the following formula: 5' n p' -N a' -Y'Y'Y'-N a' -n q' 3' (Ia).

[0252] When the antisense strand is represented by formula (IIa), each N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. Each of X', Y' and Z' can be the same as or different from each other.

[0253] Each nucleotide of the sense strand and the antisense strand may independently be modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-hydroxyl, or 2'-fluoro. For example, each nucleotide of the sense strand and the antisense strand is independently modified with 2'-O-methyl or 2'-fluoro. Each X, Y, Z, X', Y', and Z' may in particular represent a 2'-O-methyl modification or a 2'-fluoro modification.

[0254] In some embodiments, the sense strand of the dsRNAi agent, when the double-stranded region is 21 nt, may contain a YYY motif present at positions 9, 10, and 11 of the strand, starting to count from the first nucleotide from the 5'-end, or optionally, starting to count from the first base-pairing nucleotide within the double-stranded region from the 5'-end; 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 end opposite to the double-stranded region; XXX and ZZZ each independently represent a 2'-OMe modification or a 2'-F modification.

[0255] In some embodiments, the antisense strand may contain a Y'Y'Y' motif present at positions 11, 12, and 13 of the strand, starting to count from the first nucleotide from the 5'-end, or optionally, starting to count from the first base-pairing nucleotide within the double-stranded region from the 5'-end; Y' represents a 2'-O-methyl modification. The antisense strand may additionally contain an X'X'X' motif or a Z'Z'Z' motif as a wing modification at the end opposite to the double-stranded region; X'X'X' and Z'Z'Z' each independently represent a 2'-OMe modification or a 2'-F modification.

[0256] The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a double strand with the antisense strand represented by any one of the formulas (IIa), (IIb), (IIc), and (IId), respectively.

[0257] Thus, the dsRNAi agent for use in the method of the present invention may comprise a sense strand and an antisense strand each having 14 to 30 nucleotides, and the iRNA duplex is represented by formula (III): TIFF2025106281000033.tif17128wherein, 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 represent an oligonucleotide sequence comprising 0 to 25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b and N b ' independently represent an oligonucleotide sequence comprising 0 to 10 modified nucleotides; each n p ', n p n q ', and n q independently represent overhang nucleotides; XXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' each independently represent one motif having three identical modifications on three consecutive nucleotides.

[0258] In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

[0259] Exemplary combinations of the sense strand and the antisense strand forming the iRNA duplex include the following formula: TIFF2025106281000034.tif74128.

[0260] When the dsRNAi agent is represented by formula (IIIa), each N a independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0261] When the dsRNAi agent is represented by formula (IIIb), each N b independently represents an oligonucleotide sequence containing 1 to 10, 1 to 7, 1 to 5, or 1 to 4 modified nucleotides. Each N a independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0262] When the dsRNAi agent is represented by formula (IIIc), each N b , N b ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N a independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides.

[0263] When the dsRNAi agent is represented by formula (IIId), each N b , N b ' independently represents an oligonucleotide sequence containing 0 to 10, 0 to 7, 0 to 10, 0 to 7, 0 to 5, 0 to 4, 0 to 2 or 0 modified nucleotides. Each N a , N a ' independently represents an oligonucleotide sequence containing 2 to 20, 2 to 15, or 2 to 10 modified nucleotides. N a , N a ', N b, and N b ' each independently contains modifications in an alternating pattern.

[0264] Each of X, Y and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) can be the same as or different from one another.

[0265] When the dsRNAi agent is represented by formulas (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides can form a base pair with one of the Y' nucleotides. Alternatively, at least two of the Y nucleotides can form base pairs with the corresponding Y' nucleotides; or all three of the Y nucleotides can all form base pairs with the corresponding Y' nucleotides.

[0266] When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides can form a base pair with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides can form base pairs with the corresponding Z' nucleotides; or all three of the Z nucleotides can all form base pairs with the corresponding Z' nucleotides.

[0267] When the dsRNAi agent is represented by formula (IIIc) or (IIId), at least one of the X nucleotides can form a base pair with one of the X' nucleotides. Alternatively, at least two of the X nucleotides can form base pairs with the corresponding X' nucleotides; or all three of the X nucleotides can all form base pairs with the corresponding X' nucleotides.

[0268] In certain embodiments, 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.

[0269] In certain embodiments, when the dsRNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification. In other embodiments, when the RNAi agent is represented by formula (IIId), N aThe modification is a 2'-O-methyl or 2'-fluoro modification, and n p '>0, and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond. In yet other embodiments, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification, and n p '>0, and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker (described below). In other embodiments, when the RNAi agent is represented by formula (IIId), N a The modification is a 2'-O-methyl or 2'-fluoro modification, and n p '>0, and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0270] In some embodiments, when the dsRNAi agent is represented by formula (IIIa), N a The modification is a 2'-O-methyl or 2'-fluoro modification, and n p '>0, and at least one n p ' is linked to an adjacent nucleotide via a phosphorothioate bond, the sense strand contains at least one phosphorothioate bond, and the sense strand is conjugated to one or more GalNAc derivatives linked via a divalent or trivalent branched linker.

[0271] In some embodiments, the dsRNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are linked by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

[0272] In some embodiments, the dsRNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are linked by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target the same gene at two different target sites.

[0273] In one embodiment, two dsRNAi agents represented by at least one of formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at one or both of the 5'-end and the 3'-end, and optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target the same gene at two different target sites.

[0274] Multimeric iRNAs that can be used in the methods of the present invention are described in various publications. Such publications include U.S. Patent No. 7,858,769, WO2007 / 091269, WO2010 / 141511, WO2007 / 117686, WO2009 / 014887, and WO2011 / 031520, the entire contents of each of which are incorporated herein by reference.

[0275] As described in more detail below, iRNAs containing the conjugation of one or more carbohydrate moieties to the iRNA can optimize one or more properties of the iRNA. In many cases, the carbohydrate moiety is attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of the iRNA can be replaced with another moiety, such as a non-carbohydrate (preferably cyclic) carrier to which a carbohydrate ligand binds. A ribonucleotide subunit in which the ribose sugar of the subunit is so replaced is herein referred to as a ribose-substituted modified subunit (RRMS). The cyclic carrier can be a carbocyclic system (i.e., all ring atoms are carbon atoms) or a heterocyclic system (i.e., one or more ring atoms can be heteroatoms, such as nitrogen, oxygen, sulfur). The cyclic carrier can be monocyclic or can contain two or more rings, such as fused rings. The cyclic carrier can be a completely saturated ring system or it can contain one or more double bonds.

[0276] A ligand can be attached to a polynucleotide via a carrier. The carrier includes (i) at least one "backbone attachment point", preferably two "backbone attachment points", and (ii) at least one "tethering attachment point". As used herein, a "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally an attachment available for and suitable for incorporation of the carrier into the backbone of ribonucleic acid, such as phosphate, or a modified phosphate, such as a sulfur-containing backbone. A "tethering attachment point" (TAP) refers, in one aspect, to a ring-constituting atom of a cyclic carrier that connects a selected moiety, such as a carbon atom or a heteroatom (different from the atom providing the backbone attachment point). This moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected to the cyclic carrier by an intervening tether. Thus, the cyclic carrier often contains a functional group, such as an amino group, or generally provides an attachment suitable for incorporation or tethering of another chemical entity, such as a ligand, into the ring structure.

[0277] iRNA may be conjugated to a ligand via a carrier, where 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, tetrahydrofuryl, and decalin; preferably, the acyclic group is a serinol backbone or a diethanolamine backbone.

[0278] In certain embodiments, the iRNA is an agent selected from the agents listed in Tables 3 and 5. In one embodiment, the iRNA agent targets nucleotides 3221-3243 of SEQ ID NO:1. In one embodiment, the RNAi agent is AD-67635 (targeting nucleotides 3224-3243 of SEQ ID NO:1). In another embodiment, the RNAi agent is AD-67637 (targeting nucleotides 3223-3242 of SEQ ID NO:1). These agents may further comprise a ligand.

[0279] III. iRNA Conjugated to a Ligand Another modification of the RNA of the iRNA of the present invention involves chemically linking one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or, for example, cellular uptake into cells of the iRNA to the iRNA. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060).In certain embodiments, the modification can include a thioether, such as beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), thiolesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, such as dodecanediol or undecyl residue (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0280] In certain embodiments, the ligand alters the distribution, targeting, or lifespan 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 cellular or organ compartment), tissue, organ, or region of the body, as compared to a species in which such ligand is absent. Preferred ligands do not participate in duplex formation in double-stranded nucleic acids.

[0281] The ligand can include natural 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-acetylgalactosamine, or hyaluronic acid); or lipids. The ligand can also be a synthetic polymer, such as a recombinant or synthetic molecule, e.g., a synthetic polyamino acid. Examples of polyamino acids include 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-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, peptidomimetic-polyamine, peptoid polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salts of polyamines, or alpha-helical peptide.

[0282] The ligand can also include a target-directed group, such as a cell or tissue target-directed agent, such as a lectin, glycoprotein, lipid or protein, such as an antibody that binds to a specific cell type such as a renal cell. The target-directed group can be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, monovalent or polyvalent N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is monovalent or polyvalent N-acetyl-galactosamine. In certain embodiments, the ligand is cholesterol.

[0283] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl) lithocholic acid, O3-(oleoyl) cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphates, aminos, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamines, alkyls, substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption promoters (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, tetraaza macrocyclic Eu3+ complex), dinitrophenyl, HRP, or AP.

[0284] A ligand can be a molecule having specific affinity for a protein, such as a glycoprotein, or a peptide, such as a co-ligand, or an antibody, such as an antibody that binds to a specific cell type such as a hepatocyte. A ligand can also include hormones and hormone receptors. A ligand can also include non-peptide species such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. A ligand can be, for example, a lipopolysaccharide, a p38MAP kinase activator, or an NF-κB activator.

[0285] The ligand can be, for example, a substance that can increase the uptake of the iRNA agent into cells by disrupting, for example, the cytoskeleton of the cell, such as by disrupting the microtubules, microfilaments, or intermediate filaments of the cell, and can be, for example, a drug. The drug can be, for example, a taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0286] In some embodiments, the ligand conjugated to the iRNA described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glyceride, diacyl glyceride, phospholipid, sphingolipid, naproxen, ibuprofen, vitamin E, biotin, and the like. It is also known that oligonucleotides containing some phosphorothioate linkages bind to serum proteins, and thus short oligonucleotides, such as oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases that contain multiple phosphorothioate linkages in the backbone, are also suitable for the present invention as ligands (e.g., PK modulate ligands). In addition, aptamers that bind to serum components (e.g., serum proteins) are also suitable for use as PK modulate ligands in the embodiments described herein.

[0287] The ligand-conjugated iRNA of the present invention can be synthesized (described below) by using oligonucleotides having reactive pendant functional groups, such as those obtained from the attachment of a linking molecule on the oligonucleotide. This reactive oligonucleotide can be reacted directly with a commercially available ligand, a synthesized ligand having any of a variety of protecting groups, or a ligand to which a linking moiety is attached.

[0288] The oligonucleotides used in the conjugates of the present invention can be conveniently and routinely prepared by well-known techniques of solid-phase synthesis. Apparatus for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may be additionally or alternatively employed. It is also known to use similar techniques for preparing other oligonucleotides such as phosphorothioate and alkylated derivatives.

[0289] In the ligand conjugates iRNA and ligand molecules having the sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides of the present invention can be assembled using an appropriate DNA synthesizer, utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors already having a linking moiety, ligand-nucleotide or nucleoside-conjugate precursors already having a ligand molecule, or non-nucleoside ligand-containing building blocks.

[0290] When using a nucleotide conjugate precursor already having a linking moiety, the synthesis of the sequence-specific linked nucleoside is typically completed and then the ligand molecule is reacted with the linking moiety to form a ligand-conjugate oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites obtained from ligand-nucleoside conjugates in addition to standard phosphoramidites and non-standard phosphoramidites commercially available and routinely used in oligonucleotide synthesis.

[0291] A. Lipid Conjugate In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins such as human serum albumin (HSA). The HSA-binding ligand enables the distribution of the conjugate to target tissues, such as non-renal target tissues of the body. For example, the target tissue can be the liver, including hepatocytes of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) increase the degradation resistance of the conjugate, (b) increase the targeting or transport to target cells or cell membranes, or (c) modulate binding to serum proteins such as HSA.

[0292] Lipid-based ligands can be used to inhibit, e.g., control the binding of the conjugate to target tissues. For example, lipid or lipid-based ligands that bind more strongly to HSA are less likely to be targeted to the kidney and thus less likely to be removed from the body. Lipid or lipid-based ligands that do not bind as strongly to HSA can be used to target the conjugate to the kidney.

[0293] In certain embodiments, the lipid-based ligand binds to HSA. Preferably, the ligand binds to HSA with sufficient affinity such that the conjugate preferably distributes to non-renal tissues. However, it is preferred that the affinity is not so strong that the HSA-ligand binding cannot be reversed.

[0294] In other embodiments, by the lipid-based ligand binding weakly or not at all to HSA, the conjugate preferably distributes to the kidney. Other moieties that target renal cells can also be used instead of or in addition to the lipid-based ligand.

[0295] In another aspect, the ligand is a moiety, such as a vitamin, that is taken up by target cells, such as proliferating cells. These are particularly useful for treating disorders characterized by unwanted cell proliferation, such as malignant or non-malignant types, such as cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamins B, such as folic acid, B12, riboflavin, biotin, pyridoxal, or other vitamins or nutrients taken up by target cells such as hepatocytes. HSA and low density lipoprotein (LDL) are also included.

[0296] B. Cell-Penetrating Agents 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. When the agent is a peptide, it can be modified to include peptidomimetics, reverse isomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. The helical agent is preferably an alpha-helical agent having a lipophilic and a lipophobic phase.

[0297] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The binding of peptides and peptidomimetics to iRNA agents can affect the pharmacokinetic distribution of iRNA, such as by enhancing cell recognition and uptake. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0298] 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 mainly of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, a constrained peptide or a cross-linked peptide. In another alternative embodiment, the peptide moiety can contain a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:14). RFGF analogs containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO:15)) can also be targeting moieties. The peptide moiety can be a “delivery” peptide that can pass through the cell membrane and carry large polar molecules, including peptides, oligonucleotides, and proteins. For example, sequences from the TIFF2025106281000035.tif4128 and the Drosophila antennapedia protein from the TIFF2025106281000036.tif4128 have been found to be capable of functioning as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or a 1-bead 1-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of peptides or peptidomimetics tethered to the dsRNA agent via incorporated monomer units for the purpose of targeting cells are arginine-glycine-aspartic acid (RGD) peptides, or RGD mimetics. The peptide moiety can range from about 5 amino acids to about 40 amino acids in length. The peptide moiety can have structural modifications, such as to increase stability or direct conformational properties. Any of the following structural modifications can be utilized.

[0299] The RGD peptides for use in the compositions and methods of the present invention can be linear or cyclic and can be modified, for example, glycosylated or methylated, to promote targeting to a specific tissue. The RGD-containing peptides and peptidiomimetics can include not only D-amino acids but also synthetic RGD mimetics. In addition to RGD, other moieties targeting integrin ligands can be used. Preferred conjugates of this ligand target PECAM-1 or VEGF.

[0300] A "cell-penetrating peptide" has the ability to penetrate cells such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. Microbial cell-penetrating peptides can be, for example, α-helical linear peptides (such as LL-37 or Ceropin P1), disulfide bond-containing peptides (such as α-defensin, β-defensin or bacteriocin), or peptides containing only one or two dominant amino acids (such as PR-39 or indolicidin). The cell-penetrating peptide can also include a nuclear localization signal (NLS). For example, the cell-penetrating peptide can be a bipartite amphipathic peptide such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0301] C. Carbohydrate Conjugate In some aspects of the compositions and methods of the present invention, the iRNA further comprises a carbohydrate. Carbohydrate-conjugated iRNA is advantageous for in vivo delivery of compositions suitable for the in vivo therapeutic applications described herein, in addition to nucleic acids. As used herein, "carbohydrate" refers to a carbohydrate itself composed of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic), with oxygen, nitrogen or sulfur atoms bonded to each carbon atom; or a compound having as part of it a carbohydrate moiety composed of one or more monosaccharide units each having at least 6 carbon atoms (which can be linear, branched or cyclic), with oxygen, nitrogen or sulfur atoms bonded to each carbon atom. Representative carbohydrates include saccharides (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starch, glycogen, cellulose, and polysaccharide gums. Specific monosaccharides include saccharides with more than HBV (e.g., C5, C6, C7, or C8); disaccharides and trisaccharides include saccharides having 2 or 3 monosaccharide units (e.g., C5, C6, C7, or C8).

[0302] In one aspect, the carbohydrate conjugate for use in the compositions and methods of the present invention is a monosaccharide. In another aspect, the carbohydrate conjugate for use in the compositions and methods of the present invention is: selected from the group consisting of TIFF2025106281000037.tif227107TIFF2025106281000038.tif244123TIFF2025106281000039.tif242109TIFF2025106281000040.tif185128.

[0303] In one aspect, the monosaccharide is N-acetylgalactosamine as follows: TIFF2025106281000041.tif55128.

[0304] Other representative carbohydrate conjugates for use in the embodiments described herein include, without limitation, the following TIFF2025106281000042.tif72138If one of X or Y is an oligonucleotide, the other is hydrogen.

[0305] In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to the iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to the iRNA agent of the invention via a divalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to the iRNA agent of the invention via a trivalent linker.

[0306] In one embodiment, the double-stranded RNAi agent of the invention comprises one GalNAc or GalNAc derivative attached to the iRNA agent. In another embodiment, the double-stranded RNAi agent of the invention comprises a plurality (e.g., 2, 3, 4, 5, or 6) of GalNAc or GalNAc derivatives each independently attached to a plurality of nucleotides of the double-stranded RNAi agent via a plurality of monovalent linkers.

[0307] In some embodiments, for example, if the two strands of the iRNA agent of the invention are part of one larger molecule that is joined by an uninterrupted strand of nucleotides between the 3' end of one strand and the 5' end of each other strand to form a hairpin loop containing a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

[0308] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, without limitation, a PK modulator or a cell-penetrating peptide.

[0309] Additional carbohydrate conjugates suitable for use in the present invention include those described in PCT publications WO2014 / 179620 and WO2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0310] D. Linker In some embodiments, the conjugates or ligands described herein can be attached to the iRNA oligonucleotide using a variety of linkers that can be cleavable or non-cleavable.

[0311] The term "linker" or "linking group" means an organic moiety that connects two parts of a compound, e.g., covalently attaches to two parts of a compound.The linker typically contains a direct bond, or atoms such as oxygen or sulfur, units such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or, without limitation, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl (alkylhererocyclylalkynyl), alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl (alkynylhereroaryl), etc., where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocycle, where R8 is hydrogen, acyl, aliphatic or substituted aliphatic.In one embodiment, the linker is between about 1 and 24 atoms, between 2 and 24, 3 and 24, 4 and 24, 5 and 24, 6 and 24, 6 and 18, 7 and 18, 8 and 18, 7 and 17, 8 and 17, 6 and 16, 7 and 16, or 8 and 16 atoms.

[0312] The cleavable linking group is sufficiently stable outside the cell but, upon entering the target cell, is cleaved to release the two moieties held together by the linker. In a preferred embodiment, the cleavable linking group is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or about 100-fold faster inside the target cell or under a first reference condition (e.g., selected to mimic or represent intracellular conditions) or in the subject's blood or under a second reference condition (e.g., selected to mimic or represent conditions found in blood or serum) compared to outside the cell.

[0313] The cleavable linking group is sensitive to a cleavage agent, e.g., pH, redox potential, or the presence of a degradable molecule. Generally, the cleavage agent is more prevalent or found at a higher level or activity inside the cell compared to in serum or blood. Examples of such degrading agents include, for example, redox agents selected for a particular substrate or having no substrate specificity, including oxidoreductases or reducing agents such as mercaptans that can degrade a redox-cleavable linking group by oxidation or reduction by intracellularly present oxidoreductases; esterases; agents that can create an endosomal or acidic environment, e.g., an agent that brings about a pH of 5 or less; enzymes that can hydrolyze or degrade an acid-cleavable linking group by acting as a general acid, peptidases (which can have substrate specificity), and phosphatases.

[0314] Cleavable linking groups such as disulfide bonds can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, in the range of about 7.1 - 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 approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell or within a desired compartment of the cell.

[0315] The linker can include a cleavable linking group that is cleavable by a specific enzyme. The type of cleavable linking group incorporated into the linker can depend on the targeted cell. For example, a ligand targeting the liver can be linked to a cationic lipid via a linker containing an ester group. Since hepatocytes are rich in esterases, this linker is cleaved more efficiently within hepatocytes compared to cell types that are not rich in esterases. Other cell types rich in esterases include cells of the lung, renal cortex, and testis.

[0316] Linkers containing peptide bonds can be used when targeting cell types rich in peptidases such as hepatocytes and synoviocytes.

[0317] In general, the suitability of a cleavable candidate linking group can be evaluated by examining the ability of a degrading agent (or degrading conditions) to cleave the candidate linking group. It is also desirable to examine the ability of a cleavable candidate linking group to resist cleavage in blood or upon contact with other non-target tissues. Thus, a first condition is selected to exhibit cleavage within a target cell and a second condition is selected to exhibit cleavage within other tissues or in a biological fluid such as blood or serum, and the relative sensitivity to cleavage can be determined between the first condition and the second condition. This evaluation can be performed in a cell-free system, intracellularly, in cell culture, in an organ or tissue culture, or in a whole animal. It can be useful to perform an initial evaluation under cell-free or culture conditions and confirm by further evaluation in a whole animal. In a preferred embodiment, a useful candidate compound is cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0318] i. Redox-cleavable linking group In certain embodiments, the cleavable linking group is a redox-cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (-S-S-). To determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or, for example, suitable for use with a particular iRNA moiety and a particular targeting agent, one can look to the methods described herein. The candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents in the art using known reagents that mimic the cleavage rate observed intracellularly, e.g., within a target cell. The candidate can also be evaluated under conditions selected to mimic blood or serum conditions. In one of these, the candidate compound is cleaved by up to about 10% in blood. In other embodiments, a useful candidate compound is degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100-fold faster intracellularly (or under in vitro conditions selected to mimic intracellular conditions) compared to in blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetics assays under conditions selected to mimic intracellular media and compared to conditions selected to mimic extracellular media.

[0319] ii. Phosphate ester-based cleavable linking group In other embodiments, the cleavable linker comprises a phosphate ester-based cleavable linking group. The phosphate ester-based cleavable linking group is cleaved by an agent that decomposes or hydrolyzes the phosphate group. Examples of agents that cleave phosphate groups intracellularly are enzymes such as intracellular phosphatases. Examples of phosphate ester-based linking groups are -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-. Preferred embodiments are -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-, and -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidates can be evaluated using a method similar to the above method.

[0320] iii. Acid-cleavable linking group In other embodiments, 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 having a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0 or less), or by an agent such as an enzyme that can act as a general acid. Inside the cell, certain low-pH organelles such as endosomes and lysosomes can provide a cleavage environment for acid-cleavable linking groups. Examples of acid-cleavable linking groups include, without limitation, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). Preferred embodiments are those in which the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0321] iv. Ester-based linking groups In other embodiments, the cleavable linker comprises an ester-based cleavable linking group. Ester-based cleavable linking groups are cleaved by enzymes such as intracellular esterases and amylases. Examples of ester-based cleavable linking groups include, without limitation, esters of alkylene, alkenylene, and alkynylene groups. Ester-cleavable linking groups have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to the above methods.

[0322] v. Peptide-based cleavage groups In yet other embodiments, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases within the cell. The peptide-based cleavable linking group is a peptide bond formed between amino acids, giving rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. The peptide-based cleavable group does not include an amide group (-C(O)NH-). The amide group can be formed between any of alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids, giving rise to peptides and proteins. The peptide-based cleavage group is generally limited to the peptide bond (i.e., amide bond) formed between amino acids to give peptides and proteins, and does not include the entire amide functional group. The peptide-based cleavable linking group has the general formula: TIFF2025106281000043.tif4128, where RA and RB are the R groups of two adjacent amino acids. These candidates can be evaluated using a method similar to the above method.

[0323] In some embodiments, the iRNA of the present invention is conjugated to a carbohydrate via a linker. Non-limiting examples of iRNA carbohydrate conjugates with the linker of the compositions and methods of the present invention include, but are not limited to, the following: TIFF2025106281000044.tif88134TIFF2025106281000045.tif238151TIFF2025106281000046.tif49151If either X or Y is an oligonucleotide, the other is hydrogen.

[0324] In certain embodiments of the compositions and methods of the present invention, the ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached via a divalent or trivalent branched linker.

[0325] In certain embodiments, the dsRNA of the invention is conjugated to a divalent or trivalent branched linker selected from the group of structures shown in any of Formulas (XXXII)-(XXXV): TIFF2025106281000047.tif96150 wherein, q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C each independently represent from 0 to 20 each time they occur, and the repeating units can 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 independently represent absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH, or CH2O each time they occur; Q 2A 、Q 2B 、Q 3A 、Q 3B 、Q 4A 、Q 4B 、Q 5A 、Q 5B 、Q 5C each independently represent absent, alkylene, substituted alkylene, wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(R N ), C(R')=C(R"), C≡C, or C(O); R 2A 、R 2B 、R 3A 、R 3B 、R 4A 、R 4B 、R 5A 、R 5B 、R5C is, each time it appears independently, absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=N-O, TIFF2025106281000048.tif17128TIFF2025106281000049.tif14131 or is heterocyclyl; L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B , and L 5C represent ligands; that is, each time they appear independently, they are monosaccharides (e.g., GalNAc), disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides; R a is H or an amino acid side chain. Trivalent conjugate GalNAc derivatives such as the GalNAc derivatives of formula (XXXV) are particularly useful for use with RNAi agents to inhibit the expression of target genes: TIFF2025106281000050.tif39128 wherein L 5A , L 5B and L 5C represent monosaccharides such as GalNAc derivatives.

[0326] Examples of suitable divalent and trivalent branched linker groups for conjugating to GalNAc derivatives include, without limitation, the structures listed above as formulas II, VII, XI, X, and XIII.

[0327] Representative U.S. patents teaching the preparation of RNA conjugates include, without limitation, 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; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are incorporated herein by reference.

[0328] Not all positions of a given compound need to be uniformly modified, and in fact, more than one of the above modifications can be incorporated into a single compound or even into a single nucleoside within an iRNA. The invention also includes iRNA compounds that are chimeric compounds.

[0329] In the context of the present invention, a "chimeric" iRNA compound or "chimera" is an iRNA compound, preferably a dsRNA agent, containing at least two chemically distinct regions, each composed of at least one monomer unit, i.e., nucleotides in the case of a dsRNA compound. These iRNAs typically contain at least one region where the RNA is modified to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. The additional region of the iRNA can serve as a substrate for an enzyme capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby significantly enhancing the efficiency of iRNA inhibition of gene expression. As a result, when chimeric dsRNAs are used, equivalent results can often be obtained with shorter iRNAs compared to phosphorothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, by related nucleic acid hybridization techniques known in the art.

[0330] In some cases, the RNA of the iRNA can be modified by a non-ligand group. Some non-ligand molecules are conjugated to the iRNA to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for such conjugation are available from the scientific literature. Such non-ligand moieties include lipid moieties such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), and these can be used in the agents of the present invention.Other non-ligand moieties include lipid moieties, cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), thiocolesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmitoyl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such RNA conjugates are listed above.Typical conjugation protocols involve the synthesis of RNA having an amino linker at one or more positions of the array. Next, an appropriate coupling or activating reagent is used such that the amino group is reacted with the molecule to be conjugated. The conjugation reaction can be carried out either using RNA still attached to a solid support or following cleavage of the RNA in solution phase. Purification of the RNA conjugate by HPLC typically yields a pure conjugate.

[0331] IV. Delivery of the iRNA of the present invention Delivery of the iRNA of the present invention into cells, e.g., cells in a subject such as a human subject (e.g., a subject in need of iRNA such as a subject having a disease, disorder, or condition associated with the expression of the PD-L1 gene), can be carried out in several different ways. For example, delivery can be effected by contacting the cells with the iRNA of the present invention either in vitro or in vivo. In vivo delivery can also be effected directly by administering to the subject a composition comprising the iRNA, e.g., dsRNA. Alternatively, in vivo delivery can be effected indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternative embodiments are further described below.

[0332] In general, any method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the iRNAs of the invention (see, e.g., Akhtar S. and Julian RL. (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are hereby incorporated by reference in their entirety). For in vivo delivery, factors to be considered for delivering iRNA molecules include, for example, the biological stability of the molecule being delivered, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of iRNA can be minimized by local administration, for example, by direct injection or implantation into tissue, or by topical administration of the preparation. Local administration to the site of treatment maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may otherwise be adversely affected by or may degrade the agent, and allows for a lower total dose of the iRNA molecule being administered. Several studies have shown the success of gene product knockdown when dsRNAi agents are administered locally. For example, intravitreal delivery of VEGF dsRNA by injection in rhesus monkeys (Tolentino, MJ, et al (2004) Retina 24:132-138) and subretinal injection in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) have both been shown to prevent angiogenesis in experimental models of age-related macular degeneration. In addition, direct intratumoral injection of dsRNA in mice can reduce tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can extend the survival of tumor-bearing mice (Kim, WJ., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523).RNA interference has also shown success in local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, PH., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, GT., et al (2004) Neuroscience 129:521-528; Thakker, ER., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya,Y., et al (2005) J. Neurophysiol. 93:594-602) and local delivery to the lung by intranasal administration (Howard, KA., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). To systemically administer iRNA for the treatment of disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of dsRNA in vivo by endonucleases and exonucleases. Modification of the RNA or pharmaceutical carrier can allow targeting of the iRNA to the target tissue and also avoid unwanted off-target effects. The iRNA molecule can be modified by chemical conjugation to a lipophilic group such as cholesterol that enhances cellular uptake and prevents degradation. For example, an iRNA against ApoB conjugated to a lipophilic cholesterol moiety was systemically injected into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178).Conjugation of iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO, et al (2006) Nat. Biotechnol. 24:1005-1015). In alternative embodiments, the iRNA can be delivered using a drug delivery system such as a nanoparticle, dendrimer, polymer, liposome, or cationic delivery system. A positively charged cationic delivery system promotes binding of the iRNA molecule (which is negatively charged) and also enhances the interaction with the negatively charged cell membrane, enabling efficient uptake of the iRNA by the cell. Either binding a cationic lipid, dendrimer, or polymer to the iRNA or inducing the formation of vesicles or micelles that encapsulate the iRNA can be done (see, for example, Kim SH, et al (2008) Journal of Controlled Release 129(2):107-116). Formation of vesicles or micelles further protects the iRNA from degradation when administered systemically. Methods for making and administering cationic iRNA complexes are well within the capabilities of those skilled in the art (see, for example, Sorensen, DR, et al (2003) J. Mol. Biol 327:761-766; Verma, UN, et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al (2007) J. Hypertens. 25:197-205, which are hereby incorporated by reference in their entirety).Some non-limiting examples of drug delivery systems useful 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" (Zimmermann, TS, et al (2006) Nature 441:111-114), cardiolipin (Chien, PY, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet ME, et al (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, DA, et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, the iRNA forms a complex with cyclodextrin for systemic administration. Methods of administering the iRNA and cyclodextrin and pharmaceutical compositions can be found in U.S. Patent No. 7,427,605, which is hereby incorporated by reference in its entirety.

[0333] A. The iRNA of the invention encoded by the vector iRNAs targeting the PD-L1 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; International PCT Publication WO00 / 22113 by Skillern, A. et al., International PCT Publication WO00 / 22114 by Conrad, and U.S. Patent No. 6,054,299 by Conrad). Expression can be transient (from several hours to about several weeks) or persistent (from several weeks to several months or more) depending on the particular construct used and the target tissue or cell type. These transgenes can be introduced as linear constructs, circular plasmids, or viral vectors that can be either integrative or non-integrative vectors. The transgene can also be constructed to allow it to be inherited as an episomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0334] Individual one or more strands of the iRNA can be transcribed from a promoter on an expression vector. When attempting to express two separate strands, for example, to generate dsRNA, two separate expression vectors can be co-introduced into the target cells (e.g., by transfection or infection). Alternatively, each individual strand of the dsRNA can be transcribed by a promoter located 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 it has a stem-loop structure.

[0335] iRNA expression vectors are generally DNA plasmids or viral vectors. Recombinant constructs for the expression of the iRNAs described herein can be produced using expression vectors compatible with eukaryotic cells, preferably expression vectors compatible with vertebrate cells. Expression vectors for eukaryotic cells are well known in the art and are available from several commercial sources. Typically, such vectors are provided that contain convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of the iRNA expression vector can be systemic, such as by intravenous or intramuscular administration, or by administration to target cells explanted from the patient followed by reintroduction into the patient, or by any other means that allows introduction into the desired target cells.

[0336] Viral vector systems that can be utilized with the methods and compositions described herein include, without limitation, (a) adenoviral vectors; (b) retroviral vectors, including without limitation lentiviral vectors, Moloney murine leukemia virus, etc.; (c) adeno-associated viral vectors; (d) herpes simplex viral vectors; (e) SV40 vectors; (f) polyomavirus vectors; (g) papillomavirus vectors; (h) picornavirus vectors; (i) orthopox, e.g., vaccinia virus vectors or avipox, e.g., canarypox or fowlpox, etc., poxvirus vectors; and (j) helper-dependent or gutless adenoviruses. Replication-deficient viruses can also be advantageous. Different vectors may or may not become integrated into the genome of the cell. The construct can contain viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into a vector capable of episomal replication, such as EPV and EBV vectors. Constructs for the recombinant expression of iRNAs generally require regulatory elements, such as promoters, enhancers, etc., to ensure expression of the iRNA in the target cell. Other aspects to consider with respect to vectors and constructs are known in the art.

[0337] V. Pharmaceutical Compositions of the Present Invention The present invention also includes pharmaceutical compositions and formulations containing the iRNA of the present invention. In one aspect, provided herein is a pharmaceutical composition containing the iRNA described herein and a pharmaceutically acceptable carrier. The pharmaceutical composition containing iRNA is useful for treating diseases or disorders related to the expression or activity of the PD-L1 gene. Such pharmaceutical compositions are formulated based on the mode of delivery. An example is a composition formulated for systemic administration via parenteral delivery, such as subcutaneous (SC) or intravenous (IV) delivery. The pharmaceutical composition of the present invention can be administered in a dosage sufficient to inhibit the expression of the PD-L1 gene.

[0338] The pharmaceutical composition of the present invention can be administered in a dosage sufficient to inhibit the expression of the PD-L1 gene. Generally, the appropriate dosage of the iRNA of the present invention is in the range of about 0.001 to about 200.0 milligrams per day per kilogram of the recipient's body weight, and generally, it is in the range of about 1 to 50 mg / kg body weight per day. Typically, the appropriate dosage of the iRNA of the present invention is in the range of about 0.1 mg / kg to about 5.0 mg / kg, for example, in the range of about 0.3 mg / kg to about 3.0 mg / kg. The repeated dosing regimen can include administering a therapeutic amount of iRNA on a regular basis, such as once a day or once a year. In a particular aspect, the iRNA is administered about once a month to about once every quarter (i.e., about once every three months).

[0339] After the initial treatment regimen, the treatment can be administered at a lower frequency. For example, after administering weekly or biweekly for three months, the administration can be repeated once a month for six months, or for one year; or longer.

[0340] The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more partial doses at appropriate intervals throughout the day, or alternatively, can be administered using continuous infusion or delivery by a controlled release formulation. In that case, the iRNA contained in each partial dose must correspondingly be less so as to achieve the total daily dosage. For example, using a conventional sustained release formulation that provides for the sustained release of iRNA over a period of several days, the dosage unit can also be formulated for delivery over several days. Sustained release formulations are well known in the art and are particularly useful for the delivery of agents at a specific site so that they can also be used with the agents of the present invention. In this aspect, the dosage unit contains a corresponding multiple of the daily dose.

[0341] In other aspects, a single dose of the pharmaceutical composition can be of long duration such that subsequent doses are administered at intervals of 3, 4, or 5 days or less, or 1, 2, 3, or 4 weeks or less. In some aspects of the invention, a single dose of the pharmaceutical composition of the invention is administered once a week. In other aspects of the invention, a single dose of the pharmaceutical composition of the invention is administered once every two months (bi-monthly). In certain aspects, the iRNA is administered from about once a month to about once every three months (i.e., about once every quarter).

[0342] One of ordinary skill in the art will recognize that certain factors, including but not limited to, the disease or severity of the disease, previous treatments, the overall health or age of the subject, and other diseases present, can affect the dosage amount and timing required to effectively treat the subject. Further, treatment of a subject with a therapeutically effective amount of the composition can include a single treatment or a series of treatments. The determination of an effective dosage amount and the in vivo half-life estimation for an individual iRNA encompassed by the present invention can be made using conventional methodologies or based on in vivo testing using appropriate animal models known in the art.

[0343] For example, animal models of hepatitis B infection, including chimpanzee, woodchuck, and transgenic mouse models of HBV, are known in the art (Wieland, 2015. Cold Spring Harb. Perspect. Med., 5:a021469, 2015; Tennant and Gerin, 2001. ILAR Journal, 42:89-102; and Moriyama et al., 1990. Science, 248:361-364). The chimpanzee model can also be used as a model of hepatitis D infection. Numerous cancer models, including chemically induced tumors and xenograft tumors, are known in the art.

[0344] The pharmaceutical compositions of the present invention can be administered in several ways depending on whether local or systemic treatment is desired and on the area to be treated. Administration can be local (e.g., by transdermal patch), pulmonary by inhalation or insufflation of a powder or aerosol, including, for example, by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous via, for example, an implantable device; or intracranial by, for example, intrasubstantial, intrathecal or intraventricular administration.

[0345] iRNA can be delivered in a manner that targets specific tissues (e.g., hepatocytes).

[0346] Pharmaceutical compositions and formulations for topical or transdermal administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, aerosols, solutions, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickening agents, etc. may be necessary or desirable. Coated condoms, gloves, etc. can also be useful. Suitable topical formulations include those in which the iRNA taken up in the present invention is mixed with topical delivery agents such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents, and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoyl phosphatidylethanolamine DOPE, dimyristoyl phosphatidylcholine DMPC, distearoyl phosphatidylcholine), negative (e.g., dimyristoyl phosphatidylglycerol DMPG), and cationic (e.g., dioleoyl tetramethylaminopropyl DOTAP and dioleoyl phosphatidylethanolamine DOTMA). The iRNA taken up in the present invention can be encapsulated within liposomes or can form a complex with liposomes, particularly cationic liposomes. Alternatively, the iRNA can form a complex with lipids, particularly cationic lipids. Suitable fatty acids and esters include, without limitation, arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitine, acylcholine, or C 1-20 alkyl esters (e.g., isopropyl myristate IPM), monoglycerides, diglycerides, or pharmaceutically acceptable salts thereof). Topical formulations are described in detail in U.S. Patent No. 6,747,014, which is incorporated herein by reference.

[0347] A. iRNA Formulations Containing Membrane-Like Molecular Assemblies The iRNA for use in the compositions and methods of the present invention can be formulated for delivery in membranous molecular assemblies, such as liposomes or micelles. As used herein, the term "liposome" refers to vesicles composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or multiple bilayers. Liposomes include monolayer and multilamellar vesicles having a membrane formed from lipophilic materials and an aqueous interior. The aqueous portion contains the iRNA. The lipophilic material separates the aqueous exterior from the aqueous interior and typically does not contain the iRNA composition, although in some instances it may. Liposomes are useful for the transport and delivery of active ingredients to the site of action. Since the liposome membrane is structurally similar to biological membranes, when liposomes are applied to tissues, the bilayer of the liposome fuses with the bilayer of the cell membrane. As the merger of the liposome and the cell proceeds, the internal aqueous contents containing the iRNA are delivered into the cell, where the iRNA can specifically bind to the target RNA and mediate RNA interference. In some cases, the liposomes are specifically targeted, e.g., to direct the iRNA to a particular cell type.

[0348] Liposomes containing the iRNA agent can be prepared by various methods. In one example, the lipid components of the liposome are dissolved in a detergent such that micelles are formed with the lipid components. For example, the lipid components can be amphiphilic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include cholates, CHAPS, octyl glucoside, deoxycholates, and lauroyl sarcosine. Next, the iRNA agent preparation is added to the micelles containing the lipid components. The cationic groups in the lipid 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 obtain a liposome preparation of the iRNA agent.

[0349] If necessary, a carrier compound that aids condensation can be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to be favorable for condensation.

[0350] A method for producing a stable polynucleotide delivery vehicle incorporating a polynucleotide / cationic lipid complex as a structural component of the delivery vehicle is further described, for example, in WO96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of the exemplary methods described in 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. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of suitable size for use as delivery vehicles include extrusion in addition to sonication and freeze-thaw (see, for example, Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used if consistently small (50-200 nm), relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adaptable for packaging the iRNA agent preparation within liposomes.

[0351] Liposomes are divided into two major classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complex binds to the negatively charged cell surface and is internalized into the endosome. Due to the acidic pH within the endosome, the liposome ruptures and releases its contents into the cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

[0352] pH-sensitive, negatively charged liposomes encapsulate nucleic acids rather than forming complexes with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acids are encapsulated within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the foreign gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

[0353] One major type of liposome composition contains phospholipids other than naturally derived phosphatidylcholine. For example, neutral liposome compositions can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic membrane-fusion liposomes are mainly formed 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 from a mixture of two or more of phospholipids, phosphatidylcholine, and cholesterol.

[0354] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent Nos. 5,283,185 and 5,171,678; WO94 / 00569; WO93 / 24640; WO91 / 16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0355] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been tested and their usefulness in drug delivery to the skin has been determined. Using nonionic liposome formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether), cyclosporin-A was delivered to the dermis of mouse skin. The results showed that such nonionic liposome systems were effective in facilitating the accumulation of cyclosporin A within different layers of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4(6) 466).

[0356] Liposomes also include "sterically stabilized" liposomes, a term used herein to refer to liposomes that contain one or more specialized lipids which, when incorporated into the liposome, result in an increased circulation lifetime compared to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid moiety of the liposome is (A) monosialoganglioside G M1Those containing one or more glycolipids such as etc., or (B) those derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. While not wishing to be bound by any particular theory, in the art, at least with respect to sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the increased circulation half-life of these sterically stabilized liposomes is thought to be due to a reduction in uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

[0357] A variety of liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported that monosialoganglioside G M1 , galactosylcerebroside sulfate and phosphatidylinositol can improve the blood half-life of liposomes. These findings were elaborated by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). Both U.S. Patent No. 4,837,028 and International Publication No. 88 / 04924 granted to Allen et al. disclose liposomes containing (1) sphingomyelin and (2) ganglioside G M1 or galactosylcerebroside sulfate ester. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in International Publication No. 97 / 13499 (Lim et al.).

[0358] 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 being able to fuse with the plasma membrane as efficiently, can be taken up by macrophages in vivo and used to deliver iRNA agents to macrophages.

[0359] Further advantages of liposomes include the following: Liposomes derived from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; liposomes can protect the iRNA encapsulated within their inner compartment from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

[0360] Small liposomes can be formed using N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), a synthetic cationic lipid with a positive charge, which naturally interacts with nucleic acids to form lipid-nucleic acid complexes that have the ability to fuse with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in the delivery of iRNA agents (see, for example, Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Patent No. 4,897,355 for a description of DOTMA and its use with DNA).

[0361] 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), a DOTMA analog, can be used in combination with a phospholipid to form DNA-complexed vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids into living tissue culture cells, and the agent contains positively charged DOTMA liposomes that interact spontaneously with negatively charged polynucleotides to form complexes. When sufficiently positively charged liposomes are used, the resulting complexes also have a net positive charge. The positively charged complexes prepared in this way adhere spontaneously to the negatively charged cell surface, fuse with the plasma membrane, and efficiently deliver functional nucleic acids, for example, into tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonio)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana), differs from DOTMA in that the oleoyl moieties are linked by esters rather than ether bonds.

[0362] Other reported cationic lipid compounds include those conjugated to diverse moieties, including carboxyspermine conjugated to one of two lipids, for example, compounds such as 5-carboxyspermyldioctaoleyloylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermy-lamide (“DPPES”) (see, for example, U.S. Patent No. 5,171,678).

[0363] Another cationic lipid conjugate involves derivatization of lipids with cholesterol (``DC-Chol'') formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolyllysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). In certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for delivery of oligonucleotides are described in WO98 / 39359 and WO96 / 37194.

[0364] Liposomal formulations are particularly suitable for topical administration, and liposomes exhibit several advantages over other formulations. Such advantages include a reduction in side effects associated with high systemic absorption of the administered drug, an increase in the accumulation of the administered drug at the desired target, and the ability to administer the iRNA agent intradermally. In some embodiments, liposomes are used to deliver the iRNA agent to epidermal cells and also to enhance the penetration of the iRNA agent into dermal tissue, e.g., the skin. For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been demonstrated (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2, 405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

[0365] Nonionic liposomal systems, particularly systems containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in drug delivery to the skin. Nonionic liposomal formulations containing Novasome™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) have been used to deliver drugs to the dermis of mouse skin. Such formulations having an iRNA agent are useful for treating skin disorders.

[0366] Liposomes containing iRNA can be made highly deformable. Such deformability can enable the liposomes to permeate pores smaller than the average radius of the liposomes. For example, transfersomes are a type of deformable liposome. Transfersomes can be prepared by adding a surface activating agent, usually a surfactant, to a standard liposome composition. To deliver iRNA to keratinocytes of the skin, transfersomes containing iRNA can be delivered subcutaneously, for example, by infection. To pass through intact mammalian skin, the lipid vesicles must pass through a series of micropores each having a diameter of less than 50 nm under the influence of an appropriate transdermal gradient. In addition, due to their lipid properties, these transfersomes can be self-optimizing (e.g., conforming to the shape of the pores of the skin), self-healing, and can reach their target without frequent fragmentation and are often self-loading.

[0367] Other formulations suitable for the present invention are described in WO / 2008 / 042973.

[0368] Transferosomes are yet another type of liposomes and are highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transferosomes can be described as lipid droplets that, due to their high deformability, can easily penetrate through pores smaller than the droplets. Transferosomes are compatible with the environments in which they are used, e.g., they are self-optimizing (adapting to the shape of pores in the skin), self-healing, and in many cases reach their targets without fragmentation and are often self-loading. To prepare transferosomes, it is possible to add surface-activating agents, usually surfactants, to standard liposome compositions. Transferosomes have been used to deliver serum albumin to the skin. Delivery of serum albumin mediated by transferosomes has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0369] Surfactants have found wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and ranking the properties of numerous different types of surfactants, both natural and synthetic, is by use of the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for classifying the different surfactants used in formulations (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, N.Y., 1988, p.285).

[0370] When the surfactant molecules are not ionized, this surfactant is classified as a non-ionic surfactant. Non-ionic surfactants find wide applications in pharmaceuticals and cosmetics and can be used over a wide range of pH values. Generally, their HLB values range from 2 to about 18 depending on their structure. Non-ionic surfactants include non-ionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Also included in this class are non-ionic alkanolamides, and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers. Polyoxyethylene surfactants are the most common members of the class of non-ionic surfactants.

[0371] When the surfactant molecules have a negative charge when dissolved or dispersed in water, this surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, sulfate esters such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0372] When the surfactant molecules have a positive charge when dissolved or dispersed in water, this surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most commonly used members of this class.

[0373] When the surfactant molecules have the ability to have either a positive or negative charge, this surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkyl betaines, and phosphatides.

[0374] The use of surfactants in pharmaceutical products, formulations, and emulsions has been reviewed (Rieger, in "Pharmaceutical Dosage Forms", Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0375] The iRNA for use in the method of the present invention can also be provided as a micelle formulation. "Micelle" is defined herein as a particular type of molecular aggregate in which all the hydrophobic parts of the molecules are oriented inward and the amphiphilic molecules are arranged in a spherical structure with the hydrophilic parts remaining in contact with the surrounding aqueous phase. When the environment is hydrophobic, the reverse arrangement exists.

[0376] A mixed micelle formulation suitable for delivery by a transdermal membrane can be prepared by mixing an aqueous solution of iRNA, an alkali metal C8 - 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, monoolein, monooleate, monolaurate, boridi oil, evening primrose oil, menthol, trihydroxyoxocolanyl glycine and its pharmaceutically acceptable salts, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ether and its analogs, polidocanol alkyl ether and its analogs, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle-forming compound can be added simultaneously with or after the addition of the alkali metal alkyl sulfate. The mixed micelles are formed with a substantially arbitrary type of mixing of the components, but are formed with strong mixing to provide smaller-sized micelles.

[0377] In one method, a first micelle composition containing RNAi and at least an alkali metal alkyl sulfate is prepared. Next, the first micelle composition is mixed with at least three micelle-forming compounds to form a mixed micelle composition. In another method, the 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 compounds while mixing strongly.

[0378] Phenol or m-cresol can be added to the mixed micelle composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol or m-cresol may be added together with the micelle-forming components. An isotonic agent such as glycerin can also be added after the formation of the mixed micelle composition.

[0379] For delivery as a spray of the micelle formulation, the formulation can be placed in an aerosol dispenser, and the dispenser is filled with a propellant. The propellant under pressure is liquid in the dispenser. The ratio of the components is adjusted so that the aqueous phase and the propellant phase become one, that is, so that there is one phase. If two phases are present, for example, it is necessary to shake the dispenser before supplying a part of the contents via a metering valve. The dosing of the drug is sprayed in a fine spray form from the metering valve.

[0380] The propellant can include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether, and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2-tetrafluoroethane) can be used.

[0381] The specific concentration of the essential components can be determined by relatively simple experiments. For absorption via the oral cavity, it is often desirable to increase the dosage for administration by injection or via the gastrointestinal tract, for example, by at least two or three times.

[0382] B. Lipid Particles The iRNA of the present invention, i.e., the dsRNA agent, can be completely encapsulated in a lipid formulation, for example, in an LNP, or other nucleic acid-lipid particles.

[0383] As used herein, the term "LNP" refers to stable nucleic acid-lipid particles. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particles (e.g., a PEG-lipid conjugate). LNPs exhibit an extended circulation lifetime after intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site), making them extremely useful for systemic applications. LNPs include "pSPLP", which contains an encapsulated condensing agent-nucleic acid complex as shown in PCT publication WO00 / 03683. The particles of the present invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. In addition, the nucleic acid is resistant to nuclease degradation in an aqueous solution when present in the nucleic acid-lipid particles of the present invention. Nucleic acid-lipid particles, and methods for their preparation, are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Patent Application Publication No. 2010 / 0324120 and PCT publication WO96 / 40964.

[0384] In one aspect, the lipid-to-drug ratio (mass / mass ratio) (e.g., the lipid-to-dsRNA ratio) is 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. Intermediate ranges within the ranges cited above are also intended to be part of the present invention.

[0385] Cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley (Dilinoley)oxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.(Cl), 1,2-dilinoleoyl-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or an analog thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can constitute about 20 mol% to about 50 mol% or about 40 mol% of the total lipids present in the particles.

[0386] In some embodiments, the compound 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles.

[0387] In some embodiments, the lipid-siRNA particles contain 40% 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane, 10% DSPC, 40% cholesterol, and 10% PEG-C-DOMG (mole percent), have a particle size of 63.0 ± 20 nm, and an siRNA / lipid ratio of 0.027.

[0388] Ionizable / non-cationic lipids can be, without limitation, anionic or neutral lipids including, for example, 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 phosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidy(phosphatidy)ethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid can be about 5 mol% to about 90 mol%, about 10 mol%, or about 58 mol% of the total lipids present in the particles when cholesterol is included.

[0389] Conjugated lipids that inhibit aggregation of the particles can be, for example, polyethylene glycol (PEG)-lipids including, without limitation, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate can be, for example, PEG-dilauryl oxypropyl (Ci2), PEG-dimyristyl oxypropyl (Ci4), PEG-dipalmityl oxypropyl (Ci6), or PEG-distearyl oxypropyl (C]8). The conjugated lipid that prevents aggregation of the particles can be 0 mol% to about 20 mol% or about 2 mol% of the total lipids present in the particles.

[0390] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol at, for example, about 10 mol% to about 60 mol% or about 48 mol% of the total lipids present in the particles.

[0391] In one embodiment, Lipidoid ND98·4HCl (MW 1487) (see US20090023673, which is incorporated herein by reference), cholesterol (Sigma-Aldrich), and PEG-ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg / ml; cholesterol, 25 mg / ml, PEG-ceramide C16, 100 mg / ml. Next, the ND98, cholesterol, and PEG-ceramide C16 stock solutions can be mixed, for example, at a molar ratio of 42:48:10. The resulting lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate (pH 5)) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be passed through a polycarbonate membrane (e.g., 100 nm cutoff) using a thermobarrel extruder such as, for example, a Lipex Extruder (Northern Lipids, Inc) and extruded. Optionally, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration. The buffer can be exchanged for, for example, phosphate buffered saline (PBS) at about pH 7, for example, 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. TIFF2025106281000051.tif48165

[0392] The LNP01 formulation is described, for example, in International Publication No. WO 2008 / 042973, which is incorporated herein by reference in its entirety.

[0393] Additional exemplary lipid-dsRNA formulations are described in Table 1.

[0394] (Table 1) Exemplary lipid formulations TIFF2025106281000052.tif58164TIFF2025106281000053.tif242164TIFF2025106281000054.tif188164DSPC: Distearoyl phosphatidylcholine DPPC: Dipalmitoyl phosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with an average molecular weight of 2000) PEG-DSG: PEG-distearyl glycerol (C18-PEG, or PEG-C18) (PEG with an average molecular weight of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with an average molecular weight of 2000)

[0395] Formulations containing SNALP (1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) are described in International Publication No. WO 2009 / 127060, which is incorporated herein by reference in its entirety.

[0396] Formulations containing XTC are described, for example, in PCT Publication No. WO2010 / 088537, which is incorporated herein by reference in its entirety.

[0397] Formulations containing MC3 are described, for example, in U.S. Patent Application Publication No. 2010 / 0324120, filed on June 10, 2010, which is incorporated herein by reference in its entirety.

[0398] Formulations containing ALNY-100 are described, for example, in PCT Publication WO2010 / 054406, the entire content of which is incorporated herein by reference.

[0399] Formulations containing C12-200 are described in PCT Publication WO2010 / 129709, the entire content of which is incorporated herein by reference.

[0400] i. Synthesis of ionizable / cationic lipids Any of the compounds used in the nucleic acid-lipid particles of the present invention, such as cationic lipids, can be prepared by known organic synthesis techniques.

[0401] Formulations prepared by either standard or extrusion-free methods can be characterized in a similar manner. For example, formulations are typically characterized by visual inspection. The formulation should be a milky translucent solution free of aggregates or precipitates. The particle size and particle size distribution of the lipid-nanoparticles can be measured, for example, by light scattering using a Malvern Zetasizer Nano ZS (Malvern, USA). The particles should be on the order of about 20 - 300 nm, such as 40 - 100 nm. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, and the encapsulated fraction, are estimated using a dye exclusion assay. Samples of formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes), in the presence or absence of a formulation-disrupting surfactant, such as 0.5% Triton®-X100. The total dsRNA in the formulation can be determined by the signal from the surfactant-containing sample compared to a standard curve. The encapsulated fraction is determined by subtracting the "free" dsRNA content (measured by the signal in the absence of surfactant) from the total dsRNA content. The percentage of encapsulated dsRNA is typically > 85%. For SNALP formulations, the particle size is at least 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, or 120 nm. Suitable ranges are typically 50 nm - 110 nm, 60 nm - 100 nm, or 80 nm - 90 nm.

[0402] Compositions and formulations for oral administration include powders or granules, microparticles, nanoparticles, suspensions, or solutions in aqueous or non-aqueous media, capsules, gel capsules, sachets, tablets or mini-tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids or binders may be desirable. In some embodiments, the oral formulation is one in which the dsRNA taken up in the present invention is administered together with one or more permeation enhancers, surfactants and chelating agents. Suitable surfactants include fatty acids or their esters or salts, bile acids or their salts. Suitable bile acids / salts include chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), cho...

Claims

【Claim 1】 The invention described in the specification of this application.

Images