TW202504619A - Compositions and methods for inhibiting expression of pd-l1 - Google Patents

Abstract

Compositions and methods useful to reduce expression of PD-L1 gene and for treatment of PD-L1-associated diseases and conditions are provided. Provided are PD-L1 dsRNA agents, PD-L1 antisense polynucleotide agents, compositions comprising PD-L1 dsRNA agents, and compositions comprising PD-L1 antisense polynucleotide agents that can be used to reduce PD-L1 expression in cells and subjects.

Description

Compositions and methods for inhibiting PD-L1 expression

The present invention relates in part to compositions and methods that can be used to inhibit expression of the programmed cell death-ligand 1 (PD-L1) gene.

Programmed cell death-ligand 1, or programmed cell death 1 ligand 1 (PD-L1), also known as B7 homolog 1 (B7-H1), is a 272 amino acid long type 1 transmembrane protein that is present as a surface marker on many different types of cells and is encoded by the CD274 gene on chromosome 19 in mice and chromosome 9 in humans. It should be understood that one of ordinary skill in the art considers the terms PD-L1 and CD274 to be generally interchangeable when considering nucleic acids (DNA or RNA) or the corresponding translated proteins or their sequences. PD-L1 is the primary ligand for PD-1 and inhibits T cell cytotoxicity and cytokine production. CD274/PD-L1 expression has been detected in a variety of tissues and cell types, including T cells, B cells, macrophages, dendritic cells, and non-hematopoietic cells (including endothelial cells, hepatocytes, muscle cells, and placenta). CD274/PD-L1 expression is associated with suppression of anti-tumor immune activity. Cancer cells such as HCC cells exploit this immune checkpoint by upregulating PD-L1 expression, resulting in dysfunction of proximal T cells' anti-tumor immunity. Studies correlating PD-L1 expression in tumors or hematologic malignancies with disease outcomes have shown that PD-L1 expression is strongly associated with poor prognosis in all types of lymphoma/leukemia, hematologic malignancies, breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, liver cancer, salivary gland cancer, gastric cancer, neuroglioma, thyroid cancer, thymic epithelial cancer, head cancer, kidney cancer, pancreatic cancer, and neck cancer.

CD274/PD-L1 expression has also been implicated in evading immune responses involved in chronic infections, such as viruses (e.g., HIV, HBV, HCV, and HTLV, etc.), bacteria (e.g., Helicobacter pylori, etc.), and parasites (e.g., Schistosoma mansoni).

Therefore, the present invention provides novel RNAi agents to reduce CD274/PD-L1 levels and treat CD274/PD-L1 related diseases, disorders and conditions, such as infectious diseases such as chronic intracellular infectious diseases, such as viral diseases, such as hepatitis infection, or bacterial infections, such as tuberculosis infection; as well as cancer or hematological malignancies.

In general, the present disclosure introduces novel PD-L1 gene-specific RNAi agents, compositions comprising PD-L1 RNAi agents, and methods of inhibiting PD-L1 gene expression in vitro and/or in vivo using the PD-L1 RNAi agents and compositions comprising PD-L1 RNAi agents described herein. The PD-L1 RNAi agents described herein can selectively and effectively reduce, inhibit, or silence the expression of the PD-L1 gene in a subject (e.g., a human or animal subject).

According to one aspect of the present invention, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting PD-L1 expression is provided, wherein the dsRNA agent comprises a sense chain and an antisense chain, wherein the sense chain comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 or 3 by no more than 1, 2 or 3 nucleotides, and the antisense chain comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 or 4 by no more than 1, 2 or 3 nucleotides, wherein the sense chain and the antisense chain may be partially, substantially or completely complementary to each other.

In certain embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to a PD-L1 RNA transcript, the complementary region comprising at least 15 consecutive nucleotides that differ from any one of the antisense sequences listed in Tables 1-3 by no more than 1, 2, or 3 nucleotides. In certain embodiments, the dsRNA agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the antisense strand comprises a region complementary to a PD-L1 RNA transcript, the complementary region comprising at least 15 consecutive nucleotides from any one of the antisense sequences listed in Tables 1-3.

In certain embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein nucleotide positions 2 to 18 in the antisense strand comprise a region complementary to a PD-L1 RNA transcript, wherein the complementary region comprises at least 15, 16, 17, 18, 19, 20, or 21 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in any one of Tables 1-3, and optionally comprises a targeting ligand.

In certain embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting PD-L1 expression is provided, wherein the dsRNA agent comprises a sense chain and an antisense chain, wherein the sense chain comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ by 0, 1, 2 or 3 nucleotides from any one of the nucleotide sequences of nucleotides 64-94, 67-97, 71-101, 72-102, 73-103, 71-103, 498-528, 552-582, 553-583, 707-737, 713-743 in the nucleotide sequence shown in SEQ ID NO: 1, and the antisense chain comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ by 0, 1, 2 or 3 nucleotides from any one of the nucleotide sequences of nucleotides 64-94, 67-97, 71-101, 72-102, 73-103, 71-103, 498-528, 552-582, 553-583, 707-737, 713-743 in the nucleotide sequence shown in SEQ ID NO: The corresponding nucleotide sequence of NO:2 differs by 0, 1, 2 or 3 nucleotides in at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides, wherein the sense strand and the antisense strand may partially, substantially or completely complement each other.

In certain embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting PD-L1 expression is provided, wherein the sense strand comprises the same sequence as SEQ ID NO: 1, wherein the antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ by 0, 1, 2 or 3 nucleotides from any one of the nucleotide sequences of nucleotides 69-89, 71-89, 72-92, 74-92, 76-96, 78-96, 77-97, 79-97, 78-98, 80-98, 76-98, 72-98, 503-523, 505-523, 557-577, 559-577, 558-578, 560-578, 712-732, 714-732, 718-738, 720-738, and the antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ by 0, 1, 2 or 3 nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2. At least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of 0, 1, 2 or 3 nucleotides.

In certain embodiments, the PD-L1 RNA transcript is SEQ ID NO: 1.

In certain embodiments, the antisense strand of the dsRNA agent is at least substantially complementary to any one of the target regions of SEQ ID NO: 1 and is provided in any one of Tables 1-3. In certain embodiments, the antisense strand of the dsRNA agent is completely complementary to any one of the target regions of SEQ ID NO: 1 and is provided in any one of Tables 1-3. In certain embodiments, the dsRNA agent comprises a sense strand sequence listed in any one of Tables 1-3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. In certain embodiments, the dsRNA agent comprises a sense strand sequence listed in any one of Tables 1-3, wherein the sense strand sequence is completely complementary to the antisense strand sequence in the dsRNA agent. In some embodiments, the dsRNA agent comprises an antisense strand sequence listed in any one of Tables 1-3. In some embodiments, the dsRNA agent comprises a sequence listed as a duplex sequence in any one of Tables 1-3.

In some embodiments, the dsRNA reagent includes at least one modified nucleotide. In some embodiments, all or substantially all nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least one modified nucleotide includes: 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2'3'-seco nucleotide mimetic, locked nucleotide, unblocked nucleic acid nucleotide (UNA), glycol nucleic acid nucleotide (GNA), 2'-F-arabinose nucleotide, 2'-methoxyethyl nucleotide, debasic nucleotide, ribitol, reverse nucleotide, reverse debasic nucleotide, reverse 2'-Ome nucleotide, reverse 2'-deoxy nucleotide, isomannitol nucleotide, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-OMe nucleotides, nucleotides comprising a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a 2'-amino modified nucleotide, a phosphoramidite, or a nucleotide comprising an unnatural base.

In certain embodiments, the dsRNA agent comprises an E-vinylphosphonate nucleotide at the 5΄ end of the guide strand.

In some embodiments, the dsRNA agent includes at least one phosphorothioate nucleoside bond. In some embodiments, the sense strand includes at least one phosphorothioate nucleoside bond. In some embodiments, the antisense strand includes at least one phosphorothioate nucleoside bond. In some embodiments, the sense strand includes 1, 2, 3, 4, 5 or 6 phosphorothioate nucleoside bonds. In some embodiments, the antisense strand includes 1, 2, 3, 4, 5 or 6 phosphorothioate nucleoside bonds.

In some embodiments, all or substantially all nucleotides of the sense strand and the antisense strand are modified nucleotides. In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides, 2'-fluoro nucleotides and UNA modified nucleotides, wherein less than 6 modified nucleotides are 2'-fluoro nucleotides. In some embodiments, the antisense strand comprises 3 or 5 2'-fluoro nucleotides, preferably, the antisense strand comprises 5 2'-fluoro nucleotides. In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein less than 4 modified nucleotides are 2'-fluoro nucleotides. In some embodiments, the sense strand comprises 3 2'-fluoro nucleotides. In certain embodiments, the antisense chain comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least 14 modified nucleotides are 2'-O-methyl nucleotides, and the nucleotides at positions 2, 5, 7, 11, 12, 14, 16 and/or 18 from the first matching position at the 5' end of the antisense chain are independently 2'-fluoro nucleotides. In certain embodiments, the antisense chain comprises at least one UNA modified nucleotide and 5 2'-fluoro nucleotides. In certain embodiments, the antisense chain comprises one UNA modified nucleotide at position 7 and 5 2'-fluoro nucleotides at positions 2, 5, 12, 14 and 16 from the first matching position at the 5' end, and the rest are 2'-O-methyl nucleotides. In certain embodiments, the antisense chain comprises a UNA-modified nucleotide at position 7 and five 2'-fluoro nucleotides at positions 2, 5, 12, 14 and 18, starting from the first matching position at the 5' end, and the remaining 2'-O-methyl nucleotides. In certain embodiments, the antisense chain comprises five 2'-fluoro nucleotides at positions 2, 7, 12, 14 and 16, starting from the first matching position at the 5' end, and the remaining 2'-O-methyl nucleotides. In certain embodiments, the antisense chain comprises five 2'-fluoro nucleotides at positions 2, 7, 11, 14 and 16, starting from the first matching position at the 5' end, and the remaining 2'-O-methyl nucleotides. In certain embodiments, the antisense chain comprises 5 2'-fluoro nucleotides at positions 2, 5, 12, 14 and 16, respectively, counting from the first matching position at the 5' end, and the rest are 2'-O-methyl nucleotides. In certain embodiments, the antisense chain comprises 5 2'-fluoro nucleotides at positions 2, 5, 12, 14 and 18, respectively, counting from the first matching position at the 5' end, and the rest are 2'-O-methyl nucleotides. In certain embodiments, the sense chain comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, preferably, at least 18 of the modified nucleotides are 2'-O-methyl nucleotides, and the nucleotides at positions 9, 11 and/or 13, counting from the first matching position at the 3' end of the sense chain, are 2'-fluoro nucleotides. In some embodiments, the sense chain comprises at least 18 modified nucleotides that are 2'-O-methyl nucleotides, and the nucleotides at positions 8, 11 and/or 13, starting from the first matching position at the 3' end of the sense chain, are 2'-fluoro nucleotides. In some embodiments, the modified sense chain is a modified sense chain sequence shown in one of Tables 2-3. In some embodiments, the modified antisense chain is a modified antisense chain sequence shown in one of Tables 2-3.

In some embodiments, the dsRNA reagent includes at least one modified nucleotide and further includes one or more targeting groups or linking groups. In some embodiments, one or more targeting groups or linking groups are conjugated to a sense chain. In some embodiments, the targeting group or linking group includes N-acetylgalactosamine (GalNAc).

In certain embodiments, the targeting group comprises a moiety having the following structure: Formula 1; each n'' is independently selected from 1 or 2.

In certain embodiments, the targeting group has the following structure: GLO-1 GLS-1* GLO-2 GLS-2* GLO-3 GLS-3* GLO-4 GLS-4* GLO-5 GLS-5* GLO-6 GLS-6* GLO-7 GLS-7* GLO-8 GLS-8* GLO-9 GLS-9* GLO-10 GLS-10* GLO-11 GLS-11* GLO-12 GLS-12* GLO-13 GLS-13* GLO-14 GLS-14* GLO-15 GLS-15* GLO-16 GLS-16*.

In some embodiments, the dsRNA reagent comprises a targeting group attached to the 5' end of the sense strand. In some embodiments, the dsRNA reagent comprises a targeting group attached to the 3' end of the sense strand.

In certain embodiments, the antisense strand includes an inverted debasing residue at the 3' terminus.

In some embodiments, the sense chain comprises one or two reverse debasing residues and/or one or two imann residues at the 3' or/and 5' ends. In some embodiments, each end of the sense chain comprises a reverse debasing residue. In some embodiments, each end of the sense chain comprises an imann residue. In some embodiments, one or more reverse debasing residues or one or more imann residues are bound to either or both ends of the sense chain via a phosphorothioate bond. In some embodiments, the targeting group is further bound to either end of the sense chain via a phosphorothioate bond. In some embodiments, the targeting group is further bound to the 5' end of the sense chain via a phosphorothioate bond. In certain embodiments, the 5' end of the sense chain includes a reverse debasing residue or imann residue, wherein the reverse debasing residue or imann residue is linked to the adjacent nucleotide at the 5' end of the nucleotide sequence of the sense chain through a phosphorothioate bond. In certain embodiments, the sense chain further includes a targeting group linked to the reverse debasing residue or imann residue at the 5' end of the sense chain, wherein the targeting group is linked to the adjacent reverse debasing residue or imann residue through a phosphorothioate bond, and optionally the targeting group is N-acetylgalactosamine (GalNAc).

In some embodiments, the dsRNA reagent has two blunt ends. In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides.

In some embodiments, the modified sense chain has a modification pattern shown in any one of Tables 2-3. In some embodiments, the modified antisense chain has a modification pattern shown in any one of Tables 2-3. In some embodiments, the modified sense chain is a modified sense chain sequence shown in any one of Tables 2-3. In some embodiments, the modified antisense chain is a modified antisense chain sequence shown in any one of Tables 2-3.

In certain embodiments, the dsRNA comprises a duplex selected from AV01002, AV00947, AV00948, AV00949, AV00950, AV00969, AV01004, AV00974, AV00981, AV01012, wherein the duplex optionally includes a targeting ligand. In certain embodiments, the dsRNA comprises a duplex selected from the group consisting of AD00883, AD01053, AD01053-1, AD00884, AD00884-1, AD00885, AD00885-1, AD01054, AD01055, AD01055-1, AD00887, AD00888, AD00889, AD01066, AD01066-1, AD01067, AD01067-1.

According to another aspect of the present invention, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting PD-L1 expression is provided, wherein the dsRNA agent comprises a sense chain and an antisense chain, wherein the sense chain is complementary to the antisense chain, wherein the antisense chain comprises a region complementary to a portion of a PD-L1 RNA transcript, wherein each chain is about 15 to about 30 nucleotides in length, wherein the sequence comprised by the sense chain can be represented by formula (I): 5′-(N′ _L ) _n′ N′ _L N′ _L N′ _L N _{′ L} N′ _F N′ _L N′ F N′ _L N′ _N1 N′ _{N2 N} ′ _L N′ _L N′ _L N′ _L N′ L (N′ _L ) _m′ -3′ (I); wherein: each N′ _F represents a 2'-fluorine-modified nucleotide; each _N'N1 and _N'N2 independently represents a modified or unmodified nucleotide; each _N'L independently represents a modified or unmodified nucleotide but does not represent a 2'-fluorine-modified nucleotide, and m' and n' are each independently an integer from 0 to 7.

In certain embodiments, _N'N1 and _N'N2 include only one 2'-fluorine modified nucleotide.

In certain embodiments, N′ and _N1 independently represent 2′-fluoro modified nucleotides.

In certain embodiments, N' and _N2 independently represent 2'-fluoro modified nucleotides.

In some embodiments, m' is 2 and n' is 4, or m' is 2 and n' is 2. In some embodiments, m' is 1 and n' is 4, or m' is 1 and n' is 2. In some embodiments, m' is 0 and n' is 4, or m' is 0 and n' is 2.

In some embodiments, the dsRNA agent includes a targeting group fused to the 5' end of the sense chain, preferably, the targeting group is selected from any one of the aforementioned GLO-1 to GLO-16 and GLS-1* to GLS-16*, more preferably, the targeting group is the aforementioned GLS-15*. In some embodiments, the dsRNA agent includes a targeting group fused to the 3' end of the sense chain. In some embodiments, the antisense chain includes a reverse debasing residue at the 3' end. In some embodiments, the sense chain includes one or two reverse debasing residues and/or one or two imann residues at the 3' or/and 5' ends. In some embodiments, the 3' and 5' ends of the sense chain each independently include a reverse debasing residue. In certain embodiments, the 3' and 5' ends of the sense chain each independently include an imann residue. In certain embodiments, the two reverse debasing residues contained at the 3' and 5' ends of the sense chain, and the residue at the 3' or 5' end is further conjugated to a targeting group, and the targeting group is preferably the aforementioned GLS-15*. In certain embodiments, the two imann residues contained at the 3' and 5' ends of the sense chain, and the residue at the 3' or 5' end is further conjugated to a targeting group, and the targeting group is preferably the aforementioned GLS-15*. In certain embodiments, the 5' end of the sense chain includes a reverse debasing residue or imann residue, wherein the reverse debasing residue or imann residue is linked to the adjacent nucleotide at the 5' end of the nucleotide sequence of the sense chain through a phosphorothioate bond. In certain embodiments, the sense chain further includes a targeting group linked to the reverse debasing residue or imann residue at the 5' end of the sense chain, wherein the targeting group is linked to the adjacent reverse debasing residue or imann residue through a phosphorothioate bond, and optionally the targeting group is N-acetylgalactosamine (GalNAc).

According to another aspect of the present invention, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting PD-L1 expression is provided, wherein the dsRNA agent comprises a sense chain and an antisense chain, wherein the sense chain is complementary to the antisense chain, wherein the antisense chain comprises _a region complementary to the _{PD -} L1 RNA transcript, wherein the length of each chain is about 18 to 30 nucleotides, wherein the sequence comprised by the antisense chain _{can be} represented by formula _{( II): 3′-(NL)nNM1NLNM2NLNFNLNM3NM4NLNLNLNM5NLNM6NLNLNFNL - 5 ′ ( II ) ; wherein :} each _NF represents _a 2′-fluorine _- modified _nucleotide ; each _{NM1 , NM2} , _NM3 , _NM4 , N _M5 and N _M6 independently represent modified or unmodified nucleotides; each _NL independently represents a modified or unmodified nucleotide, but does not represent a 2'-fluoro-modified nucleotide, and n is an integer from 0 to 7.

In some embodiments, N _M1 , N _M2 , N _M3 , N _M4 , N _M5 and N _M6 have only three 2'-fluoro modified nucleotides.

In certain embodiments, N _M2 , N _M3 , and N _M5 each independently represent a 2'-fluoro modified nucleotide.

In certain embodiments, N _M2 , N _M4 , and N _M5 each independently represent a 2'-fluoro modified nucleotide.

In certain embodiments, N _M1 , N _M3 , and N _M6 each independently represent a 2'-fluoro modified nucleotide.

In certain embodiments, N _M2 , N _M3 , and N _M6 each independently represent a 2'-fluoro modified nucleotide.

In certain embodiments, N _M2 , N _M4 , and N _M6 each independently represent a 2'-fluoro modified nucleotide.

In certain embodiments, _NM1 , _NM3 , and _NM6 each independently represent a 2'-fluoro modified nucleotide, and _NM5 represents a UNA modified nucleotide.

In certain embodiments, N _M2 , N _M3 , and N _M6 each independently represent a 2'-fluoro modified nucleotide, and N _M5 represents a UNA modified nucleotide.

In certain embodiments, N _M2 , N _M4 , and N _M6 each independently represent a 2'-fluoro modified nucleotide, and N _M5 represents a UNA modified nucleotide.

In some embodiments, n is 1, or n is 2, or n is 3, or n is 5.

According to another aspect of the present invention, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting PD-L1 expression is provided, wherein the dsRNA agent comprises a sense chain and an antisense chain, wherein the sense chain and the antisense chain form a dsRNA duplex, wherein the sense chain is complementary to the antisense chain, wherein the antisense chain comprises a region complementary to the PD-L1 RNA transcript, wherein the complementary region comprises at least 15 consecutive nucleotides, wherein the dsRNA duplex can be represented by formula (III): Sense chain: 5′-(N′ _L ) _n′ N′ _L N′ _L N′ _{L N} ′ _L N′ _F N′ _L N′ _F N′ _L N′ _{N1 N ′} N2 N′ _L N′ _L N′ _L N′ L N′ _L (N′ _L ) _m′ -3′ Antisense chain _: 3′-( _NL ) _{nNM1NLNM2NLNFNLNM3NM4NLNLNLNM5NLNM6NLNLNFNL - 5 ′ ( III ) ;} wherein: _the length of each chain is approximately 18 to 30 nucleotides; each _{NF and N′F independently} represents a 2'-fluorine- _modified nucleotide _{; NM1, NM2, NM3, NM4, NM5, NM6 , N′N1 and N′N2 independently represent a modified or unmodified} nucleotide; each _{NL and N′L} independently represents a modified or unmodified nucleotide but does not represent a 2'-fluorine-modified nucleotide _, and m′, n′ _and n are each independently integers from 0 to 7.

In some embodiments, N _M1 , N _M2 , N _M3 , N _M4 , N _M5 and N _M6 have only three 2'-fluoro modified nucleotides, and _N'N1 and _N'N2 include only one 2'-fluoro modified nucleotide.

In some embodiments, m' is 2 and n' is 4, m' is 2 and n' is 6, or m' is 2 and n' is 2. In some embodiments, m' is 1 and n' is 4, or m' is 1 and n' is 2. In some embodiments, m' is 0 and n' is 4, or m' is 0 and n' is 2. In some embodiments, n is 1, or n is 2, or n is 3, or n is 5.

In certain embodiments, N′ and _N1 independently represent 2′-fluoro modified nucleotides.

In certain embodiments, N' and _N2 independently represent 2'-fluoro modified nucleotides.

In certain embodiments, N _M2 , N _M3 , and N _M5 each independently represent a 2'-fluoro modified nucleotide.

In certain embodiments, N _M2 , N _M4 , and N _M5 each independently represent a 2'-fluoro modified nucleotide.

In certain embodiments, N _M1 , N _M3 , and N _M6 each independently represent a 2'-fluoro modified nucleotide.

In certain embodiments, N _M2 , N _M3 , and N _M6 each independently represent a 2'-fluoro modified nucleotide.

In certain embodiments, N _M2 , N _M4 , and N _M6 each independently represent a 2'-fluoro modified nucleotide.

In certain embodiments, _NM1 , _NM3 , and _NM6 each independently represent a 2'-fluoro modified nucleotide, and _NM5 represents a UNA modified nucleotide.

In certain embodiments, N _M2 , N _M3 , and N _M6 each independently represent a 2'-fluoro modified nucleotide, and N _M5 represents a UNA modified nucleotide.

In certain embodiments, N _M2 , N _M4 , and N _M6 each independently represent a 2'-fluoro modified nucleotide, and N _M5 represents a UNA modified nucleotide.

In some embodiments, the dsRNA agent includes a targeting group fused to the 5' end of the sense chain, preferably, the targeting group is selected from any one of the aforementioned GLO-1 to GLO-16 and GLS-1* to GLS-16*, more preferably, the targeting group is the aforementioned GLS-15*. In some embodiments, the dsRNA agent includes a targeting group fused to the 5' end of the sense chain. In some embodiments, the antisense chain includes a reverse debasing residue at the 3' end. In some embodiments, the sense chain includes one or two reverse debasing residues and/or one or two imann residues at the 3' or/and 5' ends. In some embodiments, the 3' and 5' ends of the sense chain each independently include a reverse debasing residue. In some embodiments, the 3' and 5' ends of the sense chain each independently include an imann residue. In some embodiments, the two reverse debasing residues contained at the 3' and 5' ends of the sense chain, and the residue at the 3' or 5' end is further fused to a targeting group, and the targeting group is preferably the aforementioned GLS-15*. In some embodiments, the two imann residues contained at the 3' and 5' ends of the sense chain, and the residue at the 3' or 5' end is further fused to a targeting group, and the targeting group is preferably the aforementioned GLS-15*. In some embodiments, the dsRNA reagent has two blunt ends. In some embodiments, at least one chain includes a 3' overhang of at least 1 nucleotide. In some embodiments, at least one chain comprises a 3' overhang of at least 2 nucleotides. In some embodiments, one or more reverse desaccharide residues or one or more imann residues are bound to either or both ends of the sense chain through a thiophosphate bond. In some embodiments, the targeting group is further bound to either end of the sense chain through a thiophosphate bond. In some embodiments, the targeting group is further bound to the 5' end of the sense chain through a thiophosphate bond. In some embodiments, the 5' end of the sense chain includes a reverse desaccharide residue or an imann residue, wherein the reverse desaccharide residue or the imann residue is connected to the adjacent nucleotide at the 5' end of the nucleotide sequence of the sense chain through a thiophosphate bond. In certain embodiments, the sense strand further comprises a targeting group linked to the reverse debasing residue or imann residue at the 5' end of the sense strand, wherein the targeting group is linked to the adjacent reverse debasing residue or imann residue via a phosphorothioate bond, and optionally the targeting group is N-acetylgalactosamine (GalNAc). In some embodiments of the above dsRNA agent, the region complementary to a portion of the PD-L1 mRNA transcript comprises at least 15, 16, 17, 18 or 19 consecutive nucleotides, which differ from the complementary sequence of any of the above target regions of the PD-L1 mRNA transcript by no more than 0, 1, 2 or 3 nucleotides. In certain embodiments, the antisense strand of the dsRNA agent is at least substantially complementary to any of the target regions of SEQ ID NO: 1 and provided in any of Tables 1-3. In some embodiments of the above dsRNA agents, the PD-L1 mRNA transcript is SEQ ID NO: 1.

In some embodiments, any sense chain in Table 1 can be further modified according to the pattern shown in formula (I) or (III) above.

In some embodiments, any antisense chain in Table 1 can be further modified according to the pattern shown in formula (II) or (III) above.

In some embodiments, any of the bichains in Table 1 can be further modified according to the pattern shown in the above formula (III).

According to one aspect of the present invention, a composition is provided, which includes any embodiment of the above-mentioned dsRNA agent of the present invention. In certain embodiments, the composition further includes a pharmaceutically acceptable carrier. In certain embodiments, the composition further includes one or more additional therapeutic agents. In certain embodiments, the composition is packaged in a kit, container, package, dispenser, prefilled syringe or vial. In certain embodiments, the composition is formulated for subcutaneous administration or formulated for intravenous (IV) administration.

According to another aspect of the present invention, a cell is provided, which includes any embodiment of the above-mentioned dsRNA agent of the present invention. In some embodiments, the cell is a mammalian cell, optionally a human cell.

According to another aspect of the present invention, a method for inhibiting PD-L1 gene expression in a cell is provided, the method comprising: (i) preparing a cell comprising an effective amount of any embodiment of the above-mentioned dsRNA agent of the present invention or any embodiment of the above-mentioned composition of the present invention. In certain embodiments, the method further comprises: (ii) maintaining the prepared cells for a sufficient time to obtain degradation of the mRNA transcript of the PD-L1 gene, thereby inhibiting the expression of the PD-L1 gene in the cell. In certain embodiments, the cell is in a subject, and the dsRNA agent is administered to the subject subcutaneously. In certain embodiments, the cell is in a subject, and the dsRNA agent is administered to the subject by intravenous administration. In certain embodiments, the method further comprises assessing inhibition of the PD-L1 gene after administering the dsRNA agent to the subject, wherein the assessing comprises: (i) determining one or more physiological characteristics of a PD-L1-associated disease or condition in the subject, and (ii) comparing the determined physiological characteristics to a pre-treatment baseline physiological characteristic of the PD-L1-associated disease or condition and/or a control physiological characteristic of the PD-L1-associated disease or condition, wherein the comparison indicates one or more of the presence or absence of inhibition of PD-L1 gene expression in the subject. In certain embodiments, the physiological characteristic is one or more of: PD-L1 mRNA level (by liver biopsy), PD-L1 protein level, the reduction of PD-L1 expression can also be indirectly assessed by measuring the reduction of PD-L1 biological activity, or other pathologies associated with elevated PD-L1 levels (preferably in the blood or liver), or other treatments that require inhibition of PD-L1 expression. The reduction of one or more of PD-L1 mRNA level, PD-L1 protein level can be determined using methods routine to those skilled in the art (e.g., northern blot, qRT-PCR); the protein level of PD-L1 can be determined by using methods routine to those skilled in the art (e.g., Western blot, immunological techniques). The reduction of PD-L1 expression can also be indirectly assessed by measuring the reduction of PD-L1 biological activity or measuring the PD-L1 level in a subject sample (e.g., serum sample).

According to another aspect of the present invention, a method for inhibiting PD-L1 gene expression in a subject is provided, the method comprising administering to the subject an effective amount of the dsRNA agent of the present invention or the composition of the present invention. In certain embodiments, the dsRNA agent is administered to the subject subcutaneously. In certain embodiments, the dsRNA agent is administered to the subject intravenously. In certain embodiments, the method further comprises: assessing inhibition of the PD-L1 gene after administration of the dsRNA agent, wherein the assessment method comprises: (i) determining one or more physiological characteristics of a PD-L1-associated disease or disorder in the subject, and (ii) comparing the determined physiological characteristics to a pre-treatment baseline physiological characteristic of the PD-L1-associated disease or disorder and/or a control physiological characteristic of the PD-L1-associated disease or disorder, wherein the comparison indicates one or more of the presence or absence of inhibition of PD-L1 gene expression in the subject. In certain embodiments, the expression of the PD-L1 gene can be assessed by measuring the level or change in the level of any variable associated with the expression of the PD-L1 gene (e.g., PD-L1 mRNA level or PD-L1 protein level) using a conventional method for a person skilled in the art (e.g., northern blot, qRT-PCR); the protein level of PD-L1 can be determined by using a conventional method for a person skilled in the art (e.g., Western blot, immunological technique). The reduction in PD-L1 expression can also be assessed indirectly by measuring the reduction in PD-L1 biological activity or measuring the PD-L1 level in a subject sample (e.g., serum sample).

According to another aspect of the present invention, a method for treating a disease or condition associated with the presence of PD-L1 protein is provided, the method comprising: administering to a subject an effective amount of any of the above-mentioned dsRNA agent embodiments of the present invention or any of the above-mentioned composition embodiments of the present invention to inhibit PD-L1 gene expression. In certain embodiments, the disease, disorder or condition associated with PD-L1 is selected from: tumors or hematological malignancies (e.g., lymphoma/leukemia, hematological malignancies, breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, liver cancer, salivary gland cancer, gastric cancer, neuroglioma, thyroid cancer, thymic epithelial cancer, head cancer, kidney cancer, pancreatic cancer and neck cancer), infectious diseases (e.g., viral, bacterial, fungal or parasitic diseases). In certain embodiments, the infectious disease is a chronic infectious disease, such as caused by viruses (such as HIV, HBV, HCV and HTLV, etc.), bacteria (such as Helicobacter pylori, etc.) and parasites (such as Schistosoma mansoni).

In certain embodiments, the effectiveness of a treatment for an infectious disease can be demonstrated, for example, by a reduction in the amount of an infectious agent present, such as by the inability to culture the pathogen from a subject's sample. The effectiveness of a treatment for an infectious disease can be demonstrated by a reduction in the amount of an infectious agent present, such as a reduction in the amount of a protein, nucleic acid, or carbohydrate present in the infectious agent. The effectiveness of a treatment can be demonstrated, for example, by the presence of an immune response, such as the presence of antibodies or immune cells directed against the infectious agent. The effectiveness of a treatment for an infectious disease can be demonstrated by a reduction in the presence of an infectious agent, such as by a reduction in one or more symptoms or signs of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss). The specific symptoms or signs depend on the specific pathogen. In certain embodiments, the efficacy of the methods of the present invention in treating HBV infection is monitored by evaluating a combination of serological markers as described below. The treatment effect of HBV subjects can be monitored by detecting the level of hepatitis B antigen (HBsAg) HBeAg or HB cccDNA in the serum of the subject to indicate effective treatment of the disease. The treatment effect can also be determined by detecting the level of anti-HBsAg antibodies in the subject.

In certain embodiments, the efficacy of the methods of the invention in the treatment of cancer can be monitored by assessing the maintenance or preferably reduction of tumor burden or prevention of metastasis in a subject's primary or metastatic tumor. Methods for detecting and monitoring tumor burden are known in the art. The efficacy of treatment can also be determined by detecting a reduction in the severity, signs, symptoms or markers of such diseases or conditions in patients with elevated PD-L1 or PD-L1 responsive tumors.

In certain embodiments, the reduction of PD-L1 expression can also be indirectly assessed by measuring or observing a change (preferably a clinically relevant change) in at least one sign or symptom of a PD-L1-related disease. The efficacy of the treatment or prevention of a disease can be assessed, for example, by measuring disease progression, disease remission, symptom severity, pain relief, quality of life, the amount of medication required to maintain the therapeutic effect, the level of a disease marker, or any other measurable parameter suitable for the specific disease being treated or prevented. Those skilled in the art are fully capable of monitoring the efficacy of treatment or prevention by measuring any one or any combination of these parameters. For example, the therapeutic effect of a disease (e.g., an infectious disease, such as a viral disease, such as hepatitis or cancer) can be improved by enhancing the immune response. A clinically appropriate approach produces a beneficial effect on at least a statistically significant portion of patients, such as symptom improvement, cure, disease reduction, prolonged life, improved quality of life, or other effects that are generally considered positive by physicians familiar with treating PD-L1-related diseases, such as provided in the HBV diagnostic criteria provided herein.

In some embodiments, the method further comprises: administering an additional treatment regimen to the subject. In some embodiments, the additional treatment regimen comprises treating a PD-L1-related disease or condition. In some embodiments, the additional treatment regimen comprises: administering one or more PD-L1 antisense polynucleotides of the present invention to the subject, administering a non-PD-L1 dsRNA therapeutic to the subject, and performing behavioral changes on the subject. In some embodiments, the additional cancer therapeutic agent is selected from the group consisting of surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, or hormone therapy. In certain embodiments, the chemotherapeutic compound includes, but is not limited to, alemtuzumab, altretamine, azacitidine, bendamustine, bleomycin, bortezomib, busulfan, cabazitaxel, capecitabine, carboplatin, carmofur, carmustine, chlorambucil, chlormethine, cisplatin, cladribine, clofarabine, cyclophosphamide, osphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, denosumab, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, everolimus, floxuridine, fludarabine, fluorouracil, fotemustine, gemcitabine, gemtuzumab b), hydroxycarbamide, ibritumomab, idarubicin, ifosfamide, irinotecan, ixabepilone, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, nedaplatin, nelarabine, ofatumumab, oxaliplatin, paclitaxel, pemetrexed, pentathiaprine In some embodiments, the targeted therapy includes but is not limited to protein kinase inhibitors, checkpoint inhibitors, and VEGF inhibitors. The protein kinase inhibitor is a small molecule, a compound, a polysaccharide, a lipid, a peptide, a polypeptide, a protein, an antibody, a nucleoside, a nucleoside analog, a nucleotide, a nucleotide analog, a nucleic acid or an oligonucleotide. In certain embodiments, the protein kinase inhibitor includes but is not limited to acalabrutinib, adavosertib, afatinib, alectinib, axitinib, binimetinib, bosutinib, brigatinib, cediranib, ceritinib, cetuximab, cobimetinib, crizotinib, b), cabozantinib, dacomitinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, lortati nib), masitinib, momelotinib, mubritinib, neratinib, nilotinib, nintedanib, olmutinib, osimertinib, pacritinib, panitumumab, pazopanib, pegaptanib, ponatinib, radotinib ), regorafenib, rociletinib, ruxolitinib, selumetinib, semaxanib, sorafenib, sunitinib, SU6656, tivozanib, toceranib, trametinib, trastuzumab, vandetanib or vemurafenib or any combination thereof. In certain embodiments, the checkpoint inhibitor is a small molecule, a compound, a polysaccharide, a lipid, a peptide, a polypeptide, a protein, an antibody, a nucleoside, a nucleoside analog, a nucleotide, a nucleotide analog, a nucleic acid or an oligonucleotide. In certain embodiments, the immune checkpoint is a PD-1/PD-L1 checkpoint. In certain embodiments, the PD-1 checkpoint includes but is not limited to nivolumab, pembrolizumab, spartalizumab, cemiplimab, camrelizumab, sintilimab, tislelizumab, toripalimab, AMP-224 or AMP-514, or any combination thereof. In certain embodiments, the PD-L1 checkpoint inhibitor includes but is not limited to atezolizumab, avelumab, durvalumab, KN035, AUNP12, CA-170 or BMS-986189, or any combination thereof. In certain embodiments, the immune checkpoint is the CTLA-4 checkpoint. In some embodiments, the CTLA-4 checkpoint inhibitor includes but is not limited to ipilimumab or tremilimumab, or any combination thereof. In some embodiments, the VEGF inhibitor is a small molecule, a compound, a polysaccharide, a lipid, a peptide, a polypeptide, a protein, an antibody, a nucleoside, a nucleoside analog, a nucleotide, a nucleotide analog, a nucleic acid, or an oligonucleotide. In certain embodiments, VEGF inhibitors include, but are not limited to, aflibercept, axitinib, bevacizumab, brivanib, cabozantinib, cediranib, lenvatinib, linifmib, nintedanib, pazopanib, ponatinib, ramucirumab, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, or vandetanib, or any combination thereof. In certain embodiments, the additional therapeutic agent selected from the group consisting of antiviral drugs refers to a drug composition administered to treat a viral infection. In certain embodiments, the viral infection is caused by adenovirus, Ebola virus, coronavirus, EBV, Friend virus, hantavirus, HBV, HCV, herpes simplex virus, HIV, human metapneumovirus, HPV, influenza virus, Japanese encephalitis virus, Kaposi's sarcoma-associated herpesvirus, lymphocytic choriomeningitis virus, parainfluenza virus, rabies virus, respiratory syncytial virus, In some embodiments, the antiviral drug is a small molecule, a compound, a polysaccharide, a lipid, a peptide, a polypeptide, a protein, an antibody, a nucleoside, a nucleoside analog, a nucleotide, a nucleotide analog, a nucleic acid or an oligonucleotide. In some embodiments, the antiviral drug is an interferon, a capsid assembly modulator, a sequence-specific oligonucleotide, an entry inhibitor or a small molecule immunomodulator. In certain embodiments, antiviral drugs include, but are not limited to, AB-423, AB-506, ABI-H2158, ABI-H0731, acyclovir, adapromine, adefovir, alafenamide, amantadine, asunaprevir, baloxavir marboxil), beclabuvir, boceprevir, brivudine, cidofovir, ciluprevir, clevudine, cytarabine, daclatasvir, danoprevir, dasabuvir, deleobuvir, dipivoxil, edoxudine, elbasvir ), entecavir, faldaprevir, famciclovir, favipiravir, filibuvir, fomivirsen, foscamet, galidesivir, ganciclovir, glecaprevir, GLS4, grazoprevir, idoxuridine, imiquimod, IFN-a, interferon alfa 2b, JNJ-440, JNJ-6379, lamivudine, laninamivir, ledipasvir, mericitabine, methisazone, MK-608, moroxydine, narlaprevir, NITD008, NZ-4, odalasvir, ombitasvir, oseltamivir ), paritaprevir, peginterferon alpha-2a, penciclovir, peramivir, pibrentasvir, pimodivir, pleconaril, podophyllotoxin, presatovir, radalbuvir, ravidasvir, remdesivir, REP 2139, REP 2165, resiquimod, RG7907, ribavirin, rifampicin, rimantadine, ruzasvir, samatasvir, setrobuvir, simeprevir, sofosbuvir, sorivudine, sovaprevir, taribavirin, telaprevir, telbivudine, tenofovir, tenofovir disoproxil, triazavirin, trifluridine uridine, tromantadine, umifenovir, uprifosbuvir, valacilovir, valgancicovir, vaniprevir, vedroprevir, velpatasvir, vidarabine, voxilaprevir, or zanamivir, or any combination thereof.

In some embodiments, the dsRNA agent is administered to the subject subcutaneously. In some embodiments, the dsRNA agent is administered to the subject by intravenous injection. In some embodiments, the method further comprises determining the efficacy of the administered double-stranded RNA (dsRNA) agent on the subject.

In certain embodiments, a method of determining a treatment effect in a subject comprises: (i) determining one or more physiological characteristics of a PD-L1-associated disease or disorder in the subject, and (ii) comparing the determined physiological characteristics to a baseline, pre-treatment physiological characteristic of the PD-L1-associated disease or disorder, wherein the comparison indicates one or more of the presence, absence, and level of efficacy of administering a double-stranded RNA (dsRNA) agent to the subject.

In certain embodiments, the expression of the PD-L1 gene can be assessed based on the level or level change of any variable associated with the expression of the PD-L1 gene in the subject (e.g., PD-L1 mRNA level, PD-L1 protein level). Reduction in PD-L1 expression is assessed indirectly by measuring a decrease in PD-L1 biological activity or PD-L1 levels in a sample from a subject (e.g., a serum sample), by measuring a decrease in proteins, nucleic acids, or carbohydrates present in an infectious agent, by assessing an immune response against antibodies or immune cells directed against an infectious agent, by a decrease in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by measuring the level of hepatitis B antigen (HBsAg), HBeAg, or HB cccDNA in the subject's serum, or by detecting the subject's anti-HBsAg antibody level.

According to another aspect of the present invention, a method for reducing the level of PD-L1 protein in a subject compared to the pre-treatment baseline level of PD-L1 protein in the subject is provided, the method comprising administering to the subject an effective amount of any of the above-mentioned dsRNA agent embodiments of the present invention or any of the above-mentioned composition embodiments of the present invention to reduce the expression level of the PD-L1 gene. In certain embodiments, the dsRNA agent is administered to the subject subcutaneously or by intravenous injection.

According to another aspect of the present invention, a method of changing the physiological characteristics of a PD-L1-related disease or condition in a subject compared to the physiological characteristics of the PD-L1-related disease or condition before treatment of the subject is provided, the method comprising administering to the subject an effective amount of any of the above-mentioned dsRNA agent embodiments of the present invention or any of the above-mentioned composition embodiments of the present invention to change the physiological characteristics of the PD-L1-related disease or condition in the subject. In certain embodiments, the dsRNA agent is administered to the subject subcutaneously or by intravenous injection. In certain embodiments, the physiological characteristics and symptoms are one or more of the following: PD-L1 mRNA level, PD-L1 protein level in the subject, a decrease in PD-L1 expression is assessed indirectly by measuring a decrease in PD-L1 biological activity or PD-L1 level in a sample (e.g., a serum sample) from the subject, by measuring a decrease in protein, nucleic acid, or carbohydrate present in an infectious agent, by assessing an immune response to an infectious agent by assessing antibodies or immune cells, by assessing a decrease in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by measuring hepatitis B antigen (HBsAg), HBeAg, or HB in the subject's serum. The level of cccDNA is assessed by testing the level of anti-HBsAg antibodies in the subjects.

According to another aspect of the present invention, a method for treating a disease or condition associated with the PD-L1 protein using the above-mentioned dsRNA agent is provided. In certain embodiments, the disease or condition is one or more of the following: tumors or hematological malignancies (e.g., lymphoma/leukemia, hematological malignancies, breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, liver cancer, salivary gland cancer, gastric cancer, neuroglioma, thyroid cancer, thymic epithelial cancer, head cancer, kidney cancer, pancreatic cancer and neck cancer), infectious diseases (e.g., viral, bacterial, fungal or parasitic diseases). In certain embodiments, the infectious disease is a chronic infectious disease, such as caused by viruses (such as HIV, HBV, HCV and HTLV, etc.), bacteria (such as Helicobacter pylori, etc.) and parasites (such as Schistosoma mansoni).

According to another aspect of the present invention, an antisense polynucleotide agent for inhibiting PD-L1 protein expression is provided, the agent comprising 10 to 30 consecutive nucleotides, wherein at least one consecutive nucleotide is a modified nucleotide, and wherein the nucleotide sequence of the agent is about 80% complementary to the equivalent region of the nucleotide sequence of SEQ ID NO: 1 over its entire length. In certain embodiments, the equivalent region is any one of the target regions of SEQ ID NO: 1, and the complementary sequence is a sequence provided in one of Tables 1-3. In certain embodiments, the antisense polynucleotide agent comprises one of the antisense sequences provided in one of Tables 1-3.

According to another aspect of the present invention, a composition comprising any of the embodiments of the antisense polynucleotide agents described above is provided. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. In certain embodiments, the composition further comprises one or more additional therapeutic agents for treating PD-L1-related diseases or conditions. In certain embodiments, the composition is packaged in a kit, container, package, dispenser, prefilled syringe or vial. In certain embodiments, the composition is formulated for subcutaneous or intravenous administration.

According to another aspect of the present invention, a cell comprising any one of the embodiments of the antisense polynucleotide agent described above is provided. In certain embodiments, the cell is a mammalian cell, optionally a human cell.

According to another aspect of the present invention, a method for inhibiting the expression of PD-L1 gene in cells is provided, the method comprising: (i) preparing cells containing an effective amount of any of the above antisense polynucleotide agents. In certain embodiments, the method further comprises (ii) maintaining the cells prepared in (i) for a sufficient time to obtain degradation of the mRNA transcript of the PD-L1 gene, thereby inhibiting the expression of the PD-L1 gene in the cells.

According to another aspect of the present invention, a method for inhibiting PD-L1 gene expression in a subject is provided, the method comprising administering to the subject an effective amount of any of the above-mentioned antisense polynucleotide agent embodiments.

According to another aspect of the present invention, a method for treating a disease or condition associated with the presence of PD-L1 protein is provided, the method comprising administering to a subject an effective amount of any one of the above-mentioned antisense polynucleotide agent embodiments or any one of the above-mentioned compositions of the present invention embodiments to inhibit PD-L1 gene expression. In certain embodiments, the disease or condition is one or more of the following: tumors or hematological malignancies (e.g., lymphoma/leukemia, hematological malignancies, breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, liver cancer, salivary gland cancer, gastric cancer, neuroglioma, thyroid cancer, thymic epithelial cancer, head cancer, kidney cancer, pancreatic cancer and neck cancer), infectious diseases (e.g., viral, bacterial, fungal or parasitic diseases). In certain embodiments, the infectious disease is a chronic infectious disease, such as caused by viruses (such as HIV, HBV, HCV and HTLV, etc.), bacteria (such as Helicobacter pylori, etc.) and parasites (such as Schistosoma mansoni).

According to another aspect of the present invention, a method for reducing the level of PD-L1 protein in a subject compared to the pre-treatment baseline level of PD-L1 protein in the subject is provided, the method comprising administering to the subject an effective amount of any of the above-mentioned antisense polynucleotide agent embodiments or any of the above-mentioned compositions of the present invention embodiments to reduce the PD-L1 gene expression level. In certain embodiments, the antisense polynucleotide agent is administered to the subject subcutaneously or intravenously.

According to another aspect of the present invention, an antisense polynucleotide agent for inhibiting PD-L1 gene expression is provided, wherein the agent comprises 10 to 30 consecutive nucleotides, wherein at least one consecutive nucleotide is a modified nucleotide, and wherein the nucleotide sequence of the agent is complementary to the equivalent region of the nucleotide sequence of SEQ ID NO: 1 by about 80% or about 85% over its entire length.

According to another aspect of the present invention, a method for changing the physiological characteristics of a PD-L1-related disease or condition in a subject compared to the physiological characteristics of the PD-L1-related disease or condition before treatment of the subject is provided, the method comprising administering to the subject an effective amount of any of the above-mentioned antisense polynucleotide agent embodiments or any of the above-mentioned compositions of the present invention embodiments to change the physiological characteristics of the PD-L1 disease or condition in the subject. In certain embodiments, the antisense polynucleotide agent is administered to the subject by subcutaneous or intravenous injection. In certain embodiments, the physiological characteristics and symptoms are one or more of the following: a decrease in the subject's PD-L1 mRNA level, PD-L1 protein level, or PD-L1 expression is assessed indirectly by measuring a decrease in PD-L1 biological activity or PD-L1 level in a sample (e.g., a serum sample) from the subject, by measuring a decrease in the presence of a protein, nucleic acid, or carbohydrate in an infectious agent, by assessing an immune response to an infectious agent by antibodies or immune cells, by a decrease in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by measuring hepatitis B antigen (HBsAg), HBeAg, or HB in the subject's serum. The level of cccDNA is assessed by testing the level of anti-HBsAg antibodies in the subjects .

SEQ ID NO: 1 and SEQ ID NO: 2 (reverse complement) are Homo sapiens CD274 /PD-L1 transcript variant 1, mRNA [NCBI Reference Sequence: NM_014143.4].

SEQ ID NO: 3 and SEQ ID NO: 4 (reverse complement) are Mus musculus (house mouse) PD-L1 mRNA [NCBI Reference Sequence: NM_021893.3].

SEQ ID NOs: 5-168 are shown in Table 1, all of which are sense chain sequences.

SEQ ID NO: 169-332 are shown in Table 1, which are antisense chain sequences.

SEQ ID NOs: 333-496 are shown in Table 2 and are chemically modified sequences.

SEQ ID NOs: 497-602 are shown in Table 3, and the delivery molecule at the 3' end or 5' end of each sense strand is represented as "GLX-__".

The present invention includes, in part, RNAi agents, such as but not limited to double-stranded (ds) RNAi agents, which can inhibit PD-L1 gene expression. The present invention also includes, in part, compositions comprising PD-L1 RNAi agents and methods of using the compositions. The PD-L1 RNAi agents disclosed herein can be attached to a delivery compound for delivery to cells, including hepatocytes. The pharmaceutical compositions of the present invention may include at least one dsRNA PD-L1 agent and a delivery compound. In some embodiments of the compositions and methods of the present invention, the delivery compound is a GalNAc-containing delivery compound. The PD-L1 RNAi agent delivered to cells can inhibit PD-L1 gene expression, thereby reducing the activity of the PD-L1 protein product of the gene in the cell. The dsRNAi agent of the present invention can be used to treat PD-L1 related diseases and disorders.

In some embodiments of the invention, reducing PD-L1 expression in a cell or subject treats a disease or condition associated with PD-L1 expression in a cell or subject, respectively. Non-limiting examples of diseases and conditions that can be treated by reducing PD-L1 activity are: alleviation or improvement of one or more symptoms associated with unwanted or excessive PD-L1 expression (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), reduction of tumor burden in primary or metastatic tumors, or prevention of metastasis. "Treatment" may also refer to prolonging survival compared to the expected survival without treatment.

As used herein, "G", "C", "A" and "U" generally represent nucleotides containing guanine, cytosine, adenine and uracil as bases, respectively. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides (as further described below) or alternative replacement moieties. Those skilled in the art understand that guanine, cytosine, adenine and uracil can be replaced by other moieties without significantly changing the base pairing properties of the oligonucleotide containing nucleotides with such replacement moieties. For example, but not limited to, nucleotides containing inosine as a base can be paired with nucleotide bases containing adenine, cytosine or uracil. Therefore, in the nucleotide sequences of the present invention, nucleotides containing uracil, guanine or adenine can be replaced by nucleotides containing, for example, inosine. Sequences containing such replacement moieties are embodiments of the present invention.

As used herein, "CD274", "programmed cell death-ligand 1" is used interchangeably with the terms "B7 homolog 1 (B7-H1)", "programmed death ligand 1", or "PD-L1" refers to a naturally occurring gene encoding a PD-L1 protein from any vertebrate or mammalian source, including but not limited to humans, cows, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys and guinea pigs, unless otherwise indicated. The term also refers to fragments and variants of native PD-L1 that retain at least one in vivo or in vitro activity of native PD-L1. The amino acid and complete coding sequence of the reference sequence of the human PD-L1 gene can be found, for example, in GenBank Ref Seq Accession No. NM_014143.4 (SEQ ID NO: 1 and SEQ ID NO: 2), Mus musculus (house mouse) NM_021893.3 (SEQ ID NO: 3 and SEQ ID NO: 4). More examples of PD-L1 mRNA sequences can be easily obtained using publicly available databases such as GenBank, UniProt, Ensembl, and OMIM.

The following describes how to prepare and use compositions comprising PD-L1 single chain (ssRNA) and dsRNA to inhibit PD-L1 gene expression, as well as compositions and methods for treating diseases and disorders caused or regulated by PD-L1 gene expression. The term "RNAi" is also known in the art and may be referred to as "siRNA".

As used herein, the term "RNAi" refers to an agent that contains RNA and mediates targeted cleavage of RNA transcripts through an RNA-induced silencing complex (RISC) pathway. As known in the art, an RNAi target region refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during gene transcription, including product mRNAs during RNA processing of primary transcription products. The target portion of the sequence will be at least long enough to serve as a substrate for RNAi-guided cleavage near the portion or portions. The length of the target sequence may be 8-30 nucleotides (inclusive), 10-30 nucleotides (inclusive), 12-25 nucleotides (inclusive), 15-23 nucleotides (inclusive), 16-23 nucleotides (inclusive), or 18-23 nucleotides (inclusive), including all shorter lengths within each of the ranges. In some embodiments of the present invention, the target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In certain embodiments, the target sequence is 9 to 26 nucleotides in length (inclusive), including all subranges and integers therebetween. For example, although not intended to be limiting, in certain embodiments of the present invention, the target sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, and the sequence is fully complementary or at least substantially complementary to at least a portion of the RNA transcript of the PD-L1 gene. Some aspects of the invention include pharmaceutical compositions comprising one or more PD-L1 dsRNA agents and a pharmaceutically acceptable carrier. In certain embodiments of the invention, the PD-L1 RNAi as described herein inhibits the expression of PD-L1 protein.

As used herein, a "dsRNA agent" refers to a composition containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of a messenger RNA (mRNA) transcript of a target mRNA in a sequence-specific manner. Although not wishing to be limited to a particular theory, the dsRNA agents of the present invention may act through an RNA interference mechanism (i.e., by inducing RNA interference by interacting with the RNA interference pathway mechanism of mammalian cells (RNA-induced silencing complex or RISC)) or through any alternative mechanism or pathway. Methods for silencing genes in plant, invertebrate and vertebrate cells are well known in the art [see, e.g., (Sharp et al., Genes Dev. 2001, 15:485; Bernstein, et al., (2001) Nature 409:363; Nykanen, et al., (2001) Cell 107:309; and Elbashir, et al., (2001) Genes Dev. 15:188)], the disclosures of each of which are incorporated herein by reference in their entirety]. Gene silencing procedures known in the art can be used in conjunction with the disclosure provided herein to inhibit the expression of PD-L1.

The dsRNA agents disclosed herein consist of a sense strand and an antisense strand, including but not limited to: short interfering RNA (siRNA), RNAi agents, micro RNA (miRNA), short hairpin RNA (shRNA) and Dicer substrates. The antisense strand of the dsRNA agents described herein is at least partially complementary to the target mRNA. It is known in the art that dsRNA duplex structures of different lengths can be used to inhibit target gene expression. For example, dsRNAs with duplex structures of 19, 20, 21, 22 and 23 base pairs are known to effectively induce RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). It is also known in the art that shorter or longer RNA duplex structures can also effectively induce RNA interference. In certain embodiments, the length of the sense strand and the antisense strand can be the same or different. In certain embodiments, the length of each strand is no more than 40 nucleotides. In certain embodiments, the length of each strand is no more than 30 nucleotides. In certain embodiments, the length of each strand is no more than 25 nucleotides. In certain embodiments, the length of each strand is no more than 23 nucleotides. In certain embodiments, the length of each strand is no more than 21 nucleotides. In certain embodiments, the length of the sense strand and the antisense strand of the RNAi agent can be 15 to 49 nucleotides, respectively. In some embodiments, the length of the antisense strand is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the sense strand is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides. In some embodiments, the length of the sense strand and the antisense strand are both 21 nucleotides. In some embodiments, the sense strand and the antisense strand complement or substantially complement each other, and the complementary region is 15 to 23 nucleotides in length. In some embodiments, the complementary region is 19-21 nucleotides in length. In some embodiments, the complementary region is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. The PD-L1 dsRNA in certain embodiments of the present invention may include at least one strand of at least 21 nt in length, or may have a shorter duplex based on one of the sequences listed in any one of Tables 1-3, but compared with the dsRNA listed in Tables 1-3, it may also be effective to reduce 1, 2, 3 or 4 nucleotides at one or both ends, respectively. In some embodiments of the present invention, the PD-L1 dsRNA agent may have a partial sequence of at least 15, 16, 17, 18, 19, 20 or more consecutive nucleotides from one or more sequences in Tables 1-3, and its ability to inhibit PD-L1 gene expression differs from the inhibition level produced by the dsRNA containing the complete sequence by no more than 5%, 10%, 15%, 20%, 25% or 30%. The sense sequences, antisense sequences and duplexes disclosed in Tables 1-3 may be referred to herein as "parent" sequences, meaning that the sequences disclosed in Tables 1-3 may be modified, shortened, lengthened, include substitutions, etc. as described herein, and the resulting sequences retain all or at least a portion of the efficacy of their parent sequences in the methods and compositions of the present invention. The sense and antisense strands contained in the dsRNA of the present invention are independently selected. As used herein, the term "independent selection" means that each of two or more similar elements can be selected independently of the selection of other elements. For example, although not intended to be limiting, when preparing the dsRNA of the present invention, "elements" of the two strands can be selected to be included in the duplex. One selected element, the sense sequence, may be SEQ ID NO: 334 (as shown in Table 2), and the other selected element, the antisense sequence, may be SEQ ID NO: 416, or may be SEQ ID NO: 416 that has been modified, shortened, lengthened, and/or includes 1, 2, or 3 substitutions compared to its parent sequence SEQ ID NO: 416. It should be understood that the double chains of the present invention do not necessarily include the paired sense and antisense sequences in the double chains shown in Tables 1-3. Each sense and antisense chain sequence in the table is followed by its SEQ ID NO.

Certain embodiments of the compositions and methods of the present invention include single-stranded RNA in the composition and/or administered to the subject. For example, the antisense chain listed in any of Tables 1-3 can be a composition or a composition administered to the subject to reduce the PD-L1 polypeptide activity and/or PD-L1 gene expression in the subject. Table 1 shows certain PD-L1 dsRNA agent antisense chain and sense chain core extension base sequences. Single-stranded antisense molecules that may be included in certain compositions of the present invention and/or administered in certain methods of the present invention are referred to herein as "single-stranded antisense agents" or "antisense polynucleotide agents". Single-stranded sense molecules that may be included in certain compositions of the present invention and/or administered in certain methods of the present invention are referred to herein as "single-stranded sense agents" or "sense polynucleotide agents". The term "base sequence" is used herein to refer to a polynucleotide sequence without chemical modifications or delivery compounds. For example, the sense strand CCAUUCCAGAAAGAUGAGGAA (SEQ ID NO: 6) shown in Table 1 is the base sequence of SEQ ID NO: 334 in Table 2 and the base sequence of SEQ ID NO: 497 in Table 3, wherein SEQ ID NO: 334 and SEQ ID NO: 497 and chemical modifications and delivery compounds thereof are shown. Sequences disclosed herein may be assigned identifiers. For example, a single-strand sense sequence may be identified by "sense strand SS#"; a single-strand antisense sequence may be identified by "antisense strand AS#", and a double strand including a sense strand and an antisense strand may be identified by "double strand AD#/AV#".

Table 1 includes a sense chain and an antisense chain, and provides the identification number of the double chain formed by the sense chain and the antisense chain on the same line in Table 1. In certain embodiments of the present invention, the antisense sequence includes a nucleobase u or a nucleobase a at position 1 of the antisense sequence. In certain embodiments of the present invention, the antisense sequence includes a nucleobase u at position 1 of the antisense sequence. As used herein, the term "matching position" in the sense chain and the antisense chain is the position of "pairing" in each chain when the two chains are double chains. For example, in a sense chain of 21 nucleobases and an antisense chain of 21 nucleobases, the nucleobase at position 1 of the sense chain and the nucleobase at position 21 of the antisense chain are in a "matching position". In yet another non-limiting example, in a 23-nucleobase sense chain and a 23-nucleobase antisense chain, nucleobase 2 of the sense chain and nucleobase 22 of the antisense chain are in matching positions. In yet another non-limiting example, in an 18-nucleobase sense chain and an 18-nucleobase antisense chain, nucleobase 1 of the sense chain and nucleobase 18 of the antisense chain are in matching positions, and nucleobase 4 of the sense chain and nucleobase 15 of the antisense chain are in matching positions. Those skilled in the art will understand how to identify matching positions in sense chains and antisense chains that are or will be double chains and paired chains.

The first column in Table 1 indicates the duplex AV/AD # of the duplex containing the sense and antisense sequences in the same table row. For example, Table 1 discloses a duplex designated as duplex AV00938.um, which contains the sense strand SEQ ID NO: 6 and the antisense strand SEQ ID NO: 170. Thus, each row in Table 1 identifies a duplex of the present invention, each duplex containing the sense and antisense sequences shown in the same row, and the assigned identifier of each duplex is shown in the first column of the row.

In some embodiments of the methods of the present invention, an RNAi agent comprising a polynucleotide sequence shown in any one of Tables 1-3 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to a subject comprises a duplex comprising at least one base sequence listed in Table 1, comprising 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 sequence modifications. In some embodiments of the methods of the present invention, an RNAi agent comprising a polynucleotide sequence shown in any one of Tables 1-3 is attached to a delivery molecule, a non-limiting example of a delivery molecule is a delivery compound comprising a GalNAc compound or a GLS-15* compound.

Table 1: Unmodified PD-L1 RNAi Antisense and Sense Strand Sequences. All sequences are shown in 5' to 3' orientation. Duplex AV/AD # is the number assigned to the duplex for both strands in the same row in the table. Dual Link AV/AD # Sense chain Serial number. Antisense chain Serial number. Range in PD-L1 mRNA NM_014143.4 AV00937.um CGGCAUUCCAGAAAGAUGAGA 5 UCUCAUCUUUCUGGAAUGCCG 169 55-75 AV00938.um CCAUUCCAGAAAGAUGAGGAA 6 UUCCUCAUCUUUCUGGAAUGG 170 57-77 AV00939.um GAUUCCAGAAAGAUGAGGAUA 7 UAUCCUCAUCUUUCUGGAAUC 171 58-78 AV00940.um GUCCAGAAAGAUGAGGAUAUA 8 UAUAUCCUCAUCUUUCUGGAC 172 60-80 AV00941.um GAGAAAGAUGAGGAUAUUUGA 9 UCAAAUAUCCUCAUCUUUCUC 173 63-83 AV00942.um GGAAAGAUGAGGAUAUUUGCA 10 UGCAAAUAUCCUCAUCUUUCC 174 64-84 AV00943.um CAAAGAUGAGGAUAUUUGCUA 11 UAGCAAAUAUCCUCAUCUUUG 175 65-85 AV00944.um GAAGAUGAGGAUAUUUGCUGA 12 UCAGCAAAUAUCCUCAUCUUC 176 66-86 AV00945.um GAGAUGAGGAUAUUUGCUGUA 13 UACAGCAAAUAUCCUCAUCUC 177 67-87 AV00946.um GAGAUGAGGAUAUUUGCUGUA 14 UACAGCAAAUAUCCUCAUCUC 178 67-87 AV00947.um CAGGAUAUUUGCUGUCUUUAA 15 UUAAAGACAGCAAAUAUCCUG 179 72-92 AV00948.um GUAUUUGCUGUCUUUAUAUUA 16 UAAUAUAAAGACAGCAAAUAC 180 76-96 AV00949.um GAUUUGCUGUCUUUAUAUUCA 17 UGAAUAUAAAGACAGCAAAUC 181 77-97 AV00950.um GUUUGCUGUCUUUAUAUUCAA 18 UUGAAUAUAAAGACAGCAAAC 182 78-98 AV00951.um CACCUACUGGCAUUUGCUGAA 19 UUCAGCAAAUGCCAGUAGGUG 183 99-119 AV00952.um GCCUACUGGCAUUUGCUGAAA 20 UUUCAGCAAAUGCCAGUAGGC 184 100-120 AV00953.um GUGCCCCAUACAACAAAAUCA twenty one UGAUUUUGUUGUAUGGGGCAC 185 461-481 AV00954.um CCCCCAUACAACAAAAUCAAA twenty two UUUGAUUUUGUUGUAUGGGGG 186 463-483 AV00955.um GCCAUACAACAAAAUCAACA twenty three UGUUGAUUUUGUUGUAUGGGC 187 464-484 AV00956.um GUACAACAAAAUCAACCAAAA twenty four UUUUGGUUGAUUUUGUUGUAC 188 468-488 AV00957.um GCAACAAAAUCAACCAAAGAA 25 UUCUUUGGUUGAUUUUGUUGC 189 470-490 AV00958.um GAACAAAAUCAACCAAAGAAA 26 UUUCUUUUGGUUGAUUUUGUUC 190 471-491 AV00959.um GAACAAAAUCAACCAAAGAAA 27 UUUCUUUUGGUUGAUUUUGUUC 191 471-491 AV00960.um GACAAAAUCAACCAAAGAAUA 28 UAUUCUUUGGUUGAUUUUGUC 192 472-492 AV00961.um GACAAAAUCAACCAAAGAAUA 29 UAUUCUUUGGUUGAUUUUGUC 193 472-492 AV00962.um GAAAUCAACCAAAGAAUUUUA 30 UAAAAUUCUUUGGUUGAUUUC 194 475-495 AV00963.um GAUCAACCAAAGAAUUUUGGA 31 UCCAAAAUUCUUUGGUUGAUC 195 477-497 AV00964.um GUCAACCAAAGAAUUUUGGUA 32 UACCAAAAUUCUUUGGUUGAC 196 478-498 AV00965.um GCAACCAAAGAAUUUUGGUUA 33 UAACCAAAAUUCUUUGGUUGC 197 479-499 AV00966.um GAACCAAAGAAUUUUGGUUGA 34 UCAACCAAAAUUCUUUGGUUC 198 480-500 AV00967.um GACCAAAGAAUUUUGGUUGUA 35 UACAACCAAAAUUCUUUGGUC 199 481-501 AV00968.um CAUCCAGUCACCUCUGAACAA 36 UUGUUCAGAGGUGACUGGAUG 200 502-522 AV00969.um GUCCAGUCACCUCUGAACAUA 37 UAUGUUCAGAGGUGACUGGAC 201 503-523 AV00970.um GCCAGUCACCUCUGAACAUGA 38 UCAUGUUCAGAGGUGACUGGC 202 504-524 AV00971.um GCAGUCACCUCUGAACAUGAA 39 UUCAUGUUCAGAGGUGACUGC 203 505-525 AV00972.um GAGUCACCUCUGAACAUGAAA 40 UUUCAUGUUCAGAGGUGACUC 204 506-526 AV00973.um CUCACCUCUGAACAUGAACUA 41 UAGUUCAUGUUCAGAGGUGAG 205 508-528 AV00974.um GGAAGUCAUCUGGACAAGCAA 42 UUGCUUGUCCAGAUGACUUCC 206 558-578 AV00975.um CGACAAGCAGUGACCAUCAAA 43 UUUGAUGGUCACUGCUUGUCG 207 569-589 AV00976.um CAGGAGAAGCUUUUCAAUGUA 44 UACAUUGAAAAGCUUCUCCUG 208 628-648 AV00977.um CAGAAGCUUUUCAAUGUGACA 45 UGUCACAUUGAAAAGCUUCUG 209 631-651 AV00978.um CAGAUUAGAUCCUGAGGAAAA 46 UUUUCCUCAGGAUCUAAUCUG 210 705-725 AV00979.um GGAUUAGAUCCUGAGGAAAAA 47 UUUUUUCCUCAGGAUCUAAUCC 211 706-726 AV00980.um CAUUAGAUCCUGAGGAAAACA 48 UGUUUUCCUCAGGAUCUAAUG 212 707-727 AV00981.um CAUCCUGAGGAAAACCAUACA 49 UGUAUGGUUUUCCUCAGGAUG 213 712-732 AV00982.um GCCUGAGGAAAACCAUACAGA 50 UCUGUAUGGUUUUCCUCAGGC 214 714-734 AV00983.um GUGAGGAAAACCAUACAGCUA 51 UAGCUGUAUGGUUUUCCUCAC 215 716-736 AV00984.um GGGAAAACCAUACAGCUGAAA 52 UUUCAGCUGUAUGGUUUUCCC 216 719-739 AV00985.um CAAAACCAUACAGCUGAAUUA 53 UAAUUCAGCUGUAUGGUUUUG 217 721-741 AV00986.um GAAACCAUACAGCUGAAUUGA 54 UCAAUUCAGCUGUAUGGUUUC 218 722-742 AV00987.um GAACCAUACAGCUGAAUUGGA 55 UCCAAUUCAGCUGUAUGGUUC 219 723-743 AV00988.um GACCAUACAGCUGAAUUGGUA 56 UACCAAUUCAGCUGUAUGGUC 220 724-744 AV00989.um GAUACAGCUGAAUUGGUCAUA 57 UAUGACCAAUUCAGCUGUAUC 221 727-747 AV00990.um GAUUGGUCAUCCCAGAACUAA 58 UUAGUUCUGGGAUGACCAAUC 222 737-757 AV00991.um GUUGGUCAUCCCAGAACUACA 59 UGUAGUUCUGGGAUGACCAAC 223 738-758 AV00992.um GCAUCCUCCAAAUGAAAGGAA 60 UUCCUUUCAUUUGGAGGAUGC 224 765-785 AV00993.um GAUCCUCCAAAUGAAAGGACA 61 UGUCCUUUCAUUUGGAGGAUC 225 766-786 AV00994.um GUCCUCCAAAUGAAAGGACUA 62 UAGUCCUUUCAUUUGGAGGAC 226 767-787 AV00995.um GCCAAAUGAAAGGACUCACUA 63 UAGUGAGUCCUUUCAUUUGGC 227 771-791 AV00996.um GCAAAUGAAAGGACUCACUUA 64 UAAGUGAGUCCUUUCAUUUGC 228 772-792 AV00997.um GAAAUGAAAGGACUCACUUGA 65 UCAAGUGAGUCCUUUCAUUUC 229 773-793 AV00998.um GAGAUACAAACUCAAAGAAGA 66 UCUUCUUUUGAGUUUGUAUCUC 230 893-913 AV00999.um CAUACAAACUCAAAGAAGCAA 67 UUGCUUCUUUGAGUUUGUAUG 231 895-915 AV01000.um GUACAAACUCAAAGAAGCAAA 68 UUUGCUUCUUUGAGUUUGUAC 232 896-916 AV01001.um GUCCUAUUUAUUUUGAGUCUA 69 UAGACUCAAAAUAAAUAGGAC 233 1396-1416 AV01002.um CAUGAGGAUAUUUGCUGUCUA 70 UAGACAGCAAAUAUCCUCAUG 234 69-89 AV01003.um GCAUGUCAGGCUGAGGGCUAA 71 UUAGCCCUCAGCCUGACAUGC 235 529-549 AV01004.um GCGAAGUCAUCUGGACAAGCA 72 UGCUUGUCCAGAUGACUUCGC 236 557-577 AV01005.um GGACCAUCAAGUCCUGAGUGA 73 UCACUCAGGACUUGAUGGUCC 237 579-599 AV01006.um GGAAGCUUUUCAAUGUGACCA 74 UGGUCACAUUGAAAAGCUUCC 238 632-652 AV01007.um CAAGCUUUUCAAUGUGACCAA 75 UUGGUCACAUUGAAAAGCUUG 239 633-653 AV01008.um CCUUUUCAAUGUGACCAGCAA 76 UUGCUGGUCACAUUGAAAAGG 240 636-656 AV01009.um CCUUUUCAAUGUGACCAGCAA 77 UUGCUGGUCACAUUGAAAAGG 241 636-656 AV01010.um GGGAGAUUAGAUCCUGAGGAA 78 UUCCUCAGGAUCUAAUCUCCC 242 703-723 AV01011.um GGAGGAAAACCAUACAGCUGA 79 UCAGCUGUAUGGUUUUCCUCC 243 717-737 AV01012.um CAGGAAAACCAUACAGCUGA 80 UUCAGCUGUAUGGUUUUCCUG 244 718-738 AV01013.um GUACAGCUGAAUUGGUCAUCA 81 UGAUGACCAAUUCAGCUGUAC 245 728-748 AV01014.um GGAAUUGGUCAUCCCAGAACA 82 UGUUCUGGGAUGACCAAUUCC 246 735-755 AV01015.um GCCUCCAAAUGAAAGGACUCA 83 UGAGUCCUUUCAUUUGGAGGC 247 768-788 AV01016.um GUCCAAAUGAAAGGACUCACA 84 UGUGAGUCCUUUCAUUUGGAC 248 770-790 AV01017.um GAAUGAAAGGACUCACUUGGA 85 UCCAAGUGAGUCCUUUCAUUC 249 774-794 AV01018.um CAUGUGAAAAAAUGUGGCAUA 86 UAUGCCACAUUUUUUCACAUG 250 871-891 AV00937-19-1.um GCAUUCCAGAAAGAUGAGA 87 UCUCAUCUUUCUGGAAUGC 251 57-75 AV00938-19-1.um AUUCCAGAAAGAUGAGGAA 88 UUCCUCAUCUUUCUGGAAU 252 59-77 AV00939-19-1.um UUCCAGAAAGAUGAGGAUA 89 UAUCCUCAUCUUUCUGGAA 253 60-78 AV00940-19-1.um CCAGAAAGAUGAGGAUAUA 90 UAUAUCCUCAUCUUUCUGG 254 62-80 AV00941-19-1.um GAAAGAUGAGGAUAUUUGA 91 UCAAAUAUCCUCAUCUUUC 255 65-83 AV00942-19-1.um AAAGAUGAGGAUAUUUGCA 92 UGCAAAUAUCCUCAUCUUU 256 66-84 AV00943-19-1.um AAGAUGAGGAUAUUUGCUA 93 UAGCAAAUAUCCUCAUCUU 257 67-85 AV00944-19-1.um AGAUGAGGAUAUUUGCUGA 94 UCAGCAAAUAUCCUCAUCU 258 68-86 AV00945-19-1.um GAUGAGGAUAUUUGCUGUA 95 UACAGCAAAUAUCCUCAUC 259 69-87 AV00946-19-1.um GAUGAGGAUAUUUGCUGUA 96 UACAGCAAAUAUCCUCAUC 260 69-87 AV00947-19-1.um GGAUAUUUGCUGUCUUUAA 97 UUAAAGACAGCAAAUAUCC 261 74-92 AV00948-19-1.um AUUUGCUGUCUUUAUAUUA 98 UAAUAUAAAGACAGCAAAU 262 78-96 AV00949-19-1.um UUUGCUGUCUUUAUAUUCA 99 UGAAUAUAAAGACAGCAAA 263 79-97 AV00950-19-1.um UUGCUGUCUUUAUAUUCAA 100 UUGAAUAUAAAGACAGCAA 264 80-98 AV00951-19-1.um CCUACUGGCAUUUGCUGAA 101 UUCAGCAAAUGCCAGUAGG 265 101-119 AV00952-19-1.um CUACUGGCAUUUGCUGAAA 102 UUUCAGCAAAUGCCAGUAG 266 102-120 AV00953-19-1.um GCCCCCAUACAACAAAAUCA 103 UGAUUUUGUUGUAUGGGGC 267 463-481 AV00954-19-1.um CCCAUACAACAAAAUCAAA 104 UUUGAUUUUGUUGUAUGGG 268 465-483 AV00955-19-1.um CCAUACAACAAAAUCAACA 105 UGUUGAUUUUGUUGUAUGG 269 466-484 AV00956-19-1.um ACAACAAAAUCAACCAAAA 106 UUUUGGUUGAUUUUGUUGU 270 470-488 AV00957-19-1.um AACAAAAUCAACCAAAGAA 107 UUCUUUGGUUGAUUUUGUU 271 472-490 AV00958-19-1.um ACAAAAUCAACCAAAGAAA 108 UUUCUUUGGUUGAUUUUGU 272 473-491 AV00959-19-1.um ACAAAAUCAACCAAAGAAA 109 UUUCUUUGGUUGAUUUUGU 273 473-491 AV00960-19-1.um CAAAAUCAACCAAAGAAUA 110 UAUUCUUUGGUUGAUUUUG 274 474-492 AV00961-19-1.um CAAAAUCAACCAAAGAAUA 111 UAUUCUUUGGUUGAUUUUG 275 474-492 AV00962-19-1.um AAUCAACCAAAGAAUUUUA 112 UAAAAUUCUUUGGUUGAUU 276 477-495 AV00963-19-1.um UCAACCAAAGAAUUUUGGA 113 UCCAAAAUUCUUUGGUUGA 277 479-497 AV00964-19-1.um CAACCAAAGAAUUUUGGUA 114 UACCAAAAUUCUUUGGUUG 278 480-498 AV00965-19-1.um AACCAAAGAAUUUUGGUUA 115 UAACCAAAAUUCUUUGGUU 279 481-499 AV00966-19-1.um ACCAAAGAAUUUUGGUUGA 116 UCAACCAAAAUUCUUUGGU 280 482-500 AV00967-19-1.um CCAAAGAAUUUUGGUUGUA 117 UACAACCAAAAUUCUUUGG 281 483-501 AV00968-19-1.um UCCAGUCACCUCUGAACAA 118 UUGUUCAGAGGUGACUGGA 282 504-522 AV00969-19-1.um CCAGUCACCUCUGAACAUA 119 UAUGUUCAGAGGUGACUGG 283 505-523 AV00970-19-1.um CAGUCACCUCUGAACAUGA 120 UCAUGUUCAGAGGUGACUG 284 506-524 AV00971-19-1.um AGUCACCUCUGAACAUGAA 121 UUCAUGUUCAGAGGUGACU 285 507-525 AV00972-19-1.um GUCACCUCUGAACAUGAAA 122 UUUCAUGUUCAGAGGUGAC 286 508-526 AV00973-19-1.um CACCUCUGAACAUGAACUA 123 UAGUUCAUGUUCAGAGGUG 287 510-528 AV00974-19-1.um AAGUCAUCUGGACAAGCAA 124 UUGCUUGUCCAGAUGACUU 288 560-578 AV00975-19-1.um ACAAGCAGUGACCAUCAAA 125 UUUGAUGGUCACUGCUUGU 289 571-589 AV00976-19-1.um GGAGAAGCUUUUCAAUGUA 126 UACAUUGAAAAGCUUCUCC 290 630-648 AV00977-19-1.um GAAGCUUUUCAAUGUGACA 127 UGUCACAUUGAAAAGCUUC 291 633-651 AV00978-19-1.um GAUUAGAUCCUGAGGAAAA 128 UUUUCCUCAGGAUCUAAUC 292 707-725 AV00979-19-1.um AUUAGAUCCUGAGGAAAAA 129 UUUUUUCCUCAGGAUCUAAU 293 708-726 AV00980-19-1.um UUAGAUCCUGAGGAAAACA 130 UGUUUUCCUCAGGAUCUAA 294 709-727 AV00981-19-1.um UCCUGAGGAAAACCAUACA 131 UGUAUGGUUUUCCUCAGGA 295 714-732 AV00982-19-1.um CUGAGGAAAACCAUACAGA 132 UCUGUAUGGUUUUCCUCAG 296 716-734 AV00983-19-1.um GAGGAAAACCAUACAGCUA 133 UAGCUGUAUGGUUUUCCUC 297 718-736 AV00984-19-1.um GAAAACCAUACAGCUGAAA 134 UUUCAGCUGUAUGGUUUUC 298 721-739 AV00985-19-1.um AAACCAUACAGCUGAAUUA 135 UAAUUCAGCUGUAUGGUUU 299 723-741 AV00986-19-1.um AACCAUACAGCUGAAUUGA 136 UCAAUUCAGCUGUAUGGUU 300 724-742 AV00987-19-1.um ACCAUACAGCUGAAUUGGA 137 UCCAAUUCAGCUGUAUGGU 301 725-743 AV00988-19-1.um CCAUACAGCUGAAUUGGUA 138 UACCAAUUCAGCUGUAUGG 302 726-744 AV00989-19-1.um UACAGCUGAAUUGGUCAUA 139 UAUGACCAAUUCAGCUGUA 303 729-747 AV00990-19-1.um UUGGUCAUCCCAGAACUAA 140 UUAGUUCUGGGAUGACCAA 304 739-757 AV00991-19-1.um UGGUCAUCCCAGAACUACA 141 UGUAGUUCUGGGAUGACCA 305 740-758 AV00992-19-1.um AUCCUCCAAAUGAAAGGAA 142 UUCCUUUCAUUUGGAGGAU 306 767-785 AV00993-19-1.um UCCUCCAAAUGAAAGGACA 143 UGUCCUUUCAUUUGGAGGA 307 768-786 AV00994-19-1.um CCUCCAAAUGAAAGGACUA 144 UAGUCCUUUCAUUUGGAGG 308 769-787 AV00995-19-1.um CAAAUGAAAGGACUCACUA 145 UAGUGAGUCCUUUCAUUUG 309 773-791 AV00996-19-1.um AAAUGAAAGGACUCACUUA 146 UAAGUGAGUCCUUUCAUUU 310 774-792 AV00997-19-1.um AAUGAAAGGACUCACUUGA 147 UCAAGUGAGUCCUUUCAUU 311 775-793 AV00998-19-1.um GAUACAAACUCAAAGAAGA 148 UCUUCUUUUGAGUUUUGUAUC 312 895-913 AV00999-19-1.um UACAAACUCAAAGAAGCAA 149 UUGCUUCUUUGAGUUUUGUA 313 897-915 AV01000-19-1.um ACAAACUCAAAGAAGCAAA 150 UUUGCUUCUUUGAGUUUGU 314 898-916 AV01001-19-1.um CCUAUUUAUUUUUGAGUCUA 151 UAGACUCAAAAUAAAUAGG 315 1398-1416 AV01002-19-1.um UGAGGAUAUUUGCUGUCUA 152 UAGACAGCAAAUAUCCUCA 316 71-89 AV01003-19-1.um AUGUCAGGCUGAGGGCUAA 153 UUAGCCCUCAGCCUGACAU 317 531-549 AV01004-19-1.um GAAGUCAUCUGGACAAGCA 154 UGCUUGUCCAGAUGACUUC 318 559-577 AV01005-19-1.um ACCAUCAAGUCCUGAGUGA 155 UCACUCAGGACUUGAUGGU 319 581-599 AV01006-19-1.um AAGCUUUUCAAUGUGACCA 156 UGGUCACAUUGAAAAGCUU 320 634-652 AV01007-19-1.um AGCUUUUCAAUGUGACCAA 157 UUGGUCACAUUGAAAAGCU 321 635-653 AV01008-19-1.um UUUUCAAUGUGACCAGCAA 158 UUGCUGGUCACAUUGAAAA 322 638-656 AV01009-19-1.um UUUUCAAUGUGACCAGCAA 159 UUGCUGGUCACAUUGAAAA 323 638-656 AV01010-19-1.um GAGAUUAGAUCCUGAGGAA 160 UUCCUCAGGAUCUAAUCUC 324 705-723 AV01011-19-1.um AGGAAAACCAUACAGCUGA 161 UCAGCUGUAUGGUUUUCCU 325 719-737 AV01012-19-1.um GGAAAACCAUACAGCUGAA 162 UUCAGCUGUAUGGUUUUCC 326 720-738 AV01013-19-1.um ACAGCUGAAUUGGUCAUCA 163 UGAUGACCAAUUCAGCUGU 327 730-748 AV01014-19-1.um AAUUGGUCAUCCCAGAACA 164 UGUUCUGGGAUGACCAAUU 328 737-755 AV01015-19-1.um CUCCAAAUGAAAGGACUCA 165 UGAGUCCUUUCAUUUGGAG 329 770-788 AV01016-19-1.um CCAAAUGAAAGGACUCACA 166 UGUGAGUCCUUUCAUUUGG 330 772-790 AV01017-19-1.um AUGAAAGGACUCACUUGGA 167 UCCAAGUGAGUCCUUUCAU 331 776-794 AV01018-19-1.um UGUGAAAAAAUGUGGCAUA 168 UAUGCCACAUUUUUUCACA 332 873-891 AD01052.um GUGAGGAUAUUUGCUGUCUUA 603 UAAGACAGCAAAUAUCCUCAC 605 70-90 AD01052-19-1.um GAGGAUAUUUGCUGUCUUA 604 UAAGACAGCAAAUAUCCUC 606 72-90

Table 2 shows certain chemically modified PD-L1 RNAi agent antisense and sense strand sequences of the present invention. In some embodiments of the methods of the present invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a cell and/or a subject. In some embodiments of the methods of the present invention, an RNAi agent having a polynucleotide sequence shown in Table 2 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to a subject comprises a duplex identified in a row of Table 2 and comprises sequence modifications in the sense strand and antisense strand sequences shown in the third and sixth columns of the same row of Table 2, respectively. In some embodiments of the methods of the present invention, the sequences shown in Table 2 may be attached to (also referred to herein as "bound to") a compound capable of delivering the RNAi agent to cells and/or tissues of a subject. Non-limiting examples of delivery compounds that can be used in certain embodiments of the present invention are compounds containing GalNAc or compounds containing GLS-15*. In Table 2, the first column represents the double-stranded AV# of the base sequence shown in Table 1. Table 2 discloses the double-stranded AV# and also shows the chemical modifications contained in the sense and antisense sequences of the double-stranded. For example, Table 1 shows the base single strand sequences SEQ ID NO:6 (sense sequence) and SEQ ID NO:170 (antisense sequence), which together form a duplex duplex, identified as: duplex AV#AV00938.um, and Table 2 lists duplex AV#AV00938, which indicates that the duplexes of SEQ ID NO:334 and SEQ ID NO:416 include the base sequences of SEQ ID NO:6 and SEQ ID NO:170, respectively, but have the chemical modifications shown in the sense and antisense sequences shown in the third and sixth columns, respectively. The "sense strand SS#" in the second column of Table 2 is the assigned identifier for the sense sequence (including modifications) shown in the third column of the same row. The "antisense strand AS#" in the fifth column of Table 2 is the assigned identifier for the antisense sequence (including modifications) shown in the sixth column.

Table 2: Provides the sequences of the antisense and sense strands of PD-L1 RNAi agents with chemical modifications. All sequences are shown 5' to 3'. These sequences were used in certain in vitro test studies described herein. Double Link AV# Meaningful chain SS# Sense chain sequence Serial number. Antisense chain AS# Antisense chain sequence Serial number. AV00937 AV00937-SS c*g*gcauucCfaGfaaAfgauga*g*a 333 AV00937-AS u*Cf*ucAfucuuucUfgGfaauGfc*c*g 415 AV00938 AV00938-SS c*c*auuccaGfaAfagAfugagg*a*a 334 AV00938-AS u*Uf*ccUfcaucuuUfcUfggaAfu*g*g 416 AV00939 AV00939-SS g*a*uuccagAfaAfgaUfgagga*u*a 335 AV00939-AS u*Af*ucCfucaucuUfuCfuggAfa*u*c 417 AV00940 AV00940-SS g*u*ccagaaAfgAfugAfggaua*u*a 336 AV00940-AS u*Af*uaUfccucauCfuUfucuGfg*a*c 418 AV00941 AV00941-SS g*a*gaaagaUfgAfggAfuauuu*g*a 337 AV00941-AS u*Cf*aaAfuauccuCfaUfcuuUfc*u*c 419 AV00942 AV00942-SS g*g*aaagauGfaGfgaUfauuug*c*a 338 AV00942-AS u*Gf*caAfauauccUfcAfucuUfu*c*c 420 AV00943 AV00943-SS c*a*aagaugAfgGfauAfuuugc*u*a 339 AV00943-AS u*Af*gcAfaauaucCfuCfaucUfu*u*g 421 AV00944 AV00944-SS g*a*agaugaGfgAfuaUfuugcu*g*a 340 AV00944-AS u*Cf*agCfaaauauCfcUfcauCfu*u*c 422 AV00945 AV00945-SS g*a*gaugagGfaUfauUfugcug*u*a 341 AV00945-AS u*Af*caGfcaaauaUfcCfucaUfc*u*c 423 AV00946 AV00946-SS g*a*gaugagGfaUfauUfugcug*u*a 342 AV00946-AS u*Af*caGfcaaauaUfcCfucaUfc*u*c 424 AV00947 AV00947-SS c*a*ggauauUfuGfcuGfucuuu*a*a 343 AV00947-AS u*Uf*aaAfgacagcAfaAfuauCfc*u*g 425 AV00948 AV00948-SS g*u*auuugcUfgUfcuUfuauau*u*a 344 AV00948-AS u*Af*auAfuaaagaCfaGfcaaAfu*a*c 426 AV00949 AV00949-SS g*a*uuugcuGfuCfuuUfauauu*c*a 345 AV00949-AS u*Gf*aaUfauaaagAfcAfgcaAfa*u*c 427 AV00950 AV00950-SS g*u*uugcugUfcUfuuAfuauuc*a*a 346 AV00950-AS u*Uf*gaAfuauaaaGfaCfagcAfa*a*c 428 AV00951 AV00951-SS c*a*ccuacuGfgCfauUfugcug*a*a 347 AV00951-AS u*Uf*caGfcaaaugCfcAfguaGfg*u*g 429 AV00952 AV00952-SS g*c*cuacugGfcAfuuUfgcuga*a*a 348 AV00952-AS u*Uf*ucAfgcaaauGfcCfaguAfg*g*c 430 AV00953 AV00953-SS g*u*gccccaUfaCfaaCfaaaau*c*a 349 AV00953-AS u*Gf*auUfuuguugUfaUfgggGfc*a*c 431 AV00954 AV00954-SS c*c*cccauaCfaAfcaAfaauca*a*a 350 AV00954-AS u*Uf*ugAfuuuuguUfgUfaugGfg*g*g 432 AV00955 AV00955-SS g*c*ccauacAfaCfaaAfaucaa*c*a 351 AV00955-AS u*Gf*uuGfauuuugUfuGfuauGfg*g*c 433 AV00956 AV00956-SS g*u*acaacaAfaAfucAfaccaa*a*a 352 AV00956-AS u*Uf*uuGfguugauUfuUfguuGfu*a*c 434 AV00957 AV00957-SS g*c*aacaaaAfuCfaaCfcaaag*a*a 353 AV00957-AS u*Uf*cuUfugguugAfuUfuugUfu*g*c 435 AV00958 AV00958-SS g*a*acaaaaUfcAfacCfaaaga*a*a 354 AV00958-AS u*Uf*ucUfuugguuGfaUfuuuGfu*u*c 436 AV00959 AV00959-SS g*a*acaaaaUfcAfacCfaaaga*a*a 355 AV00959-AS u*Uf*ucUfuugguuGfaUfuuuGfu*u*c 437 AV00960 AV00960-SS g*a*caaaauCfaAfccAfaagaa*u*a 356 AV00960-AS u*Af*uuCfuuugguUfgAfuuuUfg*u*c 438 AV00961 AV00961-SS g*a*caaaauCfaAfccAfaagaa*u*a 357 AV00961-AS u*Af*uuCfuuugguUfgAfuuuUfg*u*c 439 AV00962 AV00962-SS g*a*aaucaaCfcAfaaGfaauuu*u*a 358 AV00962-AS u*Af*aaAfuucuuuGfgUfugaUfu*u*c 440 AV00963 AV00963-SS g*a*ucaaccAfaAfgaAfuuuug*g*a 359 AV00963-AS u*Cf*caAfaauucuUfuGfguuGfa*u*c 441 AV00964 AV00964-SS g*u*caaccaAfaGfaaUfuuugg*u*a 360 AV00964-AS u*Af*ccAfaaauucUfuUfgguUfg*a*c 442 AV00965 AV00965-SS g*c*aaccaaAfgAfauUfuuggu*u*a 361 AV00965-AS u*Af*acCfaaaauuCfuUfuggUfu*g*c 443 AV00966 AV00966-SS g*a*accaaaGfaAfuuUfugguu*g*a 362 AV00966-AS u*Cf*aaCfcaaaauUfcUfuugGfu*u*c 444 AV00967 AV00967-SS g*a*ccaaagAfaUfuuUfgguug*u*a 363 AV00967-AS u*Af*caAfccaaaaUfuCfuuuGfg*u*c 445 AV00968 AV00968-SS c*a*uccaguCfaCfcuCfugaac*a*a 364 AV00968-AS u*Uf*guUfcagaggUfgAfcugGfa*u*g 446 AV00969 AV00969-SS g*u*ccagucAfcCfucUfgaaca*u*a 365 AV00969-AS u*Af*ugUfucagagGfuGfacuGfg*a*c 447 AV00970 AV00970-SS g*c*cagucaCfcUfcuGfaacau*g*a 366 AV00970-AS u*Cf*auGfuucagaGfgUfgacUfg*g*c 448 AV00971 AV00971-SS g*c*agucacCfuCfugAfacaug*a*a 367 AV00971-AS u*Uf*caUfguucagAfgGfugaCfu*g*c 449 AV00972 AV00972-SS g*a*gucaccUfcUfgaAfcauga*a*a 368 AV00972-AS u*Uf*ucAfuguucaGfaGfgugAfc*u*c 450 AV00973 AV00973-SS c*u*caccucUfgAfacAfugaac*u*a 369 AV00973-AS u*Af*guUfcauguuCfaGfaggUfg*a*g 451 AV00974 AV00974-SS g*g*aagucaUfcUfggAfcaagc*a*a 370 AV00974-AS u*Uf*gcUfuguccaGfaUfgacUfu*c*c 452 AV00975 AV00975-SS c*g*acaagcAfgUfgaCfcauca*a*a 371 AV00975-AS u*Uf*ugAfuggucaCfuGfcuuGfu*c*g 453 AV00976 AV00976-SS c*a*ggagaaGfcUfuuUfcaaug*u*a 372 AV00976-AS u*Af*caUfugaaaaGfcUfucuCfc*u*g 454 AV00977 AV00977-SS c*a*gaagcuUfuUfcaAfuguga*c*a 373 AV00977-AS u*Gf*ucAfcauugaAfaAfgcuUfc*u*g 455 AV00978 AV00978-SS c*a*gauuagAfuCfcuGfaggaa*a*a 374 AV00978-AS u*Uf*uuCfcucaggAfuCfuaaUfc*u*g 456 AV00979 AV00979-SS g*g*auuagaUfcCfugAfggaaa*a*a 375 AV00979-AS u*Uf*uuUfccucagGfaUfcuaAfu*c*c 457 AV00980 AV00980-SS c*a*uuagauCfcUfgaGfgaaaa*c*a 376 AV00980-AS u*Gf*uuUfuccucaGfgAfucuAfa*u*g 458 AV00981 AV00981-SS c*a*uccugaGfgAfaaAfccaua*c*a 377 AV00981-AS u*Gf*uaUfgguuuuCfcUfcagGfa*u*g 459 AV00982 AV00982-SS g*c*cugaggAfaAfacCfauaca*g*a 378 AV00982-AS u*Cf*ugUfaugguuUfuCfcucAfg*g*c 460 AV00983 AV00983-SS g*u*gaggaaAfaCfcaUfacagc*u*a 379 AV00983-AS u*Af*gcUfguauggUfuUfuccUfc*a*c 461 AV00984 AV00984-SS g*g*gaaaacCfaUfacAfgcuga*a*a 380 AV00984-AS u*Uf*ucAfgcuguaUfgGfuuuUfc*c*c 462 AV00985 AV00985-SS c*a*aaaccaUfaCfagCfugaau*u*a 381 AV00985-AS u*Af*auUfcagcugUfaUfgguUfu*u*g 463 AV00986 AV00986-SS g*a*aaccauAfcAfgcUfgaauu*g*a 382 AV00986-AS u*Cf*aaUfucagcuGfuAfuggUfu*u*c 464 AV00987 AV00987-SS g*a*accauaCfaGfcuGfaauug*g*a 383 AV00987-AS u*Cf*caAfuucagcUfgUfaugGfu*u*c 465 AV00988 AV00988-SS g*a*ccauacAfgCfugAfauugg*u*a 384 AV00988-AS u*Af*ccAfauucagCfuGfuauGfg*u*c 466 AV00989 AV00989-SS g*a*uacagcUfgAfauUfgguca*u*a 385 AV00989-AS u*Af*ugAfccaauuCfaGfcugUfa*u*c 467 AV00990 AV00990-SS g*a*uuggucAfuCfccAfgaacu*a*a 386 AV00990-AS u*Uf*agUfucugggAfuGfaccAfa*u*c 468 AV00991 AV00991-SS g*u*uggucaUfcCfcaGfaacua*c*a 387 AV00991-AS u*Gf*uaGfuucuggGfaUfgacCfa*a*c 469 AV00992 AV00992-SS g*c*auccucCfaAfauGfaaagg*a*a 388 AV00992-AS u*Uf*ccUfuucauuUfgGfaggAfu*g*c 470 AV00993 AV00993-SS g*a*uccuccAfaAfugAfaagga*c*a 389 AV00993-AS u*Gf*ucCfuuucauUfuGfgagGfa*u*c 471 AV00994 AV00994-SS g*u*ccuccaAfaUfgaAfaggac*u*a 390 AV00994-AS u*Af*guCfcuuucaUfuUfggaGfg*a*c 472 AV00995 AV00995-SS g*c*caaaugAfaAfggAfcucac*u*a 391 AV00995-AS u*Af*guGfaguccuUfuCfauuUfg*g*c 473 AV00996 AV00996-SS g*c*aaaugaAfaGfgaCfucacu*u*a 392 AV00996-AS u*Af*agUfgaguccUfuUfcauUfu*g*c 474 AV00997 AV00997-SS g*a*aaugaaAfgGfacUfcacuu*g*a 393 AV00997-AS u*Cf*aaGfugagucCfuUfucaUfu*u*c 475 AV00998 AV00998-SS g*a*gauacaAfaCfucAfaagaa*g*a 394 AV00998-AS u*Cf*uuCfuuugagUfuUfguaUfc*u*c 476 AV00999 AV00999-SS c*a*uacaaaCfuCfaaAfgaagc*a*a 395 AV00999-AS u*Uf*gcUfucuuugAfgUfuugUfa*u*g 477 AV01000 AV01000-SS g*u*acaaacUfcAfaaGfaagca*a*a 396 AV01000-AS u*Uf*ugCfuucuuuGfaGfuuuGfu*a*c 478 AV01001 AV01001-SS g*u*ccuauuUfaUfuuUfgaguc*u*a 397 AV01001-AS u*Af*gaCfucaaaaUfaAfauaGfg*a*c 479 AV01002 AV01002-SS c*a*ugaggaUfaUfuuGfcuguc*u*a 398 AV01002-AS u*Af*gaCfa(gUNA)caaaUfaUfccuCfa*u*g 480 AV01003 AV01003-SS g*c*augucaGfgCfugAfgggcu*a*a 399 AV01003-AS u*Uf*agCfc(cUNA)ucagCfcUfgacAfu*g*c 481 AV01004 AV01004-SS g*c*gaagucAfuCfugGfacaag*c*a 400 AV01004-AS u*Gf*cuUfg(uUNA)ccagAfuGfacuUfc*g*c 482 AV01005 AV01005-SS g*g*accaucAfaGfucCfugagu*g*a 401 AV01005-AS u*Cf*acUfc(aUNA)ggacUfuGfaugGfu*c*c 483 AV01006 AV01006-SS g*g*aagcuuUfuCfaaUfgugac*c*a 402 AV01006-AS u*Gf*guCfa(cUNA)auugAfaAfagcUfu*c*c 484 AV01007 AV01007-SS c*a*agcuuuUfcAfauGfugacc*a*a 403 AV01007-AS u*Uf*ggUfc(aUNA)cauuGfaAfaagCfu*u*g 485 AV01008 AV01008-SS c*c*uuuucaAfuGfugAfccagc*a*a 404 AV01008-AS u*Uf*gcUfg(gUNA)ucacAfuUfgaaAfa*g*g 486 AV01009 AV01009-SS c*c*uuuucaAfuGfugAfccagc*a*a 405 AV01009-AS u*Uf*gcUfg(gUNA)ucacAfuUfgaaAfa*g*g 487 AV01010 AV01010-SS g*g*gagauuAfgAfucCfugagg*a*a 406 AV01010-AS u*Uf*ccUfc(aUNA)ggauCfuAfaucUfc*c*c 488 AV01011 AV01011-SS g*g*aggaaaAfcCfauAfcagcu*g*a 407 AV01011-AS u*Cf*agCfu(gUNA)uaugGfuUfuucCfu*c*c 489 AV01012 AV01012-SS c*a*ggaaaaCfcAfuaCfagcug*a*a 408 AV01012-AS u*Uf*caGfc(uUNA)guauGfgUfuuuCfc*u*g 490 AV01013 AV01013-SS g*u*acagcuGfaAfuuGfgucau*c*a 409 AV01013-AS u*Gf*auGfa(cUNA)caauUfcAfgcuGfu*a*c 491 AV01014 AV01014-SS g*g*aauuggUfcAfucCfcagaa*c*a 410 AV01014-AS u*Gf*uuCfu(gUNA)ggauGfaCfcaaUfu*c*c 492 AV01015 AV01015-SS g*c*cuccaaAfuGfaaAfggacu*c*a 411 AV01015-AS u*Gf*agUfc(cUNA)uuucAfuUfuggAfg*g*c 493 AV01016 AV01016-SS g*u*ccaaauGfaAfagGfacuca*c*a 412 AV01016-AS u*Gf*ugAfg(uUNA)ccuuUfcAfuuuGfg*a*c 494 AV01017 AV01017-SS g*a*augaaaGfgAfcuCfacuug*g*a 413 AV01017-AS u*Cf*caAfg(uUNA)gaguCfcUfuucAfu*u*c 495 AV01018 AV01018-SS c*a*ugugaaAfaAfauGfuggca*u*a 414 AV01018-AS u*Af*ugCfc(aUNA)cauuUfuUfucaCfa*u*g 496

Table 3 shows certain chemically modified PD-L1 RNAi agent antisense and sense strand sequences of the present invention. In some embodiments of the methods of the present invention, the RNAi agent shown in Table 3 is administered to a cell and/or a subject. In some embodiments of the methods of the present invention, an RNAi agent having a polynucleotide sequence shown in Table 3 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to a subject comprises a duplex identified in the first column of a row in Table 3 and comprises sequence modifications and/or delivery compounds of the sense and antisense strand sequences shown in the third and sixth columns of the same row in Table 3, respectively. These sequences are used in certain in vivo testing studies described elsewhere herein. In some embodiments of the methods of the present invention, the sequences shown in Table 3 can be attached to (also referred to herein as "bound to") a compound for delivery, a non-limiting example of which is a GalNAc-containing compound, wherein the delivery compound is identified as "GLX-n" on the sense strand in the third column of Table 3. As used herein, "GLX-n" is used to represent a "GLS-n*" or "GLO-n" delivery compound ("X" can be "S" or "O"), and GLX-0 can be any "GLS-n*" and "GLO-n" delivery compound that can be attached to the 3' end of an oligonucleotide during synthesis. As used herein and shown in Table 3, "GLX-n" is used to indicate that the enclosed GalNAc-containing compound is any one of compounds GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16, the structures of each of which are provided elsewhere herein. Those skilled in the art will be able to prepare and use the dsRNA compounds of the invention wherein the attached delivery compound is any one of GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16. The first column of Table 3 provides the double chain AD# assigned to the double chain of the sense and antisense sequences in that row. For example, double chain AD#AD00880 is a double chain of sense chain SEQ ID NO:497 and antisense chain SEQ ID NO:550. Each row in Table 3 provides a sense chain and an antisense chain, and discloses the double chain of the sense chain and antisense chain shown. The "Sense Chain SS#" in the second column of Table 3 is the assigned identifier of the sense sequence (including modifications) shown in the third column of the same row. The "Antisense Chain AS#" in the fifth column of Table 3 is the assigned identifier of the antisense sequence (including modifications) shown in the sixth column. The identifiers for certain linked GalNAc-containing "GLO-n" or "GLS-n*" compounds are shown as GLS-5*, GLS-15*, or GLX-0, and it should be understood that another "GLO-n" or "GLS-n*" compound can be substituted for the compound shown as GLO-0, and the resulting compounds are included in the embodiments of the methods and/or compositions of the present invention.

Table 3 provides chemically modified PD-L1 RNAi agent antisense and sense strand sequences. All sequences are represented from 5' to 3'. These sequences were used in certain in vivo testing studies described elsewhere herein. The delivery molecules used in the in vivo studies were represented as "GLO-n" or "GLS-n*" at the 3' or 5' end of each sense strand. Dual-link AD# Meaningful chain SS# Sense chain sequence Serial Number Antisense chain AS# Antisense chain sequence Serial Number AD00880 AD00880-SS (GLS-15*)(invab*)ccauuccaGfaAfaGfaugagga*a*(invab) 497 AD00880-AS u*Uf*ccucAfucuuUfcUfgGfaau*g*g 550 AD00881 AD00881-SS (GLS-15*)(invab*)guccagaaAfgAfuGfaggauau*a*(invab) 498 AD00881-AS u*Af*uaucCfucauCfuUfuCfugg*a*c 551 AD00882 AD00882-SS (GLS-15*)(invab*)gagaaagaUfgAfgGfauauuug*a*(invab) 499 AD00882-AS u*Cf*aaauAfuccuCfaUfcUfuuc*u*c 552 AD00883 AD00883-SS (GLS-15*)(invab*)caugaggaUfaUfuUfgcugucu*a*(invab) 500 AD00883-AS u*Af*gaCfa(gUNA)caaaUfaUfcCfuca*u*g 553 AD00884 AD00884-SS (GLS-15*)(invab*)guauuugcUfgUfcUfuuauauu*a*(invab) 501 AD00884-AS u*Af*auauAfaagaCfaGfcAfaau*a*c 554 AD00885 AD00885-SS (GLS-15*)(invab*)gauuugcuGfuCfuUfuauauuc*a*(invab) 502 AD00885-AS u*Gf*aauaUfaaagAfcAfgCfaaa*u*c 555 AD00886 AD00886-SS (GLS-15*)(invab*)gugccccaUfaCfaAfcaaaauc*a*(invab) 503 AD00886-AS u*Gf*auuuUfguugUfaUfgGfggc*a*c 556 AD00887 AD00887-SS (GLS-15*)(invab*)guccagucAfcCfuCfugaacau*a*(invab) 504 AD00887-AS u*Af*uguuCfagagGfuGfaCfugg*a*c 557 AD00888 AD00888-SS (GLS-15*)(invab*)gcgaagucAfuCfuGfgacaagc*a*(invab) 505 AD00888-AS u*Gf*cuUfg(uUNA)ccagAfuGfaCfuuc*g*c 558 AD00889 AD00889-SS (GLS-15*)(invab*)ggaagucaUfcUfgGfacaagca*a*(invab) 506 AD00889-AS u*Uf*gcuuGfuccaGfaUfgAfcuu*c*c 559 AD00890 AD00890-SS (GLS-15*)(invab*)cgacaagcAfgUfgAfccaucaa*a*(invab) 507 AD00890-AS u*Uf*ugauGfgucaCfuGfcUfugu*c*g 560 AD00891 AD00891-SS (GLS-15*)(invab*)gaaccauaCfaGfcUfgaauugg*a*(invab) 508 AD00891-AS u*Cf*caauUfcagcUfgUfaUfggu*u*c 561 AD00892 AD00892-SS (GLS-15*)(invab*)gauacagcUfgAfaUfuggucau*a*(invab) 509 AD00892-AS u*Af*ugacCfaauuCfaGfcUfgua*u*c 562 AD00893 AD00893-SS (GLS-15*)(invab*)guacagcuGfaAfuUfggucauc*a*(invab) 510 AD00893-AS u*Gf*auGfa(cUNA)caauUfcAfgCfugu*a*c 563 AD00894 AD00894-SS (GLS-15*)(invab*)ggaauuggUfcAfuCfccagaac*a*(invab) 511 AD00894-AS u*Gf*uuCfu(gUNA)ggauGfaCfcAfauu*c*c 564 AD00895 AD00895-SS (GLS-15*)(invab*)gccuccaaAfuGfaAfaggacuc*a*(invab) 512 AD00895-AS u*Gf*agUfc(cUNA)uuucAfuUfuGfgag*g*c 565 AD00896 AD00896-SS (GLS-15*)(invab*)guccaaauGfaAfaGfgacucac*a*(invab) 513 AD00896-AS u*Gf*ugAfg(uUNA)ccuuUfcAfuUfugg*a*c 566 AD00897 AD00897-SS (GLS-15*)(invab*)gccaaaugAfaAfgGfacucacu*a*(invab) 514 AD00897-AS u*Af*gugaGfuccuUfuCfaUfuug*g*c 567 AD01052 AD01052-SS (GLS-15*)(invab*)gugaggauAfuUfuGfcugucuu*a*(invab) 515 AD01052-AS u*Af*agacAfgcaaAfuAfuCfcuc*a*c 568 AD01053 AD01053-SS (GLS-15*)(invab*)caggauauUfuGfcUfgucuuua*a*(invab) 516 AD01053-AS u*Uf*aaagAfcagcAfaAfuAfucc*u*g 569 AD01054 AD01054-SS (GLS-15*)(invab*)guuugcugUfcUfuUfauauuca*a*(invab) 517 AD01054-AS u*Uf*gaauAfuaaaGfaCfaGfcaa*a*c 570 AD01055 AD01055-SS (GLS-15*)(invab*)guuugcugUfcUfuUfauauuca*a*(invab) 518 AD01055-AS u*Uf*gaauAfuaaaGfaCfaGfcaa*a*c 571 AD01056 AD01056-SS (GLS-15*)(invab*)gccuacugGfcAfuUfugcugaa*a*(invab) 519 AD01056-AS u*Uf*ucagCfaaauGfcCfaGfuag*g*c 572 AD01057 AD01057-SS (GLS-15*)(invab*)guacaacaAfaAfuCfaaccaaa*a*(invab) 520 AD01057-AS u*Uf*uuggUfugauUfuUfgUfugu*a*c 573 AD01058 AD01058-SS (GLS-15*)(invab*)gaacaaaaUfcAfaCfcaaagaa*a*(invab) 521 AD01058-AS u*Uf*ucuuUfgguuGfaUfuUfugu*u*c 574 AD01059 AD01059-SS (GLS-15*)(invab*)gcaaccaaAfgAfaUfuuugguu*a*(invab) 522 AD01059-AS u*Af*accaAfaauuCfuUfuGfguu*g*c 575 AD01060 AD01060-SS (GLS-15*)(invab*)gaaccaaaGfaAfuUfuuguug*a*(invab) 523 AD01060-AS u*Cf*aaccAfaaauUfcUfuUfggu*u*c 576 AD01061 AD01061-SS (GLS-15*)(invab*)gaccaaagAfaUfuUfugguugu*a*(invab) 524 AD01061-AS u*Af*caacCfaaaaUfuCfuUfugg*u*c 577 AD01062 AD01062-SS (GLS-15*)(invab*)gcagucacCfuCfuGfaacauga*a*(invab) 525 AD01062-AS u*Uf*caugUfucagAfgGfuGfacu*g*c 578 AD01063 AD01063-SS (GLS-15*)(invab*)cucaccucUfgAfaCfaugaacu*a*(invab) 526 AD01063-AS u*Af*guucAfuguuCfaGfaGfgug*a*g 579 AD01064 AD01064-SS (GLS-15*)(invab*)caggagaaGfcUfuUfucaaugu*a*(invab) 527 AD01064-AS u*Af*cauuGfaaaaGfcUfuCfucc*u*g 580 AD01065 AD01065-SS (GLS-15*)(invab*)ggauuagaUfcCfuGfaggaaaa*a*(invab) 528 AD01065-AS u*Uf*uuucCfucagGfaUfcUfaau*c*c 581 AD01066 AD01066-SS (GLS-15*)(invab*)cauccugaGfgAfaAfaccauac*a*(invab) 529 AD01066-AS u*Gf*uaugGfuuuuCfcUfcAfgga*u*g 582 AD01067 AD01067-SS (GLS-15*)(invab*)caggaaaaCfcAfuAfcagcuga*a*(invab) 530 AD01067-AS u*Uf*caGfc(uUNA)guauGfgUfuUfucc*u*g 583 AD01068 AD01068-SS (GLS-15*)(invab*)gaccauacAfgCfuGfaauuggu*a*(invab) 531 AD01068-AS u*Af*ccaaUfucagCfuGfuAfugg*u*c 584 AD01069 AD01069-SS (GLS-15*)(invab*)gcaaaugaAfaGfgAfcucacuu*a*(invab) 532 AD01069-AS u*Af*agugAfguccUfuUfcAfuuu*g*c 585 AD01070 AD01070-SS (GLS-15*)(invab*)gagauacaAfaCfuCfaaagaag*a*(invab) 533 AD01070-AS u*Cf*uucuUfugagUfuUfgUfauc*u*c 586 AD01071 AD01071-SS (GLS-15*)(invab*)guccuauuUfaUfuUfugagucu*a*(invab) 534 AD01071-AS u*Af*gacuCfaaaaUfaAfaUfagg*a*c 587 AD01211 AD01211-SS (GLS-15*)(imann*)cagaagcuUfuUfcAfaugugac*a*(imann) 535 AD01211-AS u*Gf*ucacAfuugaAfaAfgCfuuc*u*g 588 AD01212 AD01212-SS (GLS-15*)(imann*)ccuuuucaAfuGfuGfaccagca*a*(imann) 536 AD01212-AS u*Uf*gcUfg(gUNA)ucacAfuUfgAfaaa*g*g 589 AD01213 AD01213-SS (GLS-15*)(imann*)cagauuagAfuCfcUfgaggaaa*a*(imann) 537 AD01213-AS u*Uf*uuccUfcaggAfuCfuAfauc*u*g 590 AD01214 AD01214-SS (GLS-15*)(imann*)cauuagauCfcUfgAfggaaaac*a*(imann) 538 AD01214-AS u*Gf*uuuuCfcucaGfgAfuCfuaa*u*g 591 AD01215 AD01215-SS (GLS-15*)(imann*)gccugaggAfaAfaCfcauacag*a*(imann) 539 AD01215-AS u*Cf*uguaUfgguuUfuCfcUfcag*g*c 592 AD01216 AD01216-SS (GLS-15*)(imann*)gugaggaaAfaCfcAfuacagcu*a*(imann) 540 AD01216-AS u*Af*gcugUfauggUfuUfuCfcuc*a*c 593 AD01217 AD01217-SS (GLS-15*)(imann*)gggaaaacCfaUfaCfagcugaa*a*(imann) 541 AD01217-AS u*Uf*ucagCfuguaUfgGfuUfuuc*c*c 594 AD01218 AD01218-SS (GLS-15*)(imann*)caaaaccaUfaCfaGfcugaauu*a*(imann) 542 AD01218-AS u*Af*auucAfgcugUfaUfgGfuuu*u*g 595 AD01219 AD01219-SS (GLS-15*)(imann*)gaaaccauAfcAfgCfugaauug*a*(imann) 543 AD01219-AS u*Cf*aauuCfagcuGfuAfuGfguu*u*c 596 AD01053-1 AD01053-1-SS (GLS-15*)(imann*)caggauauUfuGfcUfgucuuua*a*(imann) 544 AD01053-1-AS u*Uf*aaagAfcagcAfaAfuAfucc*u*g 597 AD00884-1 AD00884-1-SS (GLS-15*)(imann*)guauuugcUfgUfcUfuuauauu*a*(imann) 545 AD00884-1-AS u*Af*auauAfaagaCfaGfcAfaau*a*c 598 AD00885-1 AD00885-1-SS (GLS-15*)(imann*)gauuugcuGfuCfuUfuauauuc*a*(imann) 546 AD00885-1-AS u*Gf*aauaUfaaagAfcAfgCfaaa*u*c 599 AD01055-1 AD01055-1-SS (GLS-15*)(imann*)guuugcugUfcUfuUfauauuca*a*(imann) 547 AD01055-1-AS u*Uf*gaauAfuaaaGfaCfaGfcaa*a*c 600 AD01066-1 AD01066-1-SS (GLS-15*)(imann*)cauccugaGfgAfaAfaccauac*a*(imann) 548 AD01066-1-AS u*Gf*uaugGfuuuuCfcUfcAfgga*u*g 601 AD01067-1 AD01067-1-SS (GLS-15*)(imann*)caggaaaaCfcAfuAfcagcuga*a*(imann) 549 AD01067-1-AS u*Uf*caGfc(uUNA)guauGfgUfuUfucc*u*g 602 Table 4: Abbreviations for nucleotide monomers used in nucleotide sequences. It will be understood that these monomers, when present in an oligonucleotide, are linked to one another via 5'-3' phosphodiester bonds. It will also be understood that when a nucleotide contains a 2'-fluorine modification, the fluorine replaces the hydroxyl group at that position of the parent nucleotide (i.e., it is a 2'-deoxy-2'-fluoro nucleotide). It will also be understood that the abbreviations correspond to omitting the phosphate at the 3'-end when it is a 3'-terminal nucleotide (i.e., they are 3'-OH). The skilled person will understand that these abbreviations are merely identification symbols and will be able to determine the connection pattern of the nucleotide sequences containing these abbreviations. Abbreviation Nucleotides A Adenosine-3'-phosphate C Cytidine-3'-phosphate G Guanosine-3'-phosphate T 5-Methyluridine-3'-phosphate U Uridine-3'-phosphate a 2'-O-methyladenosine-3'-phosphate c 2'-O-methylcytidine-3'-phosphate g 2'-O-methylguanosine-3'-phosphate t 2'-O-methyl-5-methyluridine-3'-phosphate u 2'-O-methyluridine-3'-phosphate A 2'-Fluoroadenosine-3'-phosphate Cf 2'-Fluorocytidine-3'-phosphate Gf 2'-Fluoroguanosine-3'-phosphate Tf 2'-Fluoro-5-methyluridine-3'-phosphate Uf 2'-Fluorouridine-3'-phosphate Af* 2'-Fluoroadenosine-3'-phosphorothioate Cf* 2'-Fluorocytidine-3'-phosphorothioate Gf* 2'-Fluoroguanosine-3'-phosphorothioate Tf* 2'-Fluoro-5-methyluridine-3'-phosphorothioate Uf* 2'-Fluorouridine-3'-phosphorothioate a* 2'-O-methyladenosine-3'-phosphorothioate c* 2'-O-methylcytidine-3'-phosphorothioate g* 2'-O-methylguanosine-3'-phosphorothioate t* 2'-O-methyl-5-methyluridine-3'-phosphorothioate u* 2'-O-methyluridine-3'-phosphorothioate ai 2', 3'-seco-adenosine-3'-phosphate cUA 2', 3'-seco-cytidine-3'-phosphate g 2', 3'-seco-guanosine-3'-phosphate tUNA 2', 3'-seco-5-methyluridine-3'-phosphate uUNA 2', 3'- seco-uridine-3'-phosphate Vp Vinyl-phosphonate invab Inverted abasic-5'-phosphate. When located at the 3' end of the chain, it is linked to the adjacent nucleotide via the substituent at its 3' position (3'-3'linkage); when located at the 5' end of the chain, it is linked to the adjacent nucleotide via the substituent at its 5' position (5'-5' internucleotide linkage). It should also be understood that when in the 3'-terminal position, the corresponding invabs of the abbreviation omit the 5'-phosphate (i.e., they are 5'-OH). invab* Inverted abasic-5'-phosphorothioate. When located at the 3' end of the chain, it is linked to the adjacent nucleotide via the substituent at its 3' position (3'-3'linkage); when located at the 5' end of the chain, it is linked to the adjacent nucleotide via the substituent at its 5' position (5'-5' internucleotide linkage). It should also be understood that the abbreviation corresponds to invab*, which omits the 5'-phosphorothioate (i.e., they are 5'-OH) when found at the 3'-terminal position. imann imannWhen at the end of each chain: When imann is in the middle of each chain: imann* Isomannitol sulphothioate imann*When at the end of each chain imann* When in the middle of each chain

In certain embodiments of the present invention, the dsRNA (also referred to herein as a "duplex") is a dsRNA disclosed in one of Tables 1-3. Each row in Tables 1-3 discloses a duplex comprising the sense strand sequence and the antisense strand sequence in that row of the table. In addition to the duplexes disclosed in Tables 1-3, it should be understood that in some embodiments, the duplexes of the present invention may include nucleotide sequences that differ from the sense and antisense sequences shown in Tables 1-3 by zero, one, two, or three nucleotides. Therefore, as a non-limiting example, in certain embodiments, the antisense strand in the duplex of the present invention may be a nucleotide sequence that differs from the nucleotide sequence of SEQ ID NO: 169, 180, 196, 218, or 248 by zero, one, two, or three nucleotides, respectively.

Should be understood that the sense strand sequence and the antisense strand sequence in the double strand of the present invention can be selected independently. Therefore, the dsRNA of the present invention can include the sense strand and the double strand of the antisense strand disclosed in a row in Table 1-3. Or, in the dsRNA of the present invention, one or two of the sense strand and the antisense strand selected in the dsRNA can include the sequence shown in Table 1-3, but one or two of the sense strand and the antisense strand include 1,2,3 or more nucleobase substitutions from the parent sequence. In some embodiments, the selected sequence can be longer or shorter than its parent sequence. Therefore, the dsRNA agent included in the present invention can but need not include the exact sequence of the sense strand and the antisense strand disclosed as double strands in Table 1-3.

In certain embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein nucleotide positions 2 to 18 in the antisense strand comprise a region complementary to a PD-L1 RNA transcript, wherein the complementary region comprises at least 15 consecutive nucleotides that differ by 0, 1, 2, or 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3, and optionally comprises a targeting ligand. In some cases, the region complementary to a PD-L1 RNA transcript comprises at least 15, 16, 17, 18, or 19 consecutive nucleotides that differ by no more than 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3. In some embodiments of the dsRNA agents of the present invention, the antisense strand of the dsRNA is at least substantially complementary to any one of the target regions of SEQ ID NO: 1 and is provided in any one of Tables 1-3. In certain embodiments, the antisense strand of the dsRNA agents of the present invention is completely complementary to any one of the target regions of SEQ ID NO: 1 and is provided in any one of Tables 1-3. In certain embodiments, the dsRNA agents include a sense strand sequence listed in any one of Tables 1-3, and the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. In other embodiments, the dsRNA agents of the present invention include a sense strand sequence listed in any one of Tables 1-3, and the sense strand sequence is completely complementary to the antisense strand sequence in the dsRNA agent. In some cases, the dsRNA agents of the invention include an antisense strand sequence listed in any one of Tables 1-3. Some embodiments of the dsRNA agents of the invention include a double strand of sense and antisense sequences disclosed in any one of Tables 1-3 . As described herein, it is understood that the sense strand and antisense strand in the double strand of the invention can be independently selected.

It is known to those skilled in the art that mismatches are tolerated for the therapeutic effect of dsRNA, especially mismatches in the terminal regions of dsRNA. Some mismatches are more tolerated, for example, mismatches of the dangling base pairs G:U and A:C are more tolerated for therapeutic effect (Du et el., A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites. Nucleic Acids Res. 2005 Mar 21;33(5):1671-7. Doi: 10.1093/nar/gki312. Nucleic Acids Res. 2005;33(11):3698). In some embodiments of the methods and compounds of the present invention, the PD-L1 dsRNA agent may contain one or more mismatches with the PD-L1 target sequence. In certain embodiments, the PD-L1 dsRNA agent of the present invention contains no mismatches. In certain embodiments, the PD-L1 dsRNA agent of the present invention contains no more than 1 mismatch. In certain embodiments, the PD-L1 dsRNA agent of the present invention contains no more than 2 mismatches. In certain embodiments, the PD-L1 dsRNA agent of the present invention contains no more than 3 mismatches. In some embodiments of the present invention, the antisense strand of the PD-L1 dsRNA agent contains mismatches with the PD-L1 target sequence, and these mismatches are not located in the center of the complementary region. In certain embodiments, the antisense strand of the PD-L1 dsRNA agent comprises 1, 2, 3, 4 or more mismatches located within the last 5, 4, 3, 2 or 1 nucleotides of one or both of the 5' or 3' ends of the complementary region. The methods described herein and/or methods known in the art can be used to determine whether a PD-L1 dsRNA agent that mismatches with a PD-L1 target sequence is effective in inhibiting the expression of the PD-L1 gene. Complementarity

As used herein, unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence (e.g., a PD-L1 dsRNA agent sense strand or a targeted PD-L1 mRNA) relative to a second nucleotide sequence (e.g., a PD-L1 dsRNA agent antisense strand or a single-stranded antisense polynucleotide) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize [form base-pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro )] with an oligonucleotide or polynucleotide comprising the second nucleotide sequence and form a double chain or double helical structure under certain conditions. Other conditions may apply, such as physiologically relevant conditions that may occur in an organism. Depending on the ultimate application of the hybridized nucleotides, a skilled person should be able to determine a set of conditions that are most suitable for testing the complementarity of the two sequences. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs, including natural or modified nucleotides or nucleotide mimetics, as long as the above conditions regarding their hybridization ability are met. Sequence identity or complementarity is independent of modification.

Complementary sequences, for example, within the PD-L1 dsRNA described herein, include base pairing between an oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence, the base pairing extending throughout the entire length of one of the nucleotide sequences or throughout the entire length of both nucleotide sequences. Such sequences may be referred to herein as being "fully complementary" to each other. It should be understood that, in embodiments, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered mismatches in the determination of complementarity herein. For example, a PD-L1 dsRNA agent comprising one oligonucleotide of 19 nucleotides in length and another oligonucleotide of 20 nucleotides in length, wherein the longer oligonucleotide comprises a 19-nucleotide sequence that is completely complementary to the shorter oligonucleotide, can still be referred to as "completely complementary" for the purposes described herein. Thus, "completely complementary" as used herein means that all bases (100%) in the contiguous sequence of the first polynucleotide will hybridize with the same number of bases in the contiguous sequence of the second polynucleotide. The contiguous sequence may include all or part of the first or second nucleotide sequence.

The term "substantially complementary" as used herein means that in a hybridized nucleobase sequence pair, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% (but not all) of the bases in the contiguous sequence of the first polynucleotide will hybridize with the same number of bases in the contiguous sequence of the second polynucleotide. The term "substantially complementary" may be used to refer to a first sequence relative to a second sequence if the two sequences, when hybridized, form a duplex of up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs (bp) containing one or more (e.g., at least 1, 2, 3, 4 or 5) mismatched base pairs, but at the same time retain the ability to hybridize under conditions most relevant to its ultimate application (e.g., inhibition of PD-L1 gene expression via the RISC pathway).

The term "partial complement" can be used herein to refer to a hybridized nucleobase sequence pair, wherein at least 75% (but not all) of the bases in the contiguous sequence of the first polynucleotide will hybridize with the same number of bases in the contiguous sequence of the second polynucleotide. In certain embodiments, "partial complement" refers to at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the bases in the contiguous sequence of the first polynucleotide will hybridize with the same number of bases in the contiguous sequence of the second polynucleotide.

The terms "complementary", "completely complementary", "substantially complementary" and "partially complementary" as used herein refer to base pairing between the sense and antisense strands of a PD-L1 dsRNA drug, between the antisense strand of a PD-L1 dsRNA drug and a target PD-L1 mRNA sequence, or between a single-stranded antisense oligonucleotide and a target PD-L1 mRNA sequence. It should be understood that the term "antisense strand of a PD-L1 dsRNA drug" may refer to the same sequence of a "PD-L1 antisense polynucleotide drug".

As used herein, the term "substantially identical" or "substantial identity" when used to refer to a nucleic acid sequence refers to a nucleic acid sequence comprising a sequence having at least about 85% or more sequence identity compared to a reference sequence, preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. The percent sequence identity is determined by comparing the two best aligned sequences over a comparison window. The percent is calculated by determining the number of positions where the same nucleic acid base occurs in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percent sequence identity. The invention disclosed herein encompasses nucleotide sequences substantially identical to the nucleotide sequences disclosed herein, for example, those listed in Tables 1-3. In certain embodiments, the sequences disclosed herein are identical, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences disclosed herein (e.g., Tables 1-3).

The term "chain comprising a sequence" as used herein refers to an oligonucleotide comprising a nucleotide chain described by a sequence referred to using standard nucleotide nomenclature. The term "double-stranded RNA" or "dsRNA" as used herein refers to an RNAi comprising an RNA molecule or a complex of molecules having a hybrid duplex region comprising two antiparallel and substantially or completely complementary nucleic acid chains having "sense" and "antisense" orientations relative to the target PD-L1 RNA. The duplex region can be of any length as long as it allows for specific degradation of the desired target PD-L1 RNA via the RISC pathway, but is typically in the range of 9 to 30 base pairs in length, such as 15-30 base pairs in length. Considering duplexes between 9 and 30 base pairs, the length of the duplex can be any length within this range, for example 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, and any subrange therebetween. Length, including but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20- 26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs or 21-22 base pairs. The length of the PD-L1 dsRNA agent produced in the cell by processing with Dicer and similar enzymes is generally in the range of 19-22 base pairs. One of the double-stranded regions of the PD-L1 dsRNA agent contains a sequence that is substantially complementary to the target PD-L1 RNA region. The two chains forming the duplex structure may be from a single RNA molecule having at least one self-complementary region, or may be formed by two or more separate RNA molecules. When the duplex region is formed by two chains of a single molecule, the molecule may have a duplex region separated by a single-stranded nucleotide chain (referred to herein as a "hairpin loop") between the 3' end of one chain and the 5' end of the other chain forming the duplex structure. In some embodiments of the invention, the hairpin structure comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more unpaired nucleotides. When the two substantially complementary strands of the PD-L1 dsRNA agent consist of separate RNA molecules, these molecules need not but can be covalently linked. When the two strands are covalently linked by means other than a hairpin loop, the linking structure is referred to as a "linker." The term "siRNA" is also used herein to refer to the dsRNA agents described herein.

In some embodiments of the present invention, the PD-L1 dsRNA agent may include a sense sequence and an antisense sequence without unpaired nucleotides or nucleotide analogs at one or both ends of the dsRNA agent. The ends of the sequences without unpaired nucleotides are called "blunt ends" and have no nucleotide overhangs. If both ends of the dsRNA agent are blunt ends, the dsRNA is called "blunt ends". In some embodiments of the present invention, the first end of the dsRNA agent is a blunt end, in some embodiments, the second end of the dsRNA agent is a blunt end, and in some embodiments of the present invention, both ends of the PD-L1 dsRNA agent are blunt ends.

In some embodiments of the dsRNA reagent of the present invention, the dsRNA does not have one or two blunt ends. In this case, the end of the dsRNA reagent chain has at least one unpaired nucleotide. For example, when the 3' end of one dsRNA chain extends beyond the 5' end of the other chain, or vice versa, there is a nucleotide overhang. The dsRNA can include an overhang of at least 1, 2, 3, 4, 5, 6 or more nucleotides. The nucleotide overhang can include or be composed of nucleotide/nucleoside analogs (including deoxynucleotides/nucleosides). It should be understood that in certain embodiments, the nucleotide overhang is located on the sense chain of the dsRNA reagent, on the antisense chain of the dsRNA reagent, or at both ends of the dsRNA reagent, and the nucleotides of the overhang can be present at the 5' end, 3' end or both ends of the dsRNA antisense chain or sense chain. In certain embodiments of the invention, one or more nucleotides in the overhang are substituted with nucleoside phosphorothioates.

As used herein, the term "antisense strand" or "guide strand" refers to the strand of a PD-L1 dsRNA agent that includes a region that is substantially complementary to the PD-L1 target sequence. As used herein, the term "sense strand" or "passenger strand" refers to the strand of a PD-L1 dsRNA agent that includes a region that is substantially complementary to the antisense strand region of the PD-L1 dsRNA agent. Modifications

In certain embodiments of the present invention, the RNA of the PD-L1 RNAi agent is chemically modified to enhance stability and/or one or more other beneficial properties. The nucleic acids in certain embodiments of the present invention can be synthesized and/or modified by methods that are well established in the art, for example, in "Current protocols in Nucleic Acid Chemistry," Beaucage, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y., USA, which is incorporated herein by reference. Modifications that may be present in certain embodiments of the PD-L1 dsRNA agents of the present invention include, for example, (a) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, reverse ligation, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse ligation, etc.), (b) base modifications, such as replacement of a base with a stable base, an unstable base, or a base that pairs with an expanded library of partner bases, a base (abasic nucleotides), or a conjugated base, (c) sugar modifications (e.g., at the 2' position or the 4' position) or replacement of sugars, and (d) backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of RNA compounds useful in certain embodiments of the PD-L1 dsRNA agents, PD-L1 antisense polynucleotides, and PD-L1 sense polynucleotides of the present invention include, but are not limited to, RNAs containing modified backbones or no natural internucleoside bonds. As a non-limiting example, RNAs with modified backbones may not have phosphorus atoms in the backbone. RNAs without phosphorus atoms in the internucleoside backbone may be referred to as oligonucleosides. In certain embodiments of the present invention, the modified RNA has a phosphorus atom in its internucleoside backbone.

It should be understood that the term "RNA molecule" or "RNA" or "ribonucleic acid molecule" encompasses not only RNA molecules expressed or found in nature, but also RNA analogs and derivatives comprising one or more ribonucleotide/ribonucleoside analogs or derivatives described herein or known in the art. The terms "ribonucleoside" and "ribonucleotide" are used interchangeably herein. RNA molecules can be modified in the nucleobase structure or the ribose-phosphate backbone structure, for example as described below, and the molecule comprising the ribonucleoside analog or derivative must retain the ability to form a double chain. As non-limiting examples, the RNA molecule may further include at least one modified ribonucleoside, including but not limited to a 2'-O-methyl modified nucleoside, a nucleoside containing a 5' phosphorothioate group, a terminal nucleoside linked to a cholesterol derivative or a dodecanoic acid bisdecylamide group, a locked nucleoside, a debasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, a 2'-amino modified nucleoside, a 2'-alkyl modified nucleoside, a morpholino nucleoside, a phosphoramidate, or a nucleoside containing an unnatural base, or any combination thereof. In some embodiments of the invention, the RNA molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or up to the full length of the PD-L1 dsRNA drug molecule, which are modified ribonucleosides. The modification of each of the multiple modified ribonucleosides in the RNA molecule need not be the same.

In certain embodiments of the present invention, the dsRNA agent, PD-L1 antisense polynucleotide and/or PD-L1 sense polynucleotide may contain one or more independently selected modified nucleotides and/or one or more independently selected non-phosphodiester bonds. The term "independently selected" used herein to refer to selected elements (e.g., modified nucleotides, non-phosphodiester bonds, etc.) means that two or more selected elements may, but need not, be identical to each other.

As used herein, "nucleotide bases", "nucleotides" or "nucleobases" are heterocyclic pyrimidine or purine compounds that are standard components of all nucleic acids, including bases that form the nucleotides adenine, guanine, cytosine, thymine and uracil. Nucleobases can be further modified to include, but are not limited to: universal bases, hydrophobic bases, mixed bases, size-expanded bases and fluorinated bases. The term "ribonucleotide" or "nucleotide" can be used herein to refer to unmodified nucleotides, modified nucleotides or alternative substitution moieties. Those skilled in the art will recognize that guanine, cytosine, adenine and uracil can be replaced by other moieties without significantly changing the base pairing properties of oligonucleotides containing nucleotides with such substitution moieties.

In one embodiment, the modified RNA contemplated for use in the methods and compositions described herein is a peptide nucleic acid (PNA), which has the ability to form a desired double-stranded structure and allows or mediates specific degradation of the target RNA through the RISC pathway. In certain embodiments of the present invention, the PD-L1 RNA interferor includes a single-stranded RNA that interacts with a target PD-L1 RNA sequence to direct the cleavage of the target PD-L1 RNA.

The RNA backbone of modification can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), phosphorothioates, thioalkylphosphonates, thioalkylphosphotriesters and boranephosphates with normal 3'-5' bonds, 2'-5' connection analogs of these and those with reversed polarity (wherein adjacent nucleoside unit pairs are connected with 3'-5' to 5'-3' or 2'-5' to 5'-2'). Various salts, mixed salts and free acid forms are also included. Methods for preparing phospho-bond-containing proteins are routine in the art, and such methods can be used to prepare certain modified PD-L1 dsRNA agents, certain modified PD-L1 antisense polynucleotides, and/or certain modified PD-L1 sense polynucleotides of the present invention.

The modified RNA backbone that does not contain a phosphorus atom has a backbone formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatom and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatom or heterocyclic nucleoside bonds. These include backbones with morpholine bonds (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfone, and sulfonate backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; olefin-containing backbones; sulfamate backbones; methyleneimido and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and _CH2 constituents. Methods for preparing modified RNA backbones that do not include phosphorus atoms are routine in the art, and such methods can be used to prepare certain modified PD-L1 dsRNA agents, certain modified PD-L1 antisense polynucleotides, and/or certain modified PD-L1 sense polynucleotides of the present invention.

In certain embodiments of the present invention, RNA mimetics are included in PD-L1 dsRNA, PD-L1 antisense polynucleotides and/or PD-L1 sense polynucleotides, for example but not limited to: replacing the sugar and internucleoside bonds of the nucleotide units, i.e., the backbone, with new groups. In such embodiments, the base units are retained to hybridize with appropriate PD-L1 nucleic acid target compounds. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobase is retained and is directly or indirectly bound to the nitrogen-nitrogen atom of the amide portion of the backbone. Methods for preparing RNA mimetics are routinely practiced in the art, and such methods can be used to prepare certain modified PD-L1 dsRNA agents of the present invention.

Some embodiments of the present invention include RNAs having phosphorothioate backbones and oligonucleosides having heteroatom backbones, particularly _-CH2 -NH- _CH2- , -CH2- _N ( _CH3 )-O- _CH2- [referred to as methylene (methylimino) or MMI backbones], -CH2- _ON ( _CH3 ) _-CH2- , -CH2- _N (CH3)-N( _CH3 ) _-CH2- , and -N( _CH3 ) _-CH2- [wherein the natural phosphodiester backbone is represented as -OPO- _CH2- ]. Methods for preparing RNAs having phosphorothioate _backbones and oligonucleosides having heteroatom backbones are routinely practiced in the art, and such methods can be used to prepare certain modified PD-L1 dsRNA agents, certain PD-L1 antisense polynucleotides and/or certain PD-L1 sense polynucleotides of the present invention.

The modified RNA may also contain one or more substituted sugar moieties. The PD-L1 dsRNA, PD-L1 antisense polynucleotide and/or PD-L1 sense polynucleotide of the present invention may comprise one of the following at the 2' position: OH; F; O-, S- or N-alkyl; O-, S- or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl groups may be substituted or unsubstituted C ₁ to C ₁₀ alkyl or C ₂ to C ₁₀ alkenyl and alkynyl groups. Exemplary suitable modifications include O[( _CH2 ) _nO ] _mCH3 , O( _CH2 ) _nOCH3 , O(CH2) _nNH2 , O( _CH2 ) _nCH3 , O( _{CH2 ) nONH2 ,} and O( _{CH2 ) nON} [( _{CH2 ) nCH3} )] _{2 , wherein n} and m are 1-10. In other embodiments, the dsRNA includes one of the following at the 2' position: _C1 to _C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, _SCH3 , OCN, Cl, Br, CN, _CF3 , _OCF3 , _SOCH3 , _{SO2CH3, ONO2 , NO2} , _N3 , _NH2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of PD-L1 dsRNA drug, or _group for improving the pharmacodynamic properties of PD-L1 dsRNA drug, PD-L1 antisense polynucleotide and/or PD-L1 sense polynucleotide, and other substituents with similar properties. In certain embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH ₂ CH ₂ OCH ₃ , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., alkoxy-alkoxy. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., O(CH ₂ ) ₂ ON(CH ₃ ) ₂ group, also known as 2'-DMAOE, as described in the Examples below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH ₂ -O-CH ₂ -N(CH ₂ ) ₂ . Methods for preparing such modified RNAs are routinely practiced in the art, and such methods can be used to prepare certain modified PD-L1 dsRNA agents of the present invention.

Other modifications include 2'-methoxy (2'-OCH ₃ ), 2'-aminopropoxy (2'-OCH ₂ CH ₂ CH ₂ NH ₂ ) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the PD-L1 dsRNA agent, PD-L1 antisense polynucleotide and/or PD-L1 sense polynucleotide of the present invention, particularly the 3' position of the sugar on the 3' terminal nucleotide or in the 2'-5' linked PD-L1 dsRNA, PD-L1 antisense polynucleotide or PD-L1 sense polynucleotide, and the 5' position of the 5' terminal nucleotide. The PD-L1 dsRNA agent, PD-L1 antisense polynucleotide and/or PD-L1 sense polynucleotide may also have a sugar mimetic, such as replacing the pentofuranosyl with a cyclobutyl moiety. Methods for preparing the modified RNA are routine in the art, and such methods can be used to prepare certain modified PD-L1 dsRNA agents, PD-L1 antisense polynucleotides, and/or PD-L1 sense polynucleotides of the present invention.

In certain embodiments, the PD-L1 dsRNA agent, PD-L1 antisense polynucleotide and/or PD-L1 sense polynucleotide may include nucleobase (commonly referred to as "base" in the art) modification or substitution. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine and uracil. Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-Me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halogenuracil and cytosine, 5-propargyluracil and cytosine, 6-azouracil, cytosine and thymine. adenine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogen (especially 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-deazaadenine and 3-deazaguanine and 3-deazaadenine. Other nucleobases that may be included in certain embodiments of the PD-L1 dsRNA agents of the present invention are known in the art, see, for example: Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. Ed. Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, Ed. John Wiley & Sons, 1990, English et al., Angewandte Chemie, International Edition, 1991, 30, 613, Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Methods for preparing dsRNA, PD-L1 antisense polynucleotides and/or PD-L1 sense polynucleotides comprising nucleobase modifications and/or substitutions (such as those described herein) are routinely practiced in the art, and such methods can be used to prepare certain modified PD-L1 dsRNA agents, PD-L1 sense polynucleotides and/or PD-L1 antisense polynucleotides of the present invention.

Certain embodiments of the PD-L1 dsRNA agents, PD-L1 antisense polynucleotides, and/or PD-L1 sense polynucleotides of the present invention include RNA modified to include one or more locking nucleic acids (LNAs). Locking nucleic acids are nucleotides with a modified ribose moiety that includes an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. Adding a locking nucleic acid to the PD-L1 dsRNA agent, PD-L1 antisense polynucleotide and/or PD-L1 sense polynucleotide of the present invention can increase stability in serum and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Methods for preparing dsRNA agents comprising a locking nucleic acid, PD-L1 antisense polynucleotides and/or PD-L1 sense polynucleotides are routinely practiced in the art, and such methods can be used to prepare certain modified PD-L1 dsRNA agents of the present invention.

Certain embodiments of the PD-L1 dsRNA compounds, sense polynucleotides and/or antisense polynucleotides of the present invention comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'3'-seco nucleotide mimetics, locked nucleotides, 2'-F-arabinose nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides and 3'-OMe nucleotides, nucleotides comprising a 5'-phosphorothioate group, or a terminal nucleotide linked to a cholesterol derivative or dodecanoic acid bisdecylamide group, 2'-amino modified nucleotides, phosphoramidates, or nucleotides comprising an unnatural base. In certain embodiments, the PD-L1 dsRNA compound comprises an E-vinylphosphonate nucleotide at the 5' end of the antisense strand, also referred to herein as the guide strand.

Certain embodiments of the PD-L1 dsRNA compounds, the 3' and 5' ends of the sense polynucleotides and/or the 3' end of the antisense polynucleotides of the present invention include at least one modified nucleotide, wherein the at least one modified nucleotide includes: a debasic nucleotide, ribitol, inverted nucleotide, inverted debasic nucleotide, inverted 2'-OMe nucleotide, inverted 2'-deoxynucleotide. It is known to those skilled in the art that including debasic or inverted debasic nucleotides at the end of an oligonucleotide can enhance stability (Czauderna et al. Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells. Nucleic Acids Res. 2003;31(11):2705-2716. doi:10.1093/nar/gkg393). In certain embodiments, the PD-L1 dsRNA compound comprises one or more inverted debasing residues (invab) at the 3' end or the 5' end, or at both the 3' end and the 5' end. Exemplary inverted debasing residues (invab) include, but are not limited to, the following:

Certain embodiments of the PD-L1 dsRNA compounds, the 3' and 5' ends of the sense polynucleotides and/or the 3' end of the antisense polynucleotides of the present invention include at least one modified nucleotide, wherein the at least one modified nucleotide includes: isomannitol nucleotides or stereoisomers of the isomannitol nucleotides. Specific examples of isomannitol nucleotides or stereoisomers of the isomannitol nucleotides include but are not limited to: , , , , , , , , , , and , wherein the phrase "Olig" each independently represents a polynucleotide portion. Exemplary isomannide residues (imann) include, but are not limited to, the following: or .

In certain embodiments, the isomannitol nucleotides can be further conjugated to one or more targeting groups or delivery molecules, such as a GalNAc moiety.

Certain embodiments of the PD-L1 dsRNA compounds and antisense polynucleotides of the present invention include at least one modified nucleotide, wherein the at least one modified nucleotide includes an unlocked nucleic acid nucleotide (UNA) or/and a glycol nucleic acid nucleotide (GNA). It is known to those skilled in the art that UNA and GNA are thermally unstable chemical modifications that can significantly improve the off-target properties of siRNA compounds (Janas, et al., Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun. 2018;9(1):723. doi:10.1038/s41467-018-02989-4; Laursen et al., Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo. Mol BioSyst. 2010;6:862–70).

Certain embodiments of the PD-L1 dsRNA agents, PD-L1 antisense polynucleotides and/or PD-L1 sense polynucleotides of the present invention may comprise another modification in the RNA, which includes one or more ligands, moieties or conjugates chemically linked to the RNA, wherein the ligands, moieties or conjugates enhance one or more properties of the PD-L1 dsRNA agents, PD-L1 antisense polynucleotides and/or PD-L1 sense polynucleotides, respectively. Non-limiting examples of properties that can be enhanced are: activity of PD-L1 dsRNA agents, PD-L1 antisense polynucleotides and/or PD-L1 sense polynucleotides, cellular distribution, delivery of PD-L1 dsRNA agents, pharmacokinetic properties of PD-L1 dsRNA agents, and cellular uptake of PD-L1 dsRNA agents. In some embodiments of the present invention, the PD-L1 dsRNA agent comprises one or more targeting groups or linking groups, and in certain embodiments of the PD-L1 dsRNA agent of the present invention, the targeting group or linking group is bound to the sense chain. A non-limiting example of a targeting group is a compound comprising N-acetylgalactosamine (GalNAc). The terms "targeting group", "targeting agent", "linker", "targeting compound", "delivery molecule", "delivery compound" and "targeting ligand" are used interchangeably herein. In certain embodiments of the present invention, the PD-L1 dsRNA agent comprises a targeting compound bound to the 5' end of the sense chain. In certain embodiments of the present invention, the PD-L1 dsRNA agent comprises a targeting compound bound to the 3' end of the sense chain. In certain embodiments of the present invention, the PD-L1 dsRNA agent comprises a targeting group comprising GalNAc. In certain embodiments of the present invention, the PD-L1 dsRNA agent does not include a targeting compound bound to one or both of the 3' end and the 5' end of the sense chain. In certain embodiments of the invention, the PD-L1 dsRNA agent does not include a GalNAc-containing targeting compound bound to one or both of the 5' end and the 3' end of the sense strand.

Additional targeting agents and linking agents are well known in the art, for example, targeting agents and linking agents that can be used in certain embodiments of the present invention include but are not limited to lipid moieties. For example, the cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060), thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660: 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3: 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, for example dodecanediol or an undecyl residue (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, for example di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycerol-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237); or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

Certain embodiments of compositions comprising PD-L1 dsRNA agents, PD-L1 antisense polynucleotides, and/or PD-L1 sense polynucleotides may comprise a ligand that alters the distribution, targeting, etc. of the PD-L1 dsRNA agent. In some embodiments of compositions comprising the PD-L1 dsRNA agents of the invention, the ligand increases affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment, such as a cell or organ compartment, a tissue, an organ, or a body region) compared to a species in which such ligand is absent. Ligands useful in the compositions and/or methods of the invention may be naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids or polyamines. Examples of polyamino acids are polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymers, poly(L-lactide-co-glycolic acid) copolymers, divinyl ether-maleic anhydride copolymers, N-(2-hydroxypropyl)methacrylamide copolymers (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethanes, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazenes. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

The ligands included in the compositions and/or methods of the invention may comprise a targeting group, non-limiting examples of which are cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, such as antibodies that bind to specific cell types (e.g., kidney cells or liver cells). The targeting group can be thyroid stimulating hormone, melanocyte stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetyl glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin or RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saphthrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, Geranyloxyhexyl, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl, palmitic acid, myristic acid, O3-(oleyl)cholestyric acid, O3-(oleyl)cholestyric acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., tripeptide, Tat peptide), alkylating agents, phosphates, amino, hydroxyl, PEG (e.g., PEG-40K), MPEG, [MPEG] _2. Polyamino, alkyl, substituted alkyl, radiolabel, enzyme, hapten (e.g., biotin), transport/absorption enhancer (e.g., aspirin, vitamin E, folic acid), synthetic ribonuclease (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, tetrazamacrocyclic Eu3+ complex), dinitrophenyl, HRP or AP.

The ligand included in the compositions and/or methods of the present invention can be a protein, such as a glycoprotein or a peptide, such as a molecule with a specific affinity for a co-ligand, or an antibody, such as an antibody that binds to a specific cell type (e.g., cancer cells, endothelial cells, heart cells, or bone cells). The ligand useful in embodiments of the compositions and/or methods of the present invention can be a hormone or a hormone receptor. The ligand useful in embodiments of the compositions and/or methods of the present invention can be a lipid, a lectin, a carbohydrate, a vitamin, a cofactor, a multivalent lactose, a multivalent galactose, N-acetylgalactosamine, N-acetylglucosamine, a multivalent mannose, or a multivalent fucose. Ligands useful in embodiments of the compositions and/or methods of the invention can be substances that increase the entry of a PD-L1 dsRNA agent into a cell, for example, by disrupting the cell's cytoskeleton, for example, by disrupting the cell's microtubules, microfilaments and/or intermediate filaments. Non-limiting examples of such agents are: taxon, vincristine, vinblastine, cytorelaxin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, and myoservin.

In some embodiments, the ligand linked to the PD-L1 dsRNA agent of the present invention acts as a pharmacokinetic (PK) modulator. Examples of PK modulators that can be used in the compositions and methods of the present invention include, but are not limited to, lipophilic substances, bile acid, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, cholesterol, fatty acids, bile acid, cholestyric acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, serum protein-binding aptamers, etc. Oligonucleotides containing a large number of phosphorothioate bonds are also known to bind to serum proteins, so short oligonucleotides containing multiple phosphorothioate bonds in the backbone (e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases) can also be used as ligands in the compositions and/or methods of the present invention. PD-L1 dsRNA Drug Compositions

In some embodiments of the invention, the PD-L1 dsRNA agent is present in a composition. The composition of the invention may include one or more PD-L1 dsRNA agents, and optionally one or more pharmaceutically acceptable carriers, delivery agents, targeting agents, detectable markers, etc. Non-limiting examples of targeting agents that may be useful according to some embodiments of the methods of the invention are agents that direct the PD-L1 dsRNA agent of the invention to the cells to be treated and/or into the cells to be treated. The choice of targeting agent will depend on the following factors: the nature of the PD-L1-related disease or condition, and the type of cell being targeted. In a non-limiting example, in some embodiments of the invention, it may be desirable to direct a PD-L1 dsRNA agent to and/or into hepatocytes. It should be understood that in some embodiments of the methods of the invention, the therapeutic agent includes a PD-L1 dsRNA agent with only a delivery agent, such as a delivery agent comprising N-acetylgalactosamine (GalNAc), without any additional elements. For example, in some aspects of the invention, a PD-L1 dsRNA agent can be attached to a delivery compound comprising GalNAc and contained in a composition comprising a pharmaceutically acceptable carrier and administered to a cell or subject without any detectable label or targeting agent attached thereto or the like.

When the PD-L1 dsRNA agent of the present invention is administered together with and/or attached to one or more delivery agents, targeting agents, labeling agents, etc., a person skilled in the art will understand and be able to select and use agents suitable for the methods of the present invention. Labeling agents can be used in certain methods of the present invention to determine the location of PD-L1 dsRNA agents in cells and tissues, and can be used to determine the cell, tissue or organ location of a therapeutic composition containing a PD-L1 dsRNA agent that has been administered in the methods of the present invention. Means for attaching and utilizing labeling agents such as enzyme labels, dyes, radioactive labels, etc. are well known in the art. It should be understood that in some embodiments of the compositions and methods of the present invention, the labeling agent is attached to one or both of the sense polynucleotide and the antisense polynucleotide contained in the PD-L1 dsRNA agent. Delivery of PD-L1 dsRNA Agents and PD-L1 Antisense Polynucleotide Agents

Certain embodiments of the methods of the present invention include delivering a PD-L1 dsRNA agent to a cell. The term "delivery" as used herein refers to promoting or affecting the uptake or absorption of a cell. The absorption or uptake of a PD-L1 dsRNA agent may occur through an independent diffusion or activation cell process, or through the use of a delivery agent, a targeting agent, etc. that may be associated with the PD-L1 dsRNA agent of the present invention. Delivery methods suitable for the methods of the present invention include, but are not limited to: in vivo delivery, in which the PD-L1 dsRNA agent is injected into a tissue site or administered systemically. In some embodiments of the present invention, the PD-L1 dsRNA agent is attached to a delivery agent.

Non-limiting examples of methods that can be used to deliver PD-L1 dsRNA agents to cells, tissues, and/or subjects include: PD-L1 dsRNA-GalNAc conjugates, SAMiRNA technology, LNP-based delivery methods, and naked RNA delivery. These and other delivery methods have been successfully used in the art to deliver therapeutic RNAi agents to treat various diseases and conditions, such as, but not limited to: liver disease, acute intermittent porphyria (AIP), hemophilia, pulmonary fibrosis, etc. Details of various delivery methods can be found in the following publications: Nikam, R.R. & K. R. Gore (2018) Nucleic Acid Ther, 28 (4), 209-224 Aug 2018; Springer A.D. & S.F. Dowdy (2018) Nucleic Acid Ther. Jun 1; 28(3): 109–118; Lee, K. et al., (2018) Arch Pharm Res, 41(9), 867-874; and Nair, J.K. et al., (2014) J. Am. Chem. Soc. 136:16958-16961, all of which are incorporated herein by reference.

Some embodiments of the present invention include using lipid nanoparticles (LNPs) to deliver the PD-L1 dsRNA agents of the present invention to cells, tissues and/or subjects. LNPs are generally used to deliver PD-L1 dsRNA agents in vivo , including therapeutic PD-L1 dsRNA agents. One advantage of using LNPs or other delivery agents is that when PD-L1 RNA drugs are delivered to subjects using LNPs or other delivery agents, they are more stable. In some embodiments of the present invention, LNPs include cationic LNPs carrying one or more PD-L1 RNAi molecules of the present invention. LNPs containing PD-L1 RNAi molecules are administered to subjects. LNPs and their attached PD-L1 RNAi molecules are taken up by cells via endocytosis, and their presence leads to the release of RNAi trigger molecules, thereby mediating RNAi.

Another non-limiting example of a delivery agent that can be used in embodiments of the present invention to deliver the PD-L1 dsRNA agent of the present invention to cells, tissues and/or subjects is an agent comprising GalNAc, which is linked to the PD-L1 dsRNA agent of the present invention and delivers the PD-L1 dsRNA agent to cells, tissues and/or subjects. Examples of certain additional delivery agents comprising GalNAc that can be used in certain embodiments of the methods and compositions of the present invention are disclosed in PCT application: WO2020191183A1 (the entire contents of which are incorporated herein). A non-limiting example of a GalNAc targeting ligand that can be used in the compositions and methods of the present invention to deliver a PD-L1 dsRNA agent to a cell is a cluster of targeting ligands. Examples of clusters of targeting ligands presented herein are referred to as: GalNAc ligands with phosphodiester linkages (GLO) and GalNAc ligands with phosphorothioate linkages (GLS). The term "GLX-n" may be used herein to indicate that the attached GalNAc-containing compound is compound GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*, Any one of GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16, each of which has a structure as shown below, wherein the GalNAc targeting ligand is linked to the RNAi agent of the present invention at the rightmost side of each (denoted by " ⸾ ”), GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16 phosphoramidites have been disclosed in WO2023/045995A1 (incorporated herein in its entirety). It should be understood that any RNAi and dsRNA molecules of the present invention can be linked to GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15 and GLO-16, GLO-1 to GLO-16 and GLS-1* to GLS-16* structures are shown below. GLO-1 GLS-1* GLO-2 GLS-2* GLO-3 GLS-3* GLO-4 GLS-4* GLO-5 GLS-5* GLO-6 GLS-6* GLO-7 GLS-7* GLO-8 GLS-8* GLO-9 GLS-9* GLO-10 GLS-10* GLO-11 GLS-11* GLO-12 GLS-12* GLO-13 GLS-13* GLO-14 GLS-14* GLO-15 GLS-15* GLO-16 . GLS-16*

In certain embodiments, the above-mentioned isomannose nucleotides can be further conjugated with one or more GalNAc targeting ligands. Specific examples of isomannose nucleotides conjugated with GalNAc targeting ligands include but are not limited to: , , wherein the phrases "olig" each independently represent a polynucleotide portion.

In some embodiments of the invention, in vivo delivery can also be performed by β-glucan delivery systems, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. PD-L1 RNAi agents can also be introduced into cells in vitro using methods known in the art, such as electroporation and lipofection. In certain embodiments of the methods of the invention, PD-L1 dsRNA is delivered without a targeting agent. These RNAs can be delivered as "naked" RNA molecules. As a non-limiting example, the PD-L1 dsRNA of the present invention can be administered to a subject in the form of a pharmaceutical composition comprising an RNAi agent but not a targeting agent (e.g., a GalNAc targeting compound) to treat a PD-L1-related disease or disorder (e.g., cardiovascular disease) in the subject.

In addition to certain delivery methods described herein, it will be appreciated that RNAi delivery methods (such as, but not limited to, those described herein and used in the art) can be used in conjunction with the embodiments of the PD-L1 RNAi agents and treatment methods described herein.

The PD-L1 dsRNA agents of the present invention can be administered to a subject in an amount and manner effective to reduce the level and activity of PD-L1 polypeptides in cells and/or subjects. In some embodiments of the methods of the present invention, one or more PD-L1 dsRNA agents are administered to cells and/or subjects to treat diseases or conditions associated with PD-L1 expression and activity. In certain embodiments, the methods of the present invention include administering one or more PD-L1 dsRNA agents to a subject in need of such treatment to reduce a disease or condition associated with PD-L1 expression in the subject. The PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents of the present invention can be administered to reduce PD-L1 expression and/or activity in one or more of in vitro , ex vivo , and in vivo cells .

In some embodiments of the present invention, the level of PD-L1 polypeptide in a cell is reduced by delivering (e.g., introducing) a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent into a cell, thereby reducing its activity. Targeting agents and methods can be used to help deliver PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents to specific cell types, cell subtypes, organs, spatial regions, and/or subcellular regions within cells in a subject. In certain methods of the present invention, a PD-L1 dsRNA agent can be administered alone or in combination with one or more additional PD-L1 dsRNA agents. In certain embodiments, 2, 3, 4 or more independently selected PD-L1 dsRNA agents are administered to a subject.

In certain embodiments of the present invention, a PD-L1 dsRNA agent is administered to a subject together with one or more additional treatment regimens for treating a PD-L1-related disease or condition to treat a PD-L1-related disease or condition. Non-limiting examples of additional treatment regimens are: administration of one or more PD-L1 antisense polynucleotides of the present invention, administration of non-PD-L1 dsRNA therapeutic agents, and behavioral changes. Additional treatment regimens may be administered at one or more of the following times: before, simultaneously, and after administration of the PD-L1 dsRNA agent of the present invention. It should be understood that "simultaneously" as used herein refers to within 0.5 minutes, within 0.10 minutes, within 0.30 minutes, within 0.45 minutes, and within 0.60 minutes, and "zero" refers to the time when the PD-L1 dsRNA agent of the present invention is administered to the subject. Non-limiting examples of non-PD-L1 dsRNA therapeutics include: other cancer therapeutics selected from surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, or hormonal therapy. Including but not limited to alemtuzumab, altretamine, azacitidine, bendamustine, bleomycin, bortezomib, busulfan, cabazitaxel, capecitabine, carboplatin, carmofur, carmustine, chlorambucil, chlormethine, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin cin), daunorubicin, decitabine, denosumab, docetaxel, doxorubicin, epirubicin, estramustine, etoposide, everolimus, floxuridine, fludarabine, fluorouracil, fotemustine, gemcitabine, gemtuzumab, hydroxycarbamide, ibritumomab, idarubicin, ifosfamide, irinotecan, ixabepilone, epilone, lomustine, melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, nedaplatin, nelarabine, ofatumumab, oxaliplatin, paclitaxel, pemetrexed, pentostatin, pertuzumab, procarbazine, raltitrexed, streptozotocin, tegafur, temozolomide, temsirolimus, teniposide iposide, tioguanine, topotecan, tositumomab, valrubicin, vinblastine, vincristine, vindesine, vinflunine, or vinorelbine, acalabrutinib, adavosertib, afatinib, alectinib, axitinib, binimetinib, bosutinib, brigatinib, cediranib, ceritinib, cetuximab, cobimetinib, crizotinib Crizotinib, cabozantinib, dacomitinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, lestaurtinib, lortatinib, masitinib, momelotinib, mubritinib, neratinib, nilotinib, nintedanib, olmutinib , osimertinib, pacritinib, panitumumab, pazopanib, pegaptanib, ponatinib, radotinib, regorafenib, rociletinib, ruxolitinib, selumetinib, semaxanib, sorafenib, sunitinib, SU6656, tivozanib, toceranib, trametinib, trastuzumab, vandetanib or vemurafenib, nivolumab, pembrolizumab pembrolizumab, spartalizumab, cemiplimab, camrelizumab, sintilimab, tislelizumab, toripalimab, AMP-224 or AMP-514, atezolizumab, avelumab, durvalumab, KN035, AUNP12, CA-170 or BMS-986189, ipilimumab or tremilimumab, aflibercept, axitinib, bevacizumab, brivanib, cabozantinib, cediran ib), lenvatinib, linifmib, nintedanib, pazopanib, ponatinib, ramucirumab, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib ), toceranib or vandetanib, AB-423, AB-506, ABI-H2158, ABI-H0731, acyclovir, adapromine, adefovir, alafenamide, amantadine, asunaprevir, baloxavir marboxil), beclabuvir, boceprevir, brivudine, cidofovir, ciluprevir, clevudine, cytarabine, daclatasvir, danoprevir, dasabuvir, deleobuvir, dipivoxil, edoxudine, elbasvir ), entecavir, faldaprevir, famciclovir, favipiravir, filibuvir, fomivirsen, foscamet, galidesivir, ganciclovir, glecaprevir, GLS4, grazoprevir, idoxuridine, imiquimod, IFN-a, interferon alfa 2b, JNJ-440, JNJ-6379, lamivudine, laninamivir, ledipasvir, mericitabine, methisazone, MK-608, moroxydine, narlaprevir, NITD008, NZ-4, odalasvir, ombitasvir, oseltamivir ), paritaprevir, peginterferon alpha-2a, penciclovir, peramivir, pibrentasvir, pimodivir, pleconaril, podophyllotoxin, presatovir, radalbuvir, ravidasvir, remdesivir, REP 2139, REP 2165, resiquimod, RG7907, ribavirin, rifampicin, rimantadine, ruzasvir, samatasvir, setrobuvir, simeprevir, sofosbuvir, sorivudine, sovaprevir, taribavirin, telaprevir, telbivudine, tenofovir, tenofovir disoproxil, triazavirin, trifluridine uridine), tromantadine, umifenovir, uprifosbuvir, valaciclovir, valgancicovir, vaniprevir, vedroprevir, velpatasvir, vidarabine, voxilaprevir or zanamivir, or any combination thereof. These and other therapeutic agents and behavioral changes are known in the art for treating a PD-L1-related disease or disorder in a subject and can be administered to a subject in combination with administration of one or more PD-L1 dsRNA agents of the present invention to treat a PD-L1-related disease or disorder. The PD-L1 dsRNA agent of the present invention administered to cells or subjects for the treatment of PD-L1-related diseases or disorders can act synergistically with one or more other therapeutic agents or activators and enhance the effectiveness of one or more therapeutic agents or activators and/or enhance the effectiveness of the PD-L1 dsRNA agent in treating PD-L1-related diseases or disorders.

The treatment methods of the present invention include administering a PD-L1 dsRNA agent, which can be used before the onset of a PD-L1-related disease or condition and/or while a PD-L1-related disease or condition exists, including the early, middle and late stages of the disease or condition and all times before and after these stages. The methods of the present invention can also be used to treat subjects who have previously been treated for a PD-L1-related disease or condition with one or more other therapeutic agents and/or therapeutic activities that were unsuccessful, had low success rates and/or were no longer successful in treating the subject's PD-L1-related disease or condition. Vector-Encoded dsRNAs

In certain embodiments of the present invention, a vector may be used to deliver a PD-L1 dsRNA agent to a cell. The PD-L1 dsRNA agent transcription unit may be contained in a DNA or RNA vector. The preparation and use of such vectors encoding transgenes for delivering sequences to cells and/or subjects are well known in the art. Vectors that cause transient expression of PD-L1 dsRNA may be used in the methods of the present invention, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours or more, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks or more. The length of transient expression may be determined using conventional methods based on factors such as, but not limited to, the specific vector construct and target cells and/or tissues selected. Such transgenes can be introduced as linear constructs, circular plasmids or viral vectors, which can be integrating or non-integrating vectors. Transgenes can also be constructed so that they are inherited as extrachromosomal plasmids (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

A single strand or multiple strands of a PD-L1 dsRNA agent can be transcribed from a promoter on an expression vector. In the case where two separate strands are to be expressed to produce, for example, dsRNA, two separate expression vectors can be co-introduced into cells using, for example, transfection or infection. In certain embodiments, each separate strand of a PD-L1 dsRNA agent of the present invention can be transcribed from a promoter contained on the same expression vector. In certain embodiments of the present invention, a PD-L1 dsRNA agent is expressed as an inverted repeat polynucleotide connected by a linker polynucleotide sequence, such that the PD-L1 dsRNA agent has a stem-loop structure.

Non-limiting examples of RNA expression vectors are DNA plasmids or viral vectors. Expression vectors useful in embodiments of the present invention may be compatible with eukaryotic cells. Eukaryotic cell expression vectors are routinely used in the art and are available from many commercial sources. Delivery of PD-L1 dsRNA expression vectors may be systemic, for example, by intravenous or intramuscular administration, by administration to target cells removed from a subject and then reintroduced into the subject, or by any other means that allows introduction into the desired target cells.

Viral vector systems that may be included in embodiments of the method include, but are not limited to: (a) adenoviral vectors; (b) retroviral vectors, including but not limited to lentiviral vectors, Moloney murine leukemia virus, etc.; (c) adeno-associated viral vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopoxvirus vectors or avipoxvirus vectors, such as canarypox virus or chickenpox virus; and (j) helper-dependent or intestinal adenovirus. Constructs for recombinant expression of PD-L1 dsRNA agents may include regulatory elements, such as promoters, enhancers, etc., which may be selected to provide constitutive or regulated/induced expression. The use of viral vector systems, promoters and enhancers, etc. is routine in the art and can be used in conjunction with the methods and compositions described herein.

Certain embodiments of the present invention include the use of viral vectors to deliver PD-L1 dsRNA agents to cells. A variety of adenovirus-based delivery systems are routinely used in the art for delivery to, for example, the lungs, liver, central nervous system, endothelial cells, and muscle. Non-limiting examples of viral vectors that can be used in the methods of the present invention are: AAV vectors, poxviruses (e.g., vaccinia virus), modified Ankara virus (MVA), NYVAC, avian pox such as fowlpox or canarypox virus.

Certain embodiments of the present invention include methods of delivering PD-L1 dsRNA agents to cells using vectors, and such vectors may be present in a pharmaceutically acceptable carrier that may, but need not, include a sustained release matrix into which the gene delivery vector is embedded. In certain embodiments, the vector that delivers PD-L1 dsRNA may be produced by recombinant cells, and the pharmaceutical compositions of the present invention may include one or more cells that produce the PD-L1 dsRNA delivery system. Pharmaceutical compositions containing PD-L1 dsRNA or ssRNA agents

Certain embodiments of the present invention include the use of a pharmaceutical composition containing a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent and a pharmaceutically acceptable carrier. The pharmaceutical composition containing a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent can be used in the method of the present invention to reduce PD-L1 gene expression and PD-L1 activity in cells, and can be used to treat PD-L1 related diseases or conditions. Such pharmaceutical compositions can be formulated according to the delivery method. Non-limiting examples of formulations for delivery modes are: compositions formulated for subcutaneous delivery, compositions formulated for systemic administration via parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for intrathecal delivery, compositions formulated for direct delivery to the brain, etc. Administration of the pharmaceutical compositions of the present invention to deliver PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents to cells can be performed using one or more modes, such as: topical (e.g., via a transdermal patch), pulmonary, for example, by inhalation or insufflation of a powder or aerosol, including via a nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subcutaneous, for example, by implantation; or intracranial, for example, by intraparenchymal, intrathecal or intraventricular administration. PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents can also be delivered directly to target tissues, such as directly to the liver, directly to the kidneys, etc. It should be understood that "delivering a PD-L1 dsRNA agent" or "delivering a PD-L1 antisense polynucleotide agent" into a cell includes directly delivering a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent, respectively, and expressing a PD-L1 dsRNA agent directly in a cell and expressing a PD-L1 dsRNA agent from an encoding vector delivered to a cell, or any suitable means for causing a PD-L1 dsRNA or a PD-L1 antisense polynucleotide agent to appear in a cell. The preparation and use of the formulation and the means for delivering inhibitory RNA are well known and routinely used in the art.

The "pharmaceutical composition" used herein includes a pharmacologically effective amount of the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. The term explicitly excludes cell culture media. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients, such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, coloring agents, and preservatives. Suitable inert diluents include sodium and calcium carbonates, sodium and calcium phosphates, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starches and gelatin, while lubricants, if present, are typically magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay gastrointestinal absorption. The agents included in the pharmaceutical formulations will be further described below.

As used herein, terms such as "pharmacologically effective amount", "therapeutically effective amount" and "effective amount" refer to the amount of the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention that produces the desired pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or condition is reduced by at least 10%, then the therapeutically effective amount of the drug used to treat the disease or condition is the amount required to reduce the parameter by at least 10%. For example, a therapeutically effective amount of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent can reduce the level of PD-L1 polypeptide by at least 10%. Effective amount

The methods of the present invention in certain aspects include contacting cells with an effective amount of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent to reduce PD-L1 gene expression in the contacted cells. Certain embodiments of the methods of the present invention include administering an effective amount of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent to a subject to reduce PD-L1 gene expression in the subject and treat a PD-L1-related disease or condition in the subject. An "effective amount" used in reducing PD-L1 expression and/or treating a PD-L1-related disease or condition is an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent for treating a PD-L1-related disease or disorder may be an amount necessary to (i) slow or stop the progression of the disease or disorder; or (ii) reverse, alleviate or eliminate one or more symptoms of the disease or disorder. In some aspects of the present invention, an effective amount is an amount of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent that, when administered to a subject in need of treatment for a PD-L1-related disease or disorder, results in a therapeutic response that prevents and/or treats the disease or disorder. According to some aspects of the invention, an effective amount is an amount of the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the invention that, when combined or co-administered with another treatment method for a PD-L1-related disease or condition, results in a therapeutic response that prevents and/or treats the disease or condition. In some embodiments of the invention, the biological effect of treating a subject with the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the invention may be an improvement and/or complete elimination of symptoms caused by a PD-L1-related disease or condition. In some embodiments of the invention, the biological effect is complete elimination of a PD-L1-related disease or condition, as demonstrated, for example, by a diagnostic test showing that the subject does not have a PD-L1-related disease or condition. Non-limiting examples of physiological symptoms that can be detected include a decrease in the level of PD-L1 in the liver of a subject after administration of the medicament of the present invention. Other methods known in the art for assessing the status of PD-L1-related diseases or disorders can be used to determine the effects of the medicaments and/or methods of the present invention on PD-L1-related diseases or disorders.

The effective amount of a PD-L1 dsRNA reagent or a PD-L1 antisense polynucleotide reagent that reduces the activity of a yellow PD-L1 polypeptide to a level that treats a PD-L1-related disease or condition is generally determined in a clinical trial, where an effective dose is established for a test population versus a control population in a blinded study. In certain embodiments, an effective amount is an amount that results in a desired response, for example, an amount that reduces a PD-L1-related disease or condition in cells, tissues, and/or subjects suffering from the disease or condition. Therefore, the effective amount of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent for treating a PD-L1-related disease or condition that can be treated by reducing the activity of a PD-L1 polypeptide may be an amount that, when administered, reduces the amount of PD-L1 polypeptide activity in a subject to an amount less than the amount of PD-L1 polypeptide activity present in cells, tissues, and/or subjects when the PD-L1 dsRNA agent or the PD-L1 antisense polynucleotide agent is not administered. In certain aspects of the present invention, the PD-L1 polypeptide activity and/or PD-L1 gene expression level present in cells, tissues, and/or subjects that are not contacted with or administered the PD-L1 dsRNA agent or the PD-L1 antisense polynucleotide agent of the present invention is referred to as a "control" amount. In some embodiments of the methods of the present invention, the control amount of the subject is the pre-treatment amount of the subject. In other words, the level of the subject before the administration of the PD-L1 agent can be the control level of the subject and is used to compare with its PD-L1 polypeptide activity and/or PD-L1 gene expression level after the siRNA is administered to the subject. In the case of treating a PD-L1-related disease or condition, the desired response may be to reduce or eliminate one or more symptoms of the disease or condition in cells, tissues and/or subjects. The reduction or elimination may be temporary or permanent. It should be understood that the status of PD-L1-related diseases or conditions can be monitored using methods such as determining PD-L1 polypeptide activity, PD-L1 gene expression, symptom assessment, clinical testing, etc. In some aspects of the invention, the desired response to treatment of a PD-L1-associated disease or disorder is to delay the onset of the disease or disorder or even prevent the onset of the disease or disorder.

The effective amount of a compound that reduces the activity of a PD-L1 polypeptide can also be determined by evaluating the physiological effects of administering a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent to cells or subjects (e.g., a reduction in PD-L1-related diseases or conditions after administration). Assays and/or symptom monitoring of subjects can be used to determine the efficacy of the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention (which can be administered in the form of a pharmaceutical compound of the present invention) and to determine whether there is a response to treatment. As a non-limiting example, one or more PD-L1 biological activity tests known in the art are evaluated by the presence of an immune response, which is manifested by the presence of antibodies or immune cells against an infectious agent, by the reduction of one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by detecting the level of anti-HBsAg antibodies in the subject to evaluate the hepatitis B surface antigen (HBsAg), HBeAg or HB cccDNA in the subject's serum. As another non-limiting example, one or more liver function tests known in the art can be used to determine the state of PD-L1-related lipid imbalance in the subject before and after treatment of the subject with the PD-L1 dsRNA agent of the present invention.

Some embodiments of the invention include methods of determining the efficacy of a PD-L1 dsRNA reagent or a PD-L1 antisense polynucleotide reagent of the invention administered to a subject for treating a PD-L1-related disease or disorder by assessing and/or monitoring one or more "physiological characteristics" of the PD-L1-related disease or disorder in the subject. Non-limiting examples of physiological characteristics of a PD-L1-related disease or disorder are PD-L1 mRNA levels, PD-L1 protein levels, or a decrease in PD-L1 expression assessed indirectly by measuring a decrease in PD-L1 biological activity or PD-L1 levels in a subject sample (e.g., a serum sample), by measuring a decrease in proteins, nucleic acids, or carbohydrates present in an infectious agent, by assessing an immune response against an infectious agent by antibodies or immune cells, by a decrease in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by measuring the level of hepatitis B antigen (HBsAg), HBeAg, or HB cccDNA in a subject's serum, by detecting anti-HBsAg antibody levels in a subject. Standard methods for determining such physiological characteristics are known in the art and include, but are not limited to, blood tests, imaging studies, physical examination, etc.

It should be understood that the amount of PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent administered to a subject can be adjusted based at least in part on the determination of the subject's disease and/or condition state and/or physiological characteristics. The therapeutic amount can be changed, for example, by increasing or decreasing the amount of PD-L1-dsRNA agent or PD-L1 antisense polynucleotide agent, by changing the composition of the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent, respectively, by changing the route of administration, by changing the time of administration, etc. The effective amount of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent will vary with the specific condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of co-treatment (if any), the specific route of administration, and other factors within the knowledge and expertise of a health practitioner. For example, the effective amount may depend on the desired PD-L1 polypeptide activity and/or PD-L1 gene expression level for effective treatment of a PD-L1-related disease or condition. The effective amount of a specific PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent for use in the methods of the present invention can be determined empirically by a skilled artisan without undue experimentation. In combination with the teachings provided herein, by selecting from the various PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents of the present invention and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred administration methods, an effective preventive or therapeutic regimen can be planned to effectively treat a particular subject. As used in the embodiments of the present invention, an effective amount of the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention can be an amount that produces a desired biological effect in a cell when in contact with the cell.

It should be recognized that PD-L1 gene silencing can be determined in any cell expressing PD-L1, whether constitutively or by genomic engineering, and by any appropriate assay. In some embodiments of the invention, PD-L1 gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% by administering a PD-L1 dsRNA agent of the invention. In some embodiments of the present invention, by administering the PD-L1 dsRNA agent of the present invention, PD-L1 gene expression is reduced by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100%, or 50% to 100%. Dosage

PD-L1 dsRNA agents and PD-L1 antisense polynucleotide agents are delivered in a pharmaceutical composition in an amount sufficient to inhibit PD-L1 gene expression. In certain embodiments of the present invention, the dose of PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents is in the range of 0.01 to 200.0 mg per kilogram of body weight per day of the recipient, generally 1 to 50 mg/kg body weight, 5 to 40 mg/kg body weight, 10 to 30 mg/kg body weight, 1 to 20 mg/kg body weight, 1 to 10 mg/kg body weight, 4 to 15 mg/kg body weight per day, including end values. For example, PD-L1 The dosage of each single dose of the dsRNA agent or the PD-L1 antisense polynucleotide agent can be from about 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg g, 3.4mg/kg, 3.5mg/kg, 3.6mg/kg, 3.7mg/kg, 3.8mg/kg, 3.9mg/kg, 4mg/kg, 4.1mg/kg, 4.2m g/kg, 4.3mg/kg, 4.4mg/kg, 4.5mg/kg, 4.6mg/kg, 4.7mg/kg, 4.8mg/kg, 4.9mg/kg, 5mg/kg, 5. 1mg/kg, 5.2mg/kg, 5.3mg/kg, 5.4mg/kg, 5.5mg/kg, 5.6mg/kg, 5.7mg/kg, 5.8mg/kg, 5.9mg/k g, 6mg/kg, 6.1mg/kg, 6.2mg/kg, 6.3mg/kg, 6.4mg/kg, 6.5mg/kg, 6.6mg/kg, 6.7mg/kg, 6.8mg /kg, 6.9mg/kg, 7mg/kg, 7.1mg/kg, 7.2mg/kg, 7.3mg/kg, 7.4mg/kg, 7.5mg/kg, 7.6mg/kg, 7. 7mg/kg, 7.8mg/kg, 7.9mg/kg, 8mg/kg, 8.1mg/kg, 8.2mg/kg, 8.3mg/kg, 8.4mg/kg, 8.5mg/kg, 8.6mg/kg, 8.7mg/kg, 8.8mg/kg, 8.9mg/kg, 9mg/kg, 9.1mg/kg, 9.2mg/kg, 9.3mg/kg, 9.4mg/ kg, 9.5mg/kg, 9.6mg/kg, 9.7mg/kg, 9.8mg/kg, 9.9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg/ kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg, 32mg/k g, 33mg/kg, 34mg/kg, 35mg/kg, 36mg/kg, 37mg/kg, 38mg/kg, 39mg/kg, 40mg/kg, 41mg/kg, 42m g/kg, 43mg/kg, 44mg/kg, 45mg/kg, 46mg/kg, 47mg/kg, 48mg/kg, 49mg/kg, to 50mg/kg body weight.

Various factors may be considered when determining the dosage and administration schedule of the PD-L1 dsRNA agent of the present invention. The absolute amount of the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent administered will depend on various factors, including co-treatment, number of doses, and individual subject parameters, including age, physical condition, body size, and weight. These factors are well known to those of ordinary skill in the art and can be resolved with routine experiments. In certain embodiments, a maximum dose may be used, i.e., the highest safe dose based on reasonable medical judgment.

In certain embodiments, the methods of the invention may include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent to a subject. In some cases, a drug compound (e.g., a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent) may be administered to a subject at least daily, every other day, every week, every other week, every month, etc. The dose may be administered once a day or multiple times a day, for example, 2, 3, 4, 5 or more times within a 24-hour period. The pharmaceutical composition of the present invention can be administered once a day, or the PD-L1 dsRNA agent or the PD-L1 antisense polynucleotide agent can be administered as two, three or more sub-doses at appropriate intervals throughout the day, or even by continuous infusion or by controlled release formulation. In some embodiments of the method of the present invention, the pharmaceutical composition of the present invention is administered to the subject once or more per day, once or more per week, once or more per month, or once or more per year.

The methods of the present invention include, in certain aspects, administering a drug compound alone, in combination with one or more other PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents, and/or in combination with other drug therapies or therapeutic activities or regimens administered to a subject suffering from a PD-L1-related disease or condition. The drug compound may be administered in the form of a pharmaceutical composition. The pharmaceutical composition used in the methods of the present invention may be sterile and contain an amount of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent that reduces the activity of a PD-L1 polypeptide to a level sufficient to produce the desired response in a weight or volume unit suitable for administration to a subject. The dose of the drug composition comprising a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent administered to a subject to reduce PD-L1 protein activity can be selected based on various parameters, particularly the route of administration used and the condition of the subject. Other factors include the desired duration of treatment. If a subject has an inadequate response at the initial dose, a higher dose can be used (or the dose can be effectively increased by a different, more localized route of delivery) as tolerated by the patient. Treatment

As used herein, "PD-L1-related diseases", "PD-L1-related diseases and conditions" and "diseases and conditions caused and/or regulated by PD-L1" are intended to include any disease associated with the PD-L1 gene or protein. Such diseases may be caused by, for example, overproduction of PD-L1 protein, mutation of the PD-L1 gene, abnormal cleavage of PD-L1 protein, abnormal interactions between PD-L1 and other proteins or other endogenous or exogenous substances. Exemplary PD-L1-related diseases include, but are not limited to, tumors or hematological malignancies (e.g., lymphoma/leukemia, hematological malignancies, breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, liver cancer, salivary gland cancer, gastric cancer, neuroglioma, thyroid cancer, thymic epithelial cancer, head cancer, kidney cancer, pancreatic cancer, and neck cancer), infectious diseases (e.g., viral, bacterial, fungal, or parasitic diseases). In certain embodiments, the infectious disease is a chronic infectious disease, such as caused by viruses (e.g., HIV, HBV, HCV, and HTLV, etc.), bacteria (e.g., Helicobacter pylori, etc.), and parasites (e.g., Schistosoma mansoni).

In certain aspects of the invention, the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the invention may be administered to a subject at one or more times before or after a PD-L1-related disease or condition is diagnosed. In some aspects of the invention, the subject is at risk of having or developing a PD-L1-related disease or condition. A subject at risk of developing a PD-L1-related disease or condition is a subject with an increased likelihood of developing a PD-L1-related disease or condition compared to a control risk of developing a PD-L1-related disease or condition. In some embodiments of the invention, the risk level is statistically significant compared to the control level of risk. At-risk subjects may include, for example, subjects who have or will become susceptible to a pre-existing disease and/or genetic abnormality that makes the subject more susceptible to a PD-L1-related disease or disorder than a control subject without the pre-existing disease or genetic abnormality; subjects with a family and/or personal history of a PD-L1-related disease or disorder; and subjects who have previously been treated for a PD-L1-related disease or disorder. It should be understood that a pre-existing disease and/or genetic abnormality that makes a subject more susceptible to a PD-L1-related disease or disorder may be a disease or genetic abnormality that, when present, has previously been identified as being associated with a higher likelihood of developing a PD-L1-related disease or disorder.

It should be understood that a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent may be administered to a subject according to the individual subject's medical condition. For example, the medical care provided to the subject may assess the PD-L1 level measured in a sample obtained from the subject and determine that it is desirable to reduce the subject's PD-L1 level by administering a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent of the present invention. In this example, even if the subject is not diagnosed with a PD-L1-related disease disclosed herein, the PD-L1 level may be considered a physiological characteristic of a PD-L1-related disease. A healthcare provider can monitor changes in the subject's PD-L1 level as a measure of the efficacy of the administered PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention. In a non-limiting example, a biological sample, such as a blood or serum sample, can be obtained from the subject and the subject's PD-L1 level is determined in the sample. A PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent is administered to the subject, and a blood sample is obtained from the subject after administration, the PD-L1 level is determined using the sample, and the result is compared with the result determined in the subject's sample before administration (previous). A decrease in the subject's PD-L1 level in a subsequent sample compared to the pre-administration level indicates that the administered PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent is effective in lowering the subject's lipid levels.

Certain embodiments of the methods of the invention include modulating treatment comprising administering a dsRNA agent or PD-L1 antisense polynucleotide agent of the invention to a subject, based at least in part on an assessment of changes in one or more physiological characteristics of a PD-L1-related disease or condition in the subject resulting from the treatment. For example, in some embodiments of the invention, the effect of an administered dsRNA agent or PD-L1 antisense polynucleotide agent of the invention on a subject can be determined and used to help adjust the amount of the dsRNA agent or PD-L1 antisense polynucleotide agent of the invention subsequently administered to the subject. In one non-limiting example, a dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention is administered to a subject, the subject's PD-L1 level is determined after administration, and based at least in part on the determined level, it is determined that a larger amount of the dsRNA agent or PD-L1 antisense polynucleotide agent is needed to increase the physiological effect of the administered agent, such as to reduce or further reduce the subject's PD-L1 level. In another non-limiting example, a dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention is administered to a subject, the subject's PD-L1 level is determined after administration, and based at least in part on the determined level, a lower amount of the dsRNA agent or PD-L1 antisense polynucleotide agent needs to be administered to the subject.

Therefore, some embodiments of the present invention include evaluating changes in one or more physiological characteristics of a subject caused by a previous treatment to adjust the amount of the dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention subsequently administered to the subject. Some embodiments of the methods of the present invention include 1, 2, 3, 4, 5, 6 or more PD-L1 related disease or disorder physiological characteristic measurements to evaluate and/or monitor the efficacy of the administered PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent, and optionally use these measurements to adjust one or more of the following: the dosage, administration regimen and/or administration frequency of the dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention to treat the PD-L1 related disease or disorder in the subject. In some embodiments of the present invention, the desired result of administering an effective amount of the dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention to a subject is to reduce the PD-L1 level in the subject. A decrease in mRNA levels, a decrease in PD-L1 protein levels in a subject, or a decrease in PD-L1 expression is assessed indirectly by measuring a decrease in PD-L1 biological activity or PD-L1 levels in a sample from the subject (e.g., a serum sample), by measuring a decrease in proteins, nucleic acids, or carbohydrates present in an infectious agent, by assessing an immune response to antibodies or immune cells directed against an infectious agent, by a decrease in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by measuring the level of hepatitis B antigen (HBsAg), HBeAg, or HB cccDNA in the subject's serum, or by detecting the subject's anti-HBsAg antibody level.

As used herein, when used with respect to a PD-L1-related disease or disorder, the term "treatment" or "treated" or "being treated" may refer to preventive treatment that reduces the likelihood of a subject developing a PD-L1-related disease or disorder, or may refer to treatment performed after a subject has developed a PD-L1-related disease or disorder to eliminate or reduce the level of a PD-L1-related disease or disorder, prevent the PD-L1-related disease or disorder from becoming more advanced (e.g., more severe) and/or slow down the progression of a PD-L1-related disease or disorder in a subject compared to a subject who has not received treatment, thereby reducing the activity of a PD-L1 polypeptide in the subject.

Certain embodiments of the agents, compositions and methods of the present invention can be used to inhibit PD-L1 gene expression. The terms "inhibit", "silencing", "reducing", "downregulating" and "knockdown" used herein with respect to PD-L1 gene expression refer to that when a cell, cell group, tissue, organ or subject is contacted with (e.g., treated with) the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention, the expression of the PD-L1 gene is reduced, as measured by one or more of the following in a cell, cell group, tissue, organ or subject that transcribes the PD-L1 gene: the level of RNA transcribed from the gene, the level of PD-L1 activity expressed, and the level of PD-L1 polypeptide, protein or protein subunit translated from mRNA, respectively, compared to the level of RNA transcribed from a control PD-L1 gene, the level of PD-L1 activity expressed, or the level of PD-L1 translated from mRNA. In some embodiments, the control level is the level in a cell, tissue, organ, or subject that has not been contacted with (e.g., treated with) a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent .

A variety of routes of administration of PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents can be used in the methods of the present invention. The specific mode of administration selected depends at least in part on the specific condition being treated and the dose required to achieve the therapeutic effect. In general, the methods of the present invention can be implemented using any medically acceptable mode of administration, i.e., any mode that produces an effective level of treatment for a PD-L1-related disease or condition without causing clinically unacceptable adverse reactions. In some embodiments of the present invention, PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents can be administered orally, enterally, mucosally, subcutaneously and/or parenterally. The term "parenteral" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal and intrasternal injection or infusion techniques. Other routes include, but are not limited to, nasal (e.g., through a gastric nasal tube), skin, vagina, rectum, sublingual, and inhalation. The delivery route of the present invention may include intrathecal, intraventricular, or intracranial. In some embodiments of the present invention, a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent may be placed in a sustained-release matrix and administered by placing the matrix in the subject. In some aspects of the present invention, nanoparticles coated with a delivery agent targeting specific cells or organelles may be used to deliver a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent to a subject's cells. Various delivery means, methods, and agents are known in the art. Non-limiting examples of delivery methods and delivery agents are also provided elsewhere herein. In some aspects of the present invention, the term "delivery" with respect to a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent may refer to the administration of one or more "naked" PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent sequences to a cell or a subject, and in certain aspects of the present invention, "delivery" refers to administration to a cell or a subject via transfection means, delivery of cells comprising a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent to a subject, delivery of a vector encoding a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent to a cell and/or a subject, etc. Delivery of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent using transfection means may include administering a vector to a cell and/or a subject.

In some methods of the invention, one or more PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents may be administered in a formulation that may be administered in a pharmaceutically acceptable solution, which may generally contain pharmaceutically acceptable concentrations of salt, buffers, preservatives, compatible carriers, adjuvants, and optional other therapeutic ingredients. In some embodiments of the invention, PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents may be formulated with another therapeutic agent for simultaneous administration. According to the methods of the invention, PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents may be administered in the form of a pharmaceutical composition. Typically, the pharmaceutical composition comprises a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent and an optional pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. As used herein, a pharmaceutically acceptable carrier refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient (e.g., the ability of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent to inhibit PD-L1 gene expression in a cell or subject). A variety of methods for administering and delivering dsRNA agents or PD-L1 antisense polynucleotide agents for therapeutic use are known in the art and can be used in the methods of the present invention.

Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Patent No. 5,211,657, and other carriers known to those skilled in the art. Such formulations may generally contain salts, buffers, preservatives, compatible carriers, and optional other therapeutic agents. When used in a drug, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may be conveniently used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. In addition, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium salts, potassium salts or calcium salts.

Some embodiments of the methods of the present invention include administering one or more PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents directly to a tissue. In certain embodiments, the tissue to which the compound is administered is a tissue in which a PD-L1-related disease or condition exists or may occur, a non-limiting example of which is the liver. Direct tissue administration can be achieved by direct injection or other means. Many orally delivered compounds naturally reach and pass through the liver and kidneys, and some embodiments of the treatment methods of the present invention include orally administering one or more PD-L1 dsRNA agents to a subject. PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents, whether administered alone or in combination with other therapeutic agents, can be administered once, or can be administered multiple times. If multiple administrations are given, the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent may be administered by different routes. For example, although not intended to be limiting, the first (or first few) administrations may be subcutaneous, and one or more additional administrations may be oral and/or systemic.

For embodiments of the invention in which it is desired to administer the PD-L1 dsRNA agent or the PD-L1 antisense polynucleotide agent systemically, the PD-L1 dsRNA agent or the PD-L1 antisense polynucleotide agent can be formulated for parenteral administration by injection, such as by bolus injection or continuous infusion. Injectable formulations can be in unit dose form, such as in ampoules or multi-dose containers, with or without added preservatives. The PD-L1 dsRNA agent formulation (also referred to as a pharmaceutical composition) can be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain a formulation agent, such as a suspending agent, a stabilizer and/or a dispersing agent.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or non-volatile oils. Intravenous carriers include liquids and nutritional supplements, electrolyte supplements (such as supplements based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases. Other forms of administration (such as intravenous administration) will have lower doses. If the subject does not respond adequately to the initial dose, a higher dose may be used within the patient's tolerance range (or the dose may be effectively increased by a different, more localized delivery route). Multiple doses may be used daily as needed to achieve appropriate systemic or local levels of one or more PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents and to achieve an appropriate reduction in PD-L1 protein activity.

In other embodiments, the methods of the invention include the use of a delivery vehicle, such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient, such as a subject. Exemplary bioerodible implants that may be useful according to the methods are described in PCT Publication No. WO 95/24929 (incorporated herein by reference), which describes a biocompatible, biodegradable polymer matrix for containing biomacromolecules.

In the methods of the present invention, non-biodegradable and biodegradable polymer matrices can be used to deliver one or more PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents to a subject. In certain embodiments, the matrix can be biodegradable. The matrix polymer can be a natural or synthetic polymer. The polymer can be selected based on the time period over which release is required, typically from a few hours to a year or more. Typically, release over a period of time between a few hours and three to twelve months can be used. The polymer is optionally in the form of a hydrogel that can absorb up to about 90% of its weight in water and is further optionally cross-linked with multivalent ions or other polymers.

In general, in some embodiments of the invention, PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents can be delivered by diffusion or by degradation of a polymer matrix using a bioerodible implant. Exemplary synthetic polymers for such uses are well known in the art. Biodegradable polymers and non-biodegradable polymers can be used to deliver PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents using methods known in the art. Bioadhesive polymers such as bioerodible hydrogels (see H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein by reference) can also be used to deliver PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents to treat PD-L1 related diseases or conditions. Other suitable delivery systems may include slow release, delayed release, or sustained release delivery systems. Such systems can avoid repeated administration of PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents, thereby increasing convenience for subjects and healthcare professionals. Many types of release delivery systems are available and understood by those of ordinary skill in the art. (See, e.g., U.S. Patent Nos. 5,075,109; 4,452,775; 4,675,189; 5,736,152; 3,854,480; 5,133,974; and 5,407,686 (the teachings of each of which are incorporated herein by reference). In addition, pump-based hardware delivery systems may be used, some of which are suitable for implantation.

The use of long-term sustained release implants may be appropriate for preventive treatment of subjects and for subjects at risk for recurrent PD-L1-related diseases or conditions. As used herein, long-term release refers to the construction and arrangement of the implant to continuously deliver therapeutic levels of PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents for at least 10 days, 20 days, 30 days, 60 days, 90 days, six months, a year or more. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above.

Therapeutic preparations of PD-L1 dsRNA agents or PD-L1 antisense polynucleotide agents can be prepared by mixing a molecule or compound having the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer, and stored in the form of a lyophilized preparation or an aqueous solution [Remington's Pharmaceutical Sciences 21st edition, (2006)]. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethylammonium chloride; benzoammonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as TWEEN ^® , PLURONICS ^® , or polyethylene glycol (PEG). Cells, subjects, and controls

The methods of the present invention can be used with cells, tissues, organs and/or subjects. In some aspects of the present invention, the subject is a human or a vertebrate mammal, including but not limited to dogs, cats, horses, cows, goats, mice, rats, and primates, such as monkeys. Therefore, the present invention can be used to treat PD-L1-related diseases or conditions in human and non-human subjects. In some aspects of the present invention, the subject can be a farm animal, a zoo animal, a domestic animal, or a non-domestic animal, and the methods of the present invention can be used in veterinary prevention and treatment programs. In some embodiments of the present invention, the subject is a human, and the methods of the present invention can be used in human prevention and treatment programs.

Non-limiting examples of subjects to which the present invention may be applied are subjects diagnosed with, suspected of having, or at risk of having a disease or condition associated with: higher than desired PD-L1 expression and/or activity, also referred to as "elevated PD-L1 expression levels". Non-limiting examples of diseases and conditions associated with higher than desired PD-L1 expression and/or activity are described elsewhere herein. The methods of the present invention may be applied to subjects diagnosed with a disease or condition associated with higher than desired PD-L1 expression and/or activity at the time of treatment, or subjects believed to be at risk of having or developing a disease or condition associated with higher than desired PD-L1 expression and/or activity. In some aspects of the invention, the disease or condition associated with higher than desired PD-L1 expression levels and/or activity is an acute disease or condition, while in certain aspects of the invention, the disease or condition associated with higher than desired PD-L1 expression levels and/or activity is a chronic disease or condition.

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

According to the method of the present invention, the level of PD-L1 polypeptide activity can be determined and compared with a control level of PD-L1 polypeptide activity. The control can be a predetermined value, which can take a variety of forms. It can be a single cutoff value, such as a median or mean. It can be established based on a comparison group, such as a group with normal levels of PD-L1 polypeptide and/or PD-L1 polypeptide activity and a group with increased levels of PD-L1 polypeptide and/or PD-L1 polypeptide activity. Another non-limiting example of a comparison group can be a group with one or more symptoms or diagnoses of a PD-L1-related disease or condition; a group without one or more symptoms or diagnoses of a disease or condition; a group of subjects who have been treated with the siRNA of the present invention; a group of subjects who have not been treated with the siRNA of the present invention. Typically, the control may be based on apparently healthy normal individuals of appropriate age or apparently healthy cells. It should be understood that, in addition to predetermined values, the control according to the present invention may be a sample of material tested in parallel with the experimental material. Examples include samples from a control population or control samples produced by manufacturing for parallel testing with the experimental samples. In some embodiments of the present invention, the control may include cells or subjects that have not been exposed to or treated with the PD-L1 dsRNA agent of the present invention, and in this case, the control level of PD-L1 polypeptide and/or PD-L1 polypeptide activity may be compared to the PD-L1 polypeptide and/or PD-L1 polypeptide activity level in cells or subjects exposed to the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention.

In certain embodiments of the present invention, the control level can be a PD-L1 polypeptide level determined for a subject, wherein the PD-L1 polypeptide level determined for the same subject at different times is compared to the control level. In a non-limiting example, the PD-L1 level is measured in a biological sample obtained from a subject who has not received the PD-L1 treatment of the present invention. In certain embodiments, the biological sample is a serum sample. The PD-L1 polypeptide level measured in the sample obtained from the subject can serve as a baseline or control value for the subject. After administering one or more PD-L1 dsRNA agents to a subject in the treatment methods of the present invention, one or more additional serum samples can be obtained from the subject, and the PD-L1 polypeptide level in the subsequent sample can be compared to the control/baseline level of the subject. Such a comparison can be used to assess the onset, progression or regression of a PD-L1-related disease or condition in a subject. For example, a PD-L1 polypeptide level in a baseline sample obtained from a subject is higher than the level obtained from the subject after administration of a PD-L1 dsRNA agent or a PD-L1 antisense polynucleotide agent of the present invention to the same subject, indicating that the PD-L1-related disease or condition has regressed, and indicating that the administered PD-L1 dsRNA agent of the present invention is effective for treating the PD-L1-related disease or condition.

In certain aspects of the invention, the value of PD-L1 polypeptide level and/or PD-L1 polypeptide activity can be used as a control value to which PD-L1 polypeptide level and/or PD-L1 activity can be compared later in the same subject, thereby allowing assessment of changes in the subject relative to a "baseline" PD-L1 polypeptide activity. Thus, an initial PD-L1 polypeptide level and/or initial PD-L1 polypeptide activity level may be present and/or determined in a subject, and the methods and compounds of the invention can be used to reduce the level of PD-L1 polypeptide and/or PD-L1 polypeptide activity in a subject, wherein the initial level is used as a control level for that subject.

Using the methods of the present invention, the PD-L1 dsRNA agent and/or the PD-L1 antisense polynucleotide agent of the present invention can be administered to a subject. When the level of PD-L1 polypeptide in a serum sample obtained from a subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the pre-administration level of PD-L1 polypeptide in a serum sample obtained from the subject at a previous time point, or compared to a non-contact control level (e.g., the level of PD-L1 polypeptide in a control serum sample), the effectiveness of the administration and treatment of the present invention can be assessed. It should be understood that both the level of PD-L1 polypeptide and the level of PD-L1 polypeptide activity are related to the level of PD-L1 gene expression. Certain embodiments of the methods of the present invention include administering to a subject an effective amount of the PD-L1 dsRNA and/or PD-L1 antisense of the present invention to inhibit PD-L1 gene expression and thereby reduce the subject's PD-L1 polypeptide level and PD-L1 polypeptide activity level.

Certain embodiments of the present invention include determining the presence, absence, and/or amount (also referred to herein as level) of a PD-L1 polypeptide in one or more biological samples obtained from one or more subjects. This determination can be used to assess the effectiveness of the treatment methods of the present invention. For example, the methods and compositions of the present invention can be used to determine the level of a PD-L1 polypeptide in a biological sample obtained from a subject previously treated with a PD-L1 dsRNA agent and/or a PD-L1 antisense agent of the present invention. A level of PD-L1 polypeptide measured in a serum sample obtained from a treated subject that is at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more lower than the pre-treatment PD-L1 polypeptide level determined for the subject or compared to the level in an uncontacted control biological sample indicates a level of efficacy of the treatment administered to the subject.

In some embodiments of the present invention, the physiological characteristics of a PD-L1-related disease or condition determined for a subject can be used as a control result, and the results of the determination of the physiological characteristics of the same subject at different times can be compared with the control results. In non-limiting examples, physiological characteristics such as PD-L1 mRNA levels, PD-L1 protein levels in a subject, a decrease in PD-L1 expression is assessed indirectly by measuring a decrease in PD-L1 biological activity or PD-L1 levels in a sample (e.g., a serum sample) from the subject, by measuring a decrease in proteins, nucleic acids, or carbohydrates present in an infectious agent, by assessing an immune response against an infectious agent by antibodies or immune cells, by a decrease in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by measuring hepatitis B antigen (HBsAg), HBeAg, or HB in the subject's serum. cccDNA level, by detecting the level of anti-HBsAg antibodies in the subject, by detecting the level of anti-HBsAg antibodies in the subject or plasma or tissue sample, the antibody level is determined in a biological sample (e.g., serum sample) obtained from a subject who has not received the PD-L1 treatment of the present invention. The PD-L1 mRNA level (and/or other physiological characteristics of PD-L1 disease or disorder) determined in a sample obtained from the subject can be used as a baseline or control value for the subject. After administering one or more PD-L1 dsRNA agents to a subject in the treatment methods of the present invention, one or more additional serum samples may be obtained from the subject, and the PD-L1 mRNA level and/or PD-L1 protein level in the subsequent samples may be compared to the control/baseline level and/or ratio of the subject, respectively. Such comparisons may be used to assess the onset, progression, or regression of a PD-L1-related disease or condition in a subject. For example, a PD-L1 mRNA level in a baseline sample obtained from a subject that is higher than the PD-L1 mRNA level measured in a sample obtained from the subject after administration of the PD-L1 dsRNA agent or PD-L1 antisense polynucleotide agent of the present invention to the same subject indicates that the PD-L1-related disease or condition has regressed, and indicates that the administered PD-L1 dsRNA agent of the present invention is effective for treating the PD-L1-related disease or condition.

In certain aspects of the invention, one or more physiological characteristic values determined for a subject for a PD-L1-related disease or condition can be used as a control value to later compare the physiological characteristic of the same subject, thereby allowing the subject's changes relative to a "baseline" physiological characteristic to be assessed. Thus, a subject may have and/or have an initial physiological characteristic determined, and the methods and compounds of the invention can be used to reduce the level of PD-L1 polypeptide and/or PD-L1 polypeptide activity in the subject, wherein the initial physiological characteristic measurement value is used as a control for the subject.

Using the methods of the present invention, the PD-L1 dsRNA agent and/or PD-L1 antisense polynucleotide agent of the present invention can be administered to a subject in an effective amount to treat a PD-L1 disease or disorder. The efficacy of the administration and treatment of the present invention can be assessed by determining changes in one or more physiological characteristics of a PD-L1 disease or disorder. In a non-limiting example, the PD-L1 mRNA level in a serum sample obtained from a subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to pre-dose lipids in a serum sample obtained from a subject at a previous time point, or compared to a non-contact control level (e.g., PD-L1 mRNA level in a control serum sample). It should be understood that the subject's PD-L1 mRNA level, PD-L1 protein level, or lipid level, triglyceride, cholesterol level, free fatty acid level in plasma or tissue samples are each associated with the PD-L1 gene expression level. Certain embodiments of the methods of the present invention include administering to the subject an effective amount of the PD-L1 dsRNA and/or PD-L1 antisense of the present invention to inhibit PD-L1 gene expression and thereby reduce the subject's PD-L1 mRNA level, PD-L1 protein level, or otherwise positively affect the subject's physiological characteristics of PD-L1-related diseases or disorders.

Some embodiments of the present invention include determining the presence, absence and/or changes in physiological characteristics of a PD-L1-related disease or condition using methods such as, but not limited to: (1) evaluating physiological characteristics of one or more biological samples obtained from one or more subjects; (2) imaging the subject (such as, but not limited to, obtaining liver images); and (3) performing a physical examination on the subject. This determination can be used to evaluate the effectiveness of the treatment methods of the present invention. Kits

Also included within the scope of the present invention are kits containing one or more PD-L1 dsRNA agents and/or PD-L1 antisense polynucleotide agents and instructions for use in the methods of the present invention. The kits of the present invention may include one or more PD-L1 dsRNA agents, PD-L1 sense polynucleotides, and PD-L1 antisense polynucleotide agents, which can be used to treat PD-L1 related diseases or conditions. Kits containing one or more PD-L1 dsRNA agents, PD-L1 sense polynucleotides, and PD-L1 antisense polynucleotide agents can be prepared for use in the treatment methods of the present invention. The components of the kit of the present invention can be packaged in an aqueous medium or in a lyophilized form. The kit of the present invention may include a carrier that is divided to contain one or more container devices or a series of container devices (e.g., test tubes, vials, flasks, bottles, syringes, etc.) in a sealed manner therein. The first container device or a series of container devices may contain one or more compounds, such as PD-L1 dsRNA agents and/or PD-L1 sense or antisense polynucleotide agents. The second container device or a series of container devices may contain targeting agents, labeling agents, delivery agents, etc., which can be included as part of the PD-L1 dsRNA agents and/or PD-L1 antisense polynucleotides for administration in the embodiments of the treatment methods of the present invention.

The kit of the present invention may also include instructions. The instructions are usually in written form and are used to guide how to implement the treatment contained in the kit and make decisions based on the treatment.

The following examples are used to illustrate specific examples of the practice of the present invention and are not intended to limit the scope of the present invention. A person of ordinary skill in the art will appreciate that the present invention will be applied to various compositions and methods.

Embodiment 1.

Phosphamide compound 2

DMTrCl (232 g, 684 mmol, 1.0 eq.) in pyridine (400 ml) was added to a solution of compound A isomannitol (100 g, 684 mmol, 1.0 eq.) in pyridine (600 ml), and the mixture was stirred at 25°C for 12 hours. LC-MS showed that compound A was completely consumed, and a main peak with the desired mass was detected. The resulting reaction mixture was diluted with water (500 ml), extracted with dichloromethane (500 ml*2), and the combined organic phases were washed with brine (500 ml), dried over Na ₂ SO ₄ and concentrated in vacuo to obtain a residue. The residue was purified by column chromatography (DCM/MeOH=100/1 to 50/1, 0.1% Et ₃ N) to obtain compound B (150 g, yield 48.9%) as a yellow solid.

¹ H NMR: EC4783-404-P1B1_C (400 MHz, DMSO- d ₆ ) δ ppm 7.46 (br d, J =7.63 Hz, 2 H) 7.28 - 7.37 (m, 6 H) 7.19 - 7.25 (m, 1 H) 6.90 (br d, J =7.88 Hz, 4 H) 4.70 (d, J =6.50 Hz, 1 H) 3.99 - 4.09 (m, 6 H) 3.88 - 3.96 (m, 2 H) 3.83 (br dd, J =7.82, 6.94 Hz, 1 H) 3.74 (s, 6 H) 3.41 (br t, J =8.13 Hz, 1H) 3.05 (t, J =8.44 Hz, 1 H) 2.85 (br t, J =7.50 Hz, 1 H).

To a solution of compound B (80.0 g, 178 mmol, 1.0 eq ) in DCM (800 mL) was added 2H -tetrazole (0.45 M, 436 mL, 1.1 eq ) dropwise at 25 °C under _N2 atmosphere, and then a solution of compound C (80.6 g, 267 mmol, 85.0 mL, 1.5 eq ) in DCM (200 mL) was added dropwise to the mixture. The reaction mixture was stirred at 25 °C for 1.0 h. LC-MS showed that compound B was completely consumed and a major peak with the desired mass was detected. The resulting reaction mixture was cooled to -20°C and poured into ice-cold saturated NaHCO ₃ (500 mL), extracted with DCM (500 mL*3), the combined organic layers were washed with saturated NaHCO ₃ / brine = 1:1 (300 mL/300 mL), dried over Na ₂ SO ₄ and concentrated under vacuum (35°C) to obtain a residue (100 mL). The residue was purified by column chromatography (Al ₂ O ₃ , DCM/MeOH = 100/1 to 50/1, 0.1% Et ₃ N) to give compound 2 (77 g, 119 mmol, yield 66.5%) as a white solid.

¹ H NMR: EC4783-423-P1B1_C (400 MHz, DMSO- d ₆ ) δ ppm 7.22 (br d, J=7.50 Hz, 2 H) 7.05 - 7.14 (m, 6 H) 6.96 - 7.02 (m, 1 H) 6.67 (br dd, J=8.82, 1.81 Hz, 4 H) 3.95 - 4.07 (m, 2 H) 3.73 - 3.83 (m, 1 H) 3.62 - 3.72 (m, 2 H) 3.48 - 3.53 (m, 6 H) 3.27 - 3.37 (m, 3 H) 3.11 (s, 6 H) 2.82 (td, J=8.54, 2.31 Hz, 1 H) 2.47 - 2.63 (m, 3 H) 2.28 (br d, J=1.63 Hz, 3 H) 0.82 - 1.00 (m, 13 H).

Phosphamide compound 1:

To _a solution of compound B (500 mg, 1.11 mmol, 1.0 eq) in DCM (5.0 mL) were added compound D (607 mg, 3.34 mmol, 3.0 eq) and DIEA (432 mg, 3.34 mmol, 582 μL, 3.0 eq) under N 2 atmosphere at 0-5 °C, and the mixture was stirred at 25 °C for 1.0 hour. LC-MS showed that compound B was completely consumed, several new peaks appeared on LC-MS, and about 70.9% of the desired compound was detected. The resulting reaction mixture was cooled to -20°C and poured into a cold (0-5°C) saturated NaHCO ₃ (5.0 mL) solution, extracted with DCM (5.0 mL*2), and the combined organic layers were washed with cold (0-5°C) saturated NaHCO ₃ / brine = 1:1 (5.0 mL/5.0 mL), dried over Na ₂ SO ₄ and concentrated in vacuo to obtain a residue (~5 mL). The residue was purified by column chromatography (basic Al ₂ O ₃ , petroleum ether/ethyl acetate = 10/1 to 5/1, 0.1% Et ₃ N) to obtain compound 1 (280 mg, 471 μmol, yield 42.3%) as a white solid.

¹ H NMR: EC10615-49-P1N (400 MHz, DMSO- d ₆ ) δ ppm 7.44 (br d, J =7.63 Hz, 2 H), 7.31 (br t, J =7.94 Hz, 6 H), 7.18 - 7.26 (m, 1 H), 6.89 (brd, J =8.00 Hz, 4 H), 4.08 - 4.13 (m, 1 H), 3.95 - 4.03 (m, 1 H), 3.84 - 3.93 (m, 1 H), 3.77 - 3.83 (m, 1 H), 3.74 (s, 6 H), 3.43 - 3.53 (m, 3 H), 3.38 (br d, J =6.75 Hz, 1 H), 2.94 - 3.04 (m, 1 H), 2.70 - 2.85 (m, 1 H), 1.09 - 1.15 (m, 12 H), 1.07 (br s, 3 H).

Other phosphoramidites may be prepared according to the methods described herein and/or according to prior art (e.g., but not limited to, US 426,220 and WO 02/36743).

Example 2. Preparation of a solid carrier containing the phosphoramidite monomer of the present invention Represents the macroporous aminomethyl polyethylene resin carrier part

Under nitrogen protection, add dichloromethane (19.50 kg) to a 50L glass reactor and start stirring; control the temperature at 20-30°C, add DMTrimann (1.47 kg), triethylamine (1.50 kg), 4-dimethylaminopyridine (0.164 kg) and succinic anhydride (1.34 kg) to the glass reactor; keep warm at 20-30°C for 18 hours, take a sample, and terminate the reaction. Add saturated sodium bicarbonate solution (22.50 kg) to the reaction system, stir for 10-20 minutes, and separate the layers. Separate the organic phase, extract the aqueous phase twice with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, filter, and concentrate in vacuum to obtain a residue of 1.83 kg of gray to off-white solid.

Add N, N-dimethylformamide (23.50 kg) to a 100 L glass kettle and stir. Control the temperature to 20-30 ° C. Under nitrogen protection, add the product of the previous step, O-benzotriazole tetramethyluronium hexafluorophosphate (0.33 kg) and N, N-diisopropylethylamine (0.13 kg) to the above 100 L glass kettle through a solid addition funnel, stir for 10-30 minutes, and discharge into a 50 L zinc barrel for standby use. The macroporous amine methyl resin (3.25 kg) (purchased from Tianjin Nankai Synthesis Technology Co., Ltd., batch number HA2X1209, loading 0.48 mmol/g) was added to the above 100L solid phase synthesis reactor through a solid feeding funnel, and the temperature was controlled at 20-30°C. N, N-dimethylformamide (21.00 kg + 21.00 kg) and the reaction solution in the zinc barrel in the previous step were added to the solid phase synthesis reactor. The system was kept warm and the solid loading was tracked to ≥ 250 umol/g. The loading detection method was ultraviolet. Filter under nitrogen pressure, wash the filter cake three times with N, N-dimethylformamide (26.00 kg + 26.10 kg + 26.00 kg), and leave the filter cake in the kettle. Add CAP.A (50% acetonitrile and 50% acetic anhydride, 4.40kg+4.42kg+4.30kg) and CAP.B (20% pyridine and 30% N-methylimidazole and 50% acetonitrile, 4.40kg+4.40kg+4.47kg) to a 80L glass reactor and stir for 3~8min before use. Repeat this operation three times to cap the reactor, and add acetonitrile (18.00kg+18.00kg+18.00kg+17.50kg+17.50kg) to the solid phase synthesis reactor. Bubble nitrogen for 10~30min and filter. Repeat this operation four times, purge the filter cake in the solid phase synthesis reactor with nitrogen for 2~4h and transfer it to a 50L filter press. The temperature was controlled at 15-30°C and the drying was continued. After drying, a yellow to white solid product was obtained with a weight of 3.516 kg.

The isomannitol residue is added to the 5'-end or 3'-end of the oligonucleotide chain by a method well known to those skilled in the art (e.g., the invab method), and further added to the target group.

Example 3. Synthesis of PD-L1 RNAi agent.

The PD-L1 reagent bichains listed in Table 2-3 above were synthesized according to the following general procedure:

The sense and antisense sequences of siRNA are synthesized on an oligonucleotide synthesizer using a well-established solid phase synthesis method based on phosphoramidite chemistry. Extension of the oligonucleotide chain is achieved through a 4-step cycle: deprotection, condensation, capping, and oxidation or sulfurization steps for the addition of each nucleotide. The synthesis is carried out on a solid support made of controlled pore glass (CPG, 1000 Å). Monomer phosphoramidites can be purchased from commercial sources or can be the phosphoramidite compounds in Example 1. The phosphoramidite compounds herein can be attached to the 3' end as monomeric phosphoramidites and further attached to a CPG solid support. Once attached at the 5' end, the phosphoramidite compound can be used for the final coupling reaction and further conjugated to the target ligand if desired.

Phosphoramidites with GalNAc ligand clusters (GLS-5* or GLS-15* phosphoramidites as non-limiting examples) are disclosed in WO2023/045995A1 (incorporated herein in its entirety). For siRNAs for in vitro screening (Table 2), synthesis was performed at a 2 µmol scale, while for siRNAs for in vivo testing (Table 3), synthesis was performed at a 5 µmol or greater scale. In the case where the GalNAc ligand (GLO-n phosphoramidite as a non-limiting example disclosed in WO2023/045995A1 (incorporated herein in its entirety)) is attached to the 3' end of the sense strand, a GalNAc ligand-attached CPG solid support is used. In the case where a GalNAc ligand (as a non-limiting example, GLS-5* or GLS-15* is attached to the 5' end of the sense chain, GLS-5* or GLS-15* having a GalNAc ligand cluster is disclosed in WO2023/045995A1 (incorporated herein in its entirety)) is attached to the 5' end of the sense chain, the final coupling reaction is performed using GalNAc phosphoamidite. 3% trichloroacetic acid (TCA) in dichloromethane is used for deprotection of 4,4'-dimethoxytrityl protecting group (DMT). 5-Ethylthio-1H-tetrazole is used as an activator. I ₂ in THF/Py/H ₂ O and phenylacetyl disulfide (PADS) in pyridine/MeCN are used for oxidation and sulfidation reactions, respectively. After the last solid phase synthesis step, the solid support bound oligomers were cleaved and the protecting groups were removed by treatment with a 1:1 volume of 40 wt.% aqueous methylamine and 28% ammonium hydroxide solution. To synthesize siRNA for in vitro screening, the crude mixture was concentrated. The remaining solid was dissolved in 1.0 M NaOAc and ice-cold EtOH was added to precipitate the single-chain product as the sodium salt, which was used for annealing without further purification. To synthesize siRNA for in vivo testing, the crude single-chain product was further purified by ion pairing reverse phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide product from IP-RP-HPLC was converted to sodium salt by dissolution in 1.0 M NaOAc and precipitated by adding ice-cold EtOH. Equimolar complementary sense and antisense oligonucleotides were annealed in water to form a double-stranded siRNA product, which was lyophilized to obtain a fluffy white solid.

In certain studies, methods for attaching a GalNAc-containing targeting group (also referred to herein as a GalNAc delivery compound) to the 5' end of the sense strand include using a GalNAc phosphoramidite (GLS-5* or GLS-15* phosphoramidite) in the final coupling step of a solid phase synthesis, using, for example, the same synthetic process used in performing oligonucleotide chain extension to add nucleotides to the 5' end of the sense strand.

In some studies, the method of attaching a targeting group comprising GalNAc to the 3' end of the sense chain includes using a solid support (CPG) comprising GLO-n. In some studies, the method of attaching a targeting group comprising GalNAc to the 3' end of the sense chain includes attaching the GalNAc targeting group to the CPG solid support via an ester bond, and using the aforementioned CPG solid support to which the GalNAc targeting group is attached when synthesizing the sense chain, thereby obtaining the GalNAc targeting group attached to the 3' end of the sense chain.

The imann residue can be added to the 5' or 3' end of the oligonucleotide chain by methods well known to those skilled in the art, such as the invab method, and/or further added to the targeting group targeting GalNAc.

Example 4. In vitro screening of PD-L1 siRNA duplexes

SNU-387 cells were grown to near confluence in RPMI medium containing 10% FBS, streptomycin, and glutamine at 37°C in an atmosphere of 5% CO2 and then released from the culture dishes by trypsinization. Transfection was performed by adding 14.8 uL Opti-MEM and 0.2 uL Lipofectamine® RNAiMax per well to 5uL siRNA duplex per well in a 96-well plate and incubating for 15 minutes at room temperature. 80uL complete growth medium containing ~2x10 ⁴ SNU-387 cells was then added to the siRNA mixture. Cells were incubated for 24 hours before RNA purification. Single-dose experiments were performed at 0.5 nM and 0.05 nM final siRNA concentrations. mRNA was isolated and expression of mRNA (target gene) was determined by qPCR, using the housekeeping gene GAPDH for normalization. Percent knockdown was calculated by comparing mRNA (target gene) expression in siRNA-treated samples to negative control-treated samples. The duplex sequences used correspond to those shown in Table 2, and the results are summarized in Table 5.

Table 5 provides experimental results of in vitro studies using various PD-L1 RNAi drugs to inhibit PD-L1 expression. The duplex sequences used correspond to those shown in Table 2. Double Link AV# Average inhibition% ± SD 1nM 0.1nM 1 nM 0.1 nM AV00937 -15.69 -4.92 23.89 3.58 AV00938 50.63 30.55 10.69 2.27 AV00939 26.56 13.83 9.04 3.76 AV00940 52.98 36.68 7.73 5.06 AV00941 53.58 27.03 8.82 12.05 AV00942 11.57 16.40 11.20 2.67 AV00943 54.99 22.25 6.99 9.37 AV00944 48.53 15.62 14.25 8.14 AV00946 44.17 9.27 0.53 12.93 AV00947 67.45 41.09 4.37 2.36 AV00948 21.56 13.61 1.94 4.76 AV00949 63.64 26.04 3.10 13.39 AV00950 69.47 24.97 4.05 6.24 AV00952 4.54 -3.59 3.75 2.41 AV00953 2.13 -3.13 21.19 6.67 AV00954 47.58 8.77 1.92 5.62 AV00955 -5.84 -2.83 10.09 5.90 AV00956 7.47 -12.31 2.23 12.71 AV00957 3.26 4.60 3.39 4.97 AV00958 10.45 -0.63 9.72 8.85 AV00959 13.52 2.45 4.99 9.78 AV00960 17.57 1.19 5.05 10.85 AV00961 10.92 4.24 11.02 4.22 AV00962 8.87 -9.62 5.11 9.60 AV00963 -15.73 -29.57 6.16 23.24 AV00964 4.52 3.23 0.88 9.09 AV00965 -2.24 -8.97 1.43 7.85 AV00966 13.82 5.28 1.37 11.83 AV00967 13.70 7.29 4.85 5.21 AV00968 10.60 3.21 7.81 2.61 AV00969 14.95 7.49 6.99 4.65 AV00970 34.47 9.80 1.42 3.28 AV00971 20.89 9.50 9.34 10.11 AV00972 17.98 7.65 3.09 10.56 AV00973 -3.81 7.14 5.87 8.19 AV00974 20.24 5.64 6.66 5.95 AV00975 6.76 1.58 6.68 4.94 AV00976 29.83 14.08 10.93 5.29 AV00977 60.02 10.95 1.35 6.63 AV00978 21.11 22.45 5.06 6.35 AV00979 -31.38 6.99 24.28 1.65 AV00980 -29.87 0.31 30.79 6.74 AV00981 7.46 -8.30 4.50 1.57 AV00982 -1.64 -3.09 5.30 8.45 AV00983 -14.07 1.66 7.99 12.79 AV00985 24.79 7.16 6.64 1.76 AV00984 -13.10 -21.77 12.16 4.93 AV00986 8.43 -0.96 11.36 3.86 AV00987 23.96 6.47 4.70 2.02 AV00988 19.83 -1.23 1.25 7.90 AV00989 -15.29 7.88 11.51 3.35 AV00991 2.00 6.79 4.98 8.35 AV00992 -13.06 -2.31 3.19 4.99 AV00993 7.00 7.93 3.43 15.07 AV00995 23.56 9.04 1.74 7.06 AV00996 14.32 5.29 8.53 6.66 AV00997 -2.26 0.61 3.58 10.40 AV00998 41.87 10.09 14.85 17.25 AV00999 9.76 4.56 7.56 6.87 AV01000 29.87 11.74 5.10 1.10 AV01001 40.85 15.84 6.34 4.65 AV01002 52.95 24.51 2.09 3.71 AV01003 34.20 18.65 2.85 1.54 AV01004 19.94 7.51 1.95 5.14 AV01005 16.94 1.33 5.31 8.67 AV01006 5.31 7.77 8.18 5.67 AV01007 16.63 11.37 5.09 3.99 AV01008 40.34 18.18 3.39 12.21 AV01009 27.30 27.63 14.90 2.45 AV01010 38.11 11.29 5.44 10.44 AV01011 21.66 5.49 2.70 7.26 AV01012 28.57 8.68 6.40 1.44 AV01013 -15.77 -0.31 17.89 5.29 AV01014 12.56 2.94 1.76 7.64 AV01015 9.91 -2.69 4.82 9.48 AV01016 13.40 8.52 1.43 2.59 AV01017 7.83 15.97 8.84 9.56 AV01018 14.54 15.58 6.23 9.46

Example 5. In vivo testing of PD-L1 siRNA duplex

On day 1, female C57BL/6J mice (4-6 weeks old, 4 mice per group) were infected by intravenous injection of adeno-associated virus 8 (AAV8) vector solution encoding human PD-L1 and luciferase genes. On day 8, mice were subcutaneously injected with a single dose of 3 mg/kg PD-L1 siRNA drug or PBS. Blood samples were collected on day 8, before siRNA administration, day 15, day 22, and/or day 29. Plasma samples were separated and luciferase activity was measured according to the manufacturer's recommended protocol. Since the expression of human PD-L1 levels is correlated with the expression level of luciferase, the percentage of remaining PD-L1 was calculated by comparing the luciferase activity in the samples of the siRNA treatment group before and after treatment, and normalized by the change in luciferase activity of the samples of the control treatment group during the same time period. The results are summarized in Tables 6-8.

Table 6. Screening of PD-L1 siRNA with a single 3 mpk subcutaneous dose in AAV-PD-L1 transduced mice. The percent reduction of human PD-L1 in mouse serum was normalized to PD-L1 expression before siRNA administration and to the PBS control group. Double chain AD# The remaining percentage of D15 relative to D8 (mean ± SD) The remaining percentage of D22 relative to D8 (mean ± SD) Dosage (mg/kg) AD00880 1.23±0.32 0.66±0.08 3 AD00881 0.8±0.67 0.74±0.32 3 AD00882 2.17±0.87 1.46±0.81 3 AD00883 0.60±0.40 0.18±0.09 3 AD00885 0.19±0.16 0.11±0.07 3 AD00886 1.21±0.65 0.67±0.25 3 AD00887 0.51±0.37 0.65±0.40 3 AD00888 1.12±0.39 0.38±0.12 3 AD00889 1.06±0.43 0.31±0.12 3 AD00890 1.80±0.78 0.62±0.05 3 AD00891 0.94±0.44 0.40±0.20 3 AD00892 1.13±0.80 0.56±0.16 3 AD00893 1.20±0.74 0.76±0.29 3 AD00894 1.56±0.96 0.55±0.23 3 AD00895 1.61±0.74 1.00±0.52 3 AD00896 1.17±0.23 0.45±0.15 3 AD00897 1.00±0.22 0.57±0.36 3

Table 7. Screening of PD-L1 siRNA with a single 3 mpk subcutaneous dose in AAV-PD-L1 transduced mice. The percent reduction of human PD-L1 in mouse serum was normalized to PD-L1 expression before siRNA administration and to the PBS control group. Double chain AD# The remaining percentage of D15 relative to D8 (mean ± SD) The remaining percentage of D22 relative to D8 (mean ± SD) The remaining percentage of D29 relative to D8 (mean ± SD) Dosage (mg/kg) AD01052 0.23±0.09 0.33±0.09 0.44±0.3 3 AD01053 0.13±0.03 0.12±0.03 0.22±0.06 3 AD01055 0.19±0.04 0.19±0.05 0.38±0.11 3 AD01056 0.53±0.33 0.74±0.89 0.84±0.6 3 AD01057 0.59±0.18 0.8±0.38 1.3±0.53 3 AD01058 0.82±0.67 0.83±0.66 1.21±0.57 3 AD01059 1.28±0.56 1.26±0.96 1.64±0.73 3 AD01060 0.55±0.26 0.89±0.31 1.37±0.92 3 AD01061 1.54±0.52 1.04±0.47 2.2±0.37 3 AD01062 0.54±0.15 0.43±0.15 0.91±0.47 3 AD01063 0.59±0.33 0.59±0.46 0.84±0.23 3 AD01064 0.21±0.05 0.36±0.15 0.45±0.22 3 AD01065 0.89±0.26 0.53±0.21 0.65±0.17 3 AD01068 0.58±0.25 0.64±0.28 0.83±0.16 3 AD01069 0.57±0.1 0.63±0.15 0.95±0.41 3 AD01070 0.39±0.05 0.52±0.34 0.53±0.09 3 AD01071 0.41±0.21 0.42±0.11 0.66±0.17 3 AD00883 0.37±0.14 0.2±0.08 0.45±0.16 3 AD00884 0.22±0.02 0.18±0.04 0.27±0.12 3 AD00885 0.14±0.08 0.13±0.02 0.16±0.05 3 AD00887 0.72±0.27 0.74±0.31 1.15±0.31 3 AD00888 0.39±0.13 0.56±0.32 0.83±0.26 3 AD00889 0.29±0.04 0.4±0.14 0.41±0.14 3

Table 8. Screening of PD-L1 siRNA single 3 mpk subcutaneous dose in AAV-PD-L1 transduced mice. The percent reduction of human PD-L1 in mouse serum was normalized to PD-L1 expression before siRNA administration and to the PBS control group. Double chain AD# The remaining percentage of D15 relative to D8 (mean ± SD) The remaining percentage of D22 relative to D8 (mean ± SD) The remaining percentage of D29 relative to D8 (mean ± SD) Dosage (mg/kg) AD00884 0.51±0.17 0.26±0.21 0.48±0.17 3 AD00885 0.22±0.18 0.13±0.05 0.15±0.05 3 AD01053 0.22±0.1 0.13±0.01 0.19±0.07 3 AD01066 0.18±0.08 0.14±0.06 0.21±0.05 3 AD01067 0.46±0.12 0.22±0.01 0.32±0.13 3 AD01211 0.4±0.21 0.33±0.1 0.61±0.14 3 AD01212 0.54±0.34 0.28±0.06 0.33±0.23 3 AD01213 0.5±0.07 0.29±0.15 0.44±0.05 3 AD01214 0.45±0.23 0.32±0.1 0.72±0.38 3 AD01215 0.95±0.49 0.32±0.22 0.49±0.21 3 AD01216 0.9±0.28 0.29±0.16 0.46±0.23 3 AD01217 0.65±0.27 0.25±0.05 0.77±0.39 3 AD01218 0.67±0.45 0.4±0.39 0.63±0.33 3

Example 6. In vivo testing of PD-L1 siRNA duplex

On day 1, female C57BL/6J mice (4-6 weeks old, 4 mice per group) were infected by intravenous injection of adeno-associated virus 8 (AAV8) vector solution encoding human PD-L1 and luciferase genes. On day 8, mice were subcutaneously injected with a single dose of 3 mg/kg PD-L1 siRNA drug or PBS. Blood samples were collected on day 8, before siRNA administration, on days 15 and 22, and plasma samples were separated and luciferase activity was measured in plasma samples according to the manufacturer's recommended protocol. Since the expression of human PD-L1 levels is correlated with the expression level of luciferase, the percentage of remaining PD-L1 was calculated by comparing the luciferase activity in the samples of the siRNA treatment group before and after treatment, and normalized by the change in luciferase activity of the samples of the control treatment group during the same period of time. The results are summarized in Table 9.

Table 9. Screening of PD-L1 siRNA with a single 3 mpk subcutaneous dose in AAV-PD-L1 transduced mice. The percent reduction of human PD-L1 in mouse serum was normalized to PD-L1 expression before siRNA administration and to the PBS control group. Double chain AD# Remaining percentage relative to D8 (mean ± SD) Dosage (mg/kg) D15 D22 AD01053-1 0.16±0.06 0.12±0.01 3 AD00884-1 0.32±0.16 0.32±0.07 3 AD00885-1 0.06±0.03 0.24±0.13 3 AD01055-1 0.18±0.13 0.25±0.07 3 AD01066-1 0.29±0.13 0.39±0.17 3 AD01067-1 0.26±0.24 0.71±0.46 3 Equivalent

Although several embodiments of the present invention have been described and illustrated herein, a person of ordinary skill in the art will readily devise various other means and/or structures for performing the functions and/or obtaining the results and/or one or more advantages described herein, and each of these variations and/or modifications is considered to be within the scope of the present invention. More generally, a person of ordinary skill in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are intended to be examples, and that actual parameters, dimensions, materials, and/or configurations will depend on the specific application in which the teachings of the present invention are used. A person of ordinary skill in the art will recognize, or be able to determine using only routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Therefore, it should be understood that the foregoing embodiments are presented by way of example only and are within the scope of the appended patent applications and their equivalents; the present invention may be implemented in a manner different from that specifically described and claimed. The present invention is directed to each individual feature, system, article, material and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials and/or methods, if such features, systems, articles, materials and/or methods are not mutually inconsistent, are included in the scope of the present invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles "a" and "an" used in this specification and claims should be understood to mean "at least one" unless expressly stated otherwise.

The phrase "and/or" used in the specification and claims should be understood to mean "either or both" of the combined elements, that is, the elements are present in combination in some cases and separately in other cases. In addition to the elements expressly identified in the "and/or" clause, other elements may optionally be present, whether related or unrelated to the elements expressly identified, unless expressly stated otherwise.

All references, patents and patent applications and publications cited or referred to in this application are incorporated herein by reference in their entirety.

TW202504619A_113127011_SEQL.xml

Claims (77)

A double-stranded ribonucleic acid (dsRNA) agent for inhibiting PD-L1 expression, wherein the dsRNA agent comprises a sense chain and an antisense chain, wherein the sense chain comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1 by no more than 3 nucleotides, and the antisense chain comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2 by no more than 3 nucleotides, wherein the sense chain and the antisense chain may be partially, substantially or completely complementary to each other. The dsRNA agent of claim 1, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ by 0, 1, 2 or 3 nucleotides from any one of the nucleotide sequences of nucleotides 64-94, 67-97, 71-101, 72-102, 73-103, 71-103, 498-528, 552-582, 553-583, 707-737, 713-743 of SEQ ID NO: 1, and the antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ by 0, 1, 2 or 3 nucleotides from any one of the nucleotide sequences of nucleotides 64-94, 67-97, 71-101, 72-102, 73-103, 71-103, 498-528, 552-582, 553-583, 707-737, 713-743 of SEQ ID NO: The corresponding nucleotide sequences of NO:2 differ by 0, 1, 2 or 3 nucleotides in at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides, wherein the sense strand and the antisense strand may be partially, substantially or completely complementary to each other. The dsRNA agent of claim 2, wherein the sense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from any one of the nucleotide sequences of nucleotides 69-89, 71-89, 72-92, 74-92, 76-96, 78-96, 77-97, 79-97, 78-98, 80-98, 76-98, 72-98, 503-523, 505-523, 557-577, 559-577, 558-578, 560-578, 712-732, 714-732, 718-738, 720-738 of SEQ ID NO: 1 by 0, 1, 2 or 3 nucleotides, and the antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from any one of the nucleotide sequences of nucleotides 69-89, 71-89, 72-92, 74-92, 76-96, 78-96, 77-97, 79-97, 78-98, 80-98, 76-98, 72-98, 503-523, 505-523, The corresponding nucleotide sequence of NO:2 differs by 0, 1, 2 or 3 nucleotides in at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides. The dsRNA reagent of claim 1, wherein the antisense strand comprises a region complementary to the PD-L1 RNA transcript, the region comprising at least 15 consecutive nucleotides that differ by no more than 1, 2 or 3 nucleotides from any antisense sequence listed in any one of Tables 1-3. The dsRNA agent of claim 1, wherein the antisense strand comprises a region complementary to the PD-L1 RNA transcript, the region comprising at least 15 consecutive nucleotides from any one of the antisense sequences listed in any one of Tables 1-3. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting PD-L1 expression, wherein the dsRNA agent comprises a sense chain and an antisense chain, nucleotides 2 to 18 in the antisense chain comprise a region complementary to the PD-L1 RNA transcript, wherein the complementary region comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ by 0, 1, 2 or 3 nucleotides from one of the antisense sequences listed in one of Tables 1-3, and optionally comprises a targeting ligand. The dsRNA agent of claim 6, wherein the region complementary to the PD-L1 RNA transcript comprises at least 15, 16, 17, 18 or 19 consecutive nucleotides that differ from one of the antisense sequences listed in one of Tables 1-3 by no more than 3 nucleotides. A dsRNA agent as described in any one of claims 1-7, wherein the antisense chain of the dsRNA is substantially or completely complementary to any one of the target regions of SEQ ID NO: 1, and preferably the dsRNA agent comprises the antisense chain sequence described in any one of Tables 1-3. The dsRNA agent of any one of claims 1-8, wherein the sense chain sequence is at least substantially complementary or completely complementary to the antisense chain sequence in the dsRNA agent, preferably, wherein the dsRNA agent comprises the sense chain sequence of any one of Tables 1-3. The dsRNA agent of any one of claims 1 to 9, wherein the dsRNA agent comprises a sequence listed as a duplex sequence in any one of Tables 1 to 3. The dsRNA of any one of claims 1-10, wherein the dsRNA agent comprises at least one modified nucleotide. The dsRNA agent of any one of claims 1 to 11, wherein all or substantially all nucleotides of the sense chain and/or the antisense chain are modified nucleotides. The dsRNA agent of any one of claims 1-11, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region that is complementary to a portion of a PD-L1 RNA transcript, wherein the length of each strand is about 15 to about 30 nucleotides, wherein the sense strand comprises a sequence that can be represented by formula (I): 5′-(N′ _L ) _n′ N′ _L N′ L N′ _L N′ _{L N} ′ _F N′ _L N′ _{F N′ L N} ′ _N1 N′ N2 N′ L _N ′ _L N′ _L N′ _L N′ _L (N′ _L ) _m′ -3′ ( _I ); wherein: each N′ _F represents a 2'-fluorine-modified nucleotide; each N′ _N1 and N′ _N2 independently represent a modified or unmodified nucleotide; each N′ _L independently represents a modified or unmodified nucleotide but does not represent a 2'-fluoro-modified nucleotide, and m' and n' are each independently an integer from 0 to 7. The dsRNA agent of any one of claims 1-11, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region complementary to a portion of a PD-L1 RNA transcript, wherein each strand is about 18 to about 30 nucleotides in _length , wherein the antisense strand comprises a sequence _represented by formula (II): _{3 ′} - _{( NL ) nNM1NLNM2NLNFNLNM3NM4NLNLNLNM5NLNM6NLNLNFNL} - ₅ ′ (II _{) ;} wherein: each _NF represents _a 2′ _- fluorine-modified _nucleotide ; each _{NM1 , NM2 , NM3} , _NM4 , _{NM5 and NM6 independently} represents a modified or unmodified nucleotide _; each _{N L} independently represents a modified or unmodified nucleotide but not a 2'-fluorine-modified nucleotide, and n is an integer from 0 to 7. The dsRNA agent of any one of claims 1 to 11, wherein the dsRNA agent comprises a sense chain and an antisense chain, wherein the sense chain and the antisense chain form a dsRNA duplex, wherein the sense chain is complementary to the antisense chain, wherein the antisense chain comprises a region complementary to a PD-L1 RNA transcript, wherein the complementary region comprises at least 15 consecutive nucleotides, wherein the dsRNA comprises a duplex represented by formula (III): Sense chain 5′-(N′ _L ) _n′ N′ _L N′ _L N′ _L N′ _L N′ _{F N′ L N} ′ _F N′ _L N′ _N1 N′ _{N2 N} ′ L N′ _L N′ _L N′ _L N′ _L (N′ _L ) _m′ -3′ Antisense chain: 3′-(N _L ) _n N _M1 N _L N _M2 N _L N _F N _L N _M3 N _M4 N _L N _L N _L N _M5 N _L N _M6 N _L N _L N _F N _L -5′ (III); wherein: the length of each chain is about 18 to about 30 nucleotides; each _NF and N′ _F independently represents a 2'-fluorine-modified nucleotide; N _M1 , N _M2 , N _M3 , N _M4 , N _M5 , N′ _N1 and N′ _N2 each independently represents a modified or unmodified nucleotide; N′ _N1 and N′ _N2 include only one 2'-fluorine-modified nucleotide; N _M1 , N _M2 , N _M3 , N _M4 , N _M5 and N _M6 have only three 2'-fluorine-modified nucleotides; each _NL and N′ _L independently represents a modified or unmodified nucleotide, but does not represent a 2'-fluoro-modified nucleotide, and m', n' and n are each independently an integer from 0 to 7. The dsRNA agent of any one of claims 11-15, wherein the one or more modified nucleotides are independently selected from the following group: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2'3'-seco nucleotide mimetics, locked nucleotides, unlocked nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabinose nucleotides, 2'-methoxyethyl nucleotides, deoxynucleotides, ribose nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, 3'-OMe nucleotides, 5'-phosphorothioate groups, terminal nucleotides linked to cholesterol derivatives or dodecyl bis(dodecyl)amide groups, 2'-amino modified nucleotides, phosphoamido esters, or nucleotides containing unnatural bases. A dsRNA agent as described in any of claims 1-16, which comprises an E-vinyl phosphonate nucleotide located at the 5΄ end of the guide chain. The dsRNA agent of any one of claims 1-17, wherein the dsRNA agent comprises at least one phosphorothioate nucleoside interlinkage. The dsRNA agent of any one of claims 1-17, wherein the sense strand comprises at least one phosphorothioate nucleoside bond. The dsRNA agent of any one of claims 1-17, wherein the antisense strand comprises at least one phosphorothioate nucleoside bond. The dsRNA agent of any one of claims 1-17, wherein the sense strand comprises 1, 2, 3, 4, 5 or 6 phosphorothioate internucleoside bonds. The dsRNA agent of any one of claims 1-17, wherein the antisense strand comprises 1, 2, 3, 4, 5 or 6 phosphorothioate nucleoside bonds. The dsRNA agent of any one of claims 1-22, wherein the modified sense chain is a modified sense chain sequence shown in one of Tables 2-3. The dsRNA agent of any one of claims 1 to 22, wherein the modified antisense chain is a modified antisense chain sequence shown in one of Tables 2 to 3. The dsRNA agent of any one of claims 1-24, wherein the sense strand is complementary or substantially complementary to the antisense strand, and the length of the complementary region is between 16 and 23 nucleotides. The dsRNA agent of any one of claims 1-25, wherein the complementary region is 19 to 21 nucleotides in length. The dsRNA agent of any one of claims 1-26, wherein the length of each strand does not exceed 30 nucleotides. The dsRNA agent of any one of claims 1-26, wherein the length of each strand does not exceed 25 nucleotides. The dsRNA agent of any one of claims 1-26, wherein the length of each strand does not exceed 23 nucleotides. The dsRNA agent of any one of claims 1-29, wherein the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or linking groups. The dsRNA agent of claim 30, wherein one or more targeting groups or linking groups are conjugated to the sense chain. The dsRNA agent of claim 30 or 31, wherein the targeting group or linking group comprises N-acetylgalactosamine (GalNAc). The dsRNA agent of any one of claims 30-32, wherein the targeting group comprises the following structure: ; n'' is independently selected from 1 or 2. The dsRNA agent of any one of claims 30-33, wherein the targeting group has the following structure: GLO-1 GLS-1* GLO-2 GLS-2* GLO-3 GLS-3* GLO-4 GLS-4* GLO-5 GLS-5* GLO-6 GLS-6* GLO-7 GLS-7* GLO-8 GLS-8* GLO-9 GLS-9* GLO-10 GLS-10* GLO-11 GLS-11* GLO-12 GLS-12* GLO-13 GLS-13* GLO-14 GLS-14* GLO-15 GLS-15* GLO-16 GLS-16* . The dsRNA agent of any one of claims 1-34, wherein the dsRNA agent comprises a targeting group ligated to the 5'-end of the sense strand. The dsRNA agent of any one of claims 1-34, wherein the dsRNA agent comprises a targeting group ligated to the 3' end of the sense strand. The dsRNA agent of any one of claims 1-34, wherein the antisense strand comprises an inverted debasic residue at the 3' end. The dsRNA agent of any one of claims 1 to 34, wherein the sense strand comprises one or two inverted desyl residues or imann residues at the 3' or/and 5' ends. The dsRNA agent of any one of claims 1-38, wherein the dsRNA agent has two blunt ends. The dsRNA agent of any one of claims 1-38, wherein at least one strand comprises a 3' overhang of at least 1 nucleotide. The dsRNA agent of any one of claims 1-38, wherein at least one strand comprises a 3' overhang of at least 2 nucleotides. The dsRNA agent of any one of claims 1-41, wherein the PD-L1 RNA transcript is SEQ ID NO: 1. A composition comprising the dsRNA agent of any one of claims 1-42. The composition as described in claim 43 further comprises a pharmaceutically acceptable carrier. A composition as described in claim 44, further comprising one or more additional therapeutic agents. The composition of claim 45, wherein the composition is packaged in a kit, container, package, dispenser, prefilled syringe or vial. The composition of any one of claims 43-46, wherein the composition is formulated for subcutaneous administration or formulated for intravenous (IV) administration. A cell comprising the dsRNA agent of any one of claims 1-42, optionally, the cell is a mammalian cell, optionally a human cell. A method for inhibiting PD-L1 gene expression in cells, wherein the method comprises: (i) preparing cells containing an effective amount of a double-stranded RNA (dsRNA) agent as described in any one of claims 1-42 or a composition as described in any one of claims 43-47. The method as described in claim 49, further comprising: (ii) maintaining the cells prepared in claim 49(i) for a sufficient time to obtain degradation of the mRNA transcript of the PD-L1 gene, thereby inhibiting the expression of the PD-L1 gene in the cells. The method of any one of claims 49-50, wherein the cell is located in a subject and the dsRNA agent is administered subcutaneously to the subject. The method of any of claims 49-50, wherein the cell is located in a subject and the dsRNA agent is administered to the subject by IV administration. A method as described in claim 51 or 52, further comprising evaluating inhibition of the PD-L1 gene after administering a dsRNA agent to a subject, wherein the evaluation method comprises: (i) determining one or more physiological characteristics of a PD-L1-related disease or condition in the subject, and (ii) comparing the determined physiological characteristics with pre-treatment baseline physiological characteristics of the PD-L1-related disease or condition and/or control physiological characteristics of the PD-L1-related disease or condition, wherein the comparison indicates one or more of the presence or absence of inhibition of PD-L1 gene expression in the subject. The method of claim 53, wherein the physiological characteristic determined is one or more of the following: a decrease in the subject's PD-L1 mRNA level, PD-L1 protein level, or PD-L1 expression is assessed indirectly by measuring a decrease in PD-L1 biological activity or PD-L1 level in a sample (e.g., a serum sample) from the subject, by measuring a decrease in protein, nucleic acid, or carbohydrate present in an infectious agent, by assessing an immune response against an infectious agent by antibodies or immune cells, by assessing a decrease in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by measuring hepatitis B antigen (HBsAg), HBeAg, or HB in the subject's serum. The level of cccDNA is assessed by testing the level of anti-HBsAg antibodies in the subjects. A method as described in claim 54, wherein the reduction in PD-L1 gene expression can be assessed by: a reduction in one or more of the subject's PD-L1 mRNA level, the subject's PD-L1 protein level and/or PD-L1 activity; a reduction in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss); or by detecting hepatitis B antigen (HBsAg), HBeAg or HB cccDNA in the subject's serum. A method for inhibiting PD-L1 gene expression in a subject, wherein the method comprises administering to the subject an effective amount of a double-stranded RNA (dsRNA) agent as described in any one of claims 1 to 42 or a composition as described in any one of claims 43 to 47. The method of claim 56, wherein the dsRNA agent is administered to the subject subcutaneously. The method of claim 56, wherein the dsRNA agent is administered to the subject by IV administration. A method as described in any of claims 56-58, further comprising evaluating inhibition of the PD-L1 gene after administration of a dsRNA agent, wherein the method of evaluation comprises: (i) one or more physiological characteristics of a PD-L1-related disease or condition in a subject, and (ii) comparing the determined physiological characteristics with pre-treatment baseline physiological characteristics of the PD-L1-related disease or condition and/or control physiological characteristics of a PD-L1-related disease or condition; wherein, the comparison indicates one or more of the presence or absence of inhibition of PD-L1 gene expression in the subject. A method as described in claim 59, wherein the physiological characteristics determined are one or more of: the subject's PD-L1 mRNA level, PD-L1 protein level and/or PD-L1 activity, one or more signs or symptoms of infection (fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), assessed by hepatitis B antigen (HBsAg), HBeAg or HB cccDNA in the subject's serum, and assessed by detecting the subject's anti-HBsAg antibody level. The method of claim 59, wherein the reduction in one or more of the subject's PD-L1 mRNA level, the subject's PD-L1 protein level, and/or the reduction in PD-L1 expression is assessed indirectly by measuring a reduction in PD-L1 biological activity or PD-L1 level in a sample (e.g., a serum sample) from the subject, by measuring a reduction in protein, nucleic acid, or carbohydrate present in an infectious agent, by assessing an immune response against an infectious agent by antibodies or immune cells, by a reduction in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by measuring hepatitis B antigen (HBsAg), HBeAg, or HB in the subject's serum. The level of cccDNA is assessed by testing the level of anti-HBsAg antibodies in the subjects. A method for treating a disease or condition associated with PD-L1 protein, the method comprising administering to a subject an effective amount of a double-stranded RNA (dsRNA) agent as described in any one of claims 1 to 42 or a composition as described in any one of claims 43 to 47 to inhibit PD-L1 gene expression. A method as described in claim 62, wherein the disease or condition is one or more of the following: tumors or blood malignancies (such as lymphoma/leukemia, blood malignancies, breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, liver cancer, salivary cancer, gastric cancer, neuroglioma, thyroid cancer, thymic epithelial cancer, head cancer, kidney cancer, pancreatic cancer and neck cancer), infectious diseases (such as viral, bacterial, fungal or parasitic diseases), preferably, the infectious disease is a chronic infectious disease caused by viruses (such as HIV, HBV, HCV and HTLV, etc.), bacteria (such as Helicobacter pylori, etc.) and parasites (such as Schistosoma mansoni). The method of claim 63, further comprising administering an additional treatment regimen to the subject. The method of claim 64, wherein the additional treatment regimen comprises: administering to the subject one or more PD-L1 antisense polynucleotides of the present invention, administering to the subject a non-PD-L1 dsRNA therapeutic agent, and performing a behavioral change in the subject. A method as described in claim 65, wherein the non-PD-L1 dsRNA therapeutic agent is one or more of the following: the non-PD-L1 dsRNA therapeutic agent is selected from an additional cancer treatment agent of surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy or hormone therapy; and the non-PD-L1 dsRNA therapeutic agent is selected from an additional treatment agent selected from antiviral drugs, which refers to a drug composition administered to treat viral infections. The method of any one of claims 62-66, wherein the dsRNA agent is administered subcutaneously to the subject. The method of any one of claims 62-66, wherein the dsRNA agent is administered to the subject by IV administration. The method of any of claims 62-69, further comprising determining the efficacy of the administered double-stranded RNA (dsRNA) agent in the subject. A method as described in claim 69, wherein the method of determining the efficacy of the treatment in the subject comprises: (i) determining one or more physiological characteristics of a PD-L1-related disease or condition in the subject, and (ii) comparing the determined physiological characteristics to a pre-treatment baseline physiological characteristic of the PD-L1-related disease or condition wherein the comparison indicates one or more of the presence, absence, and level of efficacy of administering a double-stranded RNA (dsRNA) agent to the subject. A method as described in claim 70, wherein the physiological characteristic determined is: the subject's PD-L1 mRNA level, PD-L1 protein level, or a reduction in PD-L1 expression, which can also be assessed indirectly by measuring a reduction in PD-L1 biological activity or measuring the PD-L1 level in a sample from the subject (e.g., a serum sample); a reduction in protein, nucleic acid, or carbohydrate present in an infectious agent; assessed by the presence of an immune response, such as evidenced by the presence of antibodies or immune cells against the infectious agent; assessed by a reduction in one or more signs or symptoms of infection, such as fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss; assessed by hepatitis B surface antigen (HBsAg), HBeAg, or HBcccDNA in the subject's serum; assessed by detecting the level of anti-HBsAg antibodies in the subject. The method of claim 70, wherein a decrease in one or more of the PD-L1 mRNA level, the PD-L1 protein level, and/or a decrease in PD-L1 expression in the subject is assessed indirectly by measuring a decrease in PD-L1 biological activity or PD-L1 level in a sample (e.g., a serum sample) from the subject, by measuring a decrease in protein, nucleic acid, or carbohydrate present in an infectious agent, by assessing an immune response against an infectious agent by antibodies or immune cells, by a decrease in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by measuring hepatitis B antigen (HBsAg), HBeAg, or HB in the subject's serum. The level of cccDNA is assessed by testing the level of anti-HBsAg antibodies in the subjects. A method for reducing the level of PD-L1 protein in a subject compared to a pre-treatment baseline level of PD-L1 protein in the subject, wherein the pre-treatment baseline comprises administering to the subject an effective amount of a double-stranded RNA (dsRNA) agent as described in any one of claims 1-42 or a composition as described in any one of claims 43-47 to reduce the level of PD-L1 gene expression. The method of claim 73, wherein the dsRNA agent is administered to the subject subcutaneously or administered to the subject via IV. A method for altering the physiological characteristics of a PD-L1-related disease or condition compared to a pre-treatment baseline physiological characteristics of the subject's PD-L1-related disease or condition, wherein the pre-treatment baseline method comprises administering to the subject an effective amount of a double-stranded RNA (dsRNA) agent as described in any one of claims 1-42 or a composition as described in any one of claims 43-47 to alter the physiological characteristics of the subject's PD-L1-related disease or condition. The method of claim 75, wherein the dsRNA agent is administered to the subject subcutaneously or administered to the subject via IV. A method as described in any of claims 75-76, wherein the physiological characteristic is one or more of the following: a reduction in the subject's PD-L1 mRNA level, PD-L1 protein level and/or PD-L1 expression is assessed indirectly by measuring a reduction in PD-L1 biological activity or PD-L1 level in a sample (e.g., a serum sample) of the subject, by measuring a reduction in protein, nucleic acid or carbohydrate present in an infectious agent, by assessing an immune response against an infectious agent by antibodies or immune cells, by assessing a reduction in one or more signs or symptoms of infection (e.g., fever, pain, nausea, vomiting, abnormal blood chemistry, weight loss), by measuring hepatitis B antigen (HBsAg), HBeAg or HB in the subject's serum The level of cccDNA is assessed by testing the level of anti-HBsAg antibodies in the subjects.