OA20581A

OA20581A - Programmed cell death 1 ligand 1 (PD-L1) iRNA compositions and methods of use thereof

Info

Publication number: OA20581A
Application number: OA1201800083
Authority: OA
Inventors: Gregory Hinkle
Original assignee: Alnylam Pharmaceuticals, Inc.
Filing date: 2016-08-22
Publication date: 2022-11-29

Abstract

The present invention relates to RNAi agents, e.g., double stranded RNAi agents, targeting the programmed cell death 1 ligand 1 (PD-L1) gene, and methods of using such RNAi agents to inhibit expression of a PD-L1 gene and methods of treating subjects having a PD-L1-associated disorder

Description

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

Related Applications

This application daims priority to U.S. Provisional Application No. 62/213,224, filed on

September 2, 2015, the entire contents of which are hereby încorporated hereîn by reference.

Sequence Listing

The instant application contains a Sequence Listing which has been submitted electronically 10 in ASCII format and is hereby încorporated by reference in its entirety. Said ASCII copy, created on July 29, 2016, is named 121301-04220_SL.txt and is 108,003 bytes in size.

Background of the Invention

Programmed cell death 1 ligand 1 (PD-L1) is a 290 amino acid type I transmembrane 15 protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. PD-Ll expression is involved in évasion of immune responses involved in chronic infection, e.g., chronic viral infection (includîng, for example, HIV, HBV, HCV and HTLV, among others), chronic bacterial infection (including, for example, Hélicobacter pylori, among others), and chronic parasitic infection (including, for example, Schistosoma mansoni). PD-Ll expression has been detected in a 20 number of tissues and cell types including T-cells, B-cells, macrophages, dendritic cells, and nonhematopoietic cells including endothélial cells, hépatocytes, muscle cells, and placenta.

PD-L1 expression is also involved in suppression of anti-tumor immune activity. Tumors express antigens that can be recognized by host T cells, but immunologie clearance of tumors is rare. Part of thîs faîlure is due to immune suppression by the tumor microenvironment. PD-Ll expression 25 on many tumors is a component of this s oppressive milieu and acts in concert with other immunosuppressive signais. PD-Ll expression has been shown in situ on a wide variety of solid tumors including breast, lung, colon, ovarian, melanoma, bladder, liver, salivary, stomach, gliomas, thyroid, thymie épithélial, 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, XuN. 2006. Acta Histochem. 108:19-24). In addition, the expression of the receptor for PD-L 1, 35 Programmed cell death protein 1 (also known as PD-1 and CD279) is upregulated on tumor infiltrating lymphocytes, and this also contributes to tumor immunosuppression (Blank C et al. 2003. J. Immunol. 171:4574—81). Most importantly, studies relating PD-Ll expression on tumors to disease outcome show that PD-L 1 expression strongly correlates with unfavorable prognosis in kîdney, ovarian, bladder, breast, gastnc, and pancreatic cancer (Hamanishi J et al. 2007. Proc. Natl. Acad. Sci. USA 104:3360-65; Inman BA étal. 2007. Cancer 109:1499-505; Konishi J étal. 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 étal. 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 facilitate advancement of tumor stage and invasion into deeper tissue structures.

The PD-1 pathway can also play a rôle in hématologie malignancies. PD-Ll is expressed on multiple myeloma cells but not 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 lymphomas (Brown JA et al., 2003. J. Immunol. 170:1257-66). PD-1 is highly expressed on the T cells of angioimmunoblastic lymphomas, and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman DM étal. 2006. Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin lymphoma, the T cells associated with lymphocytic or histiocytic (L&H) cells express PD-1. Microarray analysis using a readout of genes induced by PD-1 ligation suggests that tumor-associated T cells are responding to PD-1 signais in situ in Hodgkin lymphoma (Chemnitz JM et al. 2007. Blood 110:3226-33). PD-1 and PD-Ll are expressed on CD4 T cells in HTLV-Lmediated adult T cell leukemia and lymphoma (Shimauchî T et al. 2007. Int. J. Cancer 121: 2585-90). These tumor cells are hyporesponsive to TCR signais.

Studies in animal models demonstrate that PD-Ll on tumors inhibits T cell activation and lysis of tumor cells and in some cases le ad s to increased tumor-specifîc 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 utîlîze the PD-1 :PD-L pathway to control antîtumor T cell responses. PD-Ll expression on a population of tumor-associated myeloid DCs is upregulated by tumor environm entai factors (Curiel TJ et al. 2003. Nat. Med. 9:562-67). Plasmacytoid dendritic cells (DCs) in the tumor-draining lymph node of B16 melanoma express IDO, which strongly activâtes the suppressive activity of regulatory T cells. The suppressive activity of IDO-treated regulatory T cells required cell contact with IDOexpressing DCs (Sharma MD et al. 2007. J. Clin. Invest. 117:2570-82).

Accordingly, there is a need in the art for effective treatments for PD-Ll-associated diseases, such as an infectious disease, such as a chronic intracellular infectious disease, e.g., a viral disease, e.g, hepatitis infection, or a bacterial infection, e.g., tuberculosis infection; and cancer, e.g., a hepatic cancer, e.g., hepatocellular carcinoma.

Summary of the Invention

The présent invention provides iRNA compositions which affect the RNA-induced silencing complex (RlSC)-mediated cleavage of RNA transcripts of a PD-Ll gene. The PD-Ll gene may be within a cell, e.g., a cell within a subject, such as a human.

Accordtngly, in one aspect, the invention provides a double stranded ribonucleic acid (RNAi) agent for inhibiting expression of programmed cell death 1 ligand 1 (PD-L1), wherein the RNAi comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucieotîdes differîng by no more than 3 nucléotides from the nucléotide sequence of SEQ ID NO: 1 and the antisense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucieotîdes from the nucléotide sequence of SEQ ID NO:2.

In certain embodiments, the sense strands and antisense strands comprise sequences selected from any of the sequences in Table 3. In other embodiments, the sense strands and antisense strands comprise sequences selected from any of the sequences in Table 5.

In an aspect, the invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of programmed cell death 1 ligand 1 (PD-L1), wherein the RNAi comprises a sense strand and an antisense strand, the antisense strand comprising a région of complementarity whîch comprises at least 15 contiguous nucieotîdes differing by no more than 3 nucieotîdes from any one of the antisense sequences listed in Table 3. In certain embodiments, the sense strands and antisense strands comprise sequences selected from any of the sequences in Table. 5

In certain embodiments, the RNAi comprises at least one modified nucléotide. In some embodiments, substantially ail of the nucieotîdes of the sense strand and ail of the nucieotîdes of the antisense strand comprise a modification. In some embodiments, ail of the nucieotîdes of the sense strand and ail of the nucieotîdes of the antisense strand comprise a modification.

In one aspect, the présent invention provides double stranded ribonucleic acid (RNAi) agents for inhibiting expression of programmed cell death 1 ligand 1 (PD-L1 ), wherein the RNAi agents comprise a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucieotîdes differing by no more than 3 nucieotîdes from any one of nucieotîdes 32213243, 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, 26582679, 2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3222-3243 ofthe nucléotide sequence of SEQ ID NO: 1 and the antisense strand comprises at least 15 contiguous nucieotîdes differing by no more than 3 nucieotîdes from the complementary portion of the nucléotide sequence of SEQ ID NO:2, and wherein the RNAi agent comprises at least one modified nucléotide.

In another aspect, the présent invention provides double stranded ribonucleic acid (RNAi) agents for inhibiting expression of programmed ce h death 1 ligand 1 (PD-L1), wherein the RNAi agents comprise a sense strand and an antisense strand, the antisense strand comprising a région of complementarity whîch comprises at least 15 contiguous nucieotîdes differing by no more than 3 nucieotîdes from any one of the antisense sequences in any one of the duplexes AD-67635, AD67637, 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, AD67664, AD-67662, AD-67660, AD-67656, AD-67628, AD-67647, AD-67626, or AD-67645.

In one embodiment, the sense and antisense strands comprise nucieotide sequences seiected from the group consisting of any one of the nucieotide sequences in any one of the duplexes AD67635, 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, AD67673, AD-67664, AD-67662, AD-67660, AD-67656, AD-67628, AD-67647, AD-67626, or AD67645.

In one aspect, the invention provides a double stranded RNAi agent for inhibiting expression of programmed cell death 1 ligand 1 (PD-L1), whereîn the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded région, wherein the sense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the nucieotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the nucieotide sequence of SEQ ID NO:2, wherein substantially ail of the nucléotides of the sense strand and substantially ail of the nucléotides of the antisense strand are modified nucléotides, and wherein the sense strand is conjugated to a ligand attached at the 3’-terminus.

In one aspect, the présent invention provides double stranded RNAi agents for inhibiting expression of PD-L1, which comprise a sense strand and an antisense strand forming a double stranded région, wherein the sense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from nucléotides 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, 2220-2261,22202241, 2240-2261, 2648-2680, 2648-2669, 2658-2679, 2659-2680, 3143-3164, 3198-3219,3221-3242, or 3222-3243 of the nucieotide sequence of SEQ ID NO: 1 and the antisense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the complementary corresponding position of the nucieotide sequence of SEQ ID NO:2 such that the antisense strand îs complementary to the at least 15 contiguous nucléotides differing by no more than 3 nucléotides in the sense strand.

In certain embodiments, the sense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from nucléotides 3221-3243, 351-372, 1093-1115, 10931114, 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 nucieotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the complementary corresponding position of the nucieotide sequence of SEQ ID NO:2 such that the antisense strand is complementary to the at least 15 contiguous nucléotides differing by no more than 3 nucléotides in the sense strand.

In certain embodiments, the sense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from nucléotides 3221-3243, 1093-1115, 1093-1114, 10941115, 3221-3242, or 3222-3243 of the nucieotide sequence of SEQ ID NO: l and the antisense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the complementary correspondmg position of the nucléotide sequence of SEQ ID NO:2 such that the antisense strand is complementary to the at least 15 contiguous nucléotides differing by no more than 3 nucléotides in the sense strand.

In another aspect, the présent invention provides double stranded ribonucleic acid (RNAi) agents for inhibiting expression of programmed cell death 1 ligand 1 (PD-L1), wherein the double stranded RNAi agents comprise a sense strand and an antisense strand forming a double stranded région, wherein the sense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from any one of nucléotides 3221-3243, 351-372, 618-641, 618-639, 619-640, 620641, 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, 32213242, or 3222-3243 of the nucléotide sequence of SEQ ID NO: 1 and said antisense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the complementary portion ofthe nucléotide sequence of SEQ ID NO:2, wherein substantially ail of the nucléotides of said sense strand and substantially ail of the nucléotides of said antisense strand comprise nucléotide modifications, and wherein the sense strand is conjugated to a ligand attached at the 3’-terminus.

In certain embodiments, substantially ail of the nucléotides of the sense strand or substantially ail ofthe nucléotides ofthe antisense strand are modified nucléotides, or substantially ail ofthe nucléotides of both strands are modified; and wherein the sense strand is conjugated to a ligand attached at the 3’-terminus.

In one aspect, the présent invention provides double stranded RNAi agents for inhibiting expression of PD-L1, which comprise a sense strand and an antisense strand forming a double stranded région, wherein the sense strand comprises at least 15 contiguous nucléotides from nucléotides 3221-3243,351-372,618-641,618-639,619-640, 620-641, 1093-1115, 1093-1114, 10941115, 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 ofthe nucléotide sequence of SEQ ID NO; 1 and the antisense strand comprises at least 15 contiguous nucléotides from the complementary correspondmg position of the nucléotide sequence of SEQ ID NO:2 such that the antisense strand is complementary to the at least 15 contiguous nucléotides in the sense strand.

In certain embodimetns, the agents comprise a sense strand and an antisense strand forming a double stranded région, wherein the sense strand comprises at least 15 contiguous nucléotides from nucléotides 3221-3243, 351-372, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 15181539, 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 nucléotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucléotides from the complementary corresponding position ofthe nucléotide sequence of SEQ ID NO:2 such that the antisense strand is complementary to the at least 15 contiguous nucléotides in the sense strand.

In certan embodiments, the agents comprise a sense strand and an antisense strand forming a double stranded région, wherein the sense strand comprises at least 15 contiguous nucléotides front nucléotides 3222-3243 1093-1115, 1093-1114, 1094-1115,3221 -3243, or 3221 -3242, of the nucléotide sequence of SEQ ID NO: 1 and the antisense strand comprises at least 15 contiguous nucléotides from the complemenîary corresponding position of the nucléotide sequence of SEQ ID NO:2 such that the antisense strand is complementary to the at least 15 contiguous nucléotides in the sense strand. In certain embodiments, substantially ail of the nucléotides of the sense strand are modified nucléotides. In certain embodiments, substantially ail of the nucléotides of the antisense strand are modified nucléotides. In certain embodiments, substantially ail of the nucléotides of both strands are modified. In preferred embodiments, the sense strand is conjugated to a ligand attached at the 3’-tenninus.

In certain embodiments, the sense strand and the antisense strand comprise a région of complementarîty which comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from any one of the antisense sequences lîsted in any one of Tables 3 and 5. For example, in certain embodiment, the sense strand and the antisense strand comprise a région of complementarîty which comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from any one of the antisense sequences of the duplexes AD-67635, AD-67637, AD67658, 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, AD67662, 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 région of complementarîty which comprises at least 15 contiguous nucléotides of any one of the antisense sequences of the foregoing duplexes.

In some embodiments, ail of the nucléotides of the sense strand and ail of the nucléotides of the antisense strand comprise a modification. In one embodiment, at least one ofthe modified nucléotides is selected from the group consisting of a deoxy-nucleotide, a 3’-terminal deoxy-thymine (dT) nucléotide, a 2’-O-methyl modified nucléotide, a 2'-fluoro modified nucléotide, a 2'-deoxymodified nucléotide, a locked nucléotide, an unlocked nucléotide, a conformatîonally restricted nucléotide, a constrained ethyl nucléotide, an abasic nucléotide, a2’-amino-modified nucléotide, a 2’O-allyl-modified nucléotide, 2’-C-alkyl-modified nucléotide, 2’-hydroxly-modified nucléotide, a 2’methoxyethyl modified nucléotide, a 2’-O-alkyl-modified nucléotide, a morpholino nucléotide, a phosphoramîdate, a non-naturai base comprisîng nucléotide, a tetrahydropyran modified nucléotide, a 1,5-anhydrohexitol modified nucléotide, a cyclohexenyl modified nucléotide, a nucléotide comprising a phosphorothioate group, a nucléotide comprising a methylphosphonate group, a nucléotide comprising a 5’-phosphate, and a nucléotide comprising a 5’-phosphate mimic. In another embodiment, the modified nucléotides comprise a short sequence of 3’-terminal deoxy-thymine nucléotides (dT).

In certain einbodiments, substantially ail of the nucléotides of the sense strand are modified.

In certain embodiments, substantially ail of the nucléotides of the antisense strand are modified. In certain embodiments, substantially ail of the nucléotides of both the sense strand and the antisense strand are modified.

In certain einbodiments, the duplex comprises a modified antisense strand provided in Table

5. In certain embodiments, the duplex comprises a modified sense strand provided in Table 5. In certain embodiments, the duplex comprises a modified duplex provided in Table 5.

In certain embodiments, the région of complementarity between the antisense strand and the target is at least 17 nucléotides in length. For example, the région of complementarity between the 10 antisense strand and the target is 19 to 21 nucléotides in length, for example, the région of complementarity is 21 nucléotides in length. In preferred embodiments, each strand is no more than 30 nucléotides in length.

In some embodiments, at least one strand comprises a 3’ overhang of at least 1 nucléotide, e.g., at least one strand comprises a 3’ overhang of at least 2 nucléotides.

In many embodiments, the RNAÎ agent further comprises a ligand. The ligand can be conjugated to the 3’ end of the sense strand of the RNAi agent. The ligand can be an Nacetylgalactosamine (GalNAc) dérivative including, but not limîted to

An exemplary RNAi agent conjugated to the ligand as shown in the following schématie:

and, wherein X is O or S. In one embodiment, the X is O.

In certain embodiments, the ligand can be a cholestérol moiety.

In certain embodiments, the région of complementarity comprises one of the antisense sequences of any one of Table 3 and Table 5. In another embodiment, the région of complementarity consists of one of the antisense sequences of any one of Table 3 and Table 5.

In another aspect, the invention provides a double stranded 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, wherein the antisense strand comprises a région complementary to part of an rnRNA encoding PD-L1, wherein each strand is about 14 to about 30 nucléotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5' n_p -N_a -(X X X) _;-N_b -Y Y Y -N_b -(Z Z Z)j -N_a - n_q 3 ’ antisense: 3* n_p'-N_a'-(X'X'X')_k-N_b'-Y'Y'Y'-N_b'-(Z'Z'Z')_rN_a'- n_q' 5’ (III) wherein: i, j, k, and 1 are each independently 0 or l ; p, p’, q, and q' are each independently 06; each N_a and N_a' independently represents an oligonucleotide sequence comprising 0-25 nucléotides whîch are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucléotides; each N_b and N_b' independently represents an oligonucleotide sequence comprising 0-10 nucléotides which are either modified or unmodified or combinations thereof; each n_p, n_p', n_q, and r^’, each of which may or may not be présent, independently represents an overhang nucléotide; XXX, YYY, ZZZ, X'X'X’, ΥΎΎ', and Z'Z'Z' each independently represent one motif ofthree îdentical modifications on three consecutive nucléotides; modifications on N_b differ from the modification on Y and modifications on N_b' differ from the modification on Y'; and wherein the sense strand is conjugated to at least one ligand.

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

For example, formula (III) can be represented by formula (Ilia):

sense: 5’ n_p -N_a -YYY -N_a - n_q 3' antisense: 3' n_P'-N_a'- ΥΎΎ'- N_a- n_q· 5' (IIla).

In another embodiment, formula (III) is represented by formula (Illb): sense; 5’ n_p -N_a -YYY -N_b -Z Z Z -N_a - n, 3' antisense: 3’ n_rN_a- ΥΎΎ'-NrZ'Z'Z'- N_a- n^ 5’ (Illb) wherein each N_b andN_b' independently represents an oligonucleotide sequence comprising 15 modified nucléotides.

Altematively, formula (III) can be represented by formula (IIIc): sense: 5’ n_p -N_a -XXX -N_b -Y Y Y -N, -n, 3’ antisense: 3’ n_p-N_a- X'X'X '-N_b- ΥΎΎ'- N_a- n,- 5’ (IIIc) wherein each N_b and N_b' independently represents an oligonucleotide sequence comprising I5 modified nucléotides.

Further, formula (III) can be represented by formula (IIId):

sense: 5' n_p -N. -X X X- N_b -Y Y Y -N_b -Z Z Z -N_a - n_q 3' antisense: 3' n_p-N_a- X'X'X'- N_b-Y'Y'Y'-N_b-Z'Z'Z'- N_s- n_q. 5' (IIId) wherein each N_b and N_b' independently represents an oligonucleotide sequence comprising 15 modified nucléotides and each N_a and N_a' independently represents an oligonucleotide sequence comprising 2-10 modified nucléotides.

In certain embodiment, the double stranded région is 15-30 nucléotide pairs in length. For example, the double stranded région can be 17-23 nucléotide pairs in length. The double stranded région can be 17-25 nucléotide pairs in length. The double stranded région can be 23-27 nucléotide pairs in length. The double stranded région can be 19-21 nucléotide pairs in length. The double stranded région can be 21 -23 nucléotide pairs in length.

In certain embodîments, each strand has 15-30 nucléotides. In other embodiments, each strandhas 19-30 nucléotides.

Modifications on the nucléotides may be selected from the group inciudîng, but not limited to, 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 modifications on the nucléotides are 2'-O-methyl or 2^r-fluoro modifications.

In certain embodiments, the ligand is one or more GalNAc dérivatives attached through a monovalent linker or a bivalent or trivalent branched linker. In one embodiment, the ligand is

The ligand can be attached to the 3' end of the sense strand.

An exemplary structure of a RNAi agent conjugated to the ligand is shown in the fbilowing schematic

In certain embodiments, the ligand can be a cholestérol moiety.

In certain embodiments, the RNAi agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. For example the phosphorothioate or methylphosphonate internucleotide linkage can be at the 3’-terminus of one strand. Le., the sense strand or the antisense strand; or at the ends of both strands, the sense strand and the antisense strand.

In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is atthe 5’-terminus of one strand, Le., the sense strand or the antisense strand; or at the ends ofboth strands, the sense strand and the antisense strand.

In certain embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5’- and 3’-terminus of one strand, Le., the sense strand or the antisense strand; or at the ends ofboth strands, the sense strand and the antisense strand.

In certain embodiments, the base pair at the 1 position of the 5'-end ofthe antisense strand of the duplex is an AU base pair.

in certain embodiments, the Y nucléotides contaîn a 2'-fluoro modification. In another embodiment, the Y' nucléotides contain 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 p’ overhang nucléotides are complementary to the target mRNA. In some embodiments, q’=0, p=0, q=0, and p’ overhang nucléotides are non-complementary to the target mRNA.

In certain embodiments, the sense strand has a total of 21 nucléotides and the antisense strand has a total of 23 nucléotides.

In certain embodiments, at least one n_p' is linked to a neîghboring nucléotide via a phosphorothioate linkage. In other embodiments, ail n_p' are linked to neîghboring nucléotides via phosphorothioate linkages.

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, ail of the nucléotides of the sense strand and ail of the nucléotides of the antisense strand comprise a modification.

In one aspect, the invention provides a double stranded RNAi agent capable of inhibîting the expression of PD-L1 in a cell, wherein the double stranded RNAi agent comprises a sense strand 30 complementary to an antisense strand, wherein the antisense strand comprises a région complementary to part of an mRNA encoding PD-L1, wherein each strand is about 14 to about 30 nucléotides în length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5' n_p -N_a -(XXX) _rN_b -Y Y Y -N_b -(Z Z Z)₃ -N_a - n_q 3' antisense: 3’ n_p'-N,'-(XX'XWY^YW n_q' 5' (III) wherein i, j, k, and 1 are each independently 0 or 1 ; p, p’, q, and q' are each independently 0-6; each N_a and N_a' independently represents an oligonucleotide sequence comprising 0-25 nucléotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two dîfferently modified nucléotides; each N_b and N_b' independently represents an oligonucleotide sequence comprising 0-10 nucléotides which are either modified or unmodified or combinations thereof; each n_p, n_p', n_q, and n/, each of which may or may not be présent independently represents an overhang nucléotide; XXX, YYY, ZZZ, X'X'X', ΥΎΥ, and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucléotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; modifications on N_b differ front the modification on Y and modifications on N_b' differ from the modification on Y'; and wherein the sense strand is conjugated to at least one ligand.

In one aspect, the invention provides a double stranded RNAi agent capable of inhibiting the expression of PD-L1 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a région complementary to part of an mRNA encoding PD-H, wherein each strand is about 14 to about 30 nucléotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5' n_p -N_a -(X X X) _rN_b -YYY -N_b -(Z Z Z)j -N_a - n^ 3’ antisense: 3' n_p'-N_a'-(X'X'X')_rN_b'-Y’Y'Y'-N_b'-(Z'Z'Z')_rN₈'- n_q' 5’ (III) wherein: i, j, k, and 1 are each independently 0 or 1; each n_p, n^, and n_q', each of which may or may not be présent, independently represents an overhang nucléotide;

p, q, and q' are each independently 0-6; n_p' >0 and at least one n_p' is linked to a neighboring nucléotide via a phosphorothioate linkage; each N_a and N_a' independently represents an oligonucleotide sequence comprising 0-25 nucléotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differenily modified nucléotides; each N_b and N_b' independently represents an oligonucleotide sequence comprising 0-10 nucléotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X'X'X', ΥΎΎ', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucléotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; modifications on N_b differ from the modification on Y and modifications on N_b' differ from the modification on Y’; and wherein the sense strand is conjugated to at least one ligand.

In one aspect, the invention provides a double stranded RNAi agent capable of inhibiting the expression of PD-L1 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a région complementary to part of an mRNA encoding PD-L1, wherein each strand is about 14 to about 30 nucléotides în length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5’ n_p-N_a-(X X X) _;-N_b-Y Y Y -N_b-(Z Z Z)_rN_a- 3' antisense: 3’ n_p'-N_a'-(X'X'X')_k-N_b'-Y'Y'Y'-N_b'-(Z^FZ'Z')_rN_a'- n_q' 5’ (III) wherein i, j, k, and 1 are each îndependentîy 0 or 1; each n_p, n_q, and n_q', each of which may or may not be présent, îndependentîy représenta an overhang nucléotide; p, q, and q' are each îndependentîy 0-6; n_p' >0 and at least one n_p' is linked to a neîghboring nucléotide via a phosphorothioate linkage; each N_a and N_a' îndependentîy représenta an oligonucleotide sequence comprising 0-25 nucléotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucléotides; each N_b and N_b' îndependentîy represents an oligonucleotide sequence comprising 0-10 nucléotides which are either modified or unmodified or combinations thereof; XXX, YYY, ZZZ, X'X’X', ΥΎΎ', and Z'Z'Z' each îndependentîy represent one motif of three identicai modifications on three consecutive nucléotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; modifications on N_b differ from the modification on Y and modifications on N_b' differ from the modification on Y'; and wherein the sense strand is conj ugated to at least one ligand, wherein the ligand is one or more GalNAc dérivatives attached through a monovalent linker or a bivalent or trivalent branched linker.

In one aspect, the invention provides a double stranded RNAÎ agent capable of inhibiting the expression of PD-L1 in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a région complementary to part of an mRNA encoding PD-L l, wherein each strand is about 14 to about 30 nucléotides in length, wherein the double stranded RNAi agent is represented by formula (III);

sense: 5' n_p -N_a -(X X X) _rN_b -YYY -N_b -(Z Z Z)j -N_a - n_q 3’ antisense: 3' n_p'-N_a'-(X'XX')_k-N_b'-YTTW^ n_q’ 5’ (III) wherein i, j, k, and 1 are each îndependentîy 0 or 1 ; each n_p, n_q, and n_q', each of which may or may not be présent, îndependentîy represents an overhang nucléotide; p, q, and q' are each îndependentîy 0-6; n_p' >0 and at least one n_p' is linked to a neîghboring nucléotide via a phosphorothioate linkage; each N_a and N_a' îndependentîy represents an oligonucleotide sequence comprising 0-25 nucléotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucléotides; each N_b and N_b' îndependentîy represents an oligonucleotide sequence comprising 0-10 nucléotides which are either modified or unmodified or combinations thereof; XXX, YYY, TXL, X'X'X', ΥΎΎ', and Z’Z'Z' each îndependentîy represent one motif of three identicai modifications on three consecutive nucléotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; modifications on N_b differ from the modification on Y and modifications on N_b' differ from the modification on Y'; wherein the sense strand comprises at least one phosphorothioate linkage; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc dérivatives attached through a monovalent linker or a bivalent or trivalent branched linker.

In one aspect, the invention provides a double stranded RNAi agent capable of inhibiting the expression of PD-LI in a cell, wherein the double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein the antisense strand comprises a région complementary to part of an mRNA encoding PD-L1, wherein each strand is about 14 to about 30 nucléotides in length, wherein the double stranded RNAi agent is represented by formula (III):

sense: 5' n_p-N_a -Y Y Y - N_a- n_q 3' antisense: 3’ n_p'-N_a'- ΥΎΎ'- N_a'- n_q' 5’ (Ilia) wherein each n_p, n_q, and n_q', each of which may or may not be présent, independently represents an overhang nucléotide; p, q, and q' are each independently 0-6; n_p' >0 and at least one n_p' is linked to a neighboring nucléotide via a phosphorothioate linkage; each N_a and N_a’ independently represents an oligonucleotide sequence comprising 0-25 nucléotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucléotides; YYY and ΥΎΎ' each independently represent one motif of three identical modifications on three consecutive nucléotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications; wherein the sense strand comprises at least one phosphorothioate linkage; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc dérivatives attached through a monovalent linker or a bivalent or trivalent branched iinker.

In one aspect, the invention provides a double stranded RNAi agent for inhibiting expression of PD-L1, wherein the double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded région, wherein the sense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the nucléotide sequence of SEQ ID NO: 1 and the antisense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the nucléotide sequence of SEQ ID NO:2, wherein substantially ail of the nucléotides of the sense strand comprise a modification selected from a 2’-O-methyl modification and a 2’-fluoro modification, wherein the sense strand comprises two phosphorothioate intemucleotide linkages at the 5’-terminus, wherein substantially ail ofthe nucléotides of the antisense strand comprise a modification selected from a 2’-O-methyl modification and a 2’-fluoro modification, wherein the antisense strand comprises two phosphorothioate intemucleotide linkages at the 5’terminus and two phosphorothioate intemucleotide linkages at the 3’-termînus, and wherein the sense strand is conjugated to one or more GalNAc dérivatives attached through a monovalent linker or a branched bivalent or trivalent linker at the 3’-tenninus.

In another aspect, the présent invention provides double stranded ribonucleic acid (RNAi) agents for inhibiting expression of PD-L1, wherein the double stranded RNAi agents comprise a sense strand and an antisense strand forming a double stranded région, wherein the sense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from nucléotides 32213243,351-372,618-641,618-639,619-640,620-641, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 1518-1539, 2103-2124, 2220-2261, 2220-2241, 2240-2261, 2648-2680, 2648-2669, 26582679, 2659-2680,3143-3164,3198-3219, 3221-3242, or 3222-3243 of the nucléotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the complementary portion of the nucléotide sequence of SEQ ID

NO :2, wherein substantially ail of the nucléotides of the sense strand comprise a nucléotide modification selected from the group consisting of a 2’-O-methyl modification and a 2’-fluoro modification, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5’-terminus, wherein substantially ail of the nucléotides of the antisense strand comprise a nucléotide modification selected from the group consisting of a 2’-O-methyl modification and a 2’-fluoro modification, wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5’-terminus and two phosphorothioate internucleotide linkages at the 3’-terminus, and wherein the sense strand is conjugated to one or more GalNAc dérivatives attached through a branched bivalent or trivalent linker at the 3’-terminus.

In certain embodiments, ail of the nucléotides of the sense strand and ail of the nucléotides of the antisense strand are modified nucléotides. In certain embodiments, each strand has 19-30 nucléotides.

In certain embodiments, substantially ail of the nucléotides of the sense strand are modified. In certain embodiments, substantially ail of the nucléotides of the antisense strand are modified. In certain embodiments, substantially ail of the nucléotides of both the sense strand and the antisense strand are modified.

In one aspect, the invention provides a cell containing the RNAi agent as described herein.

In one aspect, the invention provides a vector encoding at least one strand of a RNAi agent, wherein the RNAi agent comprises a région of complementarity to at least a part of an mRNA encoding PD-LI, wherein the RNAi is 30 base pairs or less in length, and wherein the RNAi agent targets the mRNA forcleavage. In certain embodiments, the région of complementarity is at least 15 nucléotides in length. In certain embodiments, the région of complementarity is 19 to 23 nucléotides in length.

In one aspect, the invention provides a cell comprising a vector as described herein.

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

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

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

In one aspect, the invention provides a method of treating a subject having a disease or 5 disorder that would benefit from réduction in PD-L1 expression, the method comprising administering to the subject a therapeutically effective amount of the RNAi agent of the invention or a pharmaceutical composition of the invention, thereby treating the subject.

In one aspect, the invention provides a method of preventing at least one symptom in a subject having a disease or disorder that would benefit from réduction in PD-L1 expression, the 10 method comprising administering to the subject a prophylactîcally effective amount of the RNAi agent of the invention or a pharmaceutical composition of the invention, thereby preventing at least one symptom in the subject having a disorder that would benefit from réduction in PD-L1 expression.

In certain embodiments, the administration of the RNAi to the subject causes a decrease in the PD-L1 signaling pathway. In certain embodiments, the administration ofthe RNAi causes a decrease 15 in the level of PD-Llin the subject, e.g., sérum levels of PD-L1 in the subject.

In certain embodiments, the PD-Ll-associated disease is an infectious disease, such as a chronic, întracellular infectious disease, e.g., a viral disease, e.g., hepatitis infection, or a bacterial infection, e.g., tuberculosis infection.

ïn certain embodiments, the PD-Ll-associated disease is cancer, e.g., a hepatic cancer, e.g., 20 hepatocellular carcinoma.

In certain embodiments, the invention further comprises administering an anti-viral agent to a subject with a PD-Ll-associated disease. In certain embodiments, the anti-viral agent is a nucléotide or nucleoside analog. In certain embodiments, the anti-viral agent is for treatment of a hepatitis virus infection, e.g., an HBV infection, an HDV infection. In certain embodiments, the anti-viral agent is 25 not an immune stîmulatory agent.

In certain embodiments, the invention further comprises administering a chemotherapeutic agent to a subject with a PD-Ll-associated disease.

In certain embodiments wherein the PD-Ll-associated disease is cancer, the subject is further treated for cancer. In certain embodiments, the treatment for cancer includes surgery. In certain embodiments, the treatment for cancer includes radiation. In certain embodiments, the treatment for cancer includes administration of a chemotherapeutic agent.

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 35 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 RNAÎ agent is administered about once per week, once per month, once every other 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.

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

In some embodiment, the RNAi agent is administered to the subject subcutaneously.

In various embodiments, the methods of the invention further comprise measuring PDLllevels in the subject. In certain embodiments, a decrease in the levels of expression or activity of the PD-L1 signaling pathway indicates that the PD-Ll-associated disease is being treated.

In various embodiments, a surrogate marker of PD-L1 expression is measured. For example, in the treatment of infections disease, the presence ofthe pathogen is detected, e.g., a protein or nucleic acid from the pathogen, e.g, HBsAg, HBeAg, HB cccDNA. In certain embodiments, an indicator of an immune response against the pathogen is detected, e.g., anti-HBs antibody. In certain embodiments, a change, preferably a clinically relevant change in the surrogate marker indicating effective treatment ofthe infection is detected. In the treatment of cancer, a démonstration of stabilization or réduction of tumor burden using RECIST criteria can be used as a surrogate marker for a réduction of PD-Ll expression or activity.

Brief Description of the Drawings

Figure 1 is a schematic showing a PD-L1 signaling between an antigen presenting cell and a T-cell.

Detailed Description of the Invention

The présent invention provides iRNA compositions which effect the RNA-induced silencing complex (RlSC)-mediated cleavage of RNA transcripts of an programmed cell death I ligand 1 (PDLl) gene. The gene may be within a cell, e.g., a cell within a subject, such as a human. The use of these ÎRNAs enables the targeted dégradation of mRNAs ofthe correponding gene (PD-L1 gene) in mammals.

The iRNAs of the invention hâve been designed to target the human PD-L1 gene, including portions ofthe genethat are conserved inthe PD-L1 othologs of other mammalian species. Without intending to be limited by theory, it is believed that a combination or sub-combination of the foregoing properties and the spécifie target sites or the spécifie modifications in these iRNA agents confer to the ÎRNAs of the invention improved efficacy, stability, potency, durability, and safety.

Accordingly, the présent invention also provides methods for treating a subject having a disorder that would benefit from inhibiting or reducingthe expression of a PD-Ll gene, e.g., an PDLl-associated disease, such as infection, e.g., a viral infection, e.g., a hepatitis virus infection, or cancer, such as a lîver cancer, e.g., hepatic cellular carcinoma, using iRNA compositions which effect the RNA-induced silencing complex (RlSC)-mediated cleavage ofRNA transcripts of an PD-Ll gene.

Very low dosages of the iRNAs of the invention, in particular, can specifïcally and efficientiy médiate RNA interférence (RNAi), resulting in significant inhibition of expression of the correponding gene (PD-Ll gene).

The iRNAs of the invention înclude an RNA strand (the antisense strand) having a région which is about 30 nucléotides or less in iength, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 1823, 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, 2126, 21-25, 21-24, 21-23, or 21-22 nucléotides in Iength, which région is substantially complementary to at least part of an mRNA transcript of a PD-Ll gene. In certain embodiments, the iRNAs of the invention include an RNA strand (the antisense strand) which can include longer lengths, for example up to 66 nucléotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucléotides in Iength with a région of at least 19 contiguous nucléotides that is substantially complementary to at least a part of an mRNA transcript of a PD-Ll gene. These iRNAs with the longer Iength antisense strands preferably include a second RNA strand (the sense strand) of 20-60 nucléotides in Iength wherein the sense and antisense strands form a duplex of 18-30 contiguous nucléotides. The use of these iRNAs enables the targeted dégradation of mRNAs of the correponding gene (PD-H gene) in mammals. Very low dosages of the iRNAs of the invention, in particular, can specifïcally and efficiently médiate RNA interférence (RNAi), resulting in significant inhibition of expression of the correponding gene (PD-Ll gene). Using in vitro and in vivo assays, the présent inventors hâve demonstrated that iRNAs targeting a PD-Ll gene can médiate RNAi, resulting in significant inhibition of expression of PD-Ll, as well as reducing signaling through the PD-L1 pathway which will decrease one or more of the symptoms associated with a PD-L 1-associated disease, such as an infections disease, e.g., a viral disease or chronic intracellular infection; or cancer. Thus, methods and compositions including these iRNAs are useful for treating a subject having a PD-L 1 -associated disease, such as an infections disease, e.g., a viral disease or chronic intracellular infection, or cancer. The methods and compositons hereîn are useful for reducing the level of PD-Llin a subject, e.g., liver PD-Ll in a subject, especially in a subject with a chronic intracellular infection, especially a chronic hepatic infection, or tumor.

The following detailed description discloses how to mate and use compositions containing iRNAs to inhibit the expression of a PD-Ll gene as well as compositions, uses, and methods for treating subjects having diseases and disorders that would benefit from réduction of the expression of a PD-Ll gene.

I. Définitions

In order that the présent invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also mtended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (Le., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of éléments.

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

The term or îs used herein to mean, and is used interchangeably with, the term and/or, unless context clearly indicates otherwîse. For example, “sense strand or antisense strand” is understood as “sense strand or antisense strand or sense strand and antisense strand.”

The term “about” is used herein to mean within the typical ranges of tolérances in the art. For example, “about” can be understood as about 2 standard déviations from the mean. In certain embodiments, about means +10%. In certain embodiments, about means +5%. When about is présent before a sériés of numbers or a range, it is understood that “about” can modify each of the numbers in the sériés or range.

The term “at least” prior to a number or sériés of numbers is understood to include the number adjacent to the term “at least”, and ail subséquent numbers or integers that could logically be included, as clear from context. For example, the number of nucieotîdes in a nucleic acid molécule must be an integer. For example, “at least 18 nucieotîdes of a 21 nucléotide nucleic acid molécule” means that 18, 19, 20, or 21 nucieotîdes hâve the îndicated property. When at least is présent before a sériés of numbers or a range, it is understood that “at least” can modify each of the numbers in the sériés or range.

As used herein, “no more than” or “less than” îs understood as the value adjacent to the phrase and logical lower values or intergers, as logical from context, to zéro. For example, a duplex with an overhang of “no more than 2 nucieotîdes” has a 2, 1, or 0 nucléotide overhang. When “no more than” is présent before a sériés of numbers or a range, it is understood that “no more than” can modify each of the numbers in the sériés or range.

In the event of a conflict between a sequence and its îndicated site on a transcript or other sequence, the nucîeotide sequence recited in the spécification takes precedence.

Various embodiments of the invention can be combined as determined appropriate by one of skill in the art.

“Programmed cell death 1 ligand 1”, “PD-L1”, or “CD274,” also known as B7-H; B7H1; PDL1; PD-L1; PDCD1L1; PDCD1LG1, B7homolog 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. PD-L1 expression is also found on a wide range of nonhematopoietic cells and is upregulated on a number of cell types after activation. Upon IFN-γ stimulation, PD-L1 is expressed on T cells, NK cells, macrophages, myeloid DCs, B cells, épithélial cells, and vascular endothélial cells (Flies DB and Chen L (2007) JImmunother. 30 (3): 251-60). PD-L1 is notably expressed on macrophages. Further information on PD-L1 is provided, for example in the NCBI Gene database at www.ncbi.nlm.nih.gov/gene/29126 (which is incorporated hereîn by reference as ofthe date of filing this application).

As used herein, “programmed cell death 1 ligand 1” is used interchangeably with the term “PD-Ll” (and optionally any ofthe other recognized names listed above) refers to the naturally occurring gene that encodes a programmed cell death 1 ligand 1 protein. The amino acid and complété codîng sequences of the reference sequence of the human PDL-1 gene may be found in, for example, GenBank Accession No. GI: 390979638 (RefSeq Accession No. NM_001267706.1; SEQ ID NO: 1; SEQ ID NO:2) and GenBank Accession No. GI: 292658763 (RefSeq Accession No. NM_014143.3; SEQ ID NO: 9 and 10). Further splice variants are provided, for example, in Grzywnowicz et al., PLoS One. 2012;7;e35178 which is incorporated herein by reference. Mammalian orthologs ofthe human PD-Ll gene may be found in, for example, GI: 755563510 (RefSeq Accession No. XM_006527249.2, mouse; SEQ ID NO:3 and SEQ ID NO:4); GI: 672040129 (RefSeq Accession No. XM_006231248.2, rat; SEQ ID NO:5 and SEQ ID NO:6); GenBank Accession Nos. GI: 544494555 (RefSeq Accession No. XM_005581779.1, cynomolgus monkey; SEQ ID NO: 7 and SEQ ID NO:8).

A number of naturally occurring SNPs are known and can be found, for example, in the SNP database atthe NCBI at www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=29126 (which is incorporated herein by reference as of the date of filing this application) which lists SNPs in human PD-LL In preferred embodiments, such naturally occuring variants are included within the scope of the PD-Ll gene sequence.

Additional examples of PD-Ll mRNA sequences are readily avaîlable using publicly available databases, e.g., GenBank, UniProt, and 0M1M.

As used herein, “target sequence” refers to a contiguous portion of the nucléotide sequence of an mRNA molécule formed during the transcription of a PD-Ll gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotïde sequence of an mRNA molécule formed during the transcription of a PD-Ll gene. In one embodiment, the target sequence is within the protein coding région of PD-Ll.

The target sequence may be from about 9-36 nucléotides in length, e.g., about 15-30 nucléotides in length. For example, the target sequence can be from about 15-30 nucléotides, 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, 1828, 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, 2021,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucléotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence refers to an oligonucleotide comprising a chain of nucléotides that is described by the sequence referred to using the standard nucléotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucléotide that contains guanine, cytosine, adenîne, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucléotide” can also refer to a modified nucléotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 2). The skîlled person is well aware that guanine, cytosine, adenîne, and uracil can be replaced by other moieties without substantially aitering the base pairing properties of an oligonucleotide comprising a nucléotide bearing such replacement moiety. For example, without limitation, a nucléotide comprising inosine as its base can base pair with nucléotides containing adenîne, cytosine, or uracil. Hence, nucléotides containing uracil, guanine, or adenine can be replaced in the nucléotide sequences of dsRNA featured in the invention by a nucléotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” and “iRNA agent,” “RNA interférence agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which médiates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific dégradation of mRNA through a process known as RNA interférence (RNAi). The iRNA modulâtes, e.g., inhibits, the expression of a PD-LI gene in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., a PD-LI target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 1923 base pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target récognition (Nykanen, et al., (2001) Cell 107:309). Upon bindîng 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 invention relates to a single stranded RNA (siRNA) generated within a cell and which promûtes the formation of a RISC complex to effect silencing of the target gene, i.e., a PD-LI gene. Accordingly, the tenn “siRNA” is also used herein to refer to an ÎRNA as described above.

In certain embodiments, the RNAi agent may be a single-stranded siRNA (ssRNAi) that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the

RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucléotides and are chemîcally modified. The design and testing of singlestranded siRNAs are described in US Patent No. 8,101,348 and in Lima et al., (2012) Cell 150:883894, the entire contents of each of which are hereby incorporated herein by reference. Any of the S antisense nucléotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemîcally modified by the methods described in Lima étal., (2012) Cell 150:883-894.

In certain embodiments, an “iRNA” for use in the compositions, uses, and methods of the invention is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molécule,” “dsRNA agent,” or “dsRNA”. The terrn “dsRNA”, refers 10 to a complex of ribonucleic acid molécules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, Le., a PD-L1 gene. In some embodiments of the invention, a double stranded RNA (dsRNA) triggers the dégradation of a target RNA, e.g., an mRNA, through a post-transcri ptional gene-silencing mechanism referred to herein as RNA interférence or 15 RNAi.

In general, the majority of nucléotides of each strand of a dsRNA molécule are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucieotide or a modified nucléotide. In addition, as used in this spécification, an “iRNA” may include ribonucleotides with Chemical modifications; an iRNA may 20 include substantial modifications at multiple nucléotides. As used herein, the term “modified nucléotide” refers to a nucléotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or modified nucleobase, or any combination thereof. Thus, the term modified nucléotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to intemucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the 25 agents of the invention include ail types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molécule, are encompassed by “iRNA” or “RNAi agent” for the purposes of this spécification and daims.

The duplex région may be of any length that permits spécifie dégradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 1530 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, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 1827, 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, 2135 30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portions of one larger RNA molécule, or they may be separate RNA molécules. Where the two strands are part of one larger molécule, and therefore are connected by an uninterrupted chain of nucléotides between the 3’-end of one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting RNA chain is refemed to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucieotide. In some embodiments, the hairpin loop can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucléotides. In some embodiments, the hairpin loop can be 10 or fewer nucléotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucléotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucléotides. In some embodiments, the hairpin loop can be 4-8 nucléotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molécules, those molécules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucléotides between the 3’end of one strand and the 5’-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may hâve the same or a different number of nucléotides. The maximum number of base pairs is the number of nucléotides in the shortest strand of the dsRNA minus any overhangs that are présent in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucieotide overhangs.

In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which comprises 19-23 nucléotides, that interacts with atarget RNA sequence, e.g., a PD-L1 gene, without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-HI-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-înduced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target récognition (Nykanen. et al., (2001) Cell 107:309). Upon bînding 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).

In some embodiments, an iRNA of the invention is a dsRNA of 24-30 nucléotides, or possibly even longer, e.g., 25-35, 27-53, or 27-49 nucléotides, that interacts with atarget RNA sequence, e.g, a PD-L1 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target récognition (Nykanen, et al., (2001) Cell 107:309), Upon bînding 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).

As used herein, the term “nucléotide overhang” refers to at least one unpaired nucléotide that protrudes from the duplex structure of a double strainded iRNA. For example, when a 3'-end of one strand of a dsRNA extends beyond the 5’-end of the other strand, or vice versa, there is a nucléotide overhang, A dsRNA can comprise an overhang of at least one nucléotide; alternative ly the overhang can comprise at least two nucléotides, at least three nucléotides, at least four nucléotides, at least five nucléotides or more. A nucléotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside, The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof, F urthermore, the nucleotide(s) of an overhang can be présent on the 5'-end, 3'-end, or both ends of either an antisense or sense strand of a dsRNA.

In certain embodiments, the antisense strand of a dsRNA has a 1-10 nucléotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucléotide, overhang at the 3’-end or the 5’-end, In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucléotides, e.g., 1-30 nucléotides, 2-30 nucléotides, 10-30 nucléotides, or 10-15 nucléotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is présent on the 3’end of the sense strand of the duplex. In certain embodiments, an extended overhang is présent on the 5’end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is présent on the 3’end of the antisense strand of the duplex. In certain embodiments, an extended overhang is présent on the 5’end of the antisense strand of the duplex. In certain embodiments, one or more of the nucléotides in the overhang is replaced with a nucleosîde thiophosphate.

“Blunt” or “blunt end” means that there are no unpaired nucléotides at that end of the double stranded RNAi agent, i.e., no nucléotide overhang. A “blunt ended” double stranded RNAi agent is double stranded over its entire length, i.e., no nucléotide overhang at either end ofthe molécule. The RNAi agents ofthe invention include RNAi agents with no nucléotide overhang at one end (i.e., agents with one overhang and one blunt end) or with no nucieotide overhangs at either end.

The term “antisense strand” or guide strand refers to the strand of an iRNA, e.g., a dsRNA, which inciudes a région that is substantially complementary to a target sequence, e.g., a PD-L1 mRNA. As used herein, the term “région of complementarity” refers to the région on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a PDL1 nucieotide sequence, as defined herein. Where the région of complementarity is not fully complementary to the target sequence, the mîsmatches can be in the internai or terminal régions ofthe molécule. Generally, the most tolerated mîsmatches are in the terminal régions, e.g., within 5, 4, 3, 2, or 1 nucléotides ofthe 5’- or 3’-end ofthe iRNA. In some embodiments, a double stranded RNAi agent of the invention inciudes a nucieotide mismatch in the antisense strand. In some embodiments, a double stranded RNAi agent ofthe invention inciudes a nucieotide mismatch in the sense strand. in sortie embodiments, the nucléotide mismatch is, for example, within 5, 4, 3, 2, or 1 nucléotides from the 3’-end of the iRNA. In another embodiment, the nucléotide mismatch is, for example, in the 3’terminal nucléotide ofthe iRNA.

The terra “sense strand” or passenger strand as used herein, refers to the strand of an iRNA that includes a région that is substantially complementary to a région of the antisense strand as that term is deftned herein.

As used herein, the term “cieavage région” refers to a région that is located immediately adjacent to the cieavage site. The cieavage site is the site on the target at which cieavage occurs. In some embodiments, the cieavage région comprises three bases on either end of, and immediately adjacent to, the cieavage site. In some embodiments, the cieavage région comprises two bases on either end of and immediately adjacent to, the cieavage site. In some embodiments, the cieavage site specifically occurs at the site bound by nucléotides 10 and 11 of the antisense strand, and the cieavage région comprises nucléotides 11,12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucléotide sequence in relation to a second nucléotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucléotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucléotide sequence, as will be understood by the skîlled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCi, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours followed by washing (see, e.g., “Molecular Cloning; A Laboratory Manual, Sambrook, étal. (1989) Coid Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered înside an organism, can appîy. The ski lied person will be able to détermine the set of conditions most appropriate for a test of complementarîty of two sequences in accordance with the ultimate application of the hybridized nucléotides.

Complementary sequences within an ÎRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucléotide sequence to an oligonucleotide or polynucleotide comprising a second nucléotide sequence over the entire length of one or both nucléotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches witb regard to the détermination of complementarîty. For example, a dsRNA comprising one oligonucleotide 21 nucléotides in length and another oligonucleotide 23 nucléotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucléotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also înclude, or be formed entireiy from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucléotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-WatsonCrick base pairs include, but are not Limited to, G:U Wobble or Hoogsteîn base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a double stranded RNAi agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of’ a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a PD-L1 gene). For example, a polynucleotide is complementary to at Least a part of a PD-L1 mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding a PD-L1 gene.

Accordingly, in some embodiments, the sense strand polynucleotides and the antisense polynucleotides disclosed herein are fully complementary to the target PD-L 1 sequence.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target PD-L1 sequence and comprise a contiguous nucléotide sequence which is at least about 80% complementary over its entire length to the équivalent région of the nucléotide sequence of any one of SEQ ID NO: 1, or a fragment of any one of SEQ ID NO: 1, such as at least about 85%, 86%, 87%, 88%, 89%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary or 100% complementary.

In some embodiments, an iRNA of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in tum, is complementary to a target PD-L1 sequence and comprises a contiguous nucléotide sequence which is at least about 80% complementary over its entire length to the équivalent région of the nucléotide sequence of any one of the antisense strands in Table 3 or Table 5, or a fragment of any one of the antisense strands in Table 3 and Table 5, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.

In some embodiments, an iRNA of the invention includes an antisense strand that is substantially complementary to the target PD-L1 sequence and comprises a contiguous nucléotide sequence which is at least 80% complementary over its entire length to the équivalent région of the nucléotide sequence of any one of the isense strands in Table 3 or 5, or a fragment of any one of the sense strands in Table 3 and 5, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary, or 100% complementary.

ln an aspect of the invention, an agent for use in the methods and compositions of the invention is a single-stranded antisense oligonucleotide molécule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense oligonucleotide molécule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotides can inhîbit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347355. The single-stranded antisense oligonucleotide molécule may be about 14 to about 30 nucléotides in length and hâve a sequence that is complementary to a target sequence. For example, the singlestranded antisense oligonucleotide molécule may comprise a sequence that is at least 14, 15, 16, 17, 10 18,19, 20, or more contiguous nucléotides from any one of the antisense sequences described herein.

The phrase “contacting a cell with an ÎRNA,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an ÎRNA includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the iRNA may be put into physical contact with the cell by the 15 individual performing the method, or altematively, the iRNA may be put into a situation that will permit or cause ît to subsequently corne into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating the cell with the iRNA. Contacting a cell in vivo may be done, for example, by injecting the ÎRNA into or near the tissue where the cell is located, or by injecting the iRNA into another area, e.g., the bloodstream or the 20 subcutaneous space, such that the agent wil l subsequently reach the tissue where the cell to be contacted is located. For example, the ÎRNA may contain or be coupled to a ligand, e.g., GalNAc, e.g., GalNAc3, that directs the iRNA to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an iRNA and subsequently transplanted into a subject.

In certain embodiments, contacting a cell with an iRNA includes introducing” or “delivering the ÎRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusion or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered System ically. In vivo delivery can also be done by a beta-glucan delivery System, such as those described in US Patent Nos. 5,032,401 and 5,607,677, and US Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and Lipofection. Further approaches are described herein below or are known in the art.

The terni “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molécule, such as a nucleic acid molécule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, US Patent Nos. 6,858,225,

6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, a subject” is an animai, such as a mammal, including a primate (such as a human, a non-human primate, e.g, a monkey, and a chimpanzee), a non-primate (such as a cow, a 5 pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose) that expresses the target gene, eiîher endogenously or heterologously, when the target gene sequence has sufficient complementarîty to the iRNA agent to promote target knockdown. In certain embodiments, the subject is a human, such as a human beîng treated or assessed for a dîsease, disorder or condition that would benefit from réduction 10 in PD-L1 gene expression or réplication; a human at risk for a disease, disorder or condition that wouid benefit from réduction in PD-L1 gene expression; a human having a disease, disorder or condition that would benefit from réduction in PD-L1 gene expression; or human being treated for a disease, disorder or condition that would benefit from réduction in PD-L1 gene expression, as described herein. In some embodiments, the subject is a female human. In other embodiments, the 15 subject is a male human.

As used herein, the tenus Areating” or “treatment” refer to a bénéficiai or desired resuit including, but not limited to, alleviation or amelioration of one or more symptoms associated with PD-L1 gene expression or PD-L1 protein production, e.g., infection, especially a chronic, intracelluiar infection, e.g., a chronic viral infection, or cancer. Treatment can also mean prolonging survival as 20 compared to expected survival in the absence of treatment. Treatment can include prévention of development of co-morbidities, e.g., reduced liver damage in a subject with a hepatic infection.

The tenu “lower” in the context of the Jevel of PD-Lî gene expression or PD-L1 protein production in a subject, or a disease marker or symptom 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%, 25 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, as compared to an appropriate control, or to below the level of détection for the détection method. In certain embodiments, the expression of the target is normalized, i.e., decreased to a level accepted as withînthe range of normal for an individual without such disorder. In certain embodiments, the methods include a clinîcally relevant inhibition of expression of PD-LI, e.g. as demonstrated by a clinîcally relevant outcome after treatment of 30 a subject with an agent to reduce the expression of PD-LI.

As used herein, the term Programmed cell death 1 ligand 1-associated disease” or ’TD-Llassociated disease,” is adisease or disorder that is caused by, or associated with PD-LI gene expression or PD-LI protein production. The term PD-Ll-associated disease” includes a disease, disorder or condition that would benefit from a decrease in PD-LI gene expression, réplication, or 35 protein activity. Non-limîting examples of PD-Ll-associated dîseases include, for example, infection, especially a chronic intracelluiar infection, e.g., viral infection, e.g., hepatitis infection, or cancer.

In certain embodiments, aPD-Ll-associated disease is infection, especially a chronic, intracelluiar infection, e.g, viral infection, e.g., hepatitis virus infection, e.g., hepatitis B infection or hepatitis D infection. In certain embodiments, the infection is a chrome bactenal infection, e.g., tuberculosis. In certain embodiments, a PD-Ll-associated disease is cancer, especially H ver cancer, e.g., heptatocellular carcinoma (HCC).

“Therapeutically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a patient for treating a subject having an infection, especially a chronic intracellular infection, or cancer, or other PD-Ll-associated disease, is sufficient to effect treatment ofthe disease (e.g., by diminishîng, ameliorating or maintaining the existing disease or one or more symptoms of disease or its related comorbidities). The frherapeutically effective amount” may vary depending on the iRNA, how it is administered, the disease and its severity and the hîstory, âge, weight, family history, genetic makeup, stage of patbological processes mediated by PD-L1 gene expression, the types of precedîng or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” also includes an amount of an iRNA that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNAs employed in the methods ofthe présent invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment. A therapeutically effective amount includes an amount that results in a clînically relevant change or stabilization, as appropriate, of an indicator of a disease or condition.

The phrase pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergie response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase pharmaceutically-acceptable carrier as used herein means a pharmaceuticallyacceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnésium, calcium or zinc stéarate, or sterîc acid), or solvent encapsulating material, involved in carrying or transportîng the subject compound from one organ, or portion ofthe body, to another organ, or portion ofthe body. Each carrier must be acceptable in the sense of being compatible with the other ingrédients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its dérivatives, such as sodium carboxymethyi cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnésium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oîl, sesame oîl, olive oil, com oil and soybean oil;(10)glycols, suchas propylene glycol; (11) polyols, such asglycerin, sorbitol, mannitoi and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnésium hydroxide and aluminum hydroxide; (15) alginic acid; ( 16) pyrogen-free water; (17) isotonie saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) sérum component, such as sérum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutîcal formulations, The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues présent within a subject. Examples of biological fluids include blood, sérum and serosal fluids, plasma, cerebrospinai fluid, ocular fluids, lymph, urine, saliva, and the like, Tissue samples may include samples from tissues, organs, or local ized régions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hépatocytes). A “sample derived from a subject” can refer to blood drawn from the subject, or plasma derived therefrom. In certain embodiments when detecting a level of PD-L1, a “sample” preferably refers to a tissue or body fluid from a subject in which PD-L1 is détectable prior to administration of an agent of the invention, e.g., a liver biopsy from a subject with a hepatic infection, a tumor biopsy. In certain subjects, e.g, healthy subjects, the level ofPD-Ll may not be détectable in a number of body tluids, cell types, and tissues.

I. iRNAs of the Invention

The présent invention provides iRNAs which înhibit the expression of a PD-L1 gene. In preferred embodiments, the iRNA includes double stranded ribonucleic acid (dsRNA) molécules for inhibiting the expression of a PD-L1 gene in a cell, such as a cell within a subject, e.g, a mammal, such as a human having a PD-Ll-associated disease, e.g, a chronic infection. The dsRNAi agent includes an antisense strand having a région of complementarity which is complementary to at least a part of an mRNA formed in the expression of a PD-L1 gene, The région of complementarity is about 30 nucléotides or less in length (e.g, about 30, 29, 28, 27, 26, 25,24, 23, 22, 21, 20, 19, or 18 nucléotides or less in length). Upon contact with a cell expressing the PD-L1 gene, the ÎRNA inhibits the expression ofthe PD-LJ gene (e.g., a human, a primate, a non-primate, ora bird PD-L1 gene) by at least about 20%, preferably by at least 30%, as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting or flowcytometric techniques. In preferred embodiments, inhibiton of expression is deteremined by the qPCR method provided in the examples, e.g., at a 10 nM concentration of the duplex. The level of réduction can be compared to, for example, an appropriate historical control or a pooled population sample control.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a région of complementarity that is substantîally complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of a PD-Ll gene. The other strand (the sense strand) mcludes a région that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as selfcomplementary régions of a single nucleic acid molécule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is about 15 to 30 base pairs in length, e.g., 15-29, 15-28, 1527, 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, 1923, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

Similarly, the région of complementarity to the target sequence is about 15 to 30 nucléotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 1928, 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 nucléotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

In some embodiments, the dsRNA is about 15 to 23 nucléotides in length, or about 25 to 30 nucléotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, ît is well-known in the art that dsRNAs longer than about 21-23 nucléotides in length may serve as substrates for Dicer. As the ordinarîly skilled person will also recognize, the région of an RNA targeted for cleavage will most often be part of a larger RNA molécule, often an mRNA molécule. Where relevant, a “part” of an mRNA target is a contîguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage {i.e., cleavage through a RISC pathway).

One of skill in the art will aiso recognize that the duplex région is a primary functional portion of a dsRNA, e.g., a duplex région of about 9 to about 36 base pairs, e.g., 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, 1334, 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, 1525, 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, 1921, 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. Thus, in one embodiment, to the extentthat it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molécule or complex of RNA molécules having a duplex région greater than 30 base pairs îs a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA ts a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target PD-L1 gene expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucléotide overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucléotides. dsRNAs having at least one nucléotide overhang can hâve superior inhibitory propertîes relative to their blunt-ended counterparts. A nucléotide overhang can comprise or consist of a nucleotide/nucleoside analog, încluding a deoxynucleotide/nucleosîde. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be présent on the 5'-end, 3’end, or both ends of an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Bîosearch, Applied Biosystems, Inc.

Double stranded RNAi compounds ofthe invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molécule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modifled nucléotides can be easiiy prepared. Simlarly, single-stranded oligonucleotides ofthe invention can be prepared using solutionphase or solid-phase organic synthesis or both.

In an aspect, a dsRNA of the invention includes at least two nucléotide sequences, a sense sequence and an anti-sense 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 ofTables 3 and 5. In this aspect, one ofthe two sequences is complementary to the other of the two sequences, with one of the sequences being substantiaily complementary to a sequence of an mRNA generated in the expression of a PD-Ll gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Table 3 or 5, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in Table 3 or 5. In certain embodiments, the substantiaily complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantiaily complementary sequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although the sequences in Table 3 are not described as modified or conjugated sequences, the RNA ofthe iRNA ofthe invention e.g., a dsRNA ofthe invention, may comprise any one ofthe sequences set forth in Table 3, or the sequences of Table 5 that are modified, or the sequences of Table 5 that are conjugated. In other words, the invention encompasses dsRNA of Tables 3 and 5 which are un-modified, un-conjugated, modified, or conjugated, as described herein.

The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs hâve been hailed as partîcularly effective in inducing RNA interférence (Elbashir et al., EMBO 2001, 20:6877-6888). However, others hâve found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in any one of Tables 3 and 5, dsRNAs described herein can include at least one strand of a Iength of minimally 21 nucléotides. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 3 and 5 minus only a few nucléotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucléotides derived from one of the sequences of Tables 3 and 5, and differing in their ability to inhibit the expression of a PD-Ll gene by not more than about 5, 10, 15, 20,25, or 30 % inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the présent invention.

In addition, the RNAs provided in Tables 3 and 5 identify a sîte(s) in a PD-Ll transcript that is susceptible to RISC-mediated cleavage. As such, the présent invention further features 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 the iRNA promûtes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucléotides from one of the sequences provided in Tables 3 and 5 coupled to additional nucléotide sequences taken from the région contiguous to the selected sequence in a PD-Ll gene.

While a target sequence is generally about 15-30 nucléotides in Iength, îhere is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Varions software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucléotides) is literaliy or figuratively (including, e.g., in sîlîco) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucléotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complété set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art or provided herein) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, médiate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 3 and 5 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucléotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identihed, e.g., in Tables 3 and 5, further optimization could be achieved by systematîcally either adding or removing nucléotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA front that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucléotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art or discussed herein to further optîmize the molécule (e.g., increasing sérum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. if the antisense strand of the iRNA contains mismatches to a target sequence, it is préférable that the area of mismatch is not located in the center of the région of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is préférable that the mismatch be restricted to be within the last 5 nucléotides from either the 5’- or 3’-end of the région of complementarity. For example, for a 23 nucléotide iRNA agent the strand which is complementary to a région of a PD-L 1 gene, generally does not contain any mismatch within the central 13 nucléotides. The methods described herein or methods known in the art can be used to détermine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of a PD-L1 gene. Considération of the efficacy of iRNAs with mismatches in inhibiting expression of a PD-L1 gene is important, especially if the particular région of complementarity in a PD-Ll gene is known to hâve polymorphie sequence variation within the population.

Π. Modified iRNAs of the Invention

In certain embodiments, the RNA ofthe iRNA ofthe invention e.g., a dsRNA, is unmodified, and does not comprise, e.g., Chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA ofan iRNA ofthe invention, e.g., a dsRNA, is chemically modified to enhance stability or other bénéficiai characteristics. In certain embodiments of the invention, substantially ail of the nucléotides of an iRNA ofthe invention are modified. In other embodiments ofthe invention, ail ofthe nucléotides ofan iRNA or substantially ail ofthe nucléotides of an iRNA are modified, i.e., not more than 5, 4, 3, 2, or 1 unmodified nucléotides are présent in a strand of the iRNA.

The nucleic acids featured in the invention can be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,”

Beaucage, S.L. étal. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by référencé. Modifications include, for example, end modifications, e.g., 5’-end modifications (phosphorylation, conjugation, inverted linkages) or 3’-end modifications (conjugation, DNA nucléotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded répertoire of partners, removal of bases (abasic nucléotides), or conjugated bases; sugar modifications (e.g., at the 2’-position or 4’position) or replacement ofthe sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. Spécifie examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modîfied backbones or no natural intemucleoside linkages. RNAs havîng modified backbones include, among others, those that do not hâve a phosphores atom in the backbone. For the purposes of this spécification, and as sometimes referenced in the art, modified RNAs that do not hâve a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will hâve a phosphorus atom in its intemucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, rnethyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5'-linked analogs of these, and those havîng inverted polarity wherein the adjacent pairs of nucleosîde units are linked 3'-5' to 5'-3’ or 2-5' to 5'-2'. Various saits, mixed salts and free acid forms are also inciuded.

Représentative US Patents that teach the préparation of the above phosphorus-containing linkages include, but are not limited to, US 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 US Patent No. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein hâve backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleosîde); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazîno backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH₂ component parts.

Représentative US Patents that teach the préparation of the above oligonucleosides include, but are not limited to, US Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

Suitable RNA mimetics are contemplated for use in iRNAs provided herein, in which both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucléotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound in which an RNA mimetic that has been shown to hâve excellent hybridization propertîes is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms ofthe amide portion of the backbone. Représentative US patents that teach the préparation of PNA compounds include, but are not limited to, US Patent Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular -CH₂—NH—CH₂-, -CH₂N(CH₃)-O-CH₂-[known as a methylene (methylimino) orMMI backbone], -CH₂-O--N(CH₃)CH₂—, -CH₂-N(CH₃)--N(CH₃)-CH₂-- and —N(CH₃)-CH2--CH₂--[wherein the native phosphodiester backbone is re présente d as — O--P--O—CH2—] ofthe above-referenced US Patent No. 5,489,677, and the amide backbones of the above-referenced US Patent No. 5,602,240. In some embodiments, the RNAs featured herein hâve morpholino backbone structures ofthe above-referenced US Patent No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one ofthe following at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-aîkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted Ci to C_l0 alkyl or C₂ to Cio alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_nO] _mCH₃, OÎCH₂)._nOCH₃, O(CH₂)_nNH2, O(CH₂) _nCH₃, O(CH₂)_nONH₂, and O(CH₂)_nON[(CH₂)_nCH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one ofthe following atthe 2' position: Cj to Ci₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl orO-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO2, NO2, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improvmg the pharmacokmetic properties of an iRNA, or a group for împroving the pharmacodynamie properties of an iRNA, and other substituents having similar properties. In sonie embodiments, the modification includes a 2'-methoxyethoxy (2'-OCH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-M0E) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) Le., an alkoxy-alkoxy group. Another exemplary modification is 2'dimethyiaminooxyethoxy, Le., a OfCIUhONlCHjh group, also known as 2-DMAOE, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-Odimethylaminoethoxyethyl or 2-DMAEOE), λ e., 2'-O—CH2-O—CH₂—N(CH₂)₂. Further exemplary modifications include : 5’-Me-2’-F nucléotides, 5’-Me-2’-OMe nucléotides, 5’-Me-2’deoxynucleotides, (both R and S isomers in these three families); 2’-alkoxyalky); and 2’-NMA (Nmethylacetamide).

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 an iRNA, particularly the 3' position ofthe sugar on the 3' terminal nucléotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucléotide. iRNAs can also hâve sugar mimetics such as cyclobutyl moîeties in place ofthe pentofuranosyl sugar. Représentative US patents that teach the préparation of such modified sugar structures include, but are not limited to, US 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, certain of which are commonly owned with the instant application,. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrîmidine bases thymine (T), cytosîne (C), and uracîl (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxythymine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2aminoadenine, 6-methyl and other alkyl dérivatives of adenine and guanine, 2-propyl and other alkyl dérivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amîno, 8-thiol, 8-thioaIkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in US Pat. No. 3,687,808, those disclosed in Modified Nucleosîdes 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, Kroschwîtz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch étal., 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 Lebieu, B., Ed., CRC Press, 1993. Certain of these nucleobases are parti cularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidînes, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5methylcytosine substitutions hâve been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S„ Crooke, S. T. and Lebieu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

Représentative US Patents that teach the préparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted US 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 hereby incorporated herein by reference.

The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucléotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively locks the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in sérum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(l);439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

In some embodiments, the iRNA of the invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucléotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked sugar residue. In one example, UNA also encompasses monomer with bonds between C1 '-C4' hâve been removed (te. the covalent carbonoxygen-carbon bond between the Cl' and C4’ carbons). In another example, the C2-C3’ bond (Le. the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Nue. Acids Symp. Sériés, 52, 133-134 (2008) and Fluiter et al.. Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

The RNA of an iRNA can also be modified to include one or more bicyclic sugar moitiés. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A“bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connecte the 4'-carbon and the 2'-carbon ofthe sugar ring. Thus, in some embodiments an agent of the invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucléotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. In other words, an LNA is a nucléotide comprising a bicyclic sugar moiety comprïsing a 4'-CH₂-O-2' bridge. This structure effectively locks the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in sérum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833-843; Grunweller, A. étal., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprïsing a bridge between the 4' and the 2' ribosyl ring atoms. In certain embodiments, the antisense polynucieotide agents of the invention include one or more bicyclic nucleosides comprïsing a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not lîmited to 4'-(CH₂)—O-2' (LNA); 4'-(CH₂)—S2'; 4'-(CH₂)₂—0-2' (ENA); 4'-CH(CH₃)--O-2' (also referred to as “constrained ethyl” or “cEt”) and 4'-CH(CH₂OCH₃)—0-2' (and analogs thereof; see, e.g., US Patent No. 7,399,845); 4'-C(CH₃)(CH₃)— 0-2' (and analogs thereof; see e.g., US Patent No. 8,278,283); 4'-CH₂—N(OCH₃)-2' (and analogs thereof; see e.g., US Patent No. 8,278,425); 4'-CH2—O—N(CH₃)-2' (see, e.g.,US Patent Publication No. 2004/0171570); 4'-CH₂-—N(R)—0-2', wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., US 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 analogs thereof; see, e.g., US Patent No. 8,278,426). The entire contents of each of the foregoîng are hereby incorporated herein by reference.

Additional représentative US Patents and US Patent Publications that teach the préparation of locked nucleic acid nucléotides include, but are not limited to, the following: US 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; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

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

The RNA of an iRNA can also be modified to include one or more constrained ethyl nucléotides. As used herein, a constrained ethyl nucléotide or cEt is a locked nucleic acid comprising a bicyclic sugar moiety comprïsing a 4'-CH(CH₃)-O-2' bridge. In one embodiment, a constrained ethyl nucléotide is in the S conformation referred to herein as “S-cEt.”

An iRNA ofthe invention may also include one or more “conformationally restricted nucléotides” (“CRN”). CRN are nucléotide analogs with a linker connecting the C2’and C4’ carbons of ribose or the C3 and C5' carbons of ribose. CRN lock the ribose ring înto a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Représentative publications that teach the préparation of certain of the above noted CRN include, but are not limited to, US Patent Publication No. 2013/0190383; and PCT publication WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, the iRNA ofthe invention comprises one or more raonomers that are UNA (unlocked nucleic acid) nucléotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked sugar” residue. In one exampie, UNA also encompasses monomer with bonds between Cl'-C4' hâve been removed (i.e. the covalent carbonoxygen-carbon bond between the Cl' and C4' carbons). In another example, the C2'-C3' bond (i.e. the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Nue. Acids Symp. Sériés, 52, 133-134 (2008) and Fluiter et al.. Mol. Biosyst., 2009, 10, 1039 hereby incorporated by référencé).

Représentative US publications that teach the préparation of UNA include, but are not limited to, US Patent No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molécules can include N(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-0-deoxythymidine (ether), N(aminocaproyi)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3- phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No, WO 2011/005861.

Other modifications ofthe nucléotides ofan ÎRNA of the invention include a 5’ phosphate or 5’ phosphate mimic, e.g., a 5’-terminal phosphate or phosphate mimic on the antisense strand of an iRNA. Suitable phosphate mi mi es are disclosed in, for example US Patent Publication No. 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNAi agents of the invention include agents with Chemical modifications as disclosed, for example, in WO2Û13/075035, the entire contents of each of which are incorporated herein by reference. WO2013/075035 provides motifs of three identicai modifications on three consecutive nucléotides into a sense strand or antisense strand of a dsRNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the dsRNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if présent, of the sense or antisense strand. The dsRNAi agent may be optionally conjugated with a GalNAc dérivative ligand, for instance on the sense strand.

More specifically, when the sense strand and antisense strand of the double stranded RNAi agent are completely modified to hâve one or more motifs of three identicai modifications on three consecutive nucléotides at or near the cleavage site of at least one strand of a dsRNAi agent, the gene siiencing acitivity of the dsRNAi agent was observed.

Accordingly, the invention provides double stranded RNAi agents capable of inhibiting the expression of a target gene (i.e., PD-Ll gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be, mdependently, 12-30 nucléotides in length. For example, each strand may independently be 14-30 nucléotides in length, 17-30 nucléotides in length, 25-30 nucléotides in iength, 27-30 nucléotides in length, 17-23 nucléotides in length, 17-21 nucléotides in length, 17-19 nucléotides in length, 19-25 nucléotides in length, 19-23 nucléotides in length, 19-21 nucléotides in length, 21-25 nucléotides in length, or 21-23 nucléotides in length.

The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as “dsRNAi agent.” The duplex région of andsRNAi agent may be 12-30 nucléotide pairs in length. For example, the duplex région can be 14-30 nucléotide pairs in length, 17-30 nucléotide pairs in length, 27-30 nucléotide pairs in length, 17 - 23 nucléotide pairs in length, 17-21 nucléotide pairs in length, 17-19 nucléotide pairs in length, 19-25 nucléotide pairs in length, 19-23 nucléotide pairs in length, 19-21 nucléotide pairs in length, 21-25 nucléotide pairs in length, or 21-23 nucléotide pairs in length. In another example, the duplex région is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, or 30 nucléotides in length.

In certain embodiments, the sense and antisense strands may be even longer. For example, in certain embodiments, the sense strand and the antisense strand are independently 25-35 nucléotides in length. In certain embodiments, each the sense and the antisense strand are independently 27-53 nucléotides in length, e.g., 27-49, 31-49, 33-49, 35-49, 37-49, and 39-49 nucléotides in length.

In certain embodiments, the dsRNAi agent may contain one or more overhang régions or capping groups at the 3’-end, 5’-end, or both ends of one or both strands. The overhang can be, independently, 1-6 nucléotides in length, for instance 2-6 nucléotides in length, 1-5 nucléotides in length, 2-5 nucléotides in length, 1-4 nucléotides in length, 2-4 nucléotides in length, 1-3 nucléotides in length, 2-3 nucléotides in length, or 1-2 nucléotides in length. In certain embodiments, the overhang régions can include extended overhang régions as provided above. The overhangs can be the resuit of one strand being longer than the other, or the resuit of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In certain embodiments, the nucléotides in the overhang région of the dsRNAi agent can each independently be a modifîed or unmodified nucléotide including, but no limited to 2’-sugar modified, such as, 2’-F, 2’-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyiuridine (Teo), 2'-Omethoxyethyladenosine (Aeo), 2'-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. For exampie, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.

The 5’ - or 3’ - overhangs at the sense strand, antisense strand, or both strands of the dsRNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucléotides havmg a phosphorothioate between the two nucléotides, where the two nucléotides can be the same or different. In some embodiments, the overhang is présent at the 3 ’-end of the sense strand, antisense strand, or both strands. In some embodiments, this 3’-overhang is présent in the antisense strand. In some embodiments, this 3’-overhang is présent in the sense strand.

The dsRNAi agent may contain only a single overhang, which can strengthen the interférence activity of the RNAi, without affectîng its overall stabîlity. For example, the single-stranded overhang may be located at the 3’- end of the sense strand or, altematively, at the 3'-end of the antisense strand. The RNAi may also hâve a blunt end, located at the 5’-end of the antisense strand (or the 3’-end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNAi agent has a nucléotide overhang at the 3’-end, and the 5’-end is blunt. Whîle not wishing to be bound by theory, the asymmetric blunt end atthe 5’-end ofthe antisense strand and 3’-end overhang ofthe antisense strand favor the guide strand loading into RISC process.

In certain embodiments, the dsRNAi agent is a double ended bluntmer of 19 nucléotides in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucléotides at positions 7, 8, 9 from the 5’end. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucléotides at positions 11, 12, 13 from the 5’end.

In other embodiments, the dsRNAi agent is a double ended bluntmer of 20 nucléotides in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucléotides at positions 8, 9, 10 from the 5’end. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucléotides at positions 11, 12, 13 from the 5’end.

In yet other embodiments, the dsRNAi agent is a double ended bluntmer of 21 nucléotides in length, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucléotides at positions 9, 10, 11 from the 5’end. The antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucléotides at positions 11, 12, 13 from the 5’end.

In certain embodiments, the dsRNAi agent comprises a 21 nucléotide sense strand and a 23 nucléotide antisense strand, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucléotides at positions 9, 10, 11 from the 5’end; the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucléotides at positions 11, 12, 13 from the 5’end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucléotide overhang. Preferably, the 2 nucléotide overhang is at the 3’-end of the antisense strand.

When the 2 nucléotide overhang is at the 3’-end of the antisense strand, there may be two phosphorothioate intemucleotîde linkages between the terminal three nucléotides, wherein two ofthe three nucléotides are the overhang nucléotides, and the third nucléotide is a paired nucléotide next to the overhang nucléotide. In one embodiment, the RNAi agent additionaily has two phosphorothioate intemucleotide linkages between the terminal three nucléotides at both the 5’-end of the sense strand and at the 5’-end of the antisense strand. In certain embodiments, every nucléotide in the sense strand and the antisense strand of the dsRNAi agent, including the nucléotides that are part of the motifs are modified nucléotides. In certain embodiments each residue is îndependently modified with a 2’-Omethyl or 3’-fiuoro, e.g., in an altemating motif. Optionaliy, the dsRNAi agent further comprises a ligand (preferably GalNAc₃).

In certain embodiments, the dsRNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucléotide residues in length, wherein starting from the 5' terminal nucléotide (position 1 ) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand îs 36-66 nucléotide residues in length and, starting from the 3’ terminal nucléotide, comprises at least 8 ribonucleotides in the positions paired with positions 1 - 23 of sense strand to form a duplex; wherein at least the 3 ' terminal nucléotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3' terminal nucléotides are unpaired with sense strand, thereby forming a 3' single stranded overhang of 1-6 nucléotides; wherein the 5' terminus of antisense strand comprises from 10-30 consecutive nucléotides which are unpaired wîth sense strand, thereby forming a 10-30 nucléotide single stranded 5' overhang; wherein at least the sense strand 5’ terminal and 3' terminal nucléotides are base paired with nucléotides of antisense strand when sense and antisense strands are aligned for maximum complementarîty, thereby forming a substantially duplexed région between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian celi; and wherein the sense strand contai ns at least one motif of three 2’-F modifications on three consecutive nucléotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2’O-methyl modifications on three consecutive nucléotides at or near the cleavage site.

In certain embodiments, the dsRNAi agent comprises sense and antisense strands, wherein the dsRNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucléotides and a second strand having a length which is at most 30 nucléotides with at least one motif of three 2’-O-methyi modifications on three consecutive nucléotides at position 11, 12, 13 from the 5’ end; wherein the 3 ’ end of the first strand and the 5 ’ end of the second strand form a blunt end and the second strand is 1 -4 nucléotides longer at its 3 ’ end than the first strand, wherein the duplex région région which is at least 25 nucléotides in length, and the second strand is sufficiently complemenatary to a target mRNA along at least 19 nucléotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein Dîcer cleavage of the dsRNAi agent preferentially results in an siRNA comprising the 3’-end of the second strand, thereby reducing expression of the target gene in the mammal. Optionaliy, the dsRNAi agent further comprises a ligand.

In certain embodiments, the sense strand of the dsRNAi agent contams at least one motif of three identical modifications on three consecutive nucléotides, where one of the motifs occurs at the cleavage site in the sense strand.

In certain embodiments, the antisense strand of the dsRNAi agent can also contain at least one motif of three identical modifications on three consecutive nucléotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For a dsRNAi agent having a duplex région of 17-23 nucléotides in length, the cleavage site of the antisense strand is typically around the 10, 11, and 12 positions from the 5’-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; the 10, 11, 12 positions; the 11, 12, 13 positions; the 12, 13, 14 positions; or the 13, 14, 15 positions ofthe antisense strand, the count starting from the first nucléotide from the 5’-end ofthe antisense strand, or, the count starting from the fïrst paired nucléotide within the duplex région from the 5’- end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex région of the dsRNAi agent from the 5’-end.

The sense strand of the dsRNAi agent may contain at least one motif of three identical modifications on three consecutive nucléotides at the cleavage site of the strand; and the antisense strand may hâve at least one motif of three identical modifications on three consecutive nucléotides 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 so aligned that one motif of the three nucléotides on the sense strand and one motif of the three nucieotides on the antisense strand hâve at least one nucléotide overlap, Le., at least one of the three nucieotides of the motif in the sense strand forms a base pair with at least one of the three nucieotides of the motif in the antisense strand. Altematively, at least two nucieotides may overlap, or ail three nucieotides may overlap.

In some embodiments, the sense strand ofthe dsRNAi agent may contain more than one motif of three identical modifications on three consecutive nucieotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occuning at another portion ofthe strand that is separated from the motif at or near the cleavage site ofthe same strand. The wing modification is either adajacent to the first motif or is separated by at least one or more nucieotides. When the motifs are immediately adjacent to each other then the chemistries of the motifs are distinct from each other, and when the motifs are separated by one or more nucléotide than the chemistries can be the same or different. Two or more wing modifications may be présent. For instance, when two wing modifications are présent, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the dsRNAi agent may contain more than one motifs of three identical modifications on three consecutive nucieotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignaient similar to the wing modifications that may be présent on the sense strand.

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 nucléotides at the 3’-end, 5’end, or both ends of the strand.

In other embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not inchide the first one or two paired nucléotides within the duplex région at the 3’-end, 5’-end, or both ends of the strand.

When the sense strand and the antisense strand of the dsRNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex région, and hâve an overlap of one, two, or three nucléotides.

When the sense strand and the antisense strand of the dsRNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aiigned that two modifications each from one strand fall on one end of the duplex région, having an overlap of one, two, or three nucléotides; two modifications each from one strand fall on the other end of the duplex région, having an overlap of one, two or three nucléotides; two modifications one strand fall on each si de of the lead motif, having an overlap of one, two or three nucléotides in the duplex région.

In some embodiments, every nucléotide in the sense strand and antisense strand of the dsRNAi agent, including the nucléotides that are part of the motifs, may be modified. Each nucléotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., ofthe 2'-hydroxyl on the ribose sugar; Wholesale replacement ofthe phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification ofthe ribose-phosphate backbone.

As nucleic acids are polymers of subunîts, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at ail of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3’- or 5’-terminal position, may only occur in a terminal région, e.g., at a position on a terminal nucléotide or in the last 2, 3, 4, 5, or 10 nucléotides of a strand. A modification may occur in a double strand région, a single strand région, or in both. A modification may occur only in the double strand région of a dsRNAi agent or may only occur in a single strand région of a dsRNAi agent. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both ends, may only occur in a terminal région, e.g., at a position on a terminal nucléotide, or in the last 2, 3, 4, 5, or 10 nucléotides of a strand, or may occur in double strand and single strand régions, particularly at the ends. The 5’-end or ends can be phosphorylated.

It may be possible, e.g., to enhance stabihty, to include particular bases in overhangs, or to include modified nucléotides or nucléotide surrogates, in single strand overhangs, e.g., in a 5’- or 3’overhang, or in both. For example, ît can be désirable to include purine nucléotides in overhangs. In some embodiments ail or some of the bases in a 3’- or 5’-overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2’ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2’-deoxy-2’-fluoro (2’-F) or 2’-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g, phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In some embodiments, each residue ofthe sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2’-methoxyethyl, 2’- O-methyl, 2’-O-allyl, 2’C- allyl, 2’-deoxy, 2’-hydroxyl, or 2’-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2’- O-methyl or 2’-fiuoro.

At least two different modifications are typically présent on the sense strand and antisense strand. Those two modifications may be the 2’- O-methyl or 2’-fluoro modifications, or others.

In certain embodiments, the N_a or N_b comprise modifications of an altemating pattern. The term “altemating motif’ as used herein refers to a motif having one or more modifications, each modification occurring on altemating nucléotides of one strand. The altemating nucléotide may refer to one per every other nucléotide or one per every three nucléotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucléotide, the altemating motif can be “ABABABABABAB...,” “AABBAABBAABB...,” “AABAABAABAAB...,” “AAABAAABAAAB...,” “AAABBBAAABBB...,” or “ABCABCABCABC...,” etc.

The type of modifications contained in the altemating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucléotide, the altemating pattern, l.e., modifications on every other nucléotide, may be the same, but each ofthe sense strand or antisense strand can be selected from several possibilities of modifications within the altemating motif such as “ABABAB...”, “ACACAC...” “BDBDBD...” or “CDCDCD...,” etc.

In some embodiments, the dsRNAi agent of the invention comprises the modification pattern for the altemating motif on the sense strand relative to the modification pattern for the altemating motif on the antisense strand is shifted. The shift may be such that the modified group of nucléotides of the sense strand corresponds to a différé ntl y modified group of nucléotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the altemating motif in the sense strand may start with “ABABAB” from 5’to 3’ of the strand and the altemating motif in the antisense strand may start with “BABABA” from 5’ to 3’ofthe strand within the duplex région. As another example, the altemating motif in the sense strand may start with “AABBAABB” from 5’ to 3’ ofthe strand and the altemating motif in the antisenese strand may start with “BBAABBAA from 5 to 3 of the strand within the duplex région, so that there is a complété or partial shift of the modification patterns between the sense strand and the antisense strand.

In sonie embodiments, the dsRNAi agent comprises the pattern of the altemating motif of 2'O-methyl modification and 2’-F modification on the sense strand initially has a shift relative to the pattern of the altemating motif of 2'-O-methyl modification and 2’-F modification on the antisense strand initially, i.e., the 2'-O-methyl modified nucléotide on the sense strand base pairs with a 2’-F modified nucléotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2'-F modification, and the 1 position of the antisense strand may start with the 2'- Omethyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucléotides to the sense strand or antisense strand interrupts the initial modification pattern présent in the sense strand or antisense strand. This interruption of the modification pattern of the sense or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucléotides to the sense or antisense strand may enhance the gene silencing acîtivty against the target gene.

In some embodiments, when the motif of three identical modifications on three consecutive nucléotides is introduced to any of the strands, the modification of the nucléotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif is . .N_aYYYN_b..where “Y” represents the modification of the motif of three identical modifications on three consecutive nucléotide, and “N_a” and “N_b” represent a modification to the nucléotide next to the motif “YYY” that is different than the modification of Y, and where N_a and N_b can be the same or different modifications. Altnematively, N_a or N_b may be présent or absent when there is a wing modification présent.

The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate intemucleotide linkage modification may occur on any nucléotide of the sense strand, antisense strand, or both strands in any position of the strand. For instance, the intemucleotide linkage modification may occur on every nucléotide on the sense strand or antisense strand; each intemucleotide linkage modification may occur in an altemating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both intemucleotide linkage modifications in an alternating pattern. The altemating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the altemating pattern of the intemucleotide linkage modification on the sense strand may hâve a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In one embodiment, a double-stahded RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises two phosphorothioate intemucleotide linkages at the 5’-end and two phosphorothioate intemucleotide linkages at the 3’-end, and the sense strand comprises at least two phosphorothioate intemucleotide linkages at eiîherthe 5’-end or the 3’-end.

In some embodiments, the dsRNAi agent comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang région. For example, the overhang région may contain two nucléotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucléotides. Internucleotide linkage modifications also may be made to lînk the overhang nucléotides with the terminal paired nucléotides within the duplex région. For example, at least 2, 3, 4, or ail the overhang nucléotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucléotide with a paired nucléotide that is next to the overhang nucléotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucléotides, in which two of the three nucléotides are overhang nucléotides, and the third is a paired nucléotide next to the overhang nucléotide. These terminal three nucléotides 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.

In some embodiments, the 2-nucleotide overhang is atthe 3’-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucléotides, wherein two of the three nucléotides are the overhang nucléotides, and the third nucléotide is a paired nucléotide next to the overhang nucléotide. Optionally, the dsRNAi agent may additionally hâve two phosphorothioate internucleotide linkages between the terminal three nucléotides at both the 5’-end of the sense strand and at the 5’-end of the antisense strand.

In one embodiment, the dsRNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch may occur in the overhang région or the duplex région. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g, on the free energy of association or dissociation of a particular pairing, the simples! approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In ternis of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=înosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In certain embodiments, the dsRNAi agent comprises at least one of the First 1, 2, 3, 4, or 5 base pairs within the duplex régions from the 5’-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5’-end of the duplex.

In certain embodiments, the nucléotide atthe 1 position within the duplex région from the 5’end in the antisense strand is selected from A, dA, dU, U, and dT. Altematîvely, at Least one of the first 1, 2, or 3 base pair within the duplex région from the 5’- end of the antisense strand is an AU base pair. For example, the First base pair within the duplex région from the 5’-end of the antisense strand is an AU base pair.

In other embodiments, the nucléotide at the 3’-end of the sense strand is deoxy-thymine (dT) or the nucléotide at the 3’-end ofthe antisense strand is deoxy-thymine (dT). For example, there is a short sequence of deoxy-thymine nucléotides, for example, two dT nucléotides on the 3’-end ofthe sense, antisense strand, or both strands.

In certain embodiments, the sense strand sequence may be represented by formula (I):

5' n_p-N_a-(X X X )_rN_b-Y Y Y -N_b-(Z Z Z )j-N_a-n_q 3’ (I) wherein:

î andj are each independently 0 or 1;

p and q are each independently 0-6;

each N_a independently represents an oligonucleotide sequence comprising 0-25 modified nucléotides, each sequence comprising at least two differently modified nucléotides;

eachNb independently represents an oligonucleotide sequence comprising 0-10 modified nucléotides;

each n_p and riq independently represent an overhang nucléotide;

wherein Nb and Y do not hâve the same modification; and

XXX, YYY, and ZZZ each independently represent one motif of three identicai modifications on three consecutive nucléotides. Preferably YYY is ail 2’-F modified nucléotides.

In some embodiments, the N_a or N_b comprises modifications of altemating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For exampie, when the dsRNAi agent has a duplex région of 17-23 nucléotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g. : can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11,12; or 11, 12, 13) of the sense strand, the count sterling from the first nucléotide, from the 5’-end; or optionally, the count sterling at the first paired nucléotide within the duplex région, from the 5’-end.

In one embodiment, i is 1 andj is 0, or i is 0 andj is 1, or both i andj are 1. The sense strand can therefore be represented by the following formulas:

5'n_p-N_a-YYY-N_b-ZZZ-N_a-n_q 3' (Ib);

5' np-Na-XXX-Nb-YYY-Na-n, 3’ (le); or

5’ n_p-N_a-XXX-N_b-YYY-N_b-ZZZ-N_a-n_q 3' (Id).

When the sense strand is represented by formula (Ib), N_b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucléotides. Each N_a independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucléotides.

When the sense strand is represented as formula (le), N_b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucléotides. EachN_a can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucléotides.

When the sense strand is represented as formula (Id), each N_b independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucléotides. Preferably, N_b is 0, 1, 2, 3, 4, 5, or 6 Each N_a can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucléotides.

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

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

5' n_p-N_a-YYY- N_a-n_q 3’ (la).

When the sense strand is represented by formula (la), each N_a independently can represent an oligonucleotide sequence comprising 2-20, 2~ 15, or 2-10 modified nucléotides.

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

5'η,-ΝΛίΖ’Ζ'Ζ'ί^’-ΥΎΎ'-Ν^-ίΧ'Χ'Χ'ίΓΝ',-ηρ'3' (II) wherein:

k and 1 are each independently 0 or 1;

p’ and q^! are each independently 0-6;

each N/ independently represents an oligonucleotide sequence comprising 0-25 modified nucléotides, each sequence comprising at least two differently modified nucléotides;

each N_b' independently represents an oligonucleotide sequence comprising 0-10 modified nucléotides;

each n_p' and n_q' independently represent an overhang nucieotide;

wherein N_b’ and Y’ do not hâve the same modification; and

X'X'X', ΥΎΎ', and Z'Z'Z' each independently represent one motif of tliree îdentical modifications on three consecutive nucléotides.

In some embodiments, the N_a’ or N_b’ comprises modifications of altemating pattern.

The ΥΎΎ' motif occurs at or near the cleavage site of the antisense strand. For example, when the dsRNAÎ agent has a duplex région of 17-23 nucléotides in length, the ΥΎΎ motif can occurat positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 ofthe antisense strand, with the count starting from the first nucieotide, from the 5’-end; or optîonally, the count starting at the first paired nucieotide within the duplex région, from the 5’-end. Preferably, the ΥΎΎ' motif occurs at positions 11, 12, 13.

In certain embodiments, ΥΎΎ' motif is ail 2’-OMe modified nucléotides.

In certain embodiments, k is 1 and 1 is 0, or k is 0 and iis 1, or both k and 1 are 1.

The antisense strand can therefore be represented by the following formulas:

5’ n_q-N_a'-Z'Z'Z'-N_b'-Y'Y'Y'-N_a'-n_p. 3’ (Ilb);

5’ n_q-N_a'-Y'Y'Y'-N_b'-X'X'X'-n_P’ 3’ (Ile); or

5’ n^-N/- Z'Z'Z'-Nb'-YY'Y'-Nb'- X'X'X'-N_a'-n_p. 3’ (Ild).

When the antisense strand is represented by formula (Ilb), N_b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucléotides. Each N_a’ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucléotides.

When the antisense strand is represented as formula (Ile), N_b’ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucléotides. Each N_a’ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucléotides.

When the antisense strand is represented as formula (Ild), each N_b’ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucléotides. Each N_a’ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucléotides. Preferably, N_b is 0, 1, 2, 3, 4, 5, or 6.

In other embodiments, k is 0 and I is 0 and the antisense strand may be represented by the formula:

5' n_p-N_a-Y’Y’Y’- N_a-n_q· 3' (la).

When the antisense strand is represented as formula (Ha), each N_a’ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucléotides.

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

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

In some embodiments, the sense strand of the dsRNAi agent may contain ΥΥΎ motif occurring at 9, 10, and 11 positions of the strand when the duplex région is 21 nt, the count startîng from the first nucléotide from the 5’-end, or optionaliy, the count starting at the first paîred nucléotide within the duplex région, from the 5’- end; and Y represents 2’-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex région; and XXX and ZZZ each independently represents a 2’-OMe modification or 2’-F modification.

In some embodiments the antisense strand may contain ΥΎΎ' motif occurring at positions 11, 12, 13 ofthe strand, the count starting from the first nucléotide from the 5’-end, or optionaliy, the count starting at the first paired nucléotide within the duplex région, from the 5’ - end; and Y' represents 2’-O-methyl modification. The antisense strand may additionally contain X'X'X' motif or Z'Z'Z' motifs as wing modifications at the opposite end of the duplex région; and X'X'X' and Z'Z'Z' each independently represents a 2’-OMe modification or 2’-F modification.

The sense strand represented by any one of the above formulas (la), (Ib), (le), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (lia), (Hb), (Ile), and (Ild), respectively.

Accordingly, the dsRNAi agents for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucléotides, the iRNA duplex represented by formula (III):

sense: 5' n_p -N_a-(X X X)_s -N_b- Y Y Y -N_b -(Z Z Z)j-N_a-n_q 3' antisense: 3' n_p'-N_a-(X’X'X')_k-N_b-Y'Y'Y'-N_b’-(Z'Z'Z')i-N_a-n_q’ 5’ (III) wherein:

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

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

each N_a and N_a’ independently represents an oligonucleotide sequence comprising 0-25 modified nucléotides, each sequence comprising ai least two differently modified nucléotides;

each N_b and N_b’ independently represents an oligonucleotide sequence comprising 0-10 modified nucléotides;

wherein each n_p’, n_p, n_q’, and n_q, each of which may or may not be présent, independently represents an overhang nucléotide; and

XXX, YYY, ZZZ, X'X'X', ΥΎΎ', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucléotides.

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

Exemplary combinations of the sense strand and antisense strand forming an iRNA duplex include the formulas below:

5’ n_p-N_a-Y Y Y-N_a-n_q 3'

3' Πρ'-Ν,’-ΥΎΥ -NX 5' (Ilia)

5’ n_p -N_a -YYY -N_b -ZZZ -N_a-n_q 3’

3' n_p-N_a’-Y'Y'Y'-N_b-Z'Z'Z'-N_an_q 5’ (IHb)

5’ n_p-N_a- XXX -N_b-Y Y Y - N_a-n_q 3'

3' n_p -N_a’-X^fX'X'-N_b -ΥΎΎ'-Na'-n, 5' (Hic)

5' n_p-N_a-X X X -N_b-Y Y Y -N_b- ZZZ -N_a-n_q 3’

3' n_p -N_a’-X'X'X'-N_b’-Y'Y'Y'-N_b-Z'Z'Z'-N_a-n_q’ 5' (Illd)

When the dsRNAi agent is represented by formula (Ilia), each N_a independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucléotides.

When the dsRNAi agent is represented by formula (IIIb), each N_b independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5, or 1-4 modified nucléotides. EachN_aindependently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucléotides.

When the dsRNAi agent is represented as formula (IHc), eachN_b, N_b’ independently represents an oligonucleotide sequence comprising 0-10, 0-7,0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucléotides. EachN_a independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucléotides.

When the dsRNAi agent is represented as formula (Illd), each N_b, N_b’ independently represents an oligonucleotide sequence comprising 0-10, 0-7,0-10, 0-7, 0-5, 0-4, 0-2, or Omodified nucléotides. Each N„ N_a' independently represents an oligonucleotide sequence comprising 2-20, 215, or 2-10 modified nucléotides. Each of N_a, N_a’, N_b, and N_b’ independently comprises modifications of alternating pattem.

Each of X, Y, and Z in formulas (III), (Ilia), (Illb), (IIIc), and (Illd) may be the saine or different from each other.

When the dsRNAi agent is represented by formula (III), (Ilia), (Illb), (IIIc), and (Illd), at least one of the Y nucléotides may form a base pair with one of the Y' nucléotides. Altematively, at least two of the Y nucléotides form base pairs with the corresponding Y' nucléotides; or ail three of the Y nucléotides ail form base pairs with the corresponding Y' nucléotides.

When the dsRNAi agent is represented by formula (Illb) or (Illd), at least one of the Z nucléotides may form a base pair with one of the Z' nucléotides. Altematively, at least two of the Z nucléotides form base pairs with the corresponding Z' nucléotides; or ali three ofthe Z nucléotides ail form base pairs with the corresponding Z' nucléotides.

When the dsRNAi agent is represented as formula (IIIc) or (Illd), at least one of the X nucléotides may form a base pair with one of the X' nucléotides. Altematively, at least two of the X nucléotides form base pairs with the corresponding X' nucléotides; or ail three ofthe X nucléotides ail form base pairs with the corresponding X' nucléotides.

In certain embodiments, the modification on the Y nucieotide is different than the modification on the Y’ nucieotide, the modification on the Z nucieotide is different than the modification on the Z’ nucieotide, and/or the modification on the X nucieotide is different than the modification on the X’ nucieotide.

In certain embodiments, when the dsRNAi agent is represented by formula (Illd), the N_amodifications are 2'-O-methyl or 2'-fluoro modifications. In other embodiments, when the RNAi agent is represented by formula (Illd), the N_a modifications are 2'-O-methyl or 2'-fluoro modifications and n_p' >0 and at least one n_p' is linked to a neighboring nucieotide a via phosphorothioate linkage. In yet other embodiments, when the RNAi agent is represented by formula (Illd), the N_a modifications are 2'-O-methyl or 2^F-fluoro modifications, np' >0 and at least one np' is linked to a neighboring nucléotide via phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc dérivatives attached through a bivalent or trivalent branched linker (described below). In other embodiments, when the RNAi agent is represented by formula (Illd), the Na modifications are 2'-Omethyl or 2'-fluoro modifications, np^F >0 and at least one np' is linked to a neighboring nucléotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc dérivatives attached through a bivalent or trivalent branched linker.

In some embodiments, when the dsRNAi agent is represented by formula (Ilia), the N_amodifications are 2'-O-methyl or 2^F-fluoro modifications , np^F >0 and at least one np^F is linked to a neighboring nucléotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc dérivatives attached through a bivalent or trivalent branched linker.

In some embodiments, the dsRNAi agent is a multimer contaîning at least two duplexes represented by formula (III), (Ilia), (IIIb), (IIIc), and (Illd), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionaîly, the multimer further comprises a ligand. Each ofthe duplexes can target the same gene or two different genes; or each ofthe duplexes can target same gene at two different target sites.

In some embodiments, the dsRNAi agent is a multimer contaîning three, four, five, six, or more duplexes represented by formula (III), (Ilia), (Illb), (IIIc), and (Illd), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionaîly, 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 same gene at two different target sites.

In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (Ilia), (111b), (IIIc), and (Illd) are linked to each other at the 5’ end, and one or both of the 3’ ends, and are optionaîly conjugated to to a ligand. Each ofthe agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

Varions publications describe multimeric iRNAs that can be used in the methods of the invention. Such publications include US Patent No. 7,858,769, WO2007/091269, WO2010/141511, WO2Û07/117686, WO2009/014887, and WO2011/031520 the entire contents of each of which are hereby incorporated herein by reference.

As described in more detail below, the iRNA that contains conjugations of one or more carbohydrate moieties to an iRNA can optimise one or more properties of the iRNA. In many cases, the carbohydrate moiety will be attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of a iRNA can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit în which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring System, Le., ail ring atoms are carbon atoms, or a heterocychc ring System, Le., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring System, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring System, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. ahydroxyl group, or generally, a bond availabié for, and that is suitabîe for incorporation of the carrier into the backbone, e.g, the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optîonally, the selected moiety is connected by an intervening tetherto the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitabîe for incorporation or tethering of another Chemical entity, e.g., a ligand to the constituent ring.

The iRNA may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, pîperazînyl, [l,3]dioxolane, oxazolidinyl, isoxazolidinyl, morphoiinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is a serinol backbone or diethanolamine backbone.

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

III. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the tRNA one or more ligands, moieties or conjugales that enhance the activity, cellular distribution, or cellular uptake of the iRNA e.g., into a cell. Such moieties include but are not limited to lipid moieties such as a cholestérol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 65536556), choiic acid (Manoharan étal., Biorg. Med. Chem. Let., 1994, 4:1053-1060). In certain embodiments, the modification can include a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan étal., Biorg. Med. Chem. Let., 1993, 3:27652770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an alîphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBOJ, 1991, 10:1111-1118;

Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al.. Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol Chain (Manoharan et al., Nucleosides & Nucléotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra étal., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting or lifetime of an ÎRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molécule, cell orcell type, compartiment, e.g., a cellular or organ compartment, tissue, organ or région of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands do not take part în duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human sérum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molécule, such as a synthetic polymer, e.g., a synthetîc polyamino acid. Examples of polyamino acids include polyamino acid is a poly lysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactideco-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyuréthane, poly(2-ethylacryllic acid), N-îsopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamîne, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary sait of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a iectin, glycoprotein, lipid or protein, e.g, an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, Iectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, monovalent or multivalent N-acetylgaiactosamine, N-acetyl-gulucoseamine multivalent mannose, multivalent fucose, glycosyiated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid. cholestérol, a steroid, bile acid, folate, vitamin B12, vitamîn A, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is monovalent or multivalent N-acetylgalactosamine. In certain embodiments, the ligand is cholestérol.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilie molécules, e.g., cholestérol, chohc acid, adamantane acetic acid, 1-pyrene butyric acid, dîhydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecyiglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,03(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG {e.g., PEG-40K), MPEG, [MPEG]i, polyamîno, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens {e.g. biotin), transport/absorption faciiitators {e.g, aspirin, vitamin E, folie acid), synthetîc ribonucleases {e.g, imidazole, bisimidazole, histamine, imidazole clusters, acridineimidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g, glycoproteîns, or peptides, e.g., molécules having a spécifie affinity for a co-ligand, or antibodies e.g., an antibody, that bînds to a specified cell type such as a hepatîc cell. Ligands can also include hormones and hormone receptors. They can also include nonpeptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-ga!actosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-kB.

The ligand can be a substance, e.g, a drug, which can încrease the uptake ofthe ÎRNA agent into the ceil, for example, by disrupting the cell’s cytoskeleton, e.g., by disrupting the cell’s microtubuïes, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, Vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, îndanocine, or myoservin.

In some embodiments, a ligand attached to an ÎRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholestérol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bînd to sérum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the présent invention as ligands {e.g. as PK modulating ligands). In addition, aptamers that bind sérum components {e.g. sérum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated iRNAs ofthe invention may be synthesized by the use ofan oligonucleotide that beats a pendant reactive functionality, such as that derived from the attachment of a linking molécule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-avai labié ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that hâve a linking moiety attached thereto.

The oligonucleotides used in the conjugales of the présent invention may be conveniently and routînely made through the weli-known technique of solid-phase synthesis. Equipment 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 additionally or altematively be employed. It 5 is also known to use similar techniques to préparé other oligonucleotides, such as the phosphorothioates and alkylated dérivatives.

In the ligand-conjugated ÎRNAs and ligand-molécule bearing sequence-specific linked nucleosides of the présent invention, the oligonucleotides and oligonucleosîdes may be assembled on a suitable DNA synthesizer utîlîzing standard nucléotide or nucleoside precursors, or nucléotide or 10 nucleoside conjugale precursors that already bear the linkîng moiety, ligand-nucléotide or nucleosideconjugate precursors that already bear the ligand molécule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molécule is then 15 reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodîments, the oligonucleotides or linked nucleosides ofthe présent invention are synthesized by an automated synthesizer using phosphoramidites derived from lîgand-nucleoside conjugales in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routînely used in oligonucleotide synthesis.

A. Lipid Conjugales

In certain embodîments, the ligand or conjugale is a lipid or lipid-based molécule. Such a lipid or lipid-based molécule preferably binds a sérum protein, e.g., human sérum albumin (HSA). An HSA binding ligand allows for distribution ofthe conjugale to a target tissue, e.g., anon-kidney target tissue of the body. For example, the target tissue can be the lîver, including parenchymal cells 25 of the liver. Other molécules that can bind HSA can also be used as ligands. For example, naproxen or aspîrin can be used. A lipid or lipid-based ligand can (a) increase résistance to dégradation of the conjugale, (b) increase targeting or transport into a target cell or cell membrane, or (c) can be used to adjust binding to a sérum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugale to a 30 target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugale to the kidney.

In certain embodîments, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficîent affinity such that the conjugale will be preferably distrîbuted to a non-kidney tissue.

However, it is preferred that the afïïnity not be so strong that the HSA-ligand binding cannot be reversed.

Τη other embodiments, the lipid based ligand binds HSA weakly or not at ail, such that the conjugate will be preferably distributed to the kidney. Other moietîes that target to kidney cells can also be used in place of, or in addition to, the lipid based ligand.

In another aspect, the ligand is a moîety, e.g., a vitamin, which is taken up by a target cell, 5 e.g., a proliferating cell. These are particularly useful for treating disorders characterîzed by unwanted cell prolifération, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folie acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Perméation Agents

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-perméation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an 15 alpha-helical agent, which preferably has a lipophilie and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molécule capable of folding into a defrned three-dimensional structure similar to a natural peptide. The attachaient of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular récognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell perméation peptide, cationic peptide, amphipathic peptide, or hydrophobie peptide (e.g, consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another 25 alternative, the peptide moiety can include a hydrophobie membrane translocation sequence (MTS). An exemplary hydrophobie MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 14). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 15) containing a hydrophobie MTS can also be a targetîng moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molécules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQIDNO:16)and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 17) hâve been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targetîng purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in Iength from about 5 amino acids to about 40 amino acids. The peptide moietîes can hâve a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g, glycosylated or methylated, to facilitate targeting to a spécifie tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugales of this ligand target PECAM-1 or VEGF.

A “cell perméation peptide” is capable of permeating a cell, e.g, a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin PI), a disulfide bondcontaming peptide (e.g., a -defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidîn). A cell perméation peptide can also include a nuclear localization signal (NLS). For example, a cell perméation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-] gp4l and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugales

In some embodiments of the compositions and methods of the invention, an iRNA further comprises a carbohydrate. The carbohydrate conjugated iRNA is advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Représentative carbohydrates include the sugars (mono-, di-, tri-, and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Spécifie monosaccharides include HBV and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

HO

HO ^0H

O

N

Formula IIL

H

O

Formula IV.

O

Formula V.

Formula VI

NHAc

O

HO

-O

NHAc OH

HO

AcHN

HO ^OH

Ο

HO

AcHN

HO

OH

HO

AcHN

HO HO

HO

OH

HO

O

HO

O

HO

OH ^NHAC

HO

NHAc

HO

OH

HO

Formula IL

Another représentative carbohydrate conjugale for use in the embodiments described herein

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

In certain embodiments of the invention, the GalNAc or GalNAc dérivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc dérivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc dérivative is attached to an iRNA agent of the invention via a tri valent linker.

In one embodiment, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc dérivative attached to the iRNA agent. In another embodiment, the double stranded

RNAi agents ofthe invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc dérivatives, each independently attached to a plurality of nucléotides ofthe double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molécule connected by an uninterrupted chain of nucléotides between the 3’-end of one strand and the 5’-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucléotides, each unpaired nucieotide within the hairpin loop may independently comprise a GalNAc or GalNAc dérivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell perméation peptide.

Additional carbohydrate conjugales suitable for use in the présent invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by référénce.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

The term linker or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SCL, SO₂NH or a chain of atoms, such as, but not limited to, substîtuted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroaryl alkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclyl alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylaryl alkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkeny!, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyî, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, aikynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which 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 heterocyclic; where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is between about 124 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred

embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mirnic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to 5 mirnic or represent conditions found in the blood or sérum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molécules. Generally, cleavage agents are more prévalent or found at higher levels or activities inside cells than in sérum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which hâve no substrate specificity, 10 including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, présent in cells, that can dégradé a redox cleavable linking group by réduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that resuit in a pH of five or lower; enzymes that can hydrolyze or dégradé an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate spécifie), and phosphatases.

A cleavable Linkage group, such as a disulfide bond can be susceptible to pH. The pH of human sérum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes hâve a more acidic pH, in the range of 5.5-6.0, and lysosomes hâve an even more acidic pH at around 5.0. Some linkers will hâve a cleavable linking group that is cleaved at a preferred pH, îhereby releasing a cationic lipid from the ligand inside the ce H, or into the desired compartment of the cell.

A iinker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a lînker can dépend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a Iinker that includes an ester group. Liver cells are rich in esterases, and therefore the Iinker will be cleaved more effîciently 25 in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, rénal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing 30 the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be désirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can détermine the relative susceptibîlity to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second îs selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or sérum. The évaluations can be carried out in cell free Systems, in cells, in cell culture, in organ or tissue culture, or in whole animais. It can be useful to make initial évaluations in ce 11-free or culture conditions and to confinn by further évaluations in whole animais. In preferred embodiments, useful candidate compounds are cleaved at least about 2,

4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in the cell (or under in vitro conditions selected to mimîc intracellular conditions) as compared to blood or sérum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox cleavable linking groups

In certain embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon réduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (-S-S-). To détermine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cieavage which wouid be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or sérum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30,40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extraceilular conditions). The rate of cieavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-based cleavable linking groups

In other embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that dégradé or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -OP(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -OP(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-, -OP(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 methods analogous to those described above.

iii. Acid cleavable linking groups

In other embodiments, a 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 acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, spécifie low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can hâve the general formula -C=NN-, C(O)O, or ~OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substîtuted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-based linking groups

In other embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups hâve the general formula -C(O)O-, or -OC(O)-. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-based cleaving groups

In yet other embodiments, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds forrned between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the ami de group (-C(O)NH-). The amide group can be forrned between any alkylene, alkenylene or alkynelene. A peptide bond is a spécial type of amide bond forrned between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (Le., the amide bond) forrned between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups hâve the general formula — NHCHRAC(O)NHCHRBC(O)- (SEQID NO:__), where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

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

(Formula XXVII),

(Formula XXIX),

(Formula XXXI), when one of X or Y is an oligonucleotide, the other is ahydrogen.

In certain embodiments ofthe compositions and methods ofthe invention, a ligand is one or more “GalNAc” (N-acetyigalactosamine) dérivatives attached through a bivalent or trivalent branched linker.

In certain embodiments, a dsRNA of the invention is conj ugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXII) - (XXXV):

Formula XXXI11

Formula XXXII

-f2A__L2A p2B„Q2^_R2B y 26 |_28 _q2B

T^-L⁴®

Formula XXXIV

pÎA.QÎA._R5A L__t5A._l5A

Vf

V ρίί.θ5€^5< L _T^_L6Q Jq^5C

Formula XXXV wherein:

q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;

p2A p2B p3A p3B p4A p4B p5A p5B p5C ψ2Α yZB yJA yBB y4A y4B y4A

T^5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, or CH₂O;

Q^2A, Q^3B, Q^3A, Q^3B, Q^4A, Q^4B, Q^5A, Q^SB, Q^îc are independently for each occurrence absent, alkylene, substituted alkylene wherin 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, rîa, rsb, r» r4b, rsa, rîc _{are eac};₁ Î_nci_ependently for each occurrence absent,

O

NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^a)C(O), -C(0)-CH(R^a)-NH-, CO, CH=N-O,

heterocyclyl;

L^2A, L^2B, L^3A, L^3B, L^4A, L⁴⁶, L^SA, L^5B, and L^5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; andR^a is H or amino acid side chain.Trivalent conjugating GalNAc dérivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

Formula XXXV

wherein L^5A, L^5B and L^5C represent a monosaccharide, such as GalNAc dérivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc dérivatives include, but are not limited to, the structures recited above as formulas Π, VII, XI, X, and XIII.

Représentative US Patents that teach the préparation of RNA conjugales include, but are not limited to, US 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 hereby incorporated herein by reference,

It is not necessary for ali positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The présent invention also inciudes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAi agents, that contaîn two or more chemically distinct régions, each made up of at least one monomer unit, i.e., a nucieotide in the case of a dsRNA compound. These iRNAs typically contaîn at least one région wherein the RNA is modified so as to confer upon the iRNA increased résistance to nuclease dégradation, increased cellular uptake, or increased bindîng affinity for the target nucleic acid. An additiona] région of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNAiRNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNAiDNA duplex. Activation of RNase H, therefore, results in cleavage ofthe RNA target, thereby greatiy enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target région. Cleavage of the RNA target can be routinely detected by gel electrophoresîs and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molécules hâve been conj ugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake ofthe iRNA, and procedures for performing such conjugations are avaiiable in the scientific literature. Such non-ligand moietîes hâve included lipid moieties, such as cholestérol (Kubo, T. étal., Biochem. Biophys. Res. Comm., 2007, 365(1 ):54-61; Letsînger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553) can be used in the agents ofthe invention. Other nonligand moieties hâve included lipid moieties, cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic Chain, e.g., dodecandiol or undecyl residues (SaisonBehmoaraser^ÆÀffiOJ., 1991, 10:111; Kabanov et al., FEBSLett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyiammonium 1,2di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol Chain (Manoharan et al., Nucleosides & Nucléotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.

Pharmacol. Exp. Ther., 1996, 277:923), Représentative United States patents that teach the préparation ofsuch RNA conjugales hâve been listed above. Typical conjugation protocols involve the synthesis of RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molécule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugale by HPLC typically affords the pure conjugale.

IV. DeJivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disease, disorder, or condition associated with PD-LI gene expression) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administerîng a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternative!y, in vivo delivery may be performed îndirectly by administerîng one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further beîow.

In general, any method of delivering a nucleic acid molécule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian RL. (1992) Trends Cell, Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in theîr entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molécule include, for example, biological stability ofthe delivered molécule, prévention of non-specific effects, and accumulation of the delivered molécule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administerîng the préparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can dégradé the agent, and permits a lower total dose of the iRNA molécule to be administered. Several studies hâve shown successful knockdown of gene products when a dsRNAi agent is administered locally. For example, intraocular delivery ofa VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, MJ, et al (2004) Retina 24:132-138) and subretinal injections in mîce (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survivai 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 interférence has also shown success with local delivery to the CNS by direct injection (Dom, G., étal. (2004) NucleicAcids 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 to the lungs by intranasa] 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). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternative!y delivered using a drug delivery System; both methods act to prevent the rapid dégradation ofthe dsRNA by endo- and exo-nucleases in vivo. Modification ofthe RNA or the pharmaceutical carrier can also permit targeting of the iRNA to the target tissue and avoid undesirable off-target effects. iRNA molécules can be modified by Chemical conjugation to lipophilie groups such as cholestérol to enhance cellular uptake and prevent dégradation. For exampie, an iRNA directed against ApoB conjugated to a lipophilie cholestérol moiety was injected systemically into mice and resulted in knockdown ofapoB mRNA in both the liver and jéjunum (Soutschek, J., étal (2004) Nature 432:173178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and médiate tumor régression in a mouse model of prostate cancer (McNamara, JO, et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery Systems such as a nanopartîcle, a dendrimer, a polymer, liposomes, or a cationic delivery System. Positively charged cationic delivery Systems facilitate binding of an iRNA molécule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an ÎRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim SH, et al (2008) iournal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents dégradation ofthe iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, DR, et al (2003)7 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 încorporated herein by reference in their entirety). Some non-limiting examples of drug delivery Systems useful for systemîc delivery of iRNAs include DOTAP (Sorensen, DR., et al (2003), supra; Verma, UN, et al (2003), supra), Oligofectamîne, solid nucleic acid lipid particies (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)7 Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Lîu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, DA, et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in US Patent No. 7,427,605, which is herein încorporated by reference in îts entirety.

A. Vector encodediRNAs of the Invention iRNA targeting the PD-L i gene can be expressed from transcription units mserted into DNA or RNA vectors (see, e.g., Couture, A, étal., TIG. (1996), 12:5-10; Skillem, A, et al., International

PCT Publication No. WO 00/22113. Conrad. International PCT Publication No. WO 00/22114, and Conrad, US Patent No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the spécifie construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circulât plasmid, or a viral vector, which can be an integrating or non-întegrating vector. The transgene can also be constructed to permît it to be inherîted as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

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

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as descri bed hereîn. Eukaryotic cell expression vectors are weli known in the art and are available from a number of commercial sources. Typically, such vectors are provîded containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by réintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

Viral vector Systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) rétro virus vectors, including but not limited to lentiviraî vectors, moloney murine leukemia virus, etc. ; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picomavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replicationdefective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells’ genome. The constructs can include viral sequences for transfection, if desired. Altematively, the construct can be incorporated into vectors capable of episomal réplication, e.g. EPV and EB V vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory éléments, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The présent invention also încludes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of a PD-L1 gene. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are fonnulated for systemic administration via parentéral delivery, e.g., by subcutaneous (SC) or intravenous (IV) delivery. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a PD-L1 gene.

The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of aPD-Ll gene. In general, a suitabie dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the récipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. Typically, a suitabie dose of an iRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, e.g., about 0.3 mg/kg and about 3.0 mg/kg. A repeat-dose regimine may include administration of a the râpe ut ic amount of iRNA on a regular basis, such as every other day or once a year. In certain embodîments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months).

After an initial treatment regimen, the treatments can be administered on a less frequent basis. For exampie, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months, or a year; or longer.

The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a contre lied release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventionaî sustained release formulation which provides sustaîned release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the présent invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

In other embodîments, a single dose of the pharmaceutical compositions can be long lasting, such that subséquent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals. In some embodîments ofthe invention, a single dose of the pharmaceutical compositions of the invention is administered once per week. In other embodîments of the invention, a single dose of the pharmaceutical compositions of the invention is administered bimonthly. In certain embodîments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months).

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity ofthe disease or disorder, préviens treatments, the general health or âge of the subject, and other diseases présent. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a sériés of treatments. Estimâtes of effective dosages and in vivo haif-lives for the individual iRNAs encompassed by the invention can be made using conventional méthodologies or on the basis of in vivo testing using an appropriate animal model, as known in the art.

For example, animal models ofhepatitis B infection are known in the art including chimpanzee, woodchuck, and transgenic mouse models ofHBV (Wieland, 2015. Cold Spring Harb. Perspect. Med., 5:a02l469, 2015; Tennant and Gerin, 2001. ILAR Journal, 42:89-102; and Moriyama étal., 1990. Science, 248:361-364). The chimpanzeemodel canalso be used as amodel forhepatitis D infection. A large number of cancer models including chemically înduced and xenograft tumors are known in the art.

The pharmaceutical compositions ofthe présent invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aérosols, including by nebulizer; intratracheal, întranasal, epidermal and transdermal, oral or parentéral. Parentéral administration includes intravenous, intraarterial, subeutaneous, intraperitoneal, or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or întraventricular administration.

The iRNA can be delivered in a manner to target a particular tissue (e.g., liver cells).

Pharmaceutical compositions and formulations for topical or transdermal administration can include fransdemtal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oîly bases, thickeners and the like can be necessary or désirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dîoleoylphosphatidyl DOPE ethanolamine, dîmyristoyl phosphatidyl choline DMPC, distearolyphosphatidyl choline) négative (e.g., dimyristoylphosphatidyl glycerot DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dîoleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, caprîc acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, l-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a C^o alkyl ester (e.g., isopropylmyriState IPM), monoglyceride, diglycende or pharmaceutically acceptable sait thereof). Topical formulations are described in detail în US Patent No. 6,747,014, which is incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assembliez

An ÎRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g-,Ά liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilameilar vesicles that hâve a membrane formed from a lipophilie material and an aqueous interior. The aqueous portion contains the iRNA. The lipophilie material isolâtes the aqueous interior from an aqueous exterior, which typîcally does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingrédients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internai aqueous contents that include the iRNA are delîvered înto the cell where the iRNA can specifically bind to a target RNA and can médiate RNA interférence. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome contaîning an iRNA agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelies are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugale. The detergent can hâve a hîgh critical micelle concentration and may be nonionic. Exemplary détergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The ÎRNA agent préparation is then added to the micelies that include the lipid component. The cationic groups on the lipid interact with the iRNA agent and condense around the iRN A agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal préparation of iRNA agent.

If necessary a carrier compound that assists în condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; US Patent No. 4,897,355; US Patent No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Oison, et al. Biochim. Biophys. Acta ΜΊ'.9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, étal. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol.

115:757, 1984. C o mm on 1 y used techniques for preparing lipid aggregates of approprîate size for use as delivery vehîcles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al.

Biochim. Biophys. Acta 858:161, 1986). Microfluidîzation can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA agent préparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molécules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acîdîc pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap nucleic acids rather than complex with it. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes hâve been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected în the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dîoleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of two or more of phospholîpid, phosphatidylcholine, and cholestérol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include US Patent Nos. 5,283,185 and 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Naît. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBOJ. 11:417, 1992.

N on-i o nie liposomal Systems hâve also been examined to détermine their utility in the delivery of drugs to the skin, in particular Systems comprising non-ionic surfactant and cholestérol. Non-ionic liposomal formulations comprising Novasome™ J (glyceryl diiaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ Il (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal Systems were effective in facilitating the déposition of cyclosporine A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4(6) 466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, resuit in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part ofthe vesicle-forming lipid portion ofthe liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_Mi, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes dérivés from a reduced uptake into cells ofthe réticuloendothélial System (RES) (Allen étal., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Varions liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N. K Acad. Sci., 1987, 507, 64) reported the abîlity of monosialoganglioside Gmi, galactocerebrosîde sulfate and phosphatidylinositol to improve biood half-lives of liposomes. These findings were expounded upon by Gabizon étal. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). US Patent No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_Ml or a galactocerebrosîde sulfate ester. US Patent No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2sn-dimyristoylphosphatidy!choline are disclosed in WO 97/13499 (Lim étal).

In some embodiments, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficient! y with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNA agents to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodégradable; liposomes can incorporate a wide range ofwater and lipid soluble drugs; liposomes can protect encapsulated tRNAs in their internai compartments from metabolism and dégradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, volume l, p. 245). Important considérations in the préparation of liposome formulations are the lipid surface charge, vesicle size, and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[l-(2,3-dioley!oxy)propyl]-N,N,Ntrimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids ofthe cell membranes of tissue culture cells, resulting in delivery of iRNA agent (see, e.g., Feîgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and US Patent No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, l,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ (Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nudeic acids into living tissue culture cells that comprise posîtively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to forai complexes. When enough posîtively charged liposomes are used, the net charge on the resulting complexes is also positive. Posîtively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercîally available cationic lipid, 1,2bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that hâve been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (DOGS) (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5carboxyspermyl-amide (“DPPES”) (see, e.g., US Patent No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholestérol (“DCChol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopoiy lysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of sérum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercîally 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 the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes présent several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, încreased accumulation of the administered drug at the desired target, and the ability to administer iRNA agent into the skin. In some implémentations, liposomes are used for delivering iRNA agent to epidermal cells and also to enhance the pénétration of iRNA agent into dermal tissue s, e.g., into skin. For examp le, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeüng, 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 Papahadjopouios, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal Systems hâve also been examined to détermine their utility in the delivery of drugs to the skin, in particular Systems comprising non-ionic surfactant and cholestérol.

Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl dîstearate/ cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA agent are useful for treatîng a dermatological disorder.

Liposomes that include iRNA can be made hîghly déformable. Such deformability can enable the liposomes to penetrate through pore that are smalier than the average radius of the liposome. For example, transfersomes are a type of déformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNAs can be delivered, for example, subcutaneously by infection in order to deliver iRNAs to kératinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a sériés of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid propertîes, these transferosomes can be selfoptimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the présent invention are described in WO/2008/042973.

Transfersomes are yet another type of liposomes, and are hîghly déformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so hîghly déformable that they are easily abie to penetrate through pores which are smalier than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes hâve been used to deliver sérum albumin to the skin. The transfersome-mediated delivety of sérum albumin has been shown to be as effective as subcutaneous injection of a solution contaîning sérum albumin.

Surfactants find wide application in formulations such as émulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the propertîes of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the head) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molécule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molécule carries a négative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfurie acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl faurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molécule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molécule bas the ability to carry either a positive or négative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid dérivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in émulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms”, Marcel Dekker, Inc., New York, N. Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided as mîcellar formulations. “Micelles” are defmed herein as a particular type of molecular assembly in which amphipathic molécules are arranged in a spherical structure such that al! the hydrophobie portions of the molécules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobie.

A mixed micellar formulation suitable for delîvery through transdermal membranes may be prepared by mixing an aqueous solution of iRNA, an alkali métal C_g to C₂₂ alkyl sulphate, and a micelle forming compounds. 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, monooleates, monoiaurates, borage oil, evening of primrose oit, menthol, trihydroxy oxo choîanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali métal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingrédients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the RNAi and at least the alkali métal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the RNAi, the alkali métal alkyl sulphate and at least one ofthe micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phénol or m-cresol may be added to the mixed micellar composition to stabilise the formulation and protect against bacterîal growth. Altematively, phénol or m-cresol may be added with the micelle forming ingrédients. An isotonie agent such as glycerin may also be added after formation of the mixed micellar composition.

For deiivery of the micellar formulation as a spray, the formulation can be put into an aérosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingrédients are adjusted so that the aqueous and propellant phases become one, Le., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion ofthe contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The spécifie concentrations of the essential ingrédients can be determined by relatively straightforward expérimentation. For absorption through the oral cavities, it is often désirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

B. Lipid particles

ÎRNAs, e.g., dsRNAi agents of in the invention may be ftilly encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-li pid parti cle.

As used herein, the terrn LNP refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g, a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include pSPLP, which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No.

WO 00/03683. The particles of the présent invention typically hâve a mean 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, most typically about 70 nm to about 90 nm, and are substantially nontoxîc. In addition, the nucleic acids when présent in the nucleic acid-lipid particles of the présent invention are résistant in aqueous solution to dégradation with a nuclease. Nucleic acid-lipid particles and their method of préparation are disclosed in, e.g., US Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; US Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) wîll be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3; 1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermédiare to the above recited ranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I -(2,3dioJeoyloxy)propy]>N,N,N-trimethylammonium chloride (DOTAP), N-(I -(2,3- dioleyloxy)propyl)Ν,Ν,Ν-trimethylammonium chloride (DOTMA), N,N-dimethyI-2,3- dioleyloxy)propylatnine (DODMA), l,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,Ndimethylaminopropane (DLenDMA), l,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLinC-DAP), l,2-Dilinoleyoxy-3-(dimethylamino)aceîoxypropane (DLin-DAC), l,2-Dilinoleyoxy-3morpholinopropane (DLin-MA), l,2-Dîlinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2Dilinoleylthio-3-dimethylaminopropane (DLÎn-S-DMA), l-Linoleoyl-2-linoleyloxy-3dîmethylaminopropane (DLÎn-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride sait (DLÎn-TMA.Cl), l,2-DilinoÎeoyl-3-trimethylaminopropane chloride sait (DLin-TAP.Cl), 1,2Dilinoleyloxy-3-(N-methy]piperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-l,2propanediol (DLinAP), 3-(N,N-Dioleylamino)-l,2-propanedio (DOAP), l,2-Dilinoleyloxo-3-(2-N,Ndimethylamino)ethoxypropane (DLin-EG-DMA), l,2-Dilinolenyioxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dîmethy]-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aHcyclopenta[d][l,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), l,T-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2hydroxydodecyl)ammo)ethyI)piperazin-l-yl)ethylazanedîyl)didodecan-2-ol (Tech Gl), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid présent in the particle.

In some embodîments, the compound 2,2-Dilmoleyl-4-dimethylaminoethyl-[l,3]-dioxolane can be used to préparé lipîd-siRNA nanoparticles.

In some embodîments, the lîpid-siRNA particle încludes 40% 2, 2-Dilinoleyl-4dîmethylaminoethyl-[l,3]-dioxolane: 10% DSPC: 40% Cholestérol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0 ± 20 nm and a 0.027 siRNA/Lipid Ratio.

The ionizable/non-cationic lipid can be an anionîc lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dîoleoyl-phosphatidylethanolamine (DOPE), palmîtoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidyl éthanol amine (POPE), dioleoyl- phosphatidyl éthanol ami ne 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPEmal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dîmyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-rnonomethyl PE, 16-O-dîmethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), cholestérol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 moi %, about 10 mol %, or about 58 mol % if cholestérol is încluded, of the total lipid présent in the particle.

The conjugated lipid that inhibits aggregation of partie les can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEGdialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryïoxypropyl (Ci₂), a PEGdimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG- distearyloxypropyl (C]_s>. The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid présent in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholestérol at, e.g, about 10 mol % to about 60 mol % or about 48 mol % ofthe total lipid présent in the particle.

In one embodiment, the lipidoid ND98-4HC1 (MW 1487) (see US20090023673, which is incorporated herein by reference), Cholestérol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to préparé lipid-dsRNA nanopartîcles (i.e., LNP01 particles). Stock solutions of each in éthanol can be prepared as follows: ND98, 133 mg/ml; Cholestérol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholestérol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final éthanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanopartîcles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the résultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and sirnultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

ND98 Isomer I

Formula 1

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

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

Table 1. Exemplary lipid formulations

	lonizable/Cationic Lipid	cationic lipid/non-cationic lipid/choiesterol/PEG-Iipid conjugale Lipid:siRNA ratio
SNALP-1	1,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLinDMA)	DLinDMA/DPPC/Cholesterol/PEG-cDMA (57.1/7.1/34.4/1.4) !ipid:siRNA ~ 7:1
2-XTC	2,2-Dilinoleyl-4-dimethylaminoethyi-{l,3]dioxoiane (XTC)	XTC/DPPC/Cholesterol/PEG-cDMA 57.1/7.1/34.4/1.4 lipid:siRNA - 7:1
LNP05	2,2-Dilinoley[-4-dimeihy!aminoethyL[l,3]dioxolane (XTC)	XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipidisiRNA ~ 6:1
LNP06	2,2-Dilinoleyl-4-dimethylaminoethyl-[l ,3]dioxolane (XTC)	XTC/DSPC/Cholesterol/PEG-DMG 57.5/7.5/31.5/3.5 lipid:siRNA —11:1
LNP07	2,2-Dilinoleyl-4-di methy 1 amînoethy 1 - [ l, 3 ]dioxolane (XTC)	XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA - 6:1
LNP08	2,2-D i l mo ley 1 -4-d i methy lamino ethy 1 - [ 1,3 ] dioxolane (XTC)	XTC/DSPC/Cholesterol/PEG-DMG 60/7.5/31/1.5, lipid:siRNA-ll:1
LNP09	2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]dioxolane (XTC)	XTC/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1
LNPIO	(3aR,5s,6aS)-N,N-dimethy]-2,2- di((9Z,12Z)-octadeca-9,12- dienyl)tetrahydro-3aH- cy c l openta[d] [ 1,3 ] di oxo 1-5 -am ine (ALN100)	ALN100/DSPC/CholesteroPPEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1
LNP11	(6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31tetraen-19-yl 4-(dimethyIamino)butanoate (MC3)	MC-3/DSPC/Cho!esterol/PEG-DMG 50/10/38.5/1.5 Lipid:sîRNA 10:1
LNP12	l,l'-(2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethy])(2- hy dro xy dodecy l )am i no )ethy 1) p î p e razin-1 -	Tech Gl/DSPC/Cholesterol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA 10:1

	lonizable/Cationîc Lipid	cationic 1 i pi d/non-cationic lipid/cholesteroLPEG-lipid conjugale LipidisiRNA ratio
	yl)ethylazanediyl)didodecan-2-ol (Tech Gl)
LNP13	XTC	XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 LipidisiRNA: 33:1
LNP14	MC3	MC3/DSPC/Chol/PEG-DMG 40/15/40/5 LipidisiRNA: 11:1
LNP15	MC3	MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 LipidisiRNA: 11:1
LNP16	MC3	MC3/DSPC/Cho!/PEG-DMG 50/10/38.5/1.5 LipidisiRNA: 7:1
LNP17	MC3	MC3/DSPC/Choi/PEG-DSG 50/10/38.5/1.5 LipidisiRNA: 10:1
LNP18	MC3	MC3/DSPC/Choi/PEG-DMG 50/10/38.5/1.5 LipidisiRNA: 12:1
LNP19	MC3	MC3/DSPC/Chol/PEG-DMG 50/10/35/5 LipidisiRNA: 8:1
LNP20	MC3	MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 LipidisiRNA: 10:1
LNP21	Cl 2-200	C 12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 LipidisiRNA: 7:1
LNP22	XTC	XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1

DSPC: distearoyl phosphatidyl choline

DPPC: dipalmitoylphosphatidylcholine

PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)

PEG-DSG: PEG-distyryl glycerol (C 18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)

PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (l,2-Dilinoleny!oxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO 2009/127060, the entire contents of which is hereby Încorporated herein by reference.

XTC comprising formulations are described, e.g, in PCT Publication No. WO 2010/088537, the entire contents of which is hereby încorporated herein by reference.

MC3 comprising formulations are described, e.g., in US Patent Publication No. 2010/0324120, filed J une 10, 2010, the entire contents of which are hereby încorporated by reference.

ALNY-100 comprising formulations are described, e.g., PCT Publication No, WO 2010/054406, the entire contents of which are hereby încorporated herein by reference.

C12-200 comprising formulations are described in PCT Publication No, WO 2010/129709, the entire contents of which are hereby încorporated herein by reference.

i. Synthesis of ionizable/caiionic lipids

Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention can be prepared by known organic synthesis techniques.

Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sédiment. Particle sîze and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvem Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in sîze. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sampie of the 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, e.g., 0.5% Trîton®-X100. The total dsRNA in the formulation can be determined by the signal from the sampie containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, 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. The suitable range is typically 50 nm to 110 nm, 60 nm to 100 nm, or 80 nm to 90 nm.

Compositions and formulations for oral administration include powders or granules, microparti cul ates, nanoparticulates, suspensions, or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders can be désirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjonction with one or more pénétration enhancer surfactants and chelators. Suitable surfactants include fatty acids or esters or salts thereof, bile acids or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glychoiic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, paimitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1 -dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a dîglyceride or a pharmaceutically acceptable sait thereof (e.g., sodium). In some embodiments, combinations of pénétration enhancers are used, for example, fatty acids/salts în combination with bile acids/salts. One exemplary combination is the sodium sait of lauric acid, capric acid and UDCA. Further pénétration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried partîcles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, poiyalkyicyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG), and starches; poiyalkyicyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses, and starches. Suitable complexing agents include chîtosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermînes, protamine, polyvinylpyridine, polythiodîethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amîno), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamîde, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-!actic acid), poly(DL-lactic-co-glycoiÎc acid (PLGA), algînate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their préparation are described in detail in US Patent 6,887,906, US Publn. No. 20030027780, and US Patent No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parentéral, intraparenchymal (into the brain), intrathecal, intraventricular, or intrahepatic administration can include stérile aqueous solutions which can also contain buffets, diluents, and other suitable addîtives such as, but not limited to, pénétration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions ofthe présent invention include, but are not limited to, solutions, émulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Formulations include those that target the liver when treatîng hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the présent invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingrédients with the pharmaceutical carrter(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingrédients with liquid carriers or ftnely divided solid carriers or both, and then, if necessary, shaping the product,

The compositions of the présent invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the présent invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media, Aqueous suspensions can further contain substances which increase the viscosîty of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, or dextran, The suspension can also contain stabilizers.

C. Additional Formulations î. Emulsions

The iRNAs of the présent invention can be prepared and formulated as émulsions. Emulsions are typicaily heterogeneous Systems of one liquid dispersed in another in the form of droplets usually exceedîng 0.1 pm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume l, p. 245; Block în Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic Systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, émulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is fine 1 y divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition îs called a water-in-oil (w/o) émulsion. Altematively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-inwater (o/w) émulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be présent as a solution either in the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and antioxidants can also be présent in émulsions as needed. Pharmaceutical émulsions can also be multiple émulsions that are comprised of more than two phases such as, for example, in the case of oil-inwater-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) émulsions. Such complex formulations often provide certain advantages that simple binary émulsions do not. Multiple émulsions in which indîvidual oîl droplets of an o/w émulsion enclose smali water droplets constitute a w/o/w émulsion.

Likewise a System of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o émulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or dîscontinuous phase ofthe émulsion is well dispersed into the extemal or continuous phase and 5 maintained in this form through the means of emulsifïers or the viscosity of the formulation. Either of the phases of the émulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and créants. Other means of stabiiizing émulsions entai! the use of emulsifïers that can be incorporated into either phase ofthe émulsion. Emulsifïers can broadly be classified into four categories: synthetic surfactants, naturaliy occurring emulsifïers, absorption bases, and fînely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, hâve found wide applicability in 15 the formulation of émulsions and hâve been reviewed in the literature (see e.g., Ansel's

Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 20 Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobie portion. The ratio of the hydrophilic to the hydrophobie nature of the surfactant has been termed the hydrophile/Iipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the préparation of formulations. Surfactants can be classified into different classes based on the nature ofthe hydrophilic group: nonionic, anionic, 25 cationic, and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturaliy occurring emulsifïers used in émulsion formulations include lanolin, beeswax, 30 phosphatides, lecithin, and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o émulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids hâve also been used as good emulsifïers especially in combination with surfactants and in viscous préparations. These include polar inorganic solids, such as heavy métal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, 35 kaolin, montmorillonite, colloïdal aluminum silicate, and colloïdal magnésium alumînum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsitying materials are also included in émulsion formulations and contribute to the properties of émulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophî lie colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume l, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N. Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose dérivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloïdal solutions that stabilize émulsions by forming strong interfacial films around the dispersed-phase dropiets and by increasing the viscosîty of the extemal phase.

Since émulsions often contain a number of ingrédients such as carbohydrates, proteins, sterols and phosphatides that can readîly support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in émulsion formulations include methyl paraben, propyl paraben, quaternary ammonium saîts, benzalkonium chloride, esters of phydroxybenzoic acid, and boric acid. Antioxidants are also commonly addedto émulsion formulations to prevent détérioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxîdant synergists such as citric acid, tartaric acid, and lecithin.

The application of émulsion formulations via dermatological, oral, and parentéral routes, and methods for their manufacture hâve been reviewed in the lîterature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Anse) HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume I, p. 199). Emulsion formulations for oral delivery hâve been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume i, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume I, p. 199). Mineral-oil base laxatives, oil-soluble vitamins, and high fat nutritive préparations are among the materials that hâve commonly been administered orally as o/w émulsions.

ii. Microemulsions

In one embodiment of the présent invention, the iRNAs are formulated as microemulsions. A microemulsion can be defined as a System of water, oil, and amphiphile which is a single optically isotropie and thermodynamically stable liquid solution (see e.g., Ansel’s Pharmaceutical Dosage

Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rîeger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typicalty microemdisions are Systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chainlength alcohol to form a transparent System. Therefore, microemulsions hâve also been described as thermodynamicaHy stable, isotropically clear dispersions oftwo immiscible liquids that are stabilized by interfacial films of surface-active molécules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and eiectrolyte. Whetherthe microemuision is ofthe waterin-oil (w/o) or an oil-in-water (o/w) type is dépendent on the properties of the oil and surfactant used and on the structure and géométrie packing ofthe polar heads and hydrocarbon tails ofthe surfactant molécules (Schott, in Remington’s Pharmaceutical Sciences, Mack Publishîng Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansefs Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich NG., and Anse! HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional émulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamicaHy stable droplets that are formed spontaneousiy.

Surfactants used in the préparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij® 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), a!one or in combination with cosurfactants. The cosurfactant, usually a short-chaîn alcohol such as éthanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molécules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemuision Systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and dérivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex® 300, Captex® 355, Capmul® MCM, fatty acid esters, medium Chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 giycerides, vegetable oîis, and silicone oil.

Microem Usions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based mîcroemulsions (both o/w and w/o) hâve been proposed to enhance the oral bioavailability of drugs, including peptides (seee.g., US Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 13851390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Mîcroemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fl uidity and permeability, ease of préparation, ease of oral administration over solid dosage forms, improved clînical potency, and decreased toxicity (see e.g., US Patent Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho étal., J. Pharm. Sci., 1996, 85, 138-143). Often mîcroemulsions can form spontaneously when their components are brought together at ambient température. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Mîcroemulsions hâve also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsîon compositions and formulations of the présent invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as împrove the local cellular uptake of iRNAs and nucleic acids.

Mîcroemulsions ofthe présent invention can also contain additional components and additives such as sorbitan monostearate (Grill® 3), Labrasol®, and pénétration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the présent invention. Pénétration enhancers used in the mîcroemulsions of the présent invention can be classified as belonging to one of five broad categories-surfactants, fatty acids, bile salts, cheiating agents, and non-chelating non-surfactants (Lee et al., Critical Revîews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Mieroparticles

An iRNA ofthe invention may be incorporated into a particle, e.g., a microparticle. Mieroparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, évaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Pénétration Enhancers

In one embodiment, the présent invention employs various pénétration enhancers to efifect the efficient delivery of nucleic acids, particularly iRNAs, to the skîn of animais. Most drugs are présent in solution in both îonized and nonionized forms. However, usually only lipid soluble or lipophilie drugs readily cross cell membranes. It has been dîscovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a pénétration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, pénétration enhancers also enhance the permeability of lipophilie drugs.

Pénétration enhancers can be classified as belonging to one of five broad categories, Le., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Such compounds are well known in the art.

v. Carriers

Certain compositions of the présent invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess bîological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailabilîty of a nucleic acid having bîological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typicahy with an excess of the latter substance, can resuit in a substantial réduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory réservoirs, presumably due to compétition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano-stiibene-2,2'-disuifonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura étal., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatînîzed maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g.. magnésium stéarate, talc, silica, colloïdal Silicon dioxide, stearic acid, métairie stéarates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organîc or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the présent invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, sait solutions, alcohois, polyethylene glycols, gelatin, lactose, amylose, magnésium stéarate, talc, sîlicic acid, viscous paraffin, hydroxymethyl cellulose, polyvinylpyrrolidone, and the like.

Formulations for topical administration of nucleic acids can include stérile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or soîîd oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for nonparenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not lîmited to, water, sait solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnésium stéarate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

viî. Other Components

The compositions of the présent invention can additîonally contain other adjunct components conventîonally found in pharmaceuticai compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the présent invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfère with the biological activities of the components of the compositions of the présent invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., iubricants, preservatives, stabilizers, wettîng agents, emulsifiers, safts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA and (b) one or more agents which function by a non-iRNA mechanîsm and which are useful in treating a PD-L1 -associated disorder.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animais, e.g., for determining the LD5Û (the dose léthal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity.

The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeuticaily effective dose can be estimated initially from ceil culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the ÏC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately détermine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by PD-L1 expression. In any event, the administering physician can adjustthe amount and timing of iRNA administration on the basis ofresults observed using standard measures of efficacy known in the art or described herein.

VI. Methods For Inhibiting PD-Ll Expression

The présent invention also provides methods of inhibiting expression of a PD-L1 gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, in an amount effective to inhibit expression of PD-L1 in the cell, thereby inhibiting expression of PD-L1 în the cell. In certain embodiments of the invention, PD-L1 is inhibited preferentially in liver cells.

Contacting of a cell with an iRNA, e.g., a double stranded RNAÎ agent, may be done in vitro or in vivo. Contacting a cell in vivo with the iRNA includes contacting a cell or group of cells within a subject, e.g., a human subject, with the iRNA. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplîshed via a targeting ligand, including any ligand described herein or known in the art. In preferred embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc^ ligand, or any other ligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other sîmiïar ternis, and includes any level of inhibition.

The phrase “inhibiting expression of a PD-L l ” is intended to refer to inhibition of expression of any PD-L1 gene (such as, e.g., a mouse PD-L1 gene, a rat PD-L1 gene, a monkey PD-L1 gene, or a human PD-L 1 gene) as well as variants or mutants of a PD-L 1 gene. Thus, the PD-L 1 gene may be a wild-type PD-L 1 gene, a mutant PD-L 1 gene, or a transgenic PD-L 1 gene in the context of a genetîcally manipulated cell, group of cells, ororganism.

“Inhibiting expression of a PD-L1 gene” includes any level of inhibition of a PD-L1 gene, e.g., at least partial suppression of the expression of a PD-L1 gene. The expression of the PD-Ll gene may be assessed based on the level, or the change in the level, of any variable associated with PD-Ll gene expression, e.g, PD-Ll mRNA level or PD-Ll protein level. This level may be assessed în an mdividuai cell or in a group of cells, including, for example, a sample derived from a subject. It is understood that expression of PD-L1 may be near or below the ievel of détection in a normal subject in many cell types and body fluids. Therefore, the inhibition of expression of PD-L1 for example, can compare the Ievel of PD-LI in the liver of a subject infected with a hepatîtis virus prior to and after treatment with an agent for the inhibiton of PD-LI or in a tumor before or after treatment with an agent for inhibiton of PD-LI.

In certain embodiments, surrogate markers can be used to detect inhibition of PD-L1. For example, effective treatment of an infection, e.g., a hepatitis virus infection as demonstrated by acceptable diagnostic and monitoring criteria with an agent to reduce PD-L 1 expression can be understood to demonstrate a clinically relevant réduction in PD-L 1. Stabilization or réduction of tumor burden in a subject with cancer as determined by RECIST criteria after treatment with an agent to reduce PD-LI can be understood to demonstrate a clinically relevant réduction in PD-LI.

Inhibition may be assessed by a decrease in an absolute or relative Ievel of one or more variables that are associated with PD-LI expression compared with a control Ievel. The control Ievel may be any type of control Ievel that is utilized in the art, e.g., a pre-dose baseline Ievel, or a Ievel determined from a similar subject (e.g., historicai control), cell, or sample that is untreated or treated with a control (such as, e.g., buffet only control or inactive agent control).

In some embodiments of the methods of the invention, expression of a PD-L 1 gene is inhibited by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the Ievel of détection of the assay. In certain embodiments, the methods include a clinically relevant inhibition of expression of PD-LI, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of PDLI.

Inhibition of the expression of a PD-L 1 gene may be manifested by a réduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be présent, for example, in a sample derived from a subject) in which a PD-LI gene is transcribed and which has or hâve been treated (e.g., by contacting the cell or cells with an iRNA of the invention, or by administering an iRNA of the invention to a subject in which the cells are or were présent) such that the expression of a PD-L 1 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or hâve not been so treated (control cell(s) not treated with an iRNA or not treated with an iRNA targeted to the gene of interest). In preferred embodiments, the inhibition is assessed by the method provided in Example 2 in the RKO human colon carcinoma cells treated with 10 nM iRNA and expressing the Ievel of mRNA in treated cells as a percentage ofthe Ievel ofmRNA in control cells, using the following formula:

(mRNA ïn control cells) - (mRNA in treated cells) _inno/(mRNA in control cells)

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In other embodiments, inhibition ofthe expression ofa PD-Ll gene may be assessed in terms of a réduction of a parameter that is functionally linked to PD-H gene expression, e.g., PD-Ll protein expression or PD-Ll signaling pathways. PD-L1 gene sîlencing may be determined in any cell expressing PD-Ll, either endogenous or heterologous from an expression construct, and by any assay 5 known in the art.

Inhibition of the expression of a PD-Ll protein may be manifested by a réduction in the level of the PD-L1 protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As exptained above, for the assessment of mRNA suppression, the inhibiton of protein expression levels in a treated cell or group of cells may similarly be expressed as 10 a percentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess the inhibition ofthe expression of a PD-Ll gene includes acell or group of cells that has not yet been contacted with an RNAi agent of the invention. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment ofthe subject with an RNAi agent.

The level of PD-Ll mRNA that is expressed by a cell or group of cells may be detennined using any method known in the art for assessing mRNA expression. In one embodiment the level of expression of PD-Ll in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the PD-Ll gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B;

Biogenesis), RNeasy™ RNA préparation kits (Qîagen®) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating PD-Ll mRNA may be detected using methods the described in PCT Publication WO2012/177906, the entire contents of which are hereby incorporated herein by reference.

In some embodiments, the level of expression of PD-Ll is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molécule that is capable of selectively bîndîng to a spécifie PD-L 1. Probes can be synthesized by one of skill in the art, or derived from appropriate biological préparations. Probes may be specifically designed to be labeled. Examples of molécules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molécules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the détermination ofmRNA levels involves contacting the isolated mRNA with a nucleic acid molécule (probe) that can hybridize to PD-Ll mRNA. In one embodiment, the mRNA is 35 immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferrîng the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix GeneChip® array. A ski lied

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artisan can readily adapt known mRNA détection methods for use in determining the level of PD-Ll mRNA.

An alternative method for determining the level of expression of PD-L 1 in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to préparé cDNA) of for example mRNA în the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, US Patent No. 4,683,202), ligase Chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence réplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification System (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle réplication (Lizardi et al., US Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the détection of the amplified molécules using techniques well known to those of skill in the art. These détection schemes are especially useful for the détection of nucleic acid molécules tf such molécules are présent in very low numbers. In particular aspects of the invention, the level of expression of PD-Ll is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System) or the Dual-Glo® Luciferase assay in Example 2.

The expression levels of PD-Ll mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northem, Southern, dot, and the like), or microwelis, sample tubes, gels, beads or ftbers (or any solid support comprising bound nucleic acids). See US Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The détermination of PD-Ll expression level may also comprise usingnucleic acid probes in solution.

In preferred embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCR method îs described and exemplified in the Examples presented herein. Such methods can also be used for the détection of pathogen nucleic acids, e.g., hepatitis virus nucleic acids.

The level of PD-Ll protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdifïusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimétrie assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like. Such assays can also be used for the détection of proteins indicative of the presence or réplication of pathogens, e.g. viral proteins.

In some embodiments, the efficacy of the methods of the invention in the treatment of a PD-

Ll-related disease is assessed by a decrease in PD-Ll mRNA level (by liver biopsy).

In some embodiments, the efficacy of the methods of the invention in the treatment of HBV infection is monitored by evaluating combinations of serological markers as discussed below.

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Efficacy of treatment of subjects with HBV can be monitored by detectîng the level of heptatitis B s antigen (HBsAg) or HBeAg in the subject, wherein a réduction in the level of HBsAg or HBeAg, e.g., in sérum, is indicative of effective treatment of the disease. In preferred embodiments, the réduction in the level of HBsAg or HbeAg is clinîcally relevant, e.g., comparable to the level of réduction observed with the standard of care. Efficacy of treatment can also be determined by a clinîcally relevant réduction of the level of HBV DNA in the subject, e.g., comparable to the level of réduction observed with the standard of care, e.g., suppression by at least 4 log_]0 lU/mL, preferably at least 5 logio lU/mL (Dienstag, Hepatology, 2009, 49:S112-S121). Efficacy of treatment can also be determined by the presence of anti-HBsAg antibodies.

In some embodiments, the efficacy of the method ofthe invention in treatment ofcancer can be monitored by evaluating a subject for maintenance or preferably réduction of tumor burden of the primary tumor or metastatic tumor(s) or the prévention of metastasis. Methods for détection and monitoring of tumor burden are known in the art, e.g., RECIST criteria as provided in Eisenhauer et al., 2009, New response évaluation criteria in solid tumours: Revised RECIST guideline (version 1.1). Eur. J. Cancer. 45:228-247.

In some embodiments ofthe methods ofthe invention, the iRNA is administered to a subject such that the iRNA is delivered to a spécifie site within the subject. The inhibition of expression of PD-L1 may be assessed using measurements ofthe level or change in the level ofPD-Ll mRNA or PD-L1 protein in a sample derived from a spécifie site within the subject, e.g., the liver. In certain embodiments, the methods include a clinîcally relevant inhibition of expression of PD-L1, e.g. as demonstrated by a clinîcally relevant outcome after treatment of a subject with an agent to reduce the expression of PD-LI.

As used herein, the terms detecting or determining a level of an anlyte are understood to mean performing the steps to détermine if a material, e.g., protein, RNA, is présent. As used herein, methods of detecting or determining include détection or détermination of an anlyte level that is below the level of détection for the method used.

Animal models of PD-L1 associated dîseases are well known in the art. For example, animal models of hepatitis B infection are known in the art including chimpanzee, woodchuck, and transgenic mouse models of HBV (Wieland, 2015. ColdSpring Harb. Perspect. Med., 5:a02l469, 2015; Tennant and Gerin, 2001. ILAR Journal, 42:89-102; and Moriyama et al., 1990. Science, 248:361-364). The chimpanzee model may also be used as a model for hepatitis D infection. Comparative models of chronîc vs. acute lymphocytic choriomeningitis virus (LCMV) infection are useful for the study of immune exhaustion (Matloubian étal., 1994, J. Viral. 68:8056-8063). A large number of cancer models including PD-L1 expressing tumors are known in the art (Iwaî et al, 2002, PNAS, 99:12293-12297).

VIL Methods of Treating or Preventing PD-LI-Associated Dîseases

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The présent invention also provides methods of using an iRNA of the invention or a composition containing an iRNA of the invention to reduce or inhibit PD-L 1 expression in a cell. The methods include contacting the cell with a dsRNA of the invention and maintaining the cell for a time sufficient to obtain dégradation of the mRNA transcript of a PD-Ll gene, thereby inhibiting expression of the PD-Ll gene m the cell. Réduction in gene expression can be assessed by any methods known in the art. For example, a réduction in the expression of PD-Ll may be determined by determining the mRNA expression level of PD-Ll, e.g., in a liver sampie, using methods routine to one of ordinary skill in the art, e.g., northem blotting, qRT-PCR, e.g., as provided in Example 2; by determining the protein level of PD-Ll using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques. A réduction in the expression of PD-Ll may also be assessed indîrectly by measuring a decrease in biological activity of PD-Ll or measuring the level of PD-Ll in a subject sampie (e.g., a sérum sampie). A réduction in the expression of PD-Ll can also be assessed indîrectly by measuring or observing a change, preferably a clinically relevant change, in at least one sign or symptom of a PD-Ll associated disease.

In the methods ofthe invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses a PD-Ll gene, typically a liver cell. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pi g cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, atigercell, a bear cell, or a buffalo cell), a bîrd cell (e.g., a duck cell or a goose cell), or a whale cell, when the target gene sequence has sufficient complementarîty to the iRNA agent to promote target knockdown. In one embodiment, the cell is a human cell, e.g, a human liver cell.

PD-Ll expression is inhibited in the cell by at least 20%, 25%, prefereably at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to a level below the level of détection ofthe assay. In certain embodiments, the methods include a clinically relevant inhibition of expression of PD-Ll, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of PD-LL

The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucléotide sequence that is complementary to at least a part of an RNA transcript of the PD-L 1 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parentéral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, aîrway (aérosol), nasal, rectal, and topical (including buccal and sublingual)

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administration. In certain embodiments, the compositions are admînistered by intravenous infusion or injection. In certain embodiments, the compositions are admînistered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of PD-Ll, or a therapeutic effect. A depot injection may also provide more consistent sérum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an extemal pump 10 or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or épidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the présent invention also provides methods for inhibiting the expression of a PD-Ll gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets a PD-L l gene in a cell of the mammal and maîntaining the mammal for a time suffïcient to obtain dégradation ofthe mRNA transcript ofthe PD-Ll gene, thereby inhibiting expression of the PD-Ll gene in the cell. Réduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Réduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue materiai for monîtoring the réduction in the PD-Ll gene or protein expression. In certain embodiments, inhibition of PD-L 1 expression is confirmed by observation of clinically relevant outcomes.

The présent invention further provides methods of treatment of a subject in need thereof. The treatment methods of the invention include administering an ÎRNA of the invention to a subject, e.g., 30 a subject that would benefit from a réduction or inhibition of PD-Ll expression, in a therapeutîcally effective amount of an iRNA targeting a PD-Ll gene or a pharmaceutical composition comprising an iRNA targeting a PD-Ll gene.

An iRNA of the invention may be admînistered as a “free iRNA.” A free iRNA is admînistered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable 35 buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity ofthe buffer solution contaîning the iRNA can be adjusted such that it is suitable for administering to a subject.

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Altematively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a réduction or inhibition of PD-L1 gene expression are those having a disorder that would benefit from an increased immune response, e.g., an infections disease, e.g., a viral disease, e.g., hepatitis; or cancer.

The iRNA and additional therapeutic agents may be administered at the same time or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times or by another method known in the art or described herein.

In one embodiment, the method inciudes administering a composition featured herein such that expression of the target PD-L1 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 h ours, 28, 32, or abour 36 hours. In one embodiment, expression ofthe target PD-L1 gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer, e.g., about 1 month, 2 months, or 3 months.

Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) ofthe target PD-L1 gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.

Administration of the iRNA according to the methods of the invention may resuit in a réduction of the severity, signs, symptoms, or markers of such diseases or disorders in a patient with, e.g., elevated PD-L1 oraPD-Ll responsive tumor. By “réduction” in this context is meant a statistically significant decrease in such level, e.g., the level of an indicator of the presensce of a pathogen in a subject, e.g., HBsAg, HBeAg, or HB cccDNA in the sérum of a subject infected with hepatitis B. The réduction can be, for example, at least about 20%, 25%, preferably at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, orto below the level of détection of the assay used. In certain embodiment, an increase in a marker, e.g., anti-HBs antibody is indicative of a réduction of the severity of the disease. Therefore, an increase can be a statistically significant increase in the level of antibody, e.g., to a détectable level in a subject.

Efficacy of treatment or prévention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, réduction in pain, quality of life, dose of a médication required to sustain a treatment effect, level of a disease marker, or any other meas arable parameter appropriate for a gîven disease beîng treated or targeted for prévention. It is well within the abilîty of one skilled in the art to monitor efficacy of treatment or prévention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a disorder that would benefit from an increased immune response, e.g., an infectious disease, e.g., a viral disease, e.g., hepatitis, or cancer.

Efficacy of treatment of an infectious disease can be demonstrated, for example, by a decrease in the presence of the infectious agent as demonstrated by an inabihty to culture the agent from a subject sample. Efficacy of treatment of an infectious disease can be demonstrated by a decrease in

106 the presence of the infectious agent as demonstrated, for example, by a decrease in a protein, nucleic acid, or carbohydrate présent in the infectious agent. Effïcacy of treatment can be demonstrated, for example, by the presensce of an immune response as demonstrated by the presence of antibodies or immune cells targeted against the infectious agent. Efficacy of treatment of an infectious disease can be demonstrated by a decrease in the presence of the infectious agent as demonstrated, for example, by a decrease in one or more signs or symptoms of the infection, e.g., fever, pain, nausea, vomiting, abnormal blood chemîstry, weight loss. The spécifie signs or symptoms will dépend on the spécifie pathogen. Efficacy of treatment of an infectious disease can be demonstrated by the development of antibodies or immune cells targeting the pathogen.

Efficacy of treatment of cancer can be demonstrated by stabilization or a decrease in tumor burden as demonstrated by a stabilization or decrease in tumor burden of the primary tumor, metastatic tumors, or the delay or prévention of tumor metastasis. Diagnostic and monitoring methods are provided herein, e.g., RECIST criteria.

Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is weîl within the ability of one skilled in the art to monitor efficacy of treatment or prévention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting PD-L1 or pharmaceutical composition thereof, effective against a PD-L 1 related disorder indicates that administration in a clinically appropriate manner results in a bénéficiai effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a réduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating PD-L 1-related disorders as provided, for example, in the diagnostic criteria for HBV provided herein.

A treatment or préventive effect is évident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant réduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a réduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adéquate treatment using an IRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg to about 200 mg/kg.

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The iRNA can be administered by intravenous infusion over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the iRNA can reduce PD-L1 levels, e.g., in a cell or tissue ofthe patient by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or below the level of détection of the assay method used. In certain embodiments, administration results in clinical stabilization or preferably clinically relevant réduction of at least one sign or symptom of a PD-L1 associated disorder.

In certain embodiments, a full dose ofthe ÎRNA, patients can be administered a smalter dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergie reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as încreased cytokine (e.g., TNF-alpha or INF-alpha) leveis.

Altematively, the iRNA can be administered subcutaneously, i.e., by subeutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of ÎRNA on a regular basis, such as every other day or to once a year. In certain embodiments, the iRNA is administered about once per month to about once per quarter (i.e., about once every three months).

IX. Diagnostic Criteria and Treatment for PD-L1 related diseases

Exemplary diagnostic and monitoring criteria for varions PD-L1 related diseases are provided below.

A. Hepatitis B

Hepatitis is a general term meaning inflammation of the liver and can be caused by a variety of different viruses such as hepatitis A, B, C, D and E. Since the development of jaundice is a characteristic feature of liver disease, a correct diagnosis can only be made by testing patients’ sera for the presence of spécifie anti-viral antigens or antîbodies. The severe pathol ogical conséquences of persistent HBV infections include the development of chronic hepatic insufficiency, cirrhosis, and hepatocellular carcinoma (HCC). In addition, HBV carriers can transmit the disease for many years.

HBV is a large virus and does not cross the placenta, however, prégnant women who are infected with HBV can transmit their disease to their infants ai birth. If not vaccinated at birth, many of these infants develop lifelong HBV infections, and many develop liver failure or liver cancer later in life. Following acute HBV infection, the risk of developing chronic infection varies inversely with âge. Chronic HBV infection occurs among about 90% of infants infected at birth, 25-50% of children infected at 1 -5 years of âge and about 1 -5% of persons infected as older children and adults. Chronic HBV infection is also common in persons with immunodeficiency (Hepatitis B: World Health Organization. Department of Communicable Diseases Surveillance and Response, available at

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www.who.int/csr/dîsease/hepatitis/HepatitisB_whocdscsrlyo2002_2.pdf?ua=l, incorporated herein by reference).

During the incubation phase of the disease (6 to 24 weeks), patients may feel unweil with possible nausea, vomiting, diarrhea, anorexia, and headaches. Patients may then become jaundiced 5 although low grade fever and loss of appetite may improve. Sometimes HB V infection produces neither jaundice nor obvious symptoms. The asymptomatîc cases can be identified by detecting biochemical or vîrus-specific sérologie alterations in their blood. Such asymptomatîc individuals may become silent carriers of the virus and constitute a réservoir for further transmission to others.

Most adult patients recover completely from their HBV infection, but others, about 5 to i 0%, 10 will not clear the virus and will progress to become asymptomatîc carriers or develop chronic hepatitis possibly resulting in cirrhosis or liver cancer. Rarely, some patients may develop fulminant hepatitis and die. Persistent or chronic HBV infection is among the most common persistent viral infections in humans. More than 350 million people in the world today are estimated to be persistently infected with FIBV. A large fraction of these are in eastern Asia and sub-Saharan Africa, where the associated 15 complications of chronic liver disease and liver cancer are the most important health problems.

The three standard blood tests for hepatitis B (HBs antigen, antiHBs antibody, and HBc antigen) can détermine if a person is currently infected with HBV, has recovered, is a chronic carrier, or is susceptible to HBV infection.

Assay res n its	Interprétation
HBsAg	anti-HBs	anti-HBc
+	-	-	Early acute HBV infection.
+	+/-	+	Acute or chronic HBV infection. Differentiate with IgM-antîHBc. Détermine Ievel of infectivity with HBeAg or HBV DNA.
-	+	H-	Indicates préviens HBV infection and immunity to hepatitis B.
		4-	Possibilîties include: past HBV infection; low-level HBV carrier; time span between disappearance of HBsAg and appearançe of anti-HBs; or false-positive or nonspecîftc reaction. Investigate with IgM anti-HBc, and/or challenge with HBsAg vaccine. When présent, anti-HBe helps val i date the anti-HBc reactivity.
-	-	-	Another infectious agent, toxic injury to the liver, disorder of immunity, hereditary disease of the liver, or disease of the

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			biliary tract.
-	+		Vaccine-type response.

From: Hollinger FB, Liang TJ. Hepatitis B Virus. In: Knipe DM étal., eds. Fields Virology, 4th ed., Philadelphia, Lippincott Williams &Wilkins, 2001:2971-3036.

Further serological tests can be performed to differentiate subjects with chrome or acute HBV, or who may be carriers. A number of vaccines against HBV are available and are presently far 5 more effective, and cost-effective, than treatment.

Currently, there is no treatment available for acute hepatitis B. Symptomatic treatment of nausea, anorexia, vomiting, and other symptoms may be indicated.

Treatment of chronic hepatitis B is aimed at eliminating infectivity to prevent transmission and spread of HBV, at halting the progression of lîver dîsease and improving the clinical and histologie picture, and at preventing HCC from developing, by losing markers of HBV réplication in sérum and lîver like HBV DNA, HBeAg, and HBcAg. Normalization of ALT activity, resolution of hepatic inflammation and the improvement of a patient’s symptoms usually accompany these virological changes. However, presently available treatments for HBV are rarely curative. Patients must be on treatment indefînitely to suppress the dîsease and prevent transmission.

There are two main classes of treatment; antivirais; aimed at suppressing or destroying HBV by interfering with viral réplication; and immune modulators: aimed at helping the human immune System to mount a defence against the virus. Neither corticosteroids, which induce an enhanced expression of virus and viral antigens, and a suppression of T-lymphocyte fonction, nor adenine arabinoside, acyclovir, or dîdeoxyinosine, hâve been shown to be bénéficiai for the treatment of chronic hepatitis B.

Currently, chronic hepatitis B is treated with interferons to modulate immune response. The only approved ones are interferon-a-2a and interferon-a-2b. Interferons display a variety of properties that include antiviral, immunomodulatory, and antiproliférative effects. They enhance T-cell helper activity, cause maturation of B lymphocytes, inhibit T-cell suppressors, and enhance HLA type I expression. To be eligible for interferon therapy, patients should hâve infection documented for at least six months, elevated lîver enzymes (AST and ALT), and an actively dividing virus in their blood (HBeAg and/or HBV DNA positive tests). Patients with acute infection, end stage cirrhosis or other major medical problems should not be treated. Interferon-α produces a long-term, sustained remission of the disease in 35% of those with chronic hepatitis B, with normalization of liver enzymes and loss of the three markers for an active infection (HBeAg, HBV DNA, and HBsAg). Complété élimination of the virus îs achieved in some carefully selected patients.

Interferon therapy for patients with HBV-related cirrhosis decreases significantly the HCC rate, particularly in patients with a larger amount of sérum HBV DNA. In patients with HBeAgpositive compensated cirrhosis, virological and biochemical remission followîng interferon therapy is

110 associated with improved survival. In patients with chronic HBV infection, the clearance of HBeAg after treatment with interferon-α is associated with improved clinical outcomes.

Interferon-α (Intron A (interferon-a-2b), Schering Plough, and Roferon, (interferon-a-2a) Roche Labs) is the primary treatment for chronic hepatitis B. The standard duration of therapy is considered 16 weeks. Patients who exhibit a low Eeve! of viral réplication at the end of the standard regimen benefit most from prolonged treatment.

Nucieotide and nucleosîde analogs hâve long been used for the treatment of HBV. Compounds presently available and in development include lamivudine, adefovir, entecavir, telbivudine, tenofovir, emtricitabine, clevudine, ritonavir, dipivoxil, lobucavir, famvir, FTC, NAcetyl-Cysteine (NAC), PC 1323, theradigm-HBV, thymosin-alpha, and ganciclovir. Some are useful against other viral infections, e.g., HCV, HIV, whereas others are effective predomînantly in the treatment of HBV.

Permanent loss of HBV DNA and HBeAg are considered the goals of antiviral treatment, as these resuit is associated with an improvement in necro-inflammatory damage, and reduced infectivity.

B. Hepatitis D

Hepatitis Delta virus (HDV) is a defective virus that is only infections in the presence of active HBV infection. HDV infection occurs as either coinfection with HBV or superinfection of an HBV carrier. Coinfection usually résolves. Superinfection, however, causes frequently chronic HDV infection and chronic active hepatitis. Both types of infections may cause fulminant hepatitis.

Routes of transmission are simîlar to those of HBV. Preventîng acute and chronic HBV infection of susceptible persons by vaccination wiil also prevent HDV infection. Certain HBV treatments are also effective in the treatment of HDV, e.g., interferon-alpha, with or without adefovir. However others, 1 îke lamivudine, an inhibitor of HBV-DNA réplication, are not useful for the treatment of chronic hepatitis D.

C. Tuberculosis (TB)

Tuberculosis is a disease caused by Mycobacterium tuberculosis. Tuberculosis is associated with symptoms including unexplained weight loss, loss of appetite, night sweates, fever, fatigue, coughing for longer than three weeks, hemoptysis (coughing up blood), and chest pain. There are two kînds of tests that are used to détermine i f a person has been infected with TB bacteria: the tuberculin skin test and TB blood tests which include Quanti F ERON®-TB Gold In-Tube test (QFT-G1T) and TSPOT®.TB test (T-Spot). However, the tests are not indicative of an active TB infection. Diagnosîs of TB infection includes assessment of medical history, physical examination, chest radiography, and diagnostic microbiology culture assay including an analysis for drug résistance. Assessment of clinically relevant changes in signs or symptoms of TB is within the ability of those of skill in the art.

D. Cancer

Cancer refers to any of varions malignant neoplasms characterized by the prolifération of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also

111

refers to the pathological condition characterized by such malignant neoplastic growths. A cancer can be a tumor or hematologicai malignancy, and includes but is not limited to, ail types of lymphomas/leukemias, carcînomas and sarcomas. In certain embodiments, cancer includes hepatic cancer. In certain embodiments, cancer includes hepatocellular carcinoma (HCC).

RECIST criteria are clinîcally accepted assessment criteria used to provide a standard approach to solid tumor measurement and provide définitions for objective assessment of change in tumor size for use in clinical trials. Such criteria can also be used to monitor response of an individual undergoing treatment for a solid tumor. The RECIST 1.1 criteria are discussed in detail in Eisenhauer et al., New response évaluation criteria in solid tumors: Revised RECIST guideline (version 1.1). Eur.

J. Cancer. 45:228-247, 2009, which is inçorporated herein by reference. Response criteria for target lésions include:

Complété Response (CR): Disappearance of ail target lésions. Any pathological lymph nodes (whether target or non-target) must hâve a réduction in short axis to <10 mm.

Partial Response (PR): At least a 30% decrease in the sum of diameters of target lésion, taking as a reference the baseline sum diameters.

Progressive Diseases (PD): At least a 20% increase in the sum of diameters of target lésions, taking as a reference the smallest sum on the study (this includes the baseline sum if that is the smallest on the study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: the appearance of one or more new lésions is also considered progression.)

Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as a reference the smallest sum diameters while on study.

RECIST 1.1 criteria also consider non-target lésions which are defined as lésions that may be measurable, but need not be measured, and should only be assessed qualitatively at the desired time points. Response criteria for non-target lésions include:

Complété Response (CR): Disappearance of ail non-target lésions and nonnalization of tumor marker levels. Ail lymph nodes must be non-pathological in size (<10 mm short axis).

Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or maintenance of tumor marker level above the normal limîts.

Progressive Disease (PD): Unequivocal progression of existing non-target lésions. The appearance of one or more new lésions is also considered progression. To achieve unequivocal progression on the basis of non-target disease, there must be an overall level of substantial worsening of non-target disease such that, even in the presence of SD or PR in target disease, the overall tumor burden has increased sufficiently to merit discontinuation of therapy. A modest increase in the size 35 of one or more non-target lésions is usually not sufficient to qualify for unequivocal progression status. The désignation of overall progression solely on the basis of change in non-target disease in the face of SD or PR in target disease will therefore be extremeiy rare.

Clinîcally acceptable criteria for response to treatment in acute leukemias are as follows:

112

Complété remission (CR): The patient must be free of ali symptoms related to ieukemia and hâve an absolute neutrophil count of >_1.0 x 10⁹/L, platelet count >J00 x 10⁹T, and normal bone marrow with <5% blasts and no Auer rods.

Complété remission with incomplète blood count recovery (Cri): As per CE, but with residual 5 thrombocytopenia (platelet count <100 x 10⁹/L) or residual neutropenia (absolute neutrophil count <1.0 x 10⁹ L).

Partial remission (PR): A >J0% decrease in bone marrow biasts to 5 to 25% abnormal ceils in the marrow; or CR with <5% blasts if Auer rods are présent.

Treatment failure: Treatment has failed to achîeve CR, Cri, or PR. Récurrence.

Relapse after confirmed CR: Reappearance of leukemic blasts in peripheral bood or >5% blasts in the bone marrow not attributable to any other cause (e.g., bone marrow régénération after Consolidated therapy) or appearance of new dysplastic changes.

Uses ofthe compositions and methods ofthe invention include achieving at least stable disease in a subject with a solid tumor for sufficient time to meet the définition of stable disease by 15 RECIST criteria. In certain embodiments, the use of the compositions and methods of the invention include achieving at least a partial response in a subject with a solid tumor for sufficient time to meet the définition of stable disease by RECIST criteria.

Uses ofthe compositions and methods ofthe invention include achieving at least a partial remission in a subject with an acute Ieukemia for sufficient time to meet the définition of stable 20 disease by RECIST criteria. In certain embodiments, the use of the compositions and methods of the invention include achieving at least a complété remission with incomplète blood count recovery in a subject with an acute Ieukemia for sufficient time to meet the définition of stable disease by RECIST criteria.

This invention is further illustrated by the following examples which should not be construed as limiting. The entire contents of ail references, patents and pubHshed patent applications cited throughout this application, as well as the Sequence Listing, are hereby incorporated herein by reference.

EXAMPLES

Example 1. iRNA Synthesis

Source of reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained 35 from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Transcripts siRNA Design

113

A set of siRNAs targeting the human PD-Ll / CD274 (human: NCB1 refseqlD

NM_001267706; NCBI GenelD: 29126; SEQ ID NO:1), as well as toxicology-species PD-Ll orthologs (mouse: XMJXJ6527249 (SEQ ID NO:3); rat, XM_006231248 (SEQ ID NO:5); and cynomolgus monkey: XM_005581779 (SEQ ID NO; 7)) were designed using custom R and Python scripts. The human PD-Ll REFSEQ mRNA has a length of 3349 bases. The rationale and method for the set of siRNA designs is as follows: the predicted efficacy for every potential 19mer siRNA from position 109 through position 3349 (the coding région and 3’ UTR) was determined with a linear model derived the direct measure of mRNA knockdown from more than 20,000 distinct siRNA designs targeting a large number of vertebrate genes. Subsets of the PD-Ll siRNAs were designed with perfect or near-perfect matches between human and cynomolgus monkey. A further subset was designed with perfect or near-perfect matches to mouse and rat PD-Ll orthologs. A further subset was designed with perfect or near-perfect matches to human, cynomolgus monkey, mouse, and rat PD-Ll orthologs. For each strand ofthe siRNA, a custom Python script was used in a brute force search to measure the number and positions of mismatches between the siRNA and ail potential alignments in the target species transcriptome. Extra weight was given to mismatches in the seed région, defined here as positions 2-9 ofthe antisense oligonucleotide, as well the cleavage site ofthe siRNA, defined here as positions 10-11 ofthe antisense oligonucleotide. The relative weight ofthe mismatches was 2.8; 1.2: 1 for seed mismatches, cleavage site, and other positions up through antisense position 19. Mismatches in the first position were ignored. A specificity score was calculated for each strand by summing the value of each weighted mismatch. Preference was given to siRNAs whose antisense score in human and cynomolgus monkey was >= 3.0 and predicted efficacy was >= 70% knockdown of the PD-L 1 transcript.

A detailed list of the unmodified PD-Ll sense and antisense strand sequences is shown in Table 3. A detailed list of the modified PD-Ll sense and antisense strand sequences is shown in Table 5.

siRNA Synthesis

PD-L 1 siRNA sequences were synthesized at 1 pmol scale on a Mermade 192 synthesizer (BioAutomation) using the solid support mediated phosphoramidite chemistry. The solid support was controlled pore glass (500 A) loaded with custom GalNAc ligand or universal solid support (AM biochemical). Ancillary synthesis reagents, 2’-F and 2’-O-Methyl RNA and deoxy phosphoramidites were obtained from Thermo-Fisher® (Milwaukee, WI) and Hongene (China). 2’F 2’-O-Methyl, GNA (giycol nucleic acids), 5’phosphate and other modifications are introduced using the corresponding phosphoramidites. Synthesis of 3’ GalNAc conjugated single strands was performed on a GalNAc modified CPG support. Custom CPG universal solid support was used for the synthesis of antisense single strands. Coupling time for ail phosphoramidites (100 mM in acetonitrile) is 5 min employing 5-Ethylthio-1 H-tetrazole (ETT) as activator (0.6 M in acetonitrile). Phosphorothioate linkages were generated using a 50 mM solution of 3-((Dimethylamino-methylidene) amino)-3H20581

114

1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes® (Wilmington, MA, USA)) in anhydrous acetonitrile/pyridine (1:1 v/v). Oxidation time was 3 minutes. Ail sequences were synthesized with final removal of the DMT group (“DMT off’).

Upon completion ofthe solid phase synthesis, oligoribonucleotides were cleaved from the 5 solid support and deprotected in sealed 96 deep well plates using 200 pL Aqueous Methylamine reagents at 60°C for 20 minutes. For sequences contaîning 2’ rîbo residues (2’-OH) that are protected with atert-butyl dimethyl silyl (TBDMS) group, a second step deprotection is performed using TEA.3HF (triethylamine trihydro fluoride) reagent. To the methylamine deprotection solution, 200uL of dimethyl sulfoxide (DMSO) and 300ul TEA.3HF reagent were added and the solution was incubated for additional 20min at 60°C. At the end of cleavage and deprotection step, the synthesis plate was allowed to corne to room température and is precipitated by addition of ImL of acetontile: éthanol mixture (9:1). The plates are cooledat -80 C for 2 hrs, superanatant wasdecanted carefully with the aid of a mufti channel pipette. The oligonucleotide pellet was re-suspended in 20mM NaOAc buffer and is desalted using a 5 mL HiTrap® size exclusion column (GE Healthcare) on an AKTA®

Purifier System equipped with an A905 autosampler and a Frac 950 fraction collector. Desalted samples are collected in 96-well plates. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV (260 nm) for quantification and a selected set of samples by IEX chromatography to détermine purity.

Annealing of PD-Ll single strands was performed on a Tecan® lîquîd handling robot. 20 Equimoiar mixture of sense and antisense single strands were combined and annealed in 96 well plates. After combinîng the complementary single strands, the 96-well plate was sealed tightly and heated in an oven at 100°C for 10 minutes and allowed to corne slowly to room température over a period 2-3 hours. The concentration of each duplex was normalized to 10μΜ in IX PBS.

Example 2- In vitro screening:

Cell culture and plasmids/transfections for Dual-Glo® assay:

Cos 7 cells (ATCC®, Manassas, VA) were grown to near confluence at 37°C in an atmosphère of 5% CO₂ in DMEM (ATCC®) supplemented with 10% FBS, before beîng released from the plate by trypsinization. The complété human CD274 reference sequence (NM 001267706.1) 30 was cloned into the duai-lucîferase psiCHECK2™ vector using three constructs with inserts of approximately 750bp, 1.4kb,and i.4kb inlength(SEQ ID NOs: 11-13). Dual-luciferase plasmids were co-transfected with siRNA into 15x10³ cells using Lipofectamine™ 2000 (Invitrogen™, Carlsbad CA. cat # 11668-019). For each well of a 96 well plate, 0.2ul of Lipofectamine™ were added to lOng of plasmîd vector and siRNA in 14.8ul of Opti-MEM® and allowed to complex at room température for 15 minutes. The mixture was then added to the cells resuspended în 80ul of fresh complété media. Cells were incubated for 48 hours before luciferase was measured. Single dose experiments were performed at 1 OnM and 0.1 nM final duplex concentration.

Dual-Glo® Luciferase assay

115 hours after the siRNAs were transfected, Firefly (transfection control) and Rentlla (fused to PD-Ll target sequence in 3’ UTR) luciferase were measured. First, media was removed from cells, Then Firefly luciferase activity was measured by adding 75ul of Dual-Glo® Luciferase Reagent equal to the culture medium volume to each well and mixing. The mixture was incubated at room température for 30 minutes before luminescense (500nm) was measured on a Spectramax® (Molecular Devices®) to detect the Firefly luciferase signal. Renifla luciferase activity was measured by adding 75ul of room température of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 10-15 minutes before luminescence was again measured to détermine the Renifla luciferase signal, The Dual-Glo® Stop & Glo® Reagent quenches the firefly luciferase signal and sustained luminescence for the Renifla luciferase reaction, sÎRNA activity was determined by normalizing the Renifla (PD-Ll) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. Ail transfections were done intriplicate.

Cell culture and transfections for qPCR:

RKO human colon carcinoma cells (ATCC®, Manassas. VA) were grown to near confluence at 37°C in an atmosphère of 5% CO₂ in EMEM (ATCC®) supplemented with 10% FBS, before beîng released from the plate by trypsinization.

Cells were transfected by adding 4.9pl of Opti-MEM plus 0.1 μΐ of Lipofectamine™ RNAiMax per well (Invitrogen™, Carlsbad CA. cat # 13778-150) to 5μ1 of siRNA duplexes per well into a 384-well plate and incubated at room température for 15 minutes. 40μΙ of DMEM containing -5 xlO³ cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at lOnM and O.lnM final duplex concentration. Total RNA isolation using DYNABEADS® mRNA Isolation Kit:

RNA was isolated using an automated protocol on a BioTek-EL406 platform using DYNABEADs® (Invitrogen™, cat#6l012). Briefly, 50μ1 of Lysis/Binding Buffer and 25μΐ of lysis buffer containing 3μ1 of magnetîc beads were added to the plate with cells. Plates were incubated on an electromagnetic shaker for 10 minutes at room température and then magnetîc beads were captured and the supematant was removed. Bead-bound RNA was then washed 2 times with 150μ1 Wash Buffer A and once with Wash Buffer B. Beads were then washed with 150μ1 Elution Buffer, recaptured and supematant removed.

cDNA synthesis using ABI™ High capacity cDNA reverse transcription kit (Applied Biosystems®, Foster City, CA, Cat #4368813):

Ten μΐ of a master mix containing 1 μΐ 10X Buffer, 0.4μ1 25X dNTPs, 1 μΐ lOx Random primers, 0.5 μΐ Reverse Transcriptase, 0.5 μ! RNase inhibitor and 6.6μ1 of H₂O per reaction was added to RNA isolated above. Plates were sealed, mixed, and incubated on an electromagnetic shaker for 10 minutes at room température, followedby 2h 37°C. Plates were then incubated at 81°C for 5min. Real time PCR:

] 16

Two μΐ of cDNA were added to a master mix containing 0.5 μ l of GAPDH TaqMan® Probe (Hs99999905), 0.5μ1 CD274 probe (HsO 1125301_m 1, CD274) and 5μ1 Lightcycler® 480 probe master mix (Roche Cat # 04887301001) per well in a 384 well plates (Roche cat # 04887301001). Real time PCR was done in a LightCycler®480 Real Time PCR System (Roche) using the AACt(RQ) 5 assay. Each duplex was tested in four independent transfections.

To calculate relative fold change, real time data were analyzed using the AACt method and normalized to assays performed with cells transfected with lOnM AD-1955, or mock transfected cells.

Table 2. Abbreviations of nucléotide monomers used in nucleic acid sequence représentation. It will 10 be understood that these monomers, when présent in an oligonucleotide, are mutually linked by 5'-3’-

phosphodiester bonds. __________________________________________
Abbreviation	Nucleotide(s) __________
A	Adenosine-3 ’-phosphate__	__
Af	2’-fluoroadenosine-3’-phosphate ____
Afs	2’-fluoroadenosine-3’-phosphorothioate __
As	adenosine-3’-phosphorothioate ___
C	cytidine-3’-phosphate _________________________________________________.
Cf	2’-fluorocytidine-3 ’-phosphate___	____
Cfs	2’-fluorocytidine-3 ’-phosphorothioate
Cs	cytidine-3’-phosphorothioate __________________________	___
G	guanosine-3 ’-phosphate
Gf	2’-fluoroguanosine-3’-phosphate _
Gfs	2’-fluoroguanosine-3’-phosphorothioate ____
Gs	guanosine-3’-phosphorothioate
T	5’-methyiuridine-3’-phosphate __
Tf	2’-fluoro-5-methyluridine-3 ’-phosphate
Tfs	2’-fl uoro-5-methyluridine-3’-phosphorothioate
Ts	5-methyluridine-3’-phosphorothioate
U	Uridîne-3’-phosphate
Uf	2’-fluorouridine-3’-phosphate
Ufs	2’-fluorouridine -3’-phosphorothioate
Us	uridine -3’-phosphorothioate
N	any nucléotide (G, A, C, T or U)
a	2'-O-methyl adenosine-3 ’-phosphate
as	2'-O-methyladenosîne-3’- phosphorothioate
c	2'-O-methyl cytidine-3’-phosphate
CS	2'-O-methylcytidîne-3’- phosphorothioate

117

Abbreviation	Nucleotide(s)
g	2'-O-methylguanosine-3’-phosphate
gs	2'-O-methylguanosine-3’- phosphorothioate ___________
t	2’-O-methyl-5-methyluridine-3’-phosphate ___
ts	2’-O-methyl-5-methyturidme-3’-phosphorothioate ___
U	2'-O-methyluridine-3’-phosphate _______
us	2'-O-methyluridine-3’-phosphorothioate ____
s	phosphorothioate linkage ________
L96	N-ltris(GaiNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolirLol Hyp-(GalNAc-alkyl)3
dT	2' -deoxythymidine-3' -phosphate ________
dC	2'-deoxycytidine-3'-phosphate ____
Y44	inverted abasic DNA i2-hvdroxvmethyl-tetrahydrofurane-5-phosphate)_______________
(Tgn)	Thymidine-glycot nucleic acid (GNA) S-Isomer __
P	Phosphate ___
VP	Vinyl-phosphate ______
(Aam)	2' -O-(N-methyl acetamide)adenosine-3 ' -phosphate___

118 a

c

Table 3. Un modified Sense and Antisense Strand Séquences of PD-L1 dsRNAs

119

Ο

a LU ΙΛ

S

en LD

un LD

S

en LD

S

m

un

77

en

rd ce

m 00

85

□0

σι CO

rd σ

en σ

95

CH

σ en

Ç

τ—f

3

un

in rd

CÛ

oo 00

CD LD

CM un

en en

on g

LD

σ LD

œ lD

O 00 ‘D

rd

C O

a σ

16-867

s en LD

no en

rô en

A σ

en

«=r σ

ld

rd LC

en g

nn

CO rd

S!

en o

S ό CM

0

CM t» LD

00 un LD

en un LO

CM O σ LD

Q G-

WJ

00

σι

o r-1

o 1—1

o i—l

o

rd

1—1

CM

Antisense Sequence

AUUUGAAAGUAUCAAGGUCUCCC

UAGGAACUGACCCUCAAAUUAGG

UAGACUCAAAAUAAAUAGGAAAA

ACAGACUCAAAAUAAAUAG GAAA

ACAGACUCAAAAUAAAUAGGAAA

UCUACUACAAUAUAUCUUCAAAA

AAUUGUAACAUCUACUACAAUAU

UACCAAGCACCU UACAAAUACUC

AACGGUUUGAUCUUUAUGCUUCC

UAUUAAUGGGUUAAAUAAAGGUG

UACUUAAUCUGUUUGCUUCCUCA

UAUCUGUAGAUUCAAUGCCUGGC

UAUCUUUUGAAUUUUGAAUCAUG

UAAAUGUAGACUAUCUUUAGAAG

UUGCUUUUAACAU ACAUU UCCAA

UUUGACAGAAAGCAGAAAACAAA

UAAGUUUAUACUUGACAGAAAGC

UGAAGUUUAUACUUGACAGAAAG

AAAAUGUGAUUUUGCAAG UACAG

Φ

Φ E

en

σι

O

no

m

rH

en

m

σ

rn

un

en

S

en

c dt

a

rô uo

o LO

un

LO

cû

LO

un

o LD

LO

LD

LO

un

LD

un

LD

s M

un 1 /1

LO n

Antisi

Oligo

un m rd <

A-135

S i—l <

A-135

<

A-135

en <

rd <

A-135

A-13!

A-131

A-13!

A-135

SEQ1D

58

60

CM LD

64

66_____

CO CD

70

CM

74______

LD

78 J

08

CM OO

84

86

88

90______

CM en

94

LD en

86

C

rd

S

un

CO

00 oo

LD LD

un

an en

en o

S

5

LD

σι LD

en

O ce

rd rd

c O

Q 5

5 oo cô

'8-997

un σ

s

en

LP ση

σ> un

en LD

un en

en nn

rd 0

rd CO

CM un O

În

CM

CM Ô

CM 0 LD <D

CM rd LD i O

CM CM σ LD

JP

l/l

CO

σι

o rd

T—1

1—1

u 1—1

t—L

rd

1—1

CM

Sense Sequence

GAGACCUUGAUACUUUCAAAU

UAAUUUGAGGGUCAGUUCCUA

UUCCUAUUUAUUUUGAGUCUA

UCCUAUUUAUUUUGAGUCUGU

UUGAAGAUAUAUUGUAGUAGA

AUUGUAGUAGAUGUUACAAUU

GUAUUUGUAAGGUGCUUGGUA

AAGCAUAAAGAUCAAACCGUU

CCUUUAUUUAACCCAUUAAUA

1 AGGAAGCAAACAGAUUAAGUA

CAGGCAUUGAAUCUACAGAUA

UGAUUCAAAAUUCAAAAGAUA

UCUAAAGAUAG UCUACAU U UA

GGAAAUGUAUGUUAAAAGCAA

UGUUUUCUGCUUUCUGUCAAA

UUUCUGUCAAGUAUAAACUUA

UUCUGUCAAGUAUAAACUUCA

GUACUUGCAAAAUCACAUUUU

□ligo

O m

00 O

148

00 LD

00 LÛ

cm lD

CM CD

VOi

S!

015

96’

318

CM rd

Ï28

785

CM σ

526

LD O

598

CM

Φ

un

rfl

un

tn

un

c

H

rd

1—1

rd

1—I

1—1

en

T—1

Φ un

Z

<

i

«i

<

LT)

un

ΠΊ

m

en

O

on

cp

LD

σ

un

CM

en

CM

rd

X

5

LO LD

LD

LP

LD

LO

LD

iê

LD

tu

Φ E

LO

LD

LP

LÛ

LD

LO

CD

LO

3 a

œ Z

AD-

AD·

AD

AD·

AD

AD·

AD

AD-

AD

GV

AD

120

SEQID	101	103	105	107	109	111	113	115	117	119
	Γχί	LD		σι	(SJ	(SJ		A	A	A
—	LA	rn	LD		<e				4
e	O		-—\|	csj	CM	(SJ		^X1	(SI	exi
.2 a	rn	m	m	m	rn	m	en	m	rn	rn
		A	en	CO	^q			ΓΝ	cxj	^X1
’S Ci	ΓΟ			œ	(SJ		(Si	[SJ	(SJ
O uj	O				[SJ		(SJ	(SJ	rN	<si
CL 4/1	m	en	¢0	m	m	A	m	A	A	A
			4	4	4 LJ	<	3	AC	AC	AC
	o	tu	Z	4	4	<	4	Z	O	D
	<£	4. --τ'	4	<9	Z	ZD	D	tu	tu	(JD
	4	QJ	(U	4	tu	tu	tu	tu	tu	tu
	y	s	tU ω	tu	tu <	O <	tu <	3	3	LJ
	4	O		ZD		3	U	tu	tu	tu
	A	4	<c	5	tu	tu	tu	ZD	Z	Z
	<J	<J	s		Z	—)	ZI		4	4
se Sequence	JAAUUCACA	CAAAUCCAA	ü 4 tU tU ZD 3	J G AC A AU Ci*	UAAAUUAA!	UAAAUUAAI	ivvnnwvn	UUAAAUUA	.UUAAAUUA	.UUAAAUUA
c	(O	4	<	O	D	ZD	ZD	4
φ		ZD	<	<r	<1	4	<	Z	ZD	Z
	L_J	23	<	3	ZD	ZD	ZD	ZD	Z	Z
c	U	w	V?	4	ZD	_)	_î	Zl	ZD	Z
<	O	zd	Z	2	ZD		_J	Z	ZD	Z
Φ
<u E	__^		a	A	F*·,			rn	A	rxl
ιλ rfl	(S\|		σι	CO	rn	A		rn	A
= z	LD	LO	A	A	n	i n	A	A	A	A
	A	LA	A	A	n	A	A	i n	A	A
		m	en	m	m	m	rn	A	A	rn
G —	i—\|			1—1		4-4	4—1	i—l	1-4	1-4
< O	4	±	4	4	<		<	<	<	<
a

Cf	O	(SJ		LD	CO	O	<si		LO	00
LU	O	O	o	O	O	—\|				1-4
A	f—I	4-1	4—1	x—1	tH	T—i	t—1	1“1	A	iH
e	<sj	LD		œ	(SJ		ΓΜ	A	A	A
	LT]	CH	LO		rr				4	4-
e	O			^,1	n*.i	(si	nj	(SJ	(SJ	[SJ
o a	en	m	en	m	m	m	m	A	A	A
	rn	ps^	in	a	m	m	m		4’	sr
(Λ Û	m	^q		o	(SJ	<si	(SJ		n4
O S	o	^q	^q	ΓΝ	C*J		^X1	(SJ	(SJ	rxf
CL A	m	m	m	rn	m	en	en	A	A	A
	4	4 O	4	Z	<	<	<	4		4
	O LU 3	4 Z	o ZD Z»	ZD Z ZD	z>	ZD	5 2D	AAA	AAA	AAA
		(J	Z	O	<		4	“ï	Z	Z
		3)	O	<		<		4	4	4
	-J	^3	3	O	_)	_)	_J
		Z	s	Z tu	D D	nn	nn	nn	nn	uu
			ZD	Z	«r		4	Z	ZD	Z
Φ	<_/		<J	Z)	<	4	<	4	4	4
			Z	4		“1		4	4	4
φ	tU		LU	tu		O	ZD	Z	Zl	Z
□ σ φ	ÎP D	tU Z	Z Z D	nnn	3 (□	GCA	GCA	CAU	CAU	CAU
l/l	O		z>	4	Z)	O	O	tu	tu	tu
Φ	Z)	tu	U	LJ	t J	( J	l J	ZD	O	Z
	1__J	Z	O	w	LJ	1,____J	__j		(J	O
	_)	O	O	Z	4.	4	4.	LJ	LJ	(J
A	Z	z	ZD		_>	_D		4	4	4
Ο
tuo	O	LD	4		LD	LD	LD	[SJ	(SJ	(SJ
Q		_τ—	σι	oo	m	A	A	A	A	A
	A	ld	LT	A	A	A	A	A	A	A
φ φ	A	IA	A	A	i.n	i n	IJ“l	A	A	A
£ ê	rn	m	en	m	m	m	m	A	A	m
c “ a> CO <Λ Z	4	4	4	A-l	<	A-l	A-l	A-l	l-v	A-l
	O	00	c	LD	CO	1^.		LD	A	A
		LD	LD	A		m			A	4
>c	LD	LD	LD	LD	LO	LD	LO	LO	LD	LO
Q. E	LD	LD	LD	LÛ	LO	LD	LO	LO	LO	LD
□ π	Q	Q	Q	a	à	à	à	Cl	Û	ώ
a z	<1	4	4	4	<	<	4.	<	4	<

121

Table 4. CD274 Dual-Glo® Luciferase and qPCR Data

Data are expressed as percent message remaining relative to non-targeting control.

	Luc Assay Data in Cos7 ce Ils	qPCR Data in RKO cells
DupiexID	lOnM AVG	lOnM STDEV	O.lnM AVG	O.lnM STDEV	lOnM AVG	lOnM STDEV	O.lnM AVG	O.lnM STDEV	Trans start
AD-67630	7.6	0.4	17.8	1.2	53.4	3.5	63.0	15.2	330
AD-67639	16.0	2.5	62.6	2.8	44.4	2.8	71.7	5.5	330
AD-67649	12.5	1.2	32.0	10.0	70.7	6.9	65.4	11.6	330
AD-67634	19.7	1.6	25.1	1.0	73.7	13.6	78.8	5.8	332
AD-67644	67.7	5.5	107.1	3.9	79.4	5.1	85.0	4.2	332
AD-67654	28.1	1.8	65.9	5.2	79.7	4.0	77.6	9.8	332
AD-67627	12.7	2.3	27.1	0.9	52.3	3.4	67.5	1.9	333
AD-67636	33.1	6.0	82.6	5.0	56.0	3.9	67.2	23.7	333
AD-67646	21.4	1.0	70.3	8.4	62.1	5.7	85.7	4.9	333
AD-67658	11.7	0.1	16.6	0.8	35.3	5.4	58.4	3.2	351
AD-67657	18.7	3.2	55.0	3.0	53.7	8.4	61.7	15.7	469
AD-67632	6.0	0.3	10.1	0.8	25.9	4.9	29.1	15.5	618
AD-67642	33.7	3.6	79.1	0.7	52.4	4.9	85.3	0.6	618__
AD-676S2	25.9	5.9	49.3	0.9	30.6	1.7	75.3	4.0	618
AD-67629	10.4	0.6	24.9	1.9	23.1	1.9	47.6	4.8	619
AD-67638	15.9	1.2	79.5	5-1	28.6	2.9	68.3	6.7	619
AD-67648	13.6	1.2	35.4	5.3	18.9	4.3	62.6	7.0	619
AD-67631	6.8	1.0	11.4	0.8	18.5	2.8	42.9	1.2	620
AD-67641	19.2	3.2	61.2	9.4	67.3	8.7	62.3	11.4	620
AD-67651	15.2	0.9	48-5	1.1	45.3	5.9	81.8	7.6	620
AD-67675	18.5	1.1	30.7	4.2	44.4	4.8	58.6	8.6	846
AD-67665	29.9	1.1	34.3	2.8	36.4	2.0	54.0	4.4	976
AD-67633	7.4	0.8	23.2	1.0	42.5	4.9	55.3	26.1	1093
AD-67643	9.2	0.5	57.4	4.1	32.7	1.9	71.5	5.5	1093
AD-67653	7.7	0.1	16.2	1.2	22.0	1.8	59.5	4.8	1093
AD-67640	6.7	0.5	31.8	2.2	18.4	4.4	45.1	14.4	1094
AD-67650	7.3	0.7	17.3	2.3	22.0	1.7	45.6	3.2	1094
AD-67663	13.2	2.2	33.8	3.3	35.7	2.6	46.5	2.7	1157

122

	Luc Assay Data in Cos7 cells	qPCR Data in RKO cells
DuplexID	lOnM AVG	10nM STDEV	O.lnM AVG	O.lnM STDEV	lOnM AVG	lOnM STDEV	O.lnM AVG	O.lnM STDEV	Trans start
AD-67676	10.4	1.2	27.7	3.1	34.7	4.3	51.8	4.4	1167
AD-67666	9.3	0.6	14.7	1.8	47.5	4.7	63.4	6.5	1245
AD-67661	9.9	0.6	21.1	1.1	37.1	2.0	48.9	3.1	1293
AD-67669	25.4	4.2	51.9	5.8	52.0	3.3	73.3	12.0	1331
AD-67667	15.8	4.0	31.4	1.0	35.8	0.3	51.0	3.7	1518
AD-67674	13.8	2.7	21.3	3.9	43.1	10.8	61.2	15.1	1682
AD-67655	5.8	1.5	20.5	3.2	23.2	3.4	40.2	3.7	2103
AD-67672	5.7	0.3	13.5	0.5	36.1	9.0	52.5	2.6	2220
AD-67659	12.0	1.8	24.3	4.3	32.8	2.4	54.0	5.5	2240
AD-67673	8.1	0.5	44.0	5.0	33.2	1.4	58.8	8.6	2648
AD-67664	10.1	0.4	20.1	4.9	37.3	2.9	48.2	3.1	2658
AD-67662	14.6	0.9	25.9	0.6	28.6	1.8	40.3	3.0	2659
AD-67671	5.8	1.0	32.2	7.4	49.1	2.0	65.0	3.5	2690
AD-67670	9.6	0.6	28.7	3.5	41.0	2.0	63.6	3.5	3031
AD-67668	15.7	3.2	30.7	6.3	42.4	7.2	62.8	2.2	3115
AD-67660	4.9	0.6	17.1	2.7	29.2	2.0	53.5	1.7	3143
AD-67656	6.1	1.0	6.6	0.7	27.9	6.6	36.0	7.0	3198
AD-67628	11.6	0.3	26.8	3.7	38.8	4.7	53.1	8.4	3221
AD-67637	8.8	0.6	24.6	4.2	26.9	0.7	45.5	7.8	3221
AD-67647	9.1	0.6	23.7	4.1	25.2	1.3	47.3	2.2	3221
AD-67626	7.3	0.6	7.7	0.4	29.1	8.9	26.5	9.4	3222
AD-67635	9.3	1.0	28.2	4.4	26.3	3.0	49.7	4.8	3222
AD-67645	9.2	1.0	44.7	7.2	29.0	1.5	57.8	5.0	3222

123

Table 5. PD-LI Modified Sequences

Trans start în SEQID: 1

οεε

330

332

333

351

469

618

619

620

846

SEQID

121

123

125

127

σι

131

133

135

137

139

141

143

145

147

149

151

153

155

157

S

161

Antisense Oligo Sequence

UUGGUCACAUUGAAAAGCUUCUc

usUfsgguCfaCfAfuugaAfaAfgcuucsusc

Uf s Uf sgg u Cfa CfAf u uga Af a Afgc u u es u sc

UGCUGGUCACAUUGAAAAGCUUc

u sG f sc u gGfu CfAfca u uG f a Af aage ususc

UfsGfscugGfuCfAfcauuGfaAfaagcususc

UUGCUGGUCACAUUGAAAAGCUu

usUfsgcuGfgUfCfacauUfgAfaaagcsusu

UfsUfsgcuGfgUfCfacauUfgAfaaagcsusu

UUUGUGUUGAUUCUCAGUGUGCa

UAAGUGAGUCCUUUCAUUUGGAg

U ACGUCUCCUCCAAAUG UG U AUc

usAfscguCfuCfCfuccaAfa Ufguguasusc

UfsAfscguCfuCfCfuccaAfaUfguguasusc

UUACGUCUCCUCCAAAUGUGUAu

u sUf sa cg Uf cUf Cfc u cc Afa Af u gug us as u

UfsUfsacgUfcUfCfcuccAfaAfugugusasu

AUUACGUCUCCUCCAAAUGUGUa

asUfsuacGfuCfUfccucCfaAfaugugsusa

AfsUfsuacGfuCfUfccucCfaAfaugugsusa

AUUUGAAAGUAUCAAGGUCUCCc

Antisense Oligo Name

A-135541___

r-1 kD LT) LA en à

A-135576

A-135551

A-135571

A-135581

un en LA en

A-135555

m U*i un m ri <

| A-l 35591

A-135587

1 A-135547 ^!

A-135567

A-135579

A-135539

6SSSEW

A-135575

A-135545___

A-135565

A-135578

ri en LA <

SEQID

120

122

124

126

128

130

132 ____

134 |

136

138

140______

142

144

146

148

150

152

154

156

158

160

Olîgo Sequence

Y44GAAGCUUUUCAAUGUGACCAa

gsa sage u Ufu U f CfAf a u guga cca a L96

gsa s a gcu U f il U f Cf Af a u g u gacca a L9 6

Y44AGCUUUUCAAUGUGACCAGCa

asgscuu u UfcAfAfUfgugaccagcaL96

asgscuuuüfcAfAfUfgugaccagcaL96

Y44GCU U U UCAAUG UGACCAGCAa

gscs u u u u Cf a Af Uf G fuga cca gea a L96

gscsuuuuCfaAfUfGfugaccagcaaL96

CACACUGAGAAUCAACACAAa

CCAAAUGAAAGGACUCACU Ua

Y44UACACAUUUGGAGGAGACGUa

u sa sca ca Uf u UfG fG fa ggaga egua L96

usascacaUfuUfGfGfaggagacguaL96

Y44ACACAU U UGGAGGAGACGUAa

lO 3 CO Σ3 txo <0 OO Cü CkÛ £ O D ro o cG

ascsacauUfuGfGfAfggagacguaal96

Y44CACAUUUGGAGGAGACGUAAu

es asca u u UfgGfAfGfga ga eguaa u L9 6

es a sca u u UfgG fAfGfga gacguaauL96

GAGACCU UGAUACUU UCAAAu

Sense OligoName

A-135540

A-135560

A-135550

A-135S70

A-135570

A-135534

A-135554

A-135590

A-135586

A-135546

A-135566

A-135538

A-135558

A-135544

A-135564

A-135630

Duplex Name

AD-67630

AD-67639

AD-67649

AD-67634

AD-67644

AD-67654

AD-67627

AD-67636

AD-67646

AD-67658

AD-67657

AD-67632

AD-67642

AD-67652

AD-67629

AD-67638

AD-67648

AD-67631

AD-67641

AD-67651

AD-67675

124

Trans start in SEQID: 1

976

1093

1094

S O

1157

1167

1245

^! 1293

1331

1518

1682

2103

2220

2240

2648

2658

2659

2690

3031

3115

3143

SEQID

163

165

167

169

171

173

175

177

' 179

181

183

185

187

189

191

193

195

197

199

201

203

205

207

Antisense Oligo Sequence

U AGG AACUGACCCUCAAAU U AGg

UAGACUCAAAAUAAAUAGGAAAa

u s Afsga cil f cAfAf a a u a AfaU f aggaasasa

UfsAfsgacUfcAfAfaauaAfaUfaggaasasa

asCfsagaCfuCfAfaaauAfaAfuaggasasa

AfsCfsagaCfuCfAfaaauAfaAfuaggasasa

UCUACUACAAUAUAUCUUCAAAa

AAUUGUAACAUCUACUACAAUAu

UACCAAGCACCU UACAAAUACUc

AACGGUUUGAUCUUUAUGCUUCa

UAUUAAUGGGUUAAAUAAAGGUg

| UACUUAAÜCUGUUUGCUUCCUCa

UAUCUGUAGAUUCAAUGCCUGGc

UAUCUUUUGAAUUUUGAAUCAUg

UAAAUG UAGACUAUCUUUAGAAg

U UGCUUUUAACAUACAUU UCCAa

UUUGACAGAAAGCAGAAAACAAa

U AAG UUUAU ACUUGACAG AAAGc

UGAAGUUUAUACUUGACAGAAAg

AAAAUG UG AUU U UGCAAG UACAg

UCCUG U AAU U CAC AC AA AG A ACa

UCUUACAAAUCCAACACCACAAg

UGAAACAUGAGACAAAAGGGAUa

Antisense Oiigo Name

A-135609___

A-135549

A-l 35569___

A-135580

A-135563

A-135577

A-135605

A-135633

A-135611

A-135597

A-135619

A-135613

i A-135629

A-135583___

A-135625

A-135593

A-135627

A-135607

A-135599

A-135623

A-135621___

A-135617

A-135595

SEQID

162

164

166

168

170

172

174

176

178

' 180

182

184

186

188

190

192

194

196

198

200

O

206

Oligo Sequence

UAAUUUGAGGGUCAGUUCCUa

Y44UUCCUAUUUAUUUUGAGUCUa

u su scc ua U fu UfAf Uf u u uga guc ua L9 6

u su sccua Uf u UfAf U f u u uga gu cua L96

kD en 3 CbO 3 3 QO ùû □ □ 2 5c O 5 <0 Vl 3

«3 s uo 3 3 Qû ro cul 3 3 2 < 5 □ Π3 3 60 Crt 3

UUGAAGAUAUAUUGUAGUAGa

AUUGUAGUAGAUGUUACAAUu

GUAUUUGUAAGGUGCUUGGUa

AAGCAUAAAGAUCAAACCG Ua

CCU UU AUUU AACCCAU U AAUa

AGGAAGCAAACAGAUUAAGUa

CAGGCAUUGAAUCUACAGAUa

---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1 UGAU UCAMAU UCAAAAGAUa

UCUAAAGAUAGUCUACAUUUa

GGAAAUGUAUGUUAAAAGCAa

UGUUUUCUGCUUUCUGUCAAa

UUUCUGUCAAGUAUAAACUUa

UUCUG UCAAGUAUAAACU UCa

GUACUUGCAAAAUCACAUUÜU

UUCUUUGUGUGAAUUACAGGa

UGUGGUGUUGGAUUUGUAAGa

UCCCUUUUGUCUCAUGUUUCa

Sense OltgoName

A-135608

A-135548

A-135568

A-l 35562

A-135562

A-135604

A-135632

A-135610

A-135596

A-135618

A-135612

A-13S628

A-135582

A-135624

A-135592

A-135626

A-135606

A-135598

A-135622

A-135620

A-135616

A-135594

Duplex Name

AD-67665

AD-67633

AD-67643

AD-67653

AD-67640

AD-67650

AD-67663

AD-67676

AD-67666

AD-67661

AD-67669

AD-67667

AD-67674

AD-67655

AD-67672

œ LC kO LO a <

AD-67673

AD-67664

AD-67662

AD-67671

AD-67670

AD-67668

AD-67660

125

Trans start in SEQID; 1	3198	3221	3221	3221	3222	S s	3222
SEQID	209	211	213	215	217	219	221
Antisense Oligo Sequence	; AAAAGUGACAAUCAAAUGCAGAa	UUUAUUAAAUUAAUGCAGGUACa	u sllfsu a u U fa AfAf u u a a UfgCfagg u a s es a	UfsUfsuauUfaAfAfuuaaUfgCfagguascsa	UUUUAUUAAAUUAAUGCAGGUAc	usUfsuuaUfuAfAfauuaAfuGfcaggusasc	UfsUfsuuaUfuAfAfauuaAfuGfcaggusasc
Antisense Oligo Name	A-135585	1 A-135537	10 tn ro	A-135574	A-135533	A-135553___	A-135572
SEQID	208	\| 210	212	214	216	218	220
Oligo Sequence	nnnnDVDnannvonnnvûDnD	Y44UACCUGCAUUAAUUUAAUAAa	u s as cc u gCfa Uf Uf Afa u u ua a u aaa L9 6	σι ru ru ru ru (U D 0 P ru 2 5 eu 0 bû □ ru tn P	Y44ACCUGCAUUAAUUUAAUAAAa ί	a scsc u gc Af u U f Af Af u u uaa u a a a a L9 6	a scscti gcAf u Uf Af Afu u u a a ua a a a L9 6
Sense OligoName	A-135584	A-135536	A-135556	A-135556	A-135532	A-135552	A-135552
Duplex Name	L0 <0 ά	AD-67628	AD-67637	AD-67647	AD-67626	AD-67635	AD-67645

126

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine expérimentation, many équivalents to the spécifie embodiments and methods described herein. Such 5 équivalents are intended to be encompassed by the scope of the following daims.

Claims

1. A double stranded ribonucleic acid (RNAi) agent for inhibiting expression of programmed cell death l ligand 1 (PD-Ll), wherein said RNAi agent comprises a sense strand and an antisense strand, 5 wherein said sense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from any one of nudeotides 3221-3243, 351-372, 618-641, 618-639,619-640, 620-641, 1093-1115, 1093-1114, 1094-1115, 1167-1188, 1293-1314, 1518-1539,2103-2124,2220-2261,22202241, 2240-2261, 2648-2680,2648-2669,2658-2679, 2659-2680, 3143-3164, 3198-3219,3221-3242, or 3222-3243 ofthe nucieotide sequence of SEQ ID NO: 1 and said antisense strand comprises at least 15 10 contiguous nudeotides differing by no more than 3 nudeotides from the complementary portion of the nucieotide sequence of SEQ ID NO:2, and wherein the RNAi agent comprises at least one modified nucieotide.

2. A double stranded ribonucleic acid (RNAi) agent for inhibiting expression of programmed cell 15 death 1 ligand 1 (PD-Ll), wherein said RNAi agent comprises a sense strand and an antisense strand, the antisense strand comprising a région of complementarity which comprises at least 15 contiguous nudeotides differing by no more than 3 nudeotides from any one of the antisense sequences în any one ofthe duplexes AD-67635, AD-67637, AD-67658, AD-67632, AD-67629, AD-67631, AD-67633, AD67643, AD-67653, AD-67640, AD-67650, AD-67676, AD-67661, AD-67667, AD-67655, AD-67672, 20 AD-67659, AD-67673, AD-67664, AD-67662, AD-67660, AD-67656, AD-67628, AD-67647, AD-

67626, or AD-67645.

3. The dsRNA agent of daim 1 or 2, wherein the sense and antisense strands comprise nucieotide sequences selected from the group consisting of any one of the nucieotide sequences in any one of the 25 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, AD67659, AD-67673, AD-67664, AD-67662, AD-67660, AD-67656, AD-67628, AD-67647, AD-67626, or AD-67645.

30

4. The dsRNA agent of any one of daims 1 -3, wherein substantially ail of the nudeotides of said sense strand or substantially ail of the nudeotides of said antisense strand comprise a nucieotide modification.

5. The dsRNA agent of any one of claims 1 -3, wherein ail ofthe nudeotides of said sense strand 35 and ail of the nudeotides of said antisense strand comprise a modification.

128

6. A double stranded ribonucleic acid (RNAi) agent for inhîbiting expression of programmed cell death l ligand 1 (PD-Ll), wherein said double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded région, wherein said sense strand comprises at least 15 contiguous nucléotides differing by no more than 5 3 nucléotides from any one of nucléotides 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, 2220-2261,22202241,2240-2261,2648-2680,2648-2669, 2658-2679, 2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3272-3243 of the nucléotide sequence of SEQ ID NO:1 and said antisense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the complementary portion of the

10 nucléotide sequence of SEQ ID NO:2, wherein substantially ail of the nucléotides of said sense strand and substantially ail of the nucléotides of said antisense strand comprise nucléotide modifications, and wherein said sense strand is conjugated to a ligand attached at the 3’-terminus.

15

7. The double stranded RNAi agent of claim 6, wherein ail of the nucléotides of said sense strand and ail of the nucléotides of said antisense strand comprise a modification.

8. The RNAi agent of any of claim 1 -6, wherein at least one of said nucléotide modifications is selected from the group consisting of a deoxy-nucleotide, a 3’-terminal deoxy-thymine (dT) nucléotide, a 20 2'-O-methyl modified nucléotide, a 2'-fluoro modified nucléotide, a 2'-deoxy-modified nucléotide, a locked nucléotide, an unlocked nucléotide, a conformationally restricted nucléotide, a constraîned ethyl nucléotide, an abasic nucléotide, a 2’-amino-modified nucléotide, a 2’-O-allyl-modified nucléotide, 2’-Calkyl-modified nucléotide, 2’-hydroxly-modified nucléotide, a 2’-methoxyethyl modified nucléotide, a 2’O-alkyl-modified nucléotide, a morpholino nucléotide, a phosphoramidate, a non-natural base comprising 25 nucléotide, a tetrahydropyran modified nucléotide, a 1,5-anhydrohexitol modified nucléotide, a cyclohexenyl modified nucléotide, a nucléotide comprising a phosphorothioate group, a nucléotide comprising a methylphosphonate group, a nucléotide comprising a 5’-phosphate, and a nucléotide comprising a 5’-phosphate mimic.

30

9. The RNAi agent of claim 8, wherein said nucléotide modifications comprises a short sequence of

3’-terminal deoxy-thymine nucléotides (dT).

10. The RNAi agent of claim 2, wherein the région of complementarity is at least 17 nucléotides in length.

129

11. The RNAi agent ofclaim 2, wherein the région of complemeniarity is 19 -21 nucléotides in length.

12. The RNAi agent ofclaim 11. wherein the région ofcomplementarity is 19 nucléotides in length.

13. The RNAi agent of any one of daims 1, 2, and 6, wherein each strand is no more than 30 nucléotides in length.

14. The RNAi agent of any one of claims 1, 2, and 6, wherein at least one strand comprises a 3’

10 overhang of at least 1 nucléotide.

15. The RNAi agent of any one of claims 1, 2, and 6, wherein at least one strand comprises a 3’ overhang of at least 2 nucléotides.

15 16. The RNAi agent of any one of claims 1-5, further comprising a ligand.

17 The RNAi agent of claim 16, wherein the ligand is conj ugated to the 3’ end of the sense strand of the dsRNA agent.

20 1 S. The RNAi agent of claim 6 or 16, wherein the ligand is an N-acetyl galactosamine (GalNAc) dérivative.

19. The RNAi agent of claim 18, wherein the ligand is HO /^0H

20. The RNAi agent ofclaim 18, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic

and, wherein X is O or S.

21. The RNAi agent of claim 20, wherein the X is O.

22, The RNAi agent of claim 2, wherein the région of complementarity comprises any one ofthe antisense sequences in Table 3 and Table 5.

23, The RNAi agent of claim 2, wherein the région of complementarity consists of any one of the 10 antisense sequences in Table 3 or Table 5.

24. A double stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-L1, wherein said double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a région complementary to part of an rnRNA encoding PD-L 1, 15 wherein each strand is about 14 to about 30 nucieotîdes in length, wherein said double stranded RNAi agent is represented by fonnuia (III):

sense: 5’ n_p -N, -(X X X) _rN_b -Y Y Y -N_b -(Z Z Z)j -N_a - n_q 3’ antisense: 3’ n_p'-N/-(X'X'X')_k-N_b’-Y'Y^,Y'-N_b'-(Z'Z'Z')₁-N_I'- n_q' 5' (III) wherein:

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

p, p’, q, and q^r are each independently 0-6;

each N_a and N_a' independently represents an oligonucleotide sequence comprising 0-25 nucieotîdes whîch are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucieotîdes;

25 each N_b and N_b' independently represents an ol igonucleotide sequence comprising 0-10 nucieotîdes which are either modified or unmodified or combinations thereof;

each n_p, n_p', n_M, and rq', each of which may or may not be présent, independently represents an overhang nucléotide;

131

XXX, ΥΥΥ, ΖΖΖ, Χ'Χ'Χ', ΥΎΎ', and Ζ'Ζ'Ζ' each independently represent one motif of three identicai modifications on three consecutive nucléotides;

modifications on N_b differ from the modification on Y and modifications on N_b' differ from the modification on Y'; and wherein the sense strand is conjugated to at least one ligand.

25. The double stranded RNAi agent of claim 24, wherein î is 0; j is 0; i is 1 ; j is l; both i and j are 0; or both i and j are 1.

26. The double stranded RNAi agent of claim 24, wherein k is 0; 1 is 0; k is 1 ; 1 is 1 ; both k and l are 0; or both k and 1 are 1.

27. The double stranded RNAi agent of claim 24, wherein XXX is complementary to Χ'Χ'Χ', YYY is complementary to ΥΎΎ, and ZZZ is complementary to Z'Z'Z'.

28. The double stranded RNAi agent of claim 24, wherein the YYY motif occurs at or near the cleavage site of the sense strand.

29. The double stranded RNAi agent of claim 24, wherein the ΥΎΎ' motif occurs at the 11, 12 and

13 positions of the antisense strand from the 5'-end.

30. The double stranded RNAi agent of claim 29, wherein the Y' is 2'-O-methyl.

31. The double stranded RNAi agent of claim 24, wherein formula (lli) is represented by formula (Ilia):

sense: 5' n_p -N, -YYY -N_a - 3' antisense: 3’ n_p-N_a- ΥΎΎ'- N_a~ n_q· 5' (Ilia).

32. The double stranded RNAi agent of claim 24, wherein formula (III) is represented by formula (Illb):

sense: 5’ n_p -N_a -YYY -N_b -Z Z Z -N_a - n, 3' antisense: 3' n_p-N_a- Y'Y'Y'-N_b-Z'Z'Z’- N_a- n,· 5’ (Illb) wherein eachN_b and N_b' independently represents an oligonucleotide sequence comprising 1-5 modified nucléotides.

132

33. The double stranded RNAi agent of claim 24, wherein formula (III) is represented by formula (IIIc):

sense: 5’ n_p -N_a -XXX -N_b -Y Y Y -N» - n_q 3' antisense: 3' n_p-N_a- X'X'X'-N_b- ΥΎΎ'- N_a- n^ 5' (Πιο) wherein each N_b and Ni/ independently represents an oligonucleotide sequence comprising 1 -5 modified nucléotides.

34. The double stranded RNAi agent of claim 24, wherein formula (III) is represented by formula (Illd):

sense: 5’ n_p -N_a -X X X- N_b -Y Y Y -N_b -Z Z Z -N_a - n_q 3' antisense: 3' n_p-N_ÿ- X'X'X'- N_b-Y'Y’Y'-N_b-Z'Z'Z'- N_a- n_q- 5’ (Illd) wherein each N_b and N_b ^f independently represents an oligonucleotide sequence comprising 1-5 modified nucléotides and each N_a and N_a' independently represents an oligonucleotide sequence comprising 2-10 modified nucléotides.

35. The double stranded RNAi agent of claim 6 or 24, wherein the double stranded région is 15-30 nucléotide pairs in length.

36. The double stranded RNAi agent of claim 35, wherein the double stranded région Îs 17-23 nucléotide pairs in length.

37. The double stranded RNAi agent of claim 35, wherein the double stranded région is 17-25 nucléotide pairs in length.

38. The double stranded RNAi agent of claim 35, wherein the double stranded région is 23-27 nucléotide pairs in length.

39. The double stranded RNAi agent of claim 35, wherein the double stranded région is 19-21 nucléotide pairs in length.

40. The double stranded RNAi agent of claim 6 or 24, wherein the double stranded région is 21-23 nucléotide pairs in length.

41. The double stranded RNAi agent of claim 6 or 24, wherein each strand has 15-30 nucléotides.

133

42. The double stranded RNAi agent of any one of daims 6,24, and 34, wherein each strand has 1930 nudeotides.

43. The double stranded RNAi agent of claîm 6 or 24, wherein the nucléotide modifications are selected from the group consisting of LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2^r-O-allyl, 2'-Callyl, 2'-fiuoro, 2'-deoxy, 2’-hydroxyl, and combinations thereof.

44. The double stranded RNAi agent of daim 43, wherein the nucléotide modifications are 2'-Omethyl or 2'-fluoro modifications.

45. The double stranded RNAi agent of daim 6 or 24, wherein the ligand is one or more GalNAc dérivatives attached through a bivalent or trivalent branched lînker; or a cholestérol.

46. The double stranded RNAi agent of daim 24, the ligand is

47. The double stranded RNAi agent of daim 24, wherein the ligand is attached to the 3' end of the sense strand.

48. The double stranded RNAi agent of daim 47, wherein the RNAi agent is conjugated to the ligand as shown in the following schematic

134

49. The double stranded RNAi agent ofclaim 6 or 24, wherein said agent further comprises at least one phosphorothioate or methylphosphonate intemucleotide linkage.

50. The double stranded RNAi agent of claim 49, wherein the phosphorothioate or methylphosphonate intemucleotide linkage is at the 3’-terminus of one strand.

51. The double stranded RNAi agent of claim 50, wherein said strand is the antisense strand.

52. The double stranded RNAi agent of claim 50, wherein said strand is the sense strand.

53. The double stranded RNAi agent of claim 49, wherein the phosphorothioate or methylphosphonate intemucleotide linkage is at the 5’-terminus of one strand.

54. The double stranded RNAi agent of claim 53, wherein said strand is the antisense strand.

55. The double stranded RNAi agent of claim 53, wherein said strand is the sense strand.

20

56. The double stranded RNAi agent ofclaim 49, wherein the phosphorothioate or methylphosphonate intemucleotide linkage is at the both the 5’- and 3’-terminus of one strand.

57. The double stranded RNAi agent of claim 56, wherein said strand is the antisense strand.

25

58. The double stranded RNAi agent of claim 6 or 24, wherein the base pair at the 1 position of the

5'- end of the antisense strand of the duplex is an AU base pair.

59. The double stranded RNAi agent of claim 24, wherein the Y nucieotides contain a 2'-fluoro modification.

135

60. The double stranded RNAi agent of claim 24, wherein the Y' nucléotides contain a 2'-O-methyl modification.

S

61. The double stranded RNAi agent of claim 24, wherein p’>0.

62. The double stranded RNAi agent of claim 24, wherein p'=2.

63. The double stranded RNAi agent of claim 62, wherein q’=0, p=0, q=0, and p’ overhang

10 nucléotides are complementary to the target mRNA.

64. The double stranded RNAi agent of claim 62, wherein q’=0, p=0, q=0, and p’ overhang nucléotides are non-complementary to the target mRNA.

15

65. The double stranded RNAi agent of claim 56, wherein the sense strand has a total of 21 nucléotides and the antisense strand has a total of 23 nucléotides.

66. The double stranded RNAi agent of any one of claims 61-65, wherein at least one n_p' is linked to a neîghboring nucléotide via a phosphorothioate linkage.

67. The double stranded RNAi agent ofclaim 66, wherein ail n_p ^r are linked to neîghboring nucléotides via phosphorothioate linkages.

68. The double stranded RNAi agent of claim 24, wherein said RNAi agent is selected from the

25 group of RNAi agents listed in Table 3 and Table 5.

69. The double stranded RNAi agent ofclaim 24, wherein ail of the nucléotides of said sense strand and ail ofthe nucléotides of said antisense strand comprise a modification.

30

70. A double stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-L1 in a cell, wherein said double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a région complementary to part of an mRNA encoding PD-L1, wherein each strand is about 14 to about 30 nucléotides in length, wherein said double stranded RNAi agent is represented by formula (III):

sense:

antisense:

5' n_p-N_a-(X X X) i-Nb-Y Y Y -N_b-(Z Z Z)j -N_a - n^ 3'

3' n_p'-N_a'-(X'X'X')_k-N_b'-Y'Y'Y'-N_b'-(Z'Z'Z')_rN_a'- n_q' 5’ (III)

136 wherein:

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

p, p’, q, and q' are each independently 0-6;

each N_a and N_a' independently represents an oligonucleotide sequence comprising 0-25 nudeotides which are either modified or unmodifïed or combinations thereof, each sequence comprising at least two differently modified nudeotides;

each N_b and N_b' independently represents an oligonucleotide sequence comprising 0-10 nudeotides which are either modified or unmodifïed or combinations thereof;

each Πρ, n_p', n_q, and n_q’, each of which may or may not be présent independently represents an overhang nucieotide;

XXX, ΥΥΥ, ZZZ, X'X’X’, Y'Y'Y', and Z'Z'Z' each independently represent one motif ofthree identical modifications on three consecutive nudeotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications;

71. A double stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-Ll in a cell, wherein said double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a région complementary to part of an mRNA encoding PD-Ll, wherein each strand is about 14 to about 30 nudeotides in length, wherein said double stranded RNAi agent is represented by formula (III):

sense: 5’ n_p -N_a -(X X X) _rN_b - Y Y Y -N_b -(Z Z Z)j -N_a - n^ 3’ antisense: 3’ n_p'-N_a'-(X'X'X')_k-N_b'-Y'Y'Y'-N_b'-(Z'Z'Z')_rN_a'- n_q' 5' (ΠΙ) wherein:

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

each n_p rq. and rq', each of which may or may not be présent, independently represents an overhang nucieotide;

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

n_p' >0 and at least one n_p' is linked to a neighborîng nucieotide via a phosphorothioate linkage; each N_a and N,' independently represents an oligonucleotide sequence comprising 0-25 nudeotides which are either modified or unmodifïed or combinations thereof, each sequence comprising at least two differently modified nudeotides;

137

XXX, ΥΥΥ, ΖΖΖ, Χ'Χ'Χ', Υ'ΥΎ', and Ζ'Ζ'Ζ' each independently represent one motif of three identical modifications onthree consecutive nucieotîdes, and wherein the modifications are 2'-O-methyl or 2'fluoro modifications;

72. A double stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-L 1 in a cell, wherein said double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a région complementary to part of an mRNA encoding PD-L1, wherein each strand is about 14 to about 30 nucieotîdes in length, wherein said double stranded RNAi agent is represented by formula (III):

sense; 5' n_p -N_a -(X X X) _rN_b -Y Y Y -N_b -(Z Z Z)j -N_a - rq 3 ' antisense: 3' n_p'-N_a'-(X'X'X')_k-N_b'-Y'Y'Y'-N_b'-(Z'Z'Z')_rN_a'- n_q' 5’ (III) wherein:

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

each n_p, n_q, and n_q', each of which may or may not be présent, independently represents an overhang nucléotide;

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

n_p' >0 and at least one n_p' is linked to a neighboring nucléotide via a phosphorothioate lînkage; each N_a and N/ independently represents an oligonucleotide sequence comprising 0-25 nucieotîdes which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucieotîdes;

each N_b and N_b' independently represents an oligonucleotide sequence comprising 0-10 nucieotîdes which are either modified or unmodified or combinations thereof;

XXX, ΥΥΥ, ΖΖΖ, Χ'Χ'Χ', ΥΎΎ', and Z'Z'Z' each independently represent one motif of three identical modifications on three consecutive nucieotîdes, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications;

modifications on N_b differ from the modification on Y and modifications on N_b' differ from the modification on Y'; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand îs one or more GalNAc dérivatives attached through a bivalent or trivalent branched lînker.

73. A double stranded ribonucleic acid (RNAi) agent capable of inhibiting the expression of PD-L1 in a cell, wherein said double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a région complementary to part of an mRNA

138 encoding PD-Ll, wherein each strand is about 14 to about 30 nucléotides in length, wherein said double stranded RNAÎ agent is represented by formula (III):

sense : 5' n_p -N_a -(X X X) _rN_b -Y Y Y -N_b -(Z Z Z)j -N_a - 3’ antisense: 3’ n_p'-N_a'-(X'X'XVN_b'-Y^'-N^ n_q' 5' (III)

5 wherein:

i, j, k, and I are each îndependently 0 or 1 ;

each n_p, n_q, and n_q', each of which may or may not be présent, îndependently represents an overhang nucléotide;

p, q, and q' are each îndependently 0-6;

10 n_p' >0 and at least one n_p' is linked to a neighboring nucléotide via a phosphorothioate linkage;

each N_a and N_a' îndependently represents an oligonucleotide sequence comprising 0-25 nucléotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucléotides;

each N_b and N_b ^F îndependently represents an oligonucleotide sequence comprising 0-10

15 nucléotides which are either modified or unmodified or combinations thereof;

XXX, YYY, ZZZ, X'X'X', ΥΎΎ', and Z'Z'Z' each îndependently represent one motif of three identical modifications on three consecutive nucléotides, and wherein the modifications are 2'-O-methyl or 2'-fluoro modifications;

modifications on N_b differ from the modification on Y and modifications on N_b' dîffer from the 20 modification on Y';

wherein the sense strand comprises at least one phosphorothioate linkage; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc dérivatives attached through a bivalent or tri valent branched linker.

25

74. A double stranded ribonucleic acid (RNAi) agent capable of inhîbiting the expression of PD-Ll in a cell, wherein said double stranded RNAi agent comprises a sense strand complementary to an antisense strand, wherein said antisense strand comprises a région complementary to part of an mRNA encoding PD-Ll, wherein each strand is about 14 to about 30 nucléotides in length, wherein said double stranded RNAi agent is represented by formula (III):

30 sense: 5’ n_p-N_a -Y Y Y - N,-n_q 3' antisense: 3' n_p'-N/- ΥΎΎ'- N_a'- n_q ^F 5' (Ilia) wherein:

each n_p, n_q, and riq', each of which may or may not be présent, îndependently represents an overhang nucléotide;

35 p, q, and q' are each îndependently 0-6;

139 η_ρ ^Γ >0 and at least one n_p' is linked to a neighboring nucléotide via a phosphorothioate linkage; each N_a and N/ independently represents an oligonucleotide sequence comprising 0-25 nucléotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucléotides;

5 YYY and ΥΎΎ' each independently represent one motif of three identical modifications on three consecutive nucléotides, and wherein the modifications are 2^f-O-methyl or 2'-fluoro modifications; wherein the sense strand comprises at least one phosphorothioate linkage; and wherein the sense strand is conjugated to at least one ligand, wherein the ligand is one or more GalNAc dérivatives attached through a bivalent or trivalent branched linker.

75. A double stranded ribonucleic acid (RNAi) agent for inhibiting expression of PD-Ll, wherein said double stranded RNAi agent comprises a sense strand and an antisense strand forming a double stranded région, wherein said sense strand comprises at least 15 contiguous nucléotides differing by no more than 15 3 nucléotides from nucléotides 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, 2220-2261,2220-2241,22402261, 2648-2680, 2648-2669,2658-2679,2659-2680, 3143-3164, 3198-3219, 3221-3242, or 3222-3243 ofthe nucléotide sequence of SEQID NO:1 and said antisense strand comprises at least 15 contiguous nucléotides differing by no more than 3 nucléotides from the complementary portion of the nucléotide 20 sequence of SEQ ID NO:2, wherein substantially ali of the nucléotides of said sense strand comprise a nucléotide modification selected from the group consisting of a 2’-O-methyl modification and a 2’-fluoro modification, wherein said sense strand comprises two phosphorothioate intemucleotide linkages at the 5’25 terminus, wherein substantially al] of the nucléotides of said antisense strand comprise a nucléotide modification selected from the group consisting of a 2’-O-methyl modification and a 2’-fiuoro modification, wherein said antisense strand comprises two phosphorothioate internucleotide linkages at the 5’30 terminus and two phosphorothioate intemucleotide linkages at the 3’-terminus, and wherein said sense strand is conjugated to one or more GalNAc dérivatives attached through a branched bivalent or trivalent linker at the 3’-terminus.

76. The double stranded RNAi agent of claim 75, wherein ail of the nucléotides of said sense strand 35 and al] of the nucléotides of said antisense strand comprise a nucléotide modification.

140

77. The double stranded RNAi agent of claim 75, wherein each strand has 19-30 nucieotides.

78. A cell contaîning the RNAi agent of any one ofclaims 1,2, 6, 24, and 70-75.

79. A pharmaceutical composition for inhibiting expression of a PD-Ll gene comprising the RNAi agent of any one of c lai ms 1-77.

80. The pharmaceutical composition of claim 79, wherein the RNAi agent is admînistered in an unbuffered solution.

81. The pharmaceutical composition of claim 80, wherein said unbuffered solution is saline or water.

82. The pharmaceutical composition of claim 79, wherein said RNAi agent is admînistered with a buffer solution.

83. The pharmaceutical composition of claim 82, wherein said buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof.

84. The pharmaceutical composition of claim 82, wherein said buffer solution is phosphate buffered saline (PBS).

85. A pharmaceutical composition comprising the double stranded RNAi agent of any one of claims ] -5, and a lîpid formulation.

86. The pharmaceutical composition of claim 85, wherein the li pid formulation comprises a LNP.

87. The pharmaceutical composition of ciaim 85, wherein the lîpid formulation comprises a MC3.

g g The double stranded RNAi agent of any one of claims 1 -77 or a pharmaceutical composition of any one ofclaims 79-87 for use in a method of inhibiting PD-Ll expression in a cell.

gQ Use of the double stranded RNAi agent of any one of claims 1-77 in the manufacture of a pharmaceutical composition for inhibiting PD-Ll expression in a cell.

90. Use according to claim 89, wherein the pharmaceutical composition is a pharmaceutical composition according to any one ofclaims 79-87,

141

91. The agent for use of daim 88 or the use of daim 89, wherein said cell is within a subject.

92. The agent for use or the use of daim 91, wherein the subject is a human.

93. The agent for use or the use of any one of daims 88-92, wherein the PD-L 1 expression is inhibited by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%; or to below the level of détection of the assay.

10

94. The RNAi agent of any one of daims 1-77 or the pharmaceutical composition of any one of daims 79-87, for use in a method of treating a subject having a disease or disorder that would benefit from réduction in PD-Ll expression.

95. Use of the RNAi agent of any one of daims 1 -77 in the manufacture of a pharmaceutical

15 compostion for treating a subject having a disease or disorder that would benefit from réduction in PD-Ll expression.

96. Use according to daim 95, wherein the pharmaceutical composition is a pharmaceutical composition according to any one of daims 79-87.

97. The agent for use of daim 94 or the use of daim 95, wherein administration of the RNAi to the subject causes a decrease in the PD-Ll signaling pathway.

98. The agent for use of daim 94 or the use of daim 95, wherein the disorder is a PD-Ll-associated

25 disease.

99. The agent for use or the use 98, wherein the PD-L l -associated disease is an infection.

100. The agent for use or the use of claim 99, wherein the infection is a chronic, intracellular infection.

101, The agent for use or the use of any one of daims 98-100, wherein the PD-Ll -associated disease is a viral infection.

102. The agent for use or the use of any one of daims 98-101, wherein the PD-L 1 associated disease is 35 a hepatitis virus infection.

142

103. The agent for use of claim 94 or the use of claim 95, wherein the cancer is PD-L1 associated disease is cancer.

104. The agent for use or the use of any one of daims 94-103, wherein the subject is human.

105. The agent for use or the use of any one of daims 94-104, which is for administration in conjunction with an agent for the treatment of the infection or the cancer.

106. The agent for use or the use of any one of daims 94-105, wherein the dsRNA agent is for 10 administration at a dose of about 0.01 mg/kg to about 50 mg/kg.

107. The agent for use or the use of any one of daims 94-106, wherein the dsRNA agent is for administration to the subject subcutaneously.