Compositions and methods for programmed death ligand receptor (PD-L1) expression

Abstract

Provided herein are oligonucleotides that inhibit the expression of CD274. Also provided are compositions comprising the oligonucleotides and uses thereof, in particular to uses relating to the treatment of diseases, disorders and / or conditions associated with aberrant CD274 expression.

Description

[0001] Cross-Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 516,270, filed July 28, 2023, the entire contents of which are incorporated herein by reference.

[0002] Incorporation by Reference of Electronic Sequence Listing This application contains a sequence list, which has been electronically submitted in .XML format and is incorporated herein by reference in its entirety. The .XML copy was created on July 22, 2024, named “DCY-10825.xml”, and is 3,044,713 bytes in size. The sequence list contained in this .XML file is part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0003] Currently, chemotherapy is the most prevalent cancer treatment worldwide, often combined with surgery or a combination of surgery and radiation therapy, depending on the tumor type and stage (Abbas et al., An Overview of Cancer Treatment Modalities / IntechOpen, 2018). Since the discovery of several important mutations leading to carcinogenesis (e.g., adaptive immune resistance), these mutations and the proteins they represent have been widely used as targets for developing more selective drugs and drug combinations to treat cancer patients. Despite the effectiveness of these drugs, multidrug resistance (MDR) is frequently observed in patients, often leading to tumor recurrence, limited treatment options, and poor quality of life. Furthermore, cancer research often focuses on tumor cells, although the role of the tumor microenvironment and the “normal” or non-cancerous cells within it has been demonstrated to play a crucial role in tumor progression, development, and MDR (Klemm et al., TRENDS CELL BIOL (2015) 25(4): 198-213). New therapies targeting different aspects of the TME that promote tumor growth are needed. Summary of the Invention

[0004] This disclosure is based in part on the discovery of oligonucleotides that target and reduce the expression of PD-L1 mRNA. It is further based on the finding that the combination of PD-L1 oligonucleotides with CTLA-4 inhibitors provides synergistic antitumor efficacy against tumors with different tumor microenvironments. Specifically, as demonstrated herein, lipid-conjugated PD-L1 oligonucleotides (e.g., C18 hydrocarbon chains conjugated to a 5' terminal nucleotide) reduce tumor volume in vivo when delivered alone or in combination with CTLA-4 antibodies. Furthermore, as shown herein, treatment with PD-L1 oligonucleotides reduces tumor burden in inflamed tumor microenvironments. Additionally, the efficacy of PD-L1 depends on the presence of CD8+ T cells.

[0005] Therefore, in some aspects, this disclosure provides an oligonucleotide comprising an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 728, the sense strand comprising the nucleotide sequence of SEQ ID NO: 487, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide inter-bonded.

[0006] In some aspects, this disclosure provides an oligonucleotide comprising an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 728, the sense strand comprising the nucleotide sequence of SEQ ID NO: 487, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 2' carbon of the ribose ring of the 5' terminal nucleotide of the sense strand via a linker, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide interbonded.

[0007] In some aspects, this disclosure provides an oligonucleotide comprising an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 725, the sense strand comprising the nucleotide sequence of SEQ ID NO: 484, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to a 5' terminal nucleotide of the sense strand, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide inter-bonded.

[0008] In some aspects, this disclosure provides an oligonucleotide comprising an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 725, the sense strand comprising the nucleotide sequence of SEQ ID NO: 484, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 2' carbon of the ribose ring of the 5' terminal nucleotide of the sense strand via a linker, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide interbonded.

[0009] In some aspects, this disclosure provides an oligonucleotide comprising an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 732, the sense strand comprising the nucleotide sequence of SEQ ID NO: 491, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to a 5' terminal nucleotide of the sense strand, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide inter-bonded.

[0010] In some aspects, this disclosure provides an oligonucleotide comprising an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 732, the sense strand comprising the nucleotide sequence of SEQ ID NO: 491, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 2' carbon of the ribose ring of the 5' terminal nucleotide of the sense strand via a linker, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide interbonded.

[0011] In some embodiments of any of the foregoing or related aspects, the 2'-modified nucleotide comprises a 2'-modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinose.

[0012] In some embodiments of any of the foregoing or related aspects, about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand contain a 2'-fluorine modification. In some embodiments, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand contain a 2'-fluorine modification. In some embodiments, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the oligonucleotide contain a 2'-fluorine modification.

[0013] In some embodiments of any of the foregoing or related aspects, positions 8-11 of the sense strand each contain a 2'-fluorine modification. In some embodiments, positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand each contain a 2'-fluorine modification. In some embodiments, the remaining nucleotides contain a 2'-O-methyl modification, provided that the 5' terminal nucleotide of the sense strand conjugated to the saturated C18 hydrocarbon chain does not contain a 2'-O-methyl modification.

[0014] In some embodiments of any of the foregoing or related aspects, the at least one modified nucleotide bond is a phosphate thioester bond.

[0015] In some embodiments of any of the foregoing or related aspects, the sense chain contains a thiophosphate bond between positions 1 and 2 of the sense chain. In some embodiments, the sense chain contains a thiophosphate bond between positions 1 and 2, 2 and 3, and 3 and 4 of the sense chain. In some embodiments, the antisense chain contains a thiophosphate bond between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22. In some embodiments, the antisense chain contains a thiophosphate bond between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

[0016] In some embodiments of any of the foregoing or related aspects, the 4'-carbon of the sugar in the 5'-nucleotide of the antisense strand comprises a phosphate ester analog. In some embodiments, the phosphate ester analog is an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate.

[0017] In some aspects, this disclosure provides an oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 20 to 30 nucleotides in length and has a complementary region of 19 to 29 nucleotides complementary to the target sequence of CD274 shown in any of SEQ ID NO: 2, 5 and 9, wherein the sense strand is 28 to 40 nucleotides in length and comprises a stem-loop at its 3' end as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand form a double-stranded region of at least 19 nucleotides in length, and wherein the sense strand comprises a C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand.

[0018] In some aspects, this disclosure provides an oligonucleotide comprising an antisense strand of about 20 to 22 nucleotides in length and a sense strand of about 28 to 40 nucleotides in length, wherein the antisense strand and the sense strand form an asymmetric double-stranded region of about 20 to 22 base pairs, the asymmetric double-stranded region comprising a 3' terminal overhang of at least one nucleotide of the antisense strand, wherein the antisense strand comprises a nucleotide of length SEQ ID NO: The complementary region of 19 to 21 nucleotides complementary to the target sequence of CD274 shown in any of 2, 5, and 9, wherein the sense strand comprises: (i) a stem-loop at the 3' end of the sense strand, wherein the stem-loop comprises a nucleotide sequence represented by the formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, and (ii) at least one C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand, wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide interbonded.

[0019] In some embodiments of any of the foregoing or related aspects, the antisense chain comprises a sequence as shown in any of SEQ ID NO: 725, 728 and 732.

[0020] In some embodiments of any of the foregoing or related aspects, the meaningful chain comprises a sequence as shown in any of SEQ ID NO: 966, 969 and 973.

[0021] In some respects, this disclosure provides methods for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1050, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1005.

[0022] In some embodiments of any of the foregoing or related aspects, L is a tetracyclic ring. In some embodiments, L is 4 nucleotides in length. In some embodiments, L contains a sequence denoted as GAAA.

[0023] In some embodiments of any of the foregoing or related aspects, the antisense strand includes a 3' end overhang of one or more nucleotides in length. In some embodiments, the 3' end overhang is 2 nucleotides in length, and optionally the 3' end overhang sequence is GG.

[0024] In some embodiments of any of the foregoing or related aspects, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide is a 2'-modified nucleotide. In some embodiments, the 2'-modified nucleotide comprises a 2'-modification selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinose.

[0025] In some embodiments of any of the foregoing or related aspects, about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand contain a 2'-fluorine modification. In some embodiments, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand contain a 2'-fluorine modification. In some embodiments, about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the oligonucleotide contain a 2'-fluorine modification.

[0026] In some embodiments of any of the foregoing or related aspects, the sense strand comprises 36 nucleotides at positions 1-36 from 5' to 3', wherein positions 8-11 contain a 2'-fluorine modification. In some embodiments, the antisense strand comprises 22 nucleotides at positions 1-22 from 3' to 5', wherein positions 2, 3, 4, 5, 7, 10, and 14 contain a 2'-fluorine modification.

[0027] In some embodiments of any of the foregoing or related aspects, the remaining nucleotides contain a 2'-O-methyl modification, provided that the 5' terminal nucleotide of the sense chain conjugated to the saturated C18 hydrocarbon chain does not contain a 2'-O-methyl modification.

[0028] In some embodiments of any of the foregoing or related aspects, the oligonucleotide comprises at least one modified internucleotide bond. In some embodiments, the at least one modified internucleotide bond is a phosphate thioester bond.

[0029] In some embodiments of any of the foregoing or related aspects, the sense strand contains a thiophosphate bond between positions 1 and 2 of the sense strand. In some embodiments, the sense strand contains a thiophosphate bond between positions 1 and 2, 2 and 3, and 3 and 4 of the sense strand. In some embodiments, the antisense strand comprises 22 nucleotides from positions 1 to 22 from 3' to 5', wherein the antisense strand contains a thiophosphate bond between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22. In some embodiments, the antisense strand comprises 22 nucleotides from positions 1 to 22 from 3' to 5', wherein the antisense strand contains a thiophosphate bond between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22.

[0030] In some embodiments of any of the foregoing or related aspects, the 4'-carbon of the sugar in the 5'-nucleotide of the antisense strand comprises a phosphate ester analog. In some embodiments, the phosphate ester analog is an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate.

[0031] In some aspects, this disclosure provides a pharmaceutical composition comprising an oligonucleotide of any of the embodiments described above or related to the present invention and a pharmaceutically acceptable carrier, delivery agent, or excipient.

[0032] In some aspects, this disclosure provides a method for treating a subject with cancer, the method comprising administering to the subject an effective amount of an oligonucleotide or pharmaceutical composition of any of the embodiments described above or in connection with the preceding or related aspects.

[0033] In some respects, this disclosure provides treatment and activation. CD274 Methods of expressing a related disease, condition, or status, including administering an oligonucleotide or pharmaceutical composition of any of the aforementioned or related embodiments to a subject in need.

[0034] In some aspects, this disclosure provides a method of treating a subject with cancer, the method comprising administering to the subject an effective amount of an oligonucleotide or pharmaceutical composition of any of the embodiments described above or related, in combination with a CTLA4 inhibitor.

[0035] In some respects, this disclosure provides treatment and activation. CD274 Methods of expressing a related disease, condition, or status, including administering to a subject in need any of the aforementioned or related implementations of an oligonucleotide or pharmaceutical composition, in combination with a CTLA4 inhibitor.

[0036] In some implementations of any of the foregoing or related aspects, the aforementioned is related to activation. CD274The related disease, symptom, or condition is cancer. In some implementations, the cancer is selected from carcinoma, sarcoma, melanoma, lymphoma and leukemia, prostate cancer, breast cancer, hepatocellular carcinoma (HCC), colorectal cancer, pancreatic cancer, and glioblastoma.

[0037] In some embodiments of any of the foregoing or related aspects, the cancer comprises an immunosuppressive tumor microenvironment. In some embodiments, the cancer comprises an inflamed tumor microenvironment. In some embodiments, the inflamed tumor microenvironment comprises invasive T cells.

[0038] In some embodiments of any of the foregoing or related aspects, the CTLA-4 inhibitor is an antibody. In some embodiments, the antibody is an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA-4 antibody is selected from ipilimumab or tremelimumab.

[0039] In some implementations, this disclosure provides for targeting CD274 Methods for delivering oligonucleotides to the lymph nodes of a subject, including administering oligonucleotides according to any of the aforementioned or related embodiments.

[0040] In some embodiments of any of the foregoing or related aspects, the lymph node is a tumor draining lymph node.

[0041] In some embodiments, this disclosure provides the use of oligonucleotides of any of the foregoing or related embodiments in the preparation of a medicament for the treatment of [conditions related to...]. CD274 Expressing a related disease, symptom, or condition, optionally used to treat cancer.

[0042] In some respects, this disclosure provides oligonucleotides of any embodiment of the foregoing or related aspects, which are used or suitable for the treatment of [conditions related to...]. CD274 Expressing a related disease, symptom, or condition, optionally used to treat cancer.

[0043] In some aspects, this disclosure provides a kit comprising an oligonucleotide of any embodiment of the foregoing or related aspects, an optional pharmaceutically acceptable carrier, and a packaging insert, the packaging insert including information on administration to patients with... CD274 Instructions for use on subjects who express a related disease, symptom, or condition.

[0044] In some embodiments of any of the foregoing or related aspects, the above-mentioned and related CD274 The related disease, symptom, or condition is cancer.

[0045] In some aspects, this disclosure provides oligonucleotides of any of the foregoing or related embodiments in the preparation of drugs for use in combination with CTLA4 inhibitors for the treatment of patients with... CD274 The use of a drug in relation to a related disease, symptom, or condition.

[0046] In some respects, this disclosure provides oligonucleotides of any of the foregoing or related embodiments, which are used or suitable for use in combination with CTLA4 inhibitors for the treatment of patients with... CD274 It expresses the related disease, symptom, or condition.

[0047] In some aspects, this disclosure provides a kit comprising an oligonucleotide of any embodiment of the foregoing or related aspects, an optional pharmaceutically acceptable carrier, and a packaging insert, the packaging insert including information on administration to patients with... CD274 Instructions for use of RNAi oligonucleotides in combination with CTLA4 inhibitors in subjects expressing relevant diseases, symptoms, or conditions.

[0048] In some embodiments of any of the foregoing or related aspects, the above-mentioned and related CD274 The related disease, symptom, or condition is cancer. Attached Figure Description

[0049] Figure 1A The structure of an exemplary RNAi oligonucleotide molecule with chemical modifications is provided, wherein a C18 lipid is conjugated to a stem-loop, referred to as “GalXC-CD274-C18”. Throughout the text, mouse... Cd274 The specific GalXC-CD274-C18 oligonucleotide is represented as “GalXC-mCD274-C18”.

[0050] Figure 1B A structure for a lipid tail suitable for conjugation with RNAi oligonucleotide molecules was provided.

[0051] Figures 2A-2F The graph shows pancreatic tumors in checkpoint resistant Pan02 mice ( Figures 2A-2B ), checkpoint inhibitor resistant 4T1 mouse triple-negative mammary tumors ( Figures 2C-2D ), checkpoint inhibitor partially sensitive MC-38 mouse colorectal tumors ( Figures 2E-2F ) and checkpoint inhibitor-sensitive Hepa1-6 hepatocellular carcinoma ( Figure 2G In tumor-bearing mice, following subcutaneous treatment with 25 mg / kg GalXC-mCD274-C18 RNAi oligonucleotides on days 1 and 4 (q3dx2), the remaining mice in the tumor microenvironment (TME) and tumor draining lymph nodes (TDLN) showed significant improvement. Cd274mRNA. Tissue samples from TME and TDLN were collected 7 days after the last administration of RNAi oligonucleotides. Control mice were administered PBS.

[0052] Figure 3 The graph represents the remaining mouse Cd11b and Cd11c cells isolated from the tumor draining lymph nodes of Pan02 tumor-bearing mice after treatment with RNAi oligonucleotides targeting ALDH2. Aldh2 mRNA levels. Tissue samples from TDLN were collected 3 days after administration of RNAi oligonucleotides. Control mice were administered PBS.

[0053] Figures 4A-4B The image shows the treatment regimen used to treat mice carrying 4T1 tumors and the GalXC-placebo-C18 molecule ( Figure 4A These mice were treated subcutaneously with GalXC-mCD274-C18 RNAi oligonucleotide or GalXC-placebo-C18 at 25 mg / kg, or intraperitoneally with an anti-PD-L1 monoclonal antibody (mAb) at 10 mg / kg. Mice received treatment on days 1 and 4, and TDLNs were collected 7 days after the last administration of RNAi oligonucleotide or placebo and processed for immunohistochemistry of CD11c and PD-L1 expression. Figure 4B ).

[0054] Figures 5A-5C The graph shows the effect of treatment with GalXC-mCD274-C18 RNAi oligonucleotide or anti-PD-L1 mAb on Pan02 ( Figure 5A ), 4T1 ( Figure 5B ) or MC-38 ( Figure 5C The remaining mice in the TDLN of tumor-bearing mice Cd274, Ifng and Gzmb mRNA. Mice were administered 25 mg / kg subcutaneously with PBS or RNAi oligonucleotides, q3dx2 (on days 1 and 4), or 10 mg / kg intraperitoneally with anti-PD-L1 mAb, q3dx2. Tissues were collected 7 days after administration of RNAi oligonucleotides or mAb.

[0055] Figure 6Immunohistochemical images of CD8 in the tumor microenvironment (TME) of MC-38 xenografts treated with GalXC-mCD274-C18 RNAi oligonucleotides or anti-PD-L1 mAb are provided. Mice were administered PBS or RNAi oligonucleotides subcutaneously at 25 mg / kg, [q3dx2] x 2 (on days 1, 4, 8, and 12), or anti-PD-L1 mAb q3dx2 intraperitoneally at 10 mg / kg. Tissues were collected 7 days after administration of the RNAi oligonucleotides or mAb.

[0056] Figure 7 Immunohistochemical images of CD8 in the tumor microenvironment (TME) of 4T1 xenografts treated with GalXC-mCD274-C18 RNAi oligonucleotides or anti-PD-L1 monoclonal antibodies (mAbs) are provided. Mice were administered PBS or RNAi oligonucleotides subcutaneously at 25 mg / kg, q3dx2 (on days 1 and 4), or anti-PD-L1 mAb intraperitoneally at 10 mg / kg, q3dx2. Tissues were collected 7 days after administration of the RNAi oligonucleotides or mAb.

[0057] Figures 8A-8B The graph shows the antitumor effects of subcutaneous treatment with GalXC-mCD274-C18 RNAi oligonucleotides or anti-PD-L1 mAb. In patients carrying 4T1 ( Figure 8A ) and Pan02 ( Figure 8B Tumor volume was measured in mice with immunologically active tumors. Mice carrying Pan02 tumors were treated with 25 mg / kg GalXC-CD274 conjugate or PBS four times a week (q3dx2 per cycle), or mice carrying 4T1 tumors were treated with 50 mg / kg GalXC-CD274 or GalXC-placebo conjugate three times a week (q3dx3), and both tumors were treated at the same frequency but with 10 mg / kg mAb.

[0058] Figures 9A-9B Graphical representations of tumor volume were provided after treatment with i) GalXC-placebo-C18; or ii) anti-PD-L1 mAb; or iii) GalXC-mCD274-C18. Figure 9A ) and lung metastatic tumor images ( Figure 9B On days 14, 17, and 20, immunocompromised mice carrying 4T1 xenografts were administered GalXC-mCD274-C18 RNAi oligonucleotide subcutaneously at 25 mg / kg or antiPDL1 mAb intraperitoneally at 10 mg / kg. On day 24, the lungs of the mice were photographed to capture lung metastases.

[0059] Figures 10A-10B Graphical representations of tumor volume were provided after treatment with i) GalXC-placebo-C18; or ii) anti-PD-L1 mAb; or iii) GalXC-mCD274-C18. Figure 9A ) and lung metastatic tumor images ( Figure 10B On days 14, 17, and 20, immunocompetent mice carrying 4T1 xenografts were administered GalXC-mCD274-C18 RNAi oligonucleotide subcutaneously at 25 mg / kg or anti-PD-L1 mAb intraperitoneally at 10 mg / kg. On day 24, the lungs of the mice were photographed to capture lung metastases.

[0060] Figures 11A-11B The graph shows the antitumor effects of subcutaneous treatment with GalXC-mCD274-C18 RNAi oligonucleotides or anti-PD-L1 mAb. In mice carrying MC-38 colorectal tumors (… Figure 11A ) and Hepa1-6 mouse hepatocellular carcinoma ( Figure 11B Tumor volume was measured in immunocompetent mice. Mice were treated with four 25 mg / kg doses of the conjugate, once a week for 3 days x 2 weeks, followed by treatment with the same dosing frequency but with 10 mg / kg mAb. Dosing was performed on days 11, 14, 18, and 21.

[0061] Figure 12A The graph shows tumor volume after combining GalXC-mCD274-C18 RNAi oligonucleotide with anti-PD-L1 mAb. Mice carrying checkpoint inhibitor-sensitive MC-38 tumors were administered GalXC-placebo-C18, GalXC-placebo-C18 in combination with anti-PD-L1 antibody, GalXC-mCD274-C18, or GalXC-mCD274-C18 in combination with anti-PD-L1 mAb. GalXC-mCD274-C18 was administered subcutaneously at 25 mg / kg or anti-PD-L1 mAb was administered intraperitoneally at 10 mg / kg on days 8, 11, 15, and 18.

[0062] Figure 12B Images of perforin immunohistochemistry in tumors of mice carrying checkpoint inhibitor-sensitive MC-38 tumors are provided. These mice were administered GalXC-placebo-C18, GalXC-placebo-C18 in combination with anti-PD-L1 antibody, GalXC-mCD274-C18, or GalXC-mCD274-C18 in combination with anti-PD-L1 mAb. GalXC-mCD274-C18 was administered subcutaneously at 25 mg / kg, or anti-PD-L1 mAb was administered intraperitoneally at 10 mg / kg on days 8, 11, 15, and 18.

[0063] Figure 13A The graphs show tumor volumes after treatment with i) GalXC-placebo-C18; or ii) anti-PD-L1 mAb; or iii) GalXC-mCD274-C18. Mice carrying checkpoint inhibitor-resistant 4T1 tumors were administered GalXC-mCD274-C18 RNAi oligonucleotide subcutaneously at 25 mg / kg or anti-PD-L1 mAb intraperitoneally at 10 mg / kg on days 6, 9, and 12.

[0064] Figure 13B The graph shows tumor volume after combining GalXC-CD274 RNAi oligonucleotides with anti-CTLA-4 mAb. Mice carrying checkpoint inhibitor-resistant 4T1 tumors were administered GalXC-placebo-C18, GalXC-placebo-C18 in combination with anti-CTLA-4 mAb, GalXC-mCD274-C18, or GalXC-mCD274-C18 in combination with anti-CTLA-4 mAb. GalXC-mCD274-C18 was administered subcutaneously at 25 mg / kg on days 8, 11, and 14, and anti-CTLA-4 was administered intraperitoneally at 10 mg / kg.

[0065] Figure 13C Provided CD8 in tumors of mice carrying checkpoint inhibitor-resistant 4T1 tumors + Immunohistochemical images of mice administered GalXC-placebo-C18, GalXC-placebo-C18 in combination with anti-CTLA-4 mAb, GalXC-mCD274-C18, or GalXC-mCD274-C18 in combination with anti-CTLA-4 mAb. GalXC-mCD274-C18 was administered subcutaneously at 25 mg / kg on days 8, 11, and 14, and anti-CTLA-4 was administered intraperitoneally at 10 mg / kg.

[0066] Figure 14 The graph depicts the target with 1 nM CD274 GalNAc- in different regions of the gene CD274 Oligonucleotide treatment for 28 hours resulted in endogenous expression of human [cells / nucleotides]. CD274 Remaining human RKO (human colon cancer) cells CD274 Percentage of mRNA (%).

[0067] Figure 15 The graphs depict the effects of targeting with 0.3 nM, 1 nM, or 3 nM. CD274 GalNAc- in different regions of the gene CD274 Oligonucleotide treatment for 28 hours resulted in endogenous expression of human [cells / nucleotides].CD274 The remaining people in RKO cells CD274 Percentage of mRNA (%). The percentage of remaining mRNA was normalized relative to HPRT and SFRS9 housekeeping genes and a simulated transfection control.

[0068] Figures 16A-16B The provided graphic depicts the GalNAc-jointed CD274 After oligonucleotide treatment, exogenous expression of human CD274 The remaining human liver in mice (hydrodynamic injection model) CD274 Percentage of mRNA (%). Mice were subcutaneously administered 2 mg / kg of GalNAc- as shown in PBS. CD274 Oligonucleotides. Three days after administration, mice were given a hydrodynamic injection (HDI) of 50 µg of human-encoded oligonucleotides per mouse. CD274 DNA ORF plasmid. Human samples were analyzed from liver tissue collected 20 hours after injection. CD274 mRNA levels.

[0069] Figure 17 The structure of a chemically modified RNAi oligonucleotide molecule is provided, wherein GalNAc is conjugated to a stem-loop.

[0070] Figure 18 The provided graphics depict Figure 17 GalNAc suffix CD274 The dose response of oligonucleotides. Measurement using human GalNAc conjugates. CD274 Oligonucleotides were exogenously expressed after subcutaneous treatment at two doses (0.3 mg / kg or 1 mg / kg). CD274 The remaining human liver in mice (HDI model) CD274 Percentage of mRNA (%). Three days after administration, 50 µg of the encoded mRNA was injected into each mouse via hydrodynamic injection (HDI). CD274 DNA ORF plasmid. Human liver samples collected 24 hours later were analyzed. CD274 mRNA levels.

[0071] Figure 19 The provided graphics depict Figure 17 GalNAc suffix CD274 The dose response of oligonucleotides. Measurement using human GalNAc conjugates. CD274 Exogenous expression of oligonucleotides was achieved after subcutaneous treatment with two doses (0.1 mg / kg or 0.3 mg / kg). CD274 The remaining human liver in mice (HDI model) CD274Percentage of mRNA (%). Three days after administration, 50 µg of the encoded mRNA was injected into each mouse via hydrodynamic injection (HDI). CD274 DNA ORF plasmid. Human liver samples collected 24 hours later were analyzed. CD274 mRNA levels.

[0072] Figure 20 The provided figures depict endogenous expression after treatment with GalNAc-CD274-094 or GalNAc-CD274-098. CD274 H460 lung cancer cells ( CD274 The remaining human expression was expressed by reverse transfection using lipofectamine RNAiMAX overnight. CD274 Percentage of mRNA (%). H640 cells were treated with oligonucleotides at a series of dose levels (0.0032 nM, 0.16 nM, 0.08 nM, 0.04 nM, 2 nM, 10 nM, and 50 nM) for 24 hours to generate IC50. 50 curve.

[0073] Figure 21 The provided figures depict endogenous expression after treatment with GalNAc-CD274-094 or GalNAc-CD274-098. CD274 Human primary macrophages ( CD274 The remaining human expression was expressed by reverse transfection using lipofectamine RNAiMAX overnight. CD274 Percentage of mRNA (%). Human primary macrophages were polarized to the M2 immunosuppressive phase with the IL-10 cytokine and stimulated with lipopolysaccharide. Macrophages were treated with oligonucleotides at a series of dose levels (0.0032 nM, 0.16 nM, 0.08 nM, 0.04 nM, 2 nM, 10 nM, and 50 nM) for 72 hours to generate IC50. 50 curve.

[0074] Figure 22 The provided graphics depict the expressions processed with GalNAc-CD274-094 or GalNAc-CD274-098. CD274 DC immune cell culture ( CD274 The remaining human expression was expressed by reverse transfection using lipofectamine RNAiMAX overnight. CD274 Percentage of mRNA (%). Cells were first treated with the inhibitory cytokine IL-10, then with a series of doses (0.2 nM, 1 nM, and 5 nM) of oligonucleotides for 72 hours, and the mRNA percentage was measured. CD274 Plot the remaining percentage (%) of mRNA.

[0075] Figure 23 The provided graphics depict the relationship with Figure 22 The description in CD274 mRNA downregulation leads to the production of related cytokines. Supernatants were collected from the plates, and the levels of the pro-inflammatory cytokine IFN-γ were measured using the MSD V-plex assay kit. Figure 24A and Figure 24B The graphic shows a mass-like tumor from a Pan02 xenograft ( Figure 24A ) and liver ( Figure 24B The remaining mice Aldh2 mRNA. Mice were treated with 25 mg / kg of the designated GalXC-ALDH2-lipid conjugate, and mRNA was measured on day 3.

[0076] Figure 24C and Figure 24D The graph shows the mass tumors from mice with Pan02 xenografts on days 7 and 14 after treatment with the designated GalXC-ALDH2-lipid conjugate at 25 mg / kg. Figure 24C The remaining mice with tumor draining lymph nodes (TdLN) and tumor draining lymph nodes (TdLN) Aldh2 mRNA.

[0077] Figure 25 The structure of a chemically modified CD274-0098 RNAi oligonucleotide molecule is provided, wherein a C18 lipid is conjugated to a 5' terminal nucleotide. Detailed Implementation

[0078] Programmed death ligand 1 (differentiation cluster 274, CD274, or PD-L1) is a type I transmembrane inhibitory receptor ligand expressed on immune cells and some tumor cells. The interaction of this ligand with the PD-1 receptor inhibits T cell activation and subsequent cytokine production. Expression in tumor cells provides the ability to evade tumor responses by inhibiting cytotoxic T cell activation. Although tumor PD-L1 has been widely used to identify patients most likely to respond to treatment, recent evidence suggests that PD-L1 expressed by immune cells (especially antigen-presenting dendritic cells (APCs or CD11c-expressing DCs)) is a better predictor of clinical response than PD-L1 expressed by tumor cells. Furthermore, most research on PD-L1 and PD-1 has focused on their extrinsic role in suppressing the immune system, but recent studies have shown that the intrinsic tumor role of PD-L1 is associated with certain cancer types (Wu, Y et al., Front. Immunol. 10:2022, 2019, Hudson, K et al. Front. Immunol.(11:568931, 2020). Intracellular PD-L1 expressed by APCs plays a role in regulating the migration of dendritic cells (DCs) from the tumor to the tumor-draining lymph nodes. Lack of silencing of intracellular PD-L1 on DCs may impair antigen presentation mechanisms in tumors and promote resistance to immunotherapy. Monoclonal antibodies (mAbs) are engineered to primarily target extracellular / membrane PD-L1, with little chance of reaching the intracellular form. Not wishing to be bound by theory, PD-L1 RNAi oligonucleotides conjugated to GalNAc or lipids have the ability to inhibit both extracellular and intracellular PD-L1 and effectively reduce PD-L1 expression for therapeutic use. Therefore, cells with membrane or extracellular PD-L1 can be targeted by mAbs, but cells with both intracellular and extracellular PD-L1 require therapies including the PD-L1 RNAi oligonucleotides described herein to inhibit PD-L1.

[0079] According to several aspects, this disclosure provides for reducing the tumor microenvironment. CD274 Expressed oligonucleotides (e.g., RNAi oligonucleotides). In some embodiments, the oligonucleotides provided herein are designed for the treatment of tumors. CD274 Expression of related diseases. In some aspects, this disclosure provides for reducing the expression of related diseases in specific cells (e.g., tumor cells) or organs. CD274 Expression to treat and general CD274 Methods for expressing related diseases. against CD274 Oligonucleotide inhibitors expressed CD274 target sequence In some implementations, the oligonucleotides described herein (e.g., RNAi oligonucleotides) target the formation of CD274 The target sequence of mRNA. In some implementations, the oligonucleotide targeting described herein... CD274 The target sequence within the mRNA sequence. In some embodiments, the oligonucleotides described herein correspond to... CD274 The target sequence within the mRNA sequence. In some embodiments, the oligonucleotide, or a portion, fragment, or strand thereof (e.g., the antisense strand or guide strand of a double-stranded (ds)RNAi oligonucleotide), is associated with the constituent mRNA sequence. CD274 mRNA target sequence binding or annealing, thereby inhibiting CD274 Express.

[0081] In some implementation schemes, in order to inhibit in vivo CD274 The purpose of expression is for the oligonucleotide to target CD274 Target sequence. In some implementations, the target... CD274 Oligonucleotide inhibition of target sequence CD274The amount or extent of expression is related to the potency of the oligonucleotide. In some implementations, targeting... CD274 Oligonucleotide inhibition of target sequence CD274 The amount or degree of expression is related to the number of patients treated with this oligonucleotide and those suffering from [a condition]. CD274 The amount or degree of treatment benefit is related to the subject or patient expressing a relevant disease, symptom, or condition.

[0082] By checking the code CD274 The nucleotide sequences of mRNAs, including those of mRNAs from several different species (e.g., humans, cynomolgus monkeys, and mice; see, for example, Example 7), have been found as results of in vitro and in vivo tests (see, for example, Examples 2-7). CD274 Certain nucleotide sequences of mRNA are more susceptible to oligonucleotide-based repression than other sequences and can therefore be used as target sequences for the oligonucleotides described herein. In some embodiments, the sense strand of the oligonucleotides described herein (e.g., RNAi oligonucleotides) contains CD274 Target sequence. In some embodiments, a portion or region of the sense strand of the oligonucleotide described herein (e.g., RNAi oligonucleotide) contains... CD274 Target sequence. In some implementations, CD274 The target sequence comprises, or consists of, the sequences of any one of SEQ ID NO: 1-2 and 4-241. In some embodiments, CD274 The target sequence comprises, or consists of, the sequence shown in SEQ ID NO: 2, 4, 5, 6, 7, 9, or 20. In some embodiments, CD274 The target sequence comprises, or consists of, the sequence shown in SEQ ID NO: 2. In some embodiments, CD274 The target sequence comprises, or consists of, the sequence shown in SEQ ID NO: 4. In some embodiments, CD274 The target sequence comprises, or consists of, the sequence shown in SEQ ID NO: 5. In some embodiments, CD274 The target sequence comprises, or consists of, the sequence shown in SEQ ID NO: 6. In some embodiments, CD274 The target sequence comprises, or consists of, the sequence shown in SEQ ID NO: 7. In some embodiments, CD274The target sequence comprises, or consists of, the sequence shown in SEQ ID NO: 9. In some embodiments, CD274 The target sequence contains or consists of the sequence shown in SEQ ID NO:20. CD274 In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) have the same properties as... CD274 targeting sequence mRNA (e.g., in) CD274 The complementary regions within the target sequence of mRNA are used to target cells. CD274 mRNA and inhibit and / or reduce CD274 The purpose of expression. In some implementations, the oligonucleotides described herein contain... CD274 The target sequence (e.g., the antisense strand or guide strand of an RNAi oligonucleotide) has a complementary (Watson-Crick) base pairing with... CD274 The target sequence binds to or anneals to its complementary region. The target sequence or complementary region typically has an appropriate length and base content so that the oligonucleotide (or its chain) can bind to... CD274 mRNA binding or annealing, thereby inhibiting and / or reducing CD274The purpose of expression. In some embodiments, the length of the target sequence or complementary region is at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, or at least about 30 nucleotides. In some embodiments, the length of the target sequence or complementary region is about 12 to about 30 (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30) nucleotides. In some embodiments, the target sequence or complementary region is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the target sequence or complementary region is 18 nucleotides long. In some embodiments, the target sequence or complementary region is 19 nucleotides long. In some embodiments, the target sequence or complementary region is 20 nucleotides long. In some embodiments, the target sequence or complementary region is 21 nucleotides long. In some embodiments, the target sequence or complementary region is 22 nucleotides long. In some embodiments, the target sequence or complementary region is 23 nucleotides long. In some embodiments, the target sequence or complementary region is 24 nucleotides long. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to the sequence of any one of SEQ ID NO: 1-2 and 4-241, and the target sequence or complementary region is 18 nucleotides long. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to the sequence of any one of SEQ ID NO: 1-2 and 4-241, and the target sequence or complementary region is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to the sequence of SEQ ID NO: 1037, and the target sequence or complementary region is 20 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to the sequence of SEQ ID NO: 1037, and the target sequence or complementary region is 21 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to the sequence of SEQ ID NO: 1037, and the target sequence or complementary region is 22 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to the sequence of SEQ ID NO: 1037, and the target sequence or complementary region is 23 nucleotides in length.In some embodiments, the oligonucleotide includes a target sequence or complementary region that is complementary to the sequence of SEQ ID NO: 1037, and the target sequence or complementary region is 24 nucleotides in length.

[0084] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) contain [the following information / components ... CD274 A target sequence or complementary region that is completely complementary to the target sequence (e.g., the antisense strand or guide strand of a double-stranded oligonucleotide). In some embodiments, the target sequence or complementary region is... CD274 The target sequence is partially complementary. In some embodiments, the oligonucleotide contains [a specific sequence]. CD274 A target sequence or complementary region that is completely complementary to the target sequence. In some embodiments, the oligonucleotide contains a target sequence or complementary region that is completely complementary to the target sequence. CD274 The target sequence is a partially complementary target sequence or a complementary region.

[0085] In some embodiments, the oligonucleotide comprises a target sequence or complementary region that is completely complementary to the sequence of any one of SEQ ID NO: 1-2 and 4-241. In some embodiments, the oligonucleotide comprises a target sequence or complementary region that is completely complementary to the sequence shown in SEQ ID NO: 2, 4, 5, 6, 7, 9, or 20. In some embodiments, the oligonucleotide comprises a target sequence or complementary region that is partially complementary to the sequence of any one of SEQ ID NO: 1-2 and 4-241. In some embodiments, the oligonucleotide comprises a target sequence or complementary region that is partially complementary to the sequence shown in SEQ ID NO: 2. In some embodiments, the oligonucleotide comprises a target sequence or complementary region that is partially complementary to the sequence shown in SEQ ID NO: 5. In some embodiments, the oligonucleotide comprises a target sequence or complementary region that is partially complementary to the sequence shown in SEQ ID NO: 9.

[0086] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) contain [the following information / components ... CD274 A target sequence or complementary region complementary to a continuous nucleotide sequence within mRNA, wherein the continuous nucleotide sequence is about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to 22, 16 to 20, 18 to 20, or 18 to 19 nucleotides). In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a continuous nucleotide sequence within mRNA, wherein the continuous nucleotide sequence is about 12 to about 30 nucleotides in length (e.g., 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 20, 12 to 18, 12 to 16, 14 to 22, 16 CD274A target sequence or complementary region complementary to a continuous nucleotide sequence within mRNA, wherein the length of the continuous nucleotide sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the oligonucleotide comprises a target sequence or complementary region to a target sequence or complementary region within mRNA. CD274 A target sequence or complementary region complementary to a continuous nucleotide sequence within mRNA, wherein the continuous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide contains a target sequence or complementary region complementary to a target sequence or complementary region within mRNA. CD274 A target sequence or complementary region that is complementary to a continuous nucleotide sequence within mRNA, wherein the continuous nucleotide sequence is 20 nucleotides in length.

[0087] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a target sequence or complementary region complementary to a continuous nucleotide sequence of any one of SEQ ID NO: 2, 4, 5, 6, 7, 9, or 20, optionally wherein said continuous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a target sequence or complementary region complementary to a continuous nucleotide sequence of SEQ ID NO: 2, optionally wherein said continuous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a target sequence or complementary region complementary to a continuous nucleotide sequence of SEQ ID NO: 5, optionally wherein said continuous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a target sequence or complementary region complementary to a continuous nucleotide sequence of SEQ ID NO: 9, optionally wherein said continuous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a continuous nucleotide sequence of any one of SEQ ID NO: 2, 4, 5, 6, 7, 9, or 20, wherein the continuous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a continuous nucleotide sequence of SEQ ID NO: 2, wherein the continuous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a continuous nucleotide sequence of SEQ ID NO: 5, wherein the continuous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a continuous nucleotide sequence of SEQ ID NO: 9, wherein the continuous nucleotide sequence is 19 nucleotides in length. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a continuous nucleotide sequence of SEQ ID NO: 1037, wherein the continuous nucleotide sequence is 20 nucleotides in length.

[0088] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) contain the corresponding CD274 The target sequence has one or more base pair (bp) mismatches in the target sequence or complementary region. In some embodiments, the target sequence or complementary region may be associated with a corresponding CD274 The target sequence has a maximum of approximately 1, approximately 2, approximately 3, approximately 4, or approximately 5 mismatches, provided that the target sequence or complementary region, under appropriate hybridization conditions, has a mismatch with... CD274 The ability of mRNA to bind or anneal and / or the inhibition of this oligonucleotideCD274 The ability to express is maintained. Alternatively, the target sequence or complementary region can be associated with the corresponding... CD274 The target sequence has no more than one, two, three, four, or five mismatches, provided that the target sequence or complementary region, under appropriate hybridization conditions, is compatible with... CD274 The ability of mRNA to bind or anneal and / or the inhibition of this oligonucleotide CD274 The ability to express is maintained. In some embodiments, the oligonucleotide includes a target sequence or complementary region with one mismatch to the corresponding target sequence. In some embodiments, the oligonucleotide includes a target sequence or complementary region with two mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide includes a target sequence or complementary region with three mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide includes a target sequence or complementary region with four mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide includes a target sequence or complementary region with five mismatches to the corresponding target sequence. In some embodiments, the oligonucleotide includes a target sequence or complementary region with more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) to the corresponding target sequence, wherein at least two (e.g., all) mismatches are sequentially located (e.g., consecutive 2, 3, 4, 5 or more mismatches), or wherein said mismatches are scattered throughout the target sequence or complementary region. In some embodiments, the oligonucleotide comprises a target sequence or complementary region having more than one mismatch (e.g., 2, 3, 4, 5 or more mismatches) with the corresponding target sequence, wherein at least two (e.g., all) mismatches are sequentially located (e.g., consecutive 2, 3, 4, 5 or more mismatches), or wherein at least one or more non-mismatched base pairs are located between said mismatches, or combinations thereof. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a sequential nucleotide sequence of any of SEQ ID NO: 1-2 and 4-241, wherein the target sequence or complementary region may be complementary to the corresponding target sequence. CD274 The target sequence has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5 mismatches, etc. In some embodiments, the oligonucleotide includes a target sequence or complementary region that is complementary to a consecutive nucleotide sequence of either SEQ ID NO: 1-2 or 4-241, wherein the target sequence or complementary region may be compatible with the corresponding CD274 The target sequence has no more than one, two, three, four, or five mismatches. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a consecutive nucleotide sequence of either SEQ ID NO: 1-2 or 4-241, wherein the target sequence or complementary region may be compatible with the corresponding CD274The target sequence has at most about 1, at most about 2, at most about 3, at most about 4, at most about 5 mismatches, etc. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a consecutive nucleotide sequence of any one of SEQ ID NO: 2, 4, 5, 6, 7, 9, or 20, wherein the target sequence or complementary region may be compatible with the corresponding CD274 The target sequence has no more than 1, 2, 3, 4 or 5 mismatches. CD274 Multiple oligonucleotide types and / or structures can be used for targeting in the methods described in this paper. Type of oligonucleotide This includes, but is not limited to, RNAi oligonucleotides, antisense oligonucleotides (ASO), miRNAs, etc. Any oligonucleotide types described herein or elsewhere are intended to be used as a framework for inclusion in this document. CD274 Target sequence for inhibition CD274 The purpose of the expression.

[0090] In some implementations, the oligonucleotides described herein inhibit RNA interference (RNAi) pathways by being introduced upstream or downstream of the cleavage enzyme (Dicer). CD274 Expression. For example, RNAi oligonucleotides have been developed, each strand having a size of about 19-25 nucleotides and having at least one 3' overhang consisting of 1 to 5 nucleotides (see, for example, U.S. Patent No. 8,372,968). Longer oligonucleotides have also been developed, which are processed by cleavage enzymes to generate active RNAi products (see, for example, U.S. Patent No. 8,883,996). Further work has yielded extended dsRNAs, wherein at least one end of at least one strand extends beyond the double-stranded target region, including structures in which one strand comprises a thermodynamically stable tetracyclic structure (see, for example, U.S. Patents Nos. 8,513,207 and 8,927,705, and International Patent Application Publication No. WO 2010 / 033225). Such structures may include single-strand (ss) extensions (on one or both sides of the molecule) and double-strand (ds) extensions.

[0091] In some embodiments, the oligonucleotides described herein are added downstream of the cleavage enzyme (e.g., cleavage enzyme cleavage) into the RNAi pathway. In some embodiments, the oligonucleotides described herein are cleavage enzyme substrates. In some embodiments, after processing with an endogenous cleavage enzyme, a product is generated that can reduce... CD274The expressed oligonucleotide is a double-stranded nucleic acid of 19-23 nucleotides in length. In some embodiments, the oligonucleotide has a 3' overhang (e.g., 1, 2, or 3 nucleotides in length) at the 3' end of the antisense strand. In some embodiments, the oligonucleotide (e.g., siRNA) comprises a 21-nucleotide guide strand antisense to the target RNA and a complementary guest strand, wherein these two strands are annealed to form a 19-bp double helix and a 2-nucleotide overhang at either or both 3' ends. Longer oligonucleotide designs are also possible, including oligonucleotides with a 23-nucleotide guide strand and a 21-nucleotide guest strand, wherein there is a blunt end on the right side of the molecule (3' end of the guest strand / 5' end of the guide strand) and a 3'-guide strand overhang of two nucleotides on the left side of the molecule (5' end of the guest strand / 3' end of the guide strand). In such molecules, a 21 bp double-stranded region is present. See, for example, U.S. Patents 9,012,138, 9,012,621, and 9,193,753.

[0092] In some embodiments, the oligonucleotides described herein comprise a sense strand and an antisense strand, both in the range of about 17 to 36 nucleotides (e.g., 17 to 36, 20 to 25, or 21-23). ​​In some embodiments, the oligonucleotides described herein comprise an antisense strand of 19-30 nucleotides in length and a sense strand of 19-50 nucleotides in length, wherein the antisense strand and the sense strand are separate strands forming an asymmetric double-stranded region at the 3' end of the antisense strand with a 1-4 nucleotide overhang. In some embodiments, the oligonucleotides described herein comprise a sense strand and an antisense strand, both in the range of about 19-22 nucleotides in length. In some embodiments, the sense strand and the antisense strand are of equal length. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand such that a 3' overhang is present on the sense strand or the antisense strand, or on both the sense strand and the antisense strand. In some embodiments, for oligonucleotides having sense and antisense strands, each approximately 21-23 nucleotides in length, the 3' overhang on the sense strand, antisense strand, or both is 1 or 2 nucleotides long. In some embodiments, the oligonucleotide has a 22-nucleotide guiding strand and a 20-nucleotide guest strand, with a blunt end on the right side of the molecule (3' end of the guest strand / 5' end of the guiding strand) and a 2-nucleotide 3'-guiding strand overhang on the left side of the molecule (5' end of the guest strand / 3' end of the guiding strand). In such molecules, a 20 bp double-stranded region is present.

[0093] Other oligonucleotide designs that can be used with the compositions and methods described herein include: 16-meric siRNAs (see, for example, NUCLEIC ACIDS IN CHEMISTRY AND BIOLOGY, Blackburn (ed.), ROYAL SOCIETY OF CHEMISTRY, 2006), and shRNAs (e.g., those with a stem of 19 bp or less; see, for example, Moore et al. (2010)). . 629:141-158), blunt-ended siRNA (e.g., 19 bp in length; see, for example, Kraynack & Baker (2006)). 12:163-176), asymmetric siRNA (aiRNA; see, for example, Sun et al. (2008) Nat. Biotechnol . 26:1379-82), asymmetric short double-stranded siRNA (see, e.g., Chang et al. (2009) Mol. Ther. 17:725-32), forked siRNA (see, e.g., Hohjoh (2004) FEBS Lett. 557:193-98), single-stranded siRNA (Elsner (2012) Nat. Biotechnol). . 30:1063), dumbbell-shaped circular siRNAs (see, e.g., Abe et al. (2007) J. Am. Chem. Soc. 129:15108-09), and small internally segmented interfering RNAs (siRNAs; see, e.g., Bramsen et al. (2007) Nucleic Acids Res. 35:5886-97). These can be used in some implementations to reduce or inhibit [the virus / organization]. CD274 Other non-restricted examples of expressed oligonucleotide structures are microRNAs (miRNAs), short hairpin RNAs (shRNAs), and short siRNAs (see, for example, Hamilton et al. (2002) EMBO J). . 21:4671-79; see also U.S. Patent Application Publication No. 2009 / 0099115).

[0094] Additionally, in some implementations, the methods described herein for reducing or inhibiting CD274 The expressed oligonucleotides are single-stranded (ss). Such structures can include, but are not limited to, single-stranded RNAi molecules. Recent efforts have demonstrated the activity of single-stranded RNAi molecules (see, for example, Matsui et al. (2016)). 24:946-55). However, in some embodiments, the oligonucleotides herein are antisense oligonucleotides (ASOs). An antisense oligonucleotide is a single-stranded oligonucleotide having a nucleobase sequence that, when written in the 5' to 3' orientation, contains the inverse complementary sequence of the targeted segment of a specific nucleic acid and is appropriately modified (e.g., as a gapmer) to induce RNase H-mediated cleavage of its target RNA in the cell, or (e.g., as a mixmer) to inhibit the translation of the target mRNA in the cell. The ASOs used herein can be modified in any suitable manner known in the art, including, for example, as shown in U.S. Patent No. 9,567,587 (including, for example, changes in length, the sugar portion of the nucleobase (pyrimidine, purine), and the heterocyclic portion of the nucleobase). Furthermore, ASOs have been used for decades to reduce the expression of specific target genes (see, for example, Bennett et al. (2017)). . 57:81-105).

[0095] In some implementations, antisense oligonucleotides and CD274 mRNAs share complementary regions. In some implementations, antisense oligonucleotides target individuals identified as NM_014143.4. CD274 Various regions of the gene. In some implementations, the antisense oligonucleotide targets the cynomolgus monkey identified as XM_005581779.2. CD274 Various regions of the gene. In some implementations, the antisense oligonucleotide targets mice identified as NM_021893.3. CD274 Various regions of the gene. In some embodiments, the antisense oligonucleotide is 15-50 nucleotides long. In some embodiments, the antisense oligonucleotide is 15-25 nucleotides long. In some embodiments, the antisense oligonucleotide is 22 nucleotides long. In some embodiments, the antisense oligonucleotide is complementary to any of SEQ ID NO: 1-2 and 4-241. In some embodiments, the antisense oligonucleotide is at least 15 consecutive nucleotides long. In some embodiments, the antisense oligonucleotide is at least 19 consecutive nucleotides long. In some embodiments, the antisense oligonucleotide is at least 20 consecutive nucleotides long. In some embodiments, the antisense oligonucleotide differs from the target sequence by 1, 2, or 3 nucleotides.

[0096] CD274 In some respects, this disclosure provides for targeting Double-stranded oligonucleotide mRNA and inhibit CD274A double-stranded (ds) RNAi oligonucleotide expressed (e.g., via the RNAi pathway) comprising a sense strand (also referred to herein as a guest strand) and an antisense strand (also referred herein as a guide strand). In some embodiments, the sense and antisense strands are separate strands and are not covalently linked. In some embodiments, the sense and antisense strands are covalently linked. In some embodiments, the sense and antisense strands form a bistranded region, wherein the sense and antisense strands, or portions thereof, are linked to each other in a complementary manner (e.g., via Watson-Crick base pairing).

[0097] In some embodiments, the sense strand has a first region (R1) and a second region (R2), wherein R2 comprises a first subregion (S1), a four-ring or three-ring (L), and a second subregion (S2), wherein L is located between S1 and S2, and wherein S1 and S2 form a second double strand (D2). D2 may have different lengths. In some embodiments, the length of D2 is about 1-6 bp. In some embodiments, the length of D2 is 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5, or 4-5 bp. In some embodiments, the length of D2 is 1, 2, 3, 4, 5, or 6 bp. In some embodiments, the length of D2 is 6 bp.

[0098] In some embodiments, the sense strand R1 and the antisense strand form a first double helix (D1). In some embodiments, the length of D1 is at least about 15 nucleotides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21). In some embodiments, the length of D1 is in the range of about 12 to 30 nucleotides (e.g., 12 to 30, 12 to 27, 15 to 22, 18 to 22, 18 to 25, 18 to 27, 18 to 30, or 21 to 30 nucleotides). In some embodiments, the length of D1 is at least 12 nucleotides (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides). In some embodiments, the length of D1 is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of D1 is 20 nucleotides. In some embodiments, D1 containing both sense and antisense strands does not span the entire length of the sense and / or antisense strands. In some embodiments, D1 containing both sense and antisense strands spans the entire length of the sense or antisense strand, or both. In some embodiments, D1 containing both sense and antisense strands spans the entire length of both the sense and antisense strands.

[0099] In some embodiments, the oligonucleotides provided herein comprise a sense strand having a sequence having any one of SEQ ID NO: 1-2 and 4-241 and an antisense strand comprising a complementary sequence selected from SEQ ID NO: 242-243 and 245-482. In some embodiments, the oligonucleotides provided herein comprise a sense strand having a sequence having SEQ ID NO: 2 and an antisense strand comprising a complementary sequence having SEQ ID NO: 245. In some embodiments, the oligonucleotides provided herein comprise a sense strand having a sequence having SEQ ID NO: 5 and an antisense strand comprising a complementary sequence having SEQ ID NO: 246. In some embodiments, the oligonucleotides provided herein comprise a sense strand having a sequence having SEQ ID NO: 9 and an antisense strand comprising a complementary sequence having SEQ ID NO: 250.

[0100] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a sense strand having a sequence of any one of SEQ ID NO: 483-484 and 486-723 and an antisense strand comprising a complementary sequence selected from SEQ ID NO: 724-725 and 727-964.

[0101] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, the sense strand and antisense strand comprising nucleotide sequences selected from: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively.

[0102] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, the sense strand and antisense strand comprising nucleotide sequences selected from: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively.

[0103] In some embodiments, the sense strand contains the sequence of SEQ ID NO: 484 and the antisense strand contains the sequence of SEQ ID NO: 725. In some embodiments, the sense strand contains the sequence of SEQ ID NO: 486 and the antisense strand contains the sequence of SEQ ID NO: 727. In some embodiments, the sense strand contains the sequence of SEQ ID NO: 487 and the antisense strand contains the sequence of SEQ ID NO: 728. In some embodiments, the sense strand contains the sequence of SEQ ID NO: 488 and the antisense strand contains the sequence of SEQ ID NO: 729. In some embodiments, the sense strand contains the sequence of SEQ ID NO: 489 and the antisense strand contains the sequence of SEQ ID NO: 730. In some embodiments, the sense strand contains the sequence of SEQ ID NO: 491 and the antisense strand contains the sequence of SEQ ID NO: 732. In some embodiments, the sense strand contains the sequence of SEQ ID NO: 502 and the antisense strand contains the sequence of SEQ ID NO: 743.

[0104] It should be understood that in some embodiments, the sequence presented in the sequence listing may be referenced when describing the structure of an oligonucleotide (e.g., RNAi oligonucleotide) or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and / or one or more modified nucleotides and / or one or more modified internucleotide bonds and / or one or more other modifications, while still retaining substantially the same or similar complementary properties as the specified sequence.

[0105] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise a 25-nucleotide sense strand and a 27-nucleotide antisense strand, which, upon cleavage, produces an antisense strand incorporated into the mature RISC. In some embodiments, the 25-nucleotide sense strand comprises a sequence selected from SEQ ID NOs: 1-2 and 4-241. In some embodiments, the 27-nucleotide antisense strand comprises a sequence selected from SEQ ID NOs: 242-243 and 245-482. In some embodiments, the sense strand of the oligonucleotide is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the sense strand of the oligonucleotide is longer than 25 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides). In some embodiments, the sense strand of the oligonucleotide comprises a nucleotide sequence selected from SEQ ID NO: 483-484 and 486-723, wherein the nucleotide sequence is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the sense strand of the oligonucleotide comprises a nucleotide sequence selected from SEQ ID NO: 483-484 and 486-723, wherein the nucleotide sequence is longer than 25 nucleotides (e.g., 26, 27, 28, 29, or 30 nucleotides).

[0106] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) have a 5' end that is thermodynamically less stable than the other 5' end. In some embodiments, an asymmetric oligonucleotide is provided that includes a blunt end at the 3' end of the sense strand and a 3'-protrusion at the 3' end of the antisense strand. In some embodiments, the 3'-protrusion on the antisense strand is about 1-8 nucleotides long (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length). In some embodiments, the oligonucleotide has a protrusion comprising two (2) nucleotides at the 3' end of the antisense (guide) strand. However, other protrusions are also possible. In some embodiments, the overhang is a 3' overhang comprising 1 to 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or a length of 1, 2, 3, 4, 5, or 6 nucleotides. However, in some embodiments, the overhang is a 5' overhang comprising 1 to 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or a length of 1, 2, 3, 4, 5, or 6 nucleotides. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to the continuous nucleotide sequence of any of SEQ ID NO: 1-2 and 4-241, and a 5' overhang of 1 to 6 nucleotides in length. In some embodiments, the oligonucleotide comprises a sense strand comprising a nucleotide sequence selected from SEQ ID NO: 483-484 and 486-723, wherein the oligonucleotide includes a 5'-protrusion of 1 to 6 nucleotides in length. In some embodiments, the oligonucleotide comprises an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 724-725 and 727-964, wherein the oligonucleotide includes a 5'-protrusion of 1 to 6 nucleotides in length. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence selected from SEQ ID NO: 483-484 and 486-723, and the antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 724-725 and 727-964, wherein the oligonucleotide includes a 5'-protrusion of 1 to 6 nucleotides in length.

[0107] In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand are modified. In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand are modified with the target mRNA (e.g., CD274In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand are not complementary to the target mRNA. In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotide described herein are unpaired. In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotide described herein contain unpaired purines or pyrimidines. In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotide described herein contain unpaired purines. In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotide described herein contain unpaired GG, AA, AG, or GA. In some embodiments, the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotide described herein contain unpaired GG. In some embodiments, one or both of the two (2) terminal GG nucleotides at each 3' end of the oligonucleotide described herein are not complementary to the target mRNA. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a consecutive nucleotide sequence of any of SEQ ID NO: 1-2 and 4-241, wherein the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotide comprise unpaired GG. In some embodiments, the oligonucleotide comprises an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 242-243 and 245-482, wherein the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotide comprise unpaired GG. In some embodiments, the oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence selected from SEQ ID NO: 483-484 and 486-723, and the antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 724-725 and 727-964, wherein the two (2) terminal nucleotides at the 3' end of the antisense strand of the oligonucleotide comprise unpaired GG.

[0108] In some embodiments, one or more (e.g., 1, 2, 3, 4, or 5) mismatches exist between the sense and antisense strands of the oligonucleotide (e.g., RNAi oligonucleotide) constituting this article. If there are more than one mismatch between the sense and antisense strands, they may be sequentially located (e.g., consecutive 2, 3, or more) or scattered throughout the complementary region. In some embodiments, the 3' end of the sense strand contains one or more mismatches. In some embodiments, two (2) mismatches are incorporated at the 3' end of the sense strand. In some embodiments, base mismatches or segment destabilization at the 3' end of the sense strand of the oligonucleotide improves or increases the potency of the oligonucleotide. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. There are one or more mismatches (e.g., 1, 2, 3, 4 or 5) between the meaningful chain and the antisense chain.

[0109] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise sense and antisense strands, which contain nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. There are one or more mismatches (e.g., 1, 2, 3, 4 or 5) between the meaningful chain and the antisense chain.

[0110] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise sense and antisense strands, which contain the following nucleotide sequences: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (b) are SEQ ID NO: 491 and 732, respectively. There are one or more mismatches (e.g., 1, 2, 3, 4 or 5) between the meaningful chain and the antisense chain. CD274 In some implementations, the antisense strand of the oligonucleotide (e.g., RNAi oligonucleotide) is referred to as the "guide strand." For example, the antisense strand binds to the RNA-induced silencing complex (RISC) and... Antisense strand When a protein, such as Ago2, binds to or interacts with one or more similar factors and directs the silencing of a target gene, the antisense strand is called the guide strand. In some embodiments, the sense strand containing a complementary region to the guide strand is referred to herein as a "passenger strand".

[0112] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise an antisense strand of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotides comprise an antisense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, the oligonucleotide comprises an antisense strand of about 12 to about 40 nucleotides (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40). In some embodiments, the oligonucleotide comprises an antisense strand of 15 to 30 nucleotides. In some embodiments, the antisense strand of any of the oligonucleotides disclosed herein is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the oligonucleotide comprises an antisense strand of 22 nucleotides in length.

[0113] In some implementations, the methods disclosed herein for targeting ArgonauteThe oligonucleotide (e.g., RNAi oligonucleotide) contains an antisense strand that comprises or consists of sequences as shown in any of SEQ ID NO: 242-243 and 245-482. In some embodiments, the methods disclosed herein for targeting CD274 The oligonucleotide (e.g., RNAi oligonucleotide) contains an antisense strand that comprises or consists of a sequence as shown in SEQ ID NO: 243. In some embodiments, the methods disclosed herein for targeting CD274 The oligonucleotide (e.g., RNAi oligonucleotide) contains an antisense strand that comprises or consists of a sequence as shown in SEQ ID NO:246. In some embodiments, the methods disclosed herein for targeting CD274The oligonucleotide (e.g., RNAi oligonucleotide) comprises an antisense strand that contains or consists of a sequence as shown in SEQ ID NO: 250. In some embodiments, the oligonucleotide herein comprises an antisense strand that contains at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence as shown in any of SEQ ID NO: 242-243 and 245-482. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in SEQ ID NO: 243. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in SEQ ID NO: 246. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in SEQ ID NO: 250. In some embodiments, the oligonucleotides disclosed herein are for targeting CD274 The oligonucleotides herein contain an antisense strand comprising or consisting of a sequence as shown in any of SEQ ID NO: 724-725 and 727-964. In some embodiments, the oligonucleotides herein contain an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence as shown in any of SEQ ID NO: 724-725 and 727-964. In some embodiments, the oligonucleotides herein disclosed for targeting CD274The oligonucleotides herein comprise an antisense strand comprising or consisting of a sequence as shown in any of SEQ ID NO: 724-725 and 727-964. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of a sequence as shown in any of SEQ ID NO: 725, 727, 728, 729, 730, 732, and 743. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in SEQ ID NO: 725. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in SEQ ID NO: 728. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising at least about 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in SEQ ID NO: 732. In some embodiments, the oligonucleotides disclosed herein are for targeting CD274 The oligonucleotide contains an antisense strand comprising a sequence as shown in any of SEQ ID NO: 725, 727, 728, 729, 730, 732, and 743, or composed of sequences as shown in any of SEQ ID NO: 725, 727, 728, 729, 730, 732, and 743. In some embodiments, the oligonucleotide disclosed herein is used for targeting CD274 The oligonucleotide contains an antisense strand, which comprises or consists of a sequence as shown in SEQ ID NO: 725. In some embodiments, the oligonucleotides disclosed herein are used for targeting CD274The oligonucleotide contains an antisense strand, which comprises or consists of a sequence as shown in SEQ ID NO: 728. In some embodiments, the oligonucleotides disclosed herein are used for targeting CD274 CD274 The oligonucleotide contains an antisense strand, which contains or consists of a sequence as shown in SEQ ID NO: 732.

[0114] In some embodiments, the oligonucleotides herein comprise an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 242-243 and 245-482. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising a nucleotide sequence selected from SEQ ID NO: 243, 245, 246, 247, 248, 250, or 261. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising the nucleotide sequence of SEQ ID NO: 243. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising the nucleotide sequence of SEQ ID NO: 246. In some embodiments, the oligonucleotides herein comprise an antisense strand comprising the nucleotide sequence of SEQ ID NO: 250. There is a chain of righteousness In some implementations, the methods disclosed herein for targeting CD274 mRNA and inhibit CD274 The expressed oligonucleotide (e.g., RNAi oligonucleotide) comprises a sense strand sequence as shown in any of SEQ ID NO: 1-2 and 4-241. In some embodiments, the oligonucleotides herein have a sense strand consisting of at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19) consecutive nucleotides of the sequence shown in any of SEQ ID NO: 1-2 and 4-241. In some embodiments, the oligonucleotides disclosed herein are for targeting... CD274 mRNA and inhibit CD274The expressed oligonucleotide comprises a sense strand sequence as shown in any of SEQ ID NO: 483-484 and 486-723. In some embodiments, the oligonucleotides herein have a sense strand consisting of at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in any of SEQ ID NO: 483-484 and 486-723. In some embodiments, the oligonucleotides disclosed herein are for targeting CD274 mRNA and inhibit CD274 The expressed oligonucleotides comprise sense strand sequences as shown in any of SEQ ID NO: 484, 486, 487, 488, 489, 491, and 502. In some embodiments, the oligonucleotides disclosed herein are for targeting CD274 mRNA and inhibit CD274 The expressed oligonucleotides contain a sense strand sequence as shown in SEQ ID NO: 484. In some embodiments, the oligonucleotides disclosed herein are used for targeting CD274 mRNA and inhibit CD274 The expressed oligonucleotides contain a sense strand sequence as shown in SEQ ID NO: 487. In some embodiments, the oligonucleotides disclosed herein are used for targeting CD274 mRNA and inhibit CD274The expressed oligonucleotide comprises a sense strand sequence as shown in SEQ ID NO: 491. In some embodiments, the oligonucleotides herein have a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in any of SEQ ID NO: 484, 486, 487, 488, 489, 491, and 502. In some embodiments, the oligonucleotides herein have a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in SEQ ID NO: 484. In some embodiments, the oligonucleotides herein have a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in SEQ ID NO: 487. In some embodiments, the oligonucleotides herein have a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in SEQ ID NO: 491. In some embodiments, the oligonucleotides herein have a sense strand comprising at least about 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19) consecutive nucleotides of the sequence shown in any of SEQ ID NO: 2, 4, 5, 6, 7, 9, or 20.

[0116] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a sense strand (or guest strand) of up to about 50 nucleotides in length (e.g., up to 50, up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotides herein comprise a sense strand of at least about 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, the oligonucleotides herein comprise a sense strand of about 12 to about 50 nucleotides (e.g., 12 to 50, 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40). In some embodiments, the oligonucleotides herein comprise a sense strand of 15 to 50 nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand of 18 to 36 nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the oligonucleotides herein comprise a sense strand of 36 nucleotides in length.

[0117] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a sense strand with a stem-loop structure at its 3' end. In some embodiments, the stem-loop is formed by intra-chain base pairing. In some embodiments, the sense strand comprises a stem-loop structure at its 5' end. In some embodiments, the stem of the stem-loop comprises a double strand of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a double strand of 2 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a double strand of 3 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a double strand of 4 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a double strand of 5 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a double strand of 6 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a double strand of 7 nucleotides in length. In some embodiments, the stem of the stem-loop contains a double strand of 8 nucleotides in length. In some embodiments, the stem of the stem-loop contains a double strand of 9 nucleotides in length. In some embodiments, the stem of the stem-loop contains a double strand of 10 nucleotides in length. In some embodiments, the stem of the stem-loop contains a double strand of 11 nucleotides in length. In some embodiments, the stem of the stem-loop contains a double strand of 12 nucleotides in length. In some embodiments, the stem of the stem-loop contains a double strand of 13 nucleotides in length. In some embodiments, the stem of the stem-loop contains a double strand of 14 nucleotides in length.

[0118] In some embodiments, the stem-loop provides protection against oligonucleotide degradation (e.g., enzymatic degradation), promotes or improves targeting and / or delivery to target cells, tissues, or organs, or both. For example, in some embodiments, the loop of the stem-loop consists of one or more modified nucleotides that promote, improve, or increase targeting of target mRNA (e.g., CD274 Targeting mRNA, targeting the expression of target genes (e.g., CD274The oligonucleotide inhibits gene expression repression and / or delivery, uptake, and / or penetration into target cells, tissues, or organs, or combinations thereof. In some embodiments, the stem-loop itself or modifications to the stem-loop do not affect or substantially do not affect the intrinsic gene expression repressive activity of the oligonucleotide, but promote, improve, or increase the stability of the oligonucleotide (e.g., provide protection against degradation) and / or its delivery, uptake, and / or penetration into target cells, tissues, or organs. In some embodiments, the oligonucleotide herein comprises a sense chain containing (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop of up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length) between S1 and S2. In some embodiments, the loop (L) is 3 nucleotides in length. In some embodiments, the loop (L) is 4 nucleotides in length. In some embodiments, the loop (L) is 5 nucleotides in length. In some embodiments, the ring (L) is 6 nucleotides long. In some embodiments, the ring (L) is 7 nucleotides long. In some embodiments, the ring (L) is 8 nucleotides long. In some embodiments, the ring (L) is 9 nucleotides long. In some embodiments, the ring (L) is 10 nucleotides long.

[0119] In some embodiments, the quadruple ring comprises the sequence 5'-GAAA-3'. In some embodiments, the stem ring comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO: 856).

[0120] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a target sequence or complementary region complementary to a consecutive nucleotide sequence of any of SEQ ID NO: 1-2 and 4-241, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop between S1 and S2 with a length of up to about 10 nucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a consecutive nucleotide sequence of any of SEQ ID NO: 1-2 and 4-241, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop between S1 and S2 with a length of 4 nucleotides.

[0121] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a target sequence or complementary region complementary to the sequential nucleotide sequence of SEQ ID NO: 2, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop between S1 and S2 with a length of up to about 10 nucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to the sequential nucleotide sequence of SEQ ID NO: 2, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop between S1 and S2 with a length of 4 nucleotides. In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a target sequence or complementary region complementary to the sequential nucleotide sequence of SEQ ID NO: 5, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop between S1 and S2 with a length of up to about 10 nucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to the sequential nucleotide sequence of SEQ ID NO: 5, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop between S1 and S2 with a length of 4 nucleotides. In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a target sequence or complementary region complementary to the sequential nucleotide sequence of SEQ ID NO: 9, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop between S1 and S2 with a length of up to about 10 nucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to the sequential nucleotide sequence of SEQ ID NO: 9, and the oligonucleotide comprises a sense strand comprising (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a single-stranded loop between S1 and S2 with a length of 4 nucleotides.

[0122] In some embodiments, the loop (L) having the stem-loop structure S1-L-S2 as described herein is a tricyclic ring. In some embodiments, the oligonucleotide comprises a target sequence or complementary region complementary to a consecutive nucleotide sequence of any of SEQ ID NO: 1-2 and 4-241, and a tricyclic ring. In some embodiments, the tricyclic ring comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (e.g., delivery ligands), and combinations thereof.

[0123] In some embodiments, the stem-loop (L) having the structure S1-L-S2 as described above is a tetraloop as described in U.S. Patent No. 10,131,912, which is incorporated herein by reference. In some embodiments, the oligonucleotide herein comprises a target sequence or complementary region complementary to a consecutive nucleotide sequence of any of SEQ ID NOs: 1-2 and 4-241, and a tetraloop. In some embodiments, the tetraloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (e.g., delivery ligands), and combinations thereof. Double chain length In some embodiments, the length of the double strand formed between the sense and antisense strands is at least 12 nucleotides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides). In some embodiments, the length of the double strand formed between the sense and antisense strands is in the range of 12-30 nucleotides (e.g., 12-30, 12-27, 12-22, 15-25, 18-30, 18-22, 18-25, 18-27, 18-30, 19-30, or 21-30 nucleotides). In some embodiments, the length of the double strand formed between the sense and antisense strands is 12, 13, 14, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 12 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 13 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 14 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 15 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 16 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 17 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 18 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 19 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 20 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 21 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 22 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 23 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 24 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 25 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 26 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 27 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 28 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 29 nucleotides. In some embodiments, the length of the duplex formed between the sense and antisense strands is 30 nucleotides.In some embodiments, the double strand formed between the sense and antisense strands does not span the entire length of the sense and / or antisense strands. In some embodiments, the double strand between the sense and antisense strands spans the entire length of either the sense or antisense strand. In some embodiments, the double strand between the sense and antisense strands spans the entire length of both the sense and antisense strands. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. The length of the double helix formed between the sense and antisense strands is in the range of 12-30 nucleotides (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides).

[0125] In some embodiments, the double helix between the sense and antisense strands spans the entire length of both the sense and antisense strands. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The length of the double helix formed between the sense and antisense strands is in the range of 12-30 nucleotides (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides).

[0126] In some embodiments, the duplex between the sense and antisense strands spans the entire length of both the sense and antisense strands. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The length of the double helix formed between the sense and antisense strands is in the range of 12-30 nucleotides (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 18 to 30, 18 to 22, 18 to 25, 18 to 27, 18 to 30, 19 to 30 or 21 to 30 nucleotides). oligonucleotide ends In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the ends of any one or both strands comprise blunt ends. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, which are separate strands forming an asymmetric double-stranded region with a protruding end at the 3' end of the antisense strand. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the ends of any one or both strands comprise a protruding end containing one or more nucleotides. In some embodiments, the one or more nucleotides constituting the protruding end are unpaired nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 3' end of the sense strand and the 5' end of the antisense strand comprise blunt ends. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 5' end of the sense strand and the 3' end of the antisense strand comprise blunt ends.

[0128] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 3' end of any one or both strands comprises a 3'-protrusion containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the sense strand comprises a 3'-protrusion containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 3'-protrusion containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprise a 3'-protrusion containing one or more nucleotides.

[0129] In some implementations, the length of the 3'-protrusion is about one (1) to twenty (20) nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides). In some embodiments, the length of the 3' overhang is about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to two (2) nucleotides. In some embodiments, the length of the 3' overhang is (1) nucleotide. In some embodiments, the length of the 3' overhang is two (2) nucleotides. In some embodiments, the length of the 3' overhang is three (3) nucleotides. In some embodiments, the 3'-overhead is four (4) nucleotides long. In some embodiments, the 3'-overhead is five (5) nucleotides long. In some embodiments, the 3'-overhead is six (6) nucleotides long. In some embodiments, the 3'-overhead is seven (7) nucleotides long. In some embodiments, the 3'-overhead is eight (8) nucleotides long. In some embodiments, the 3'-overhead is nine (9) nucleotides long. In some embodiments, the 3'-overhead is ten (10) nucleotides long. In some embodiments, the 3'-overhead is eleven (11) nucleotides long. In some embodiments, the 3'-overhead is twelve (12) nucleotides long. In some embodiments, the 3'-overhead is thirteen (13) nucleotides long. In some embodiments, the 3'-overhead is fourteen (14) nucleotides long. In some embodiments, the 3'-overhead is fifteen (15) nucleotides long. In some embodiments, the 3'-overhead is sixteen (16) nucleotides long. In some embodiments, the 3'-protrusion is seventeen (17) nucleotides long. In some embodiments, the 3'-protrusion is eighteen (18) nucleotides long. In some embodiments, the 3'-protrusion is nineteen (19) nucleotides long. In some embodiments, the 3'-protrusion is twenty (20) nucleotides long.

[0130] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 3'-overhang, and wherein the sense strand and antisense strand of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. And the antisense strand therein contains a 3'-protrusion of about one (1) to twenty (20) nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides), optionally wherein the 3'-protrusion is two (2) nucleotides long.

[0131] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 3'-overhang, and wherein the sense strand and antisense strand of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. And the antisense strand therein contains a 3'-protrusion of about one (1) to twenty (20) nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides), optionally wherein the 3'-protrusion is two (2) nucleotides long.

[0132] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 3'-overhead terminus, and wherein the sense strand and antisense strand of the oligonucleotide comprise the following nucleotide sequences: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. And the antisense strand therein contains a 3'-protrusion of about one (1) to twenty (20) nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides), optionally wherein the 3'-protrusion is two (2) nucleotides long.

[0133] In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the 5' end of any one or both strands comprises a 5'-protrusion containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the sense strand comprises a 5'-protrusion containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 5'-protrusion containing one or more nucleotides. In some embodiments, the oligonucleotides herein comprise a sense strand and an antisense strand, wherein both the sense strand and the antisense strand comprise a 5'-protrusion containing one or more nucleotides.

[0134] In some implementations, the 5'-protrusion is about one (1) to twenty (20) nucleotides long (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides long). In some embodiments, the length of the 5' overhang is about one (1) to nineteen (19), one (1) to eighteen (18), one (1) to seventeen (17), one (1) to sixteen (16), one (1) to fifteen (15), one (1) to fourteen (14), one (1) to thirteen (13), one (1) to twelve (12), one (1) to eleven (11), one (1) to ten (10), one (1) to nine (9), one (1) to eight (8), one (1) to seven (7), one (1) to six (6), one (1) to five (5), one (1) to four (4), one (1) to three (3), or about one (1) to two (2) nucleotides. In some embodiments, the length of the 5' overhang is (1) nucleotide. In some embodiments, the length of the 5' overhang is two (2) nucleotides. In some embodiments, the length of the 5' overhang is three (3) nucleotides. In some embodiments, the 5'-overhead is four (4) nucleotides long. In some embodiments, the 5'-overhead is five (5) nucleotides long. In some embodiments, the 5'-overhead is six (6) nucleotides long. In some embodiments, the 5'-overhead is seven (7) nucleotides long. In some embodiments, the 5'-overhead is eight (8) nucleotides long. In some embodiments, the 5'-overhead is nine (9) nucleotides long. In some embodiments, the 5'-overhead is ten (10) nucleotides long. In some embodiments, the 5'-overhead is eleven (11) nucleotides long. In some embodiments, the 5'-overhead is twelve (12) nucleotides long. In some embodiments, the 5'-overhead is thirteen (13) nucleotides long. In some embodiments, the 5'-overhead is fourteen (14) nucleotides long. In some embodiments, the 5'-overhead is fifteen (15) nucleotides long. In some embodiments, the 5'-overhead is sixteen (16) nucleotides long. In some embodiments, the 5'-overhead is seventeen (17) nucleotides long. In some embodiments, the 5'-overhead is eighteen (18) nucleotides long. In some embodiments, the 5'-overhead is nineteen (19) nucleotides long. In some embodiments, the 5'-overhead is twenty (20) nucleotides long.

[0135] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 5'-overhead terminus, and wherein the sense strand and antisense strand of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. And the antisense strand therein contains a 3'-protrusion of about one (1) to twenty (20) nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides), optionally wherein the 3'-protrusion is two (2) nucleotides long.

[0136] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 5'-overhead terminus, and wherein the sense strand and antisense strand of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. And the antisense strand therein contains a 5'-protrusion of about one (1) to twenty (20) nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides), optionally wherein the 5'-protrusion is two (2) nucleotides long.

[0137] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 5'-overhead terminus, and wherein the sense strand and antisense strand of the oligonucleotide comprise the following nucleotide sequences: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. And the antisense strand therein contains a 5'-protrusion of about one (1) to twenty (20) nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or about 20 nucleotides), optionally wherein the 5'-protrusion is two (2) nucleotides long.

[0138] In some embodiments, one or more (e.g., 2, 3, 4, 5 or more) nucleotides constituting the 3' or 5' end of the sense strand and / or antisense strand are modified. For example, in some embodiments, one or both terminal nucleotides of the 3' end of the antisense strand are modified. In some embodiments, the last nucleotide of the 3' end of the antisense strand is modified such that it contains a 2' modification or contains 2'-O-methoxyethyl. In some embodiments, the last or two terminal nucleotides of the 3' end of the antisense strand are complementary to the target. In some embodiments, the last or two nucleotides of the 3' end of the antisense strand are not complementary to the target.

[0139] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the 3' end of the sense strand comprises a stem-loop as described herein, and the 3' end of the antisense strand comprises a 3'-protrusion as described herein. In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand forming a notched tetracyclic structure as described herein, wherein the 3' end of the sense strand comprises a stem-loop, wherein the loop is a tetracyclic structure as described herein, and wherein the 3' end of the antisense strand comprises a 3'-protrusion as described herein. In some embodiments, the 3'-protrusion is two (2) nucleotides in length. In some embodiments, both (2) nucleotides constituting the 3'-protrusion contain guanine (G) nucleotides. Typically, one or both nucleotides constituting the 3'-protrusion of the antisense strand are not complementary to the target mRNA.

[0140] Oligonucleotide modification In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) contain modifications. Oligonucleotides (e.g., RNAi oligonucleotides) can be modified in a variety of ways to improve or control specificity, stability, delivery, bioavailability, resistance to nuclease degradation, immunogenicity, base pairing properties, RNA distribution and cellular uptake, and other characteristics relevant to therapeutic or research use.

[0141] In some embodiments, the modification is a modified sugar. In some embodiments, the modification is a 5'-terminal phosphate group. In some embodiments, the modification is a modified intermolecular bond. In some embodiments, the modification is a modified base. In some embodiments, the oligonucleotide described herein may contain any of the modifications described herein or any combination thereof. For example, in some embodiments, the oligonucleotide described herein contains at least one modified sugar, a 5'-terminal phosphate group, at least one modified intermolecular bond, and at least one modified base. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide comprises at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleotide bond, and at least one modified base.

[0142] In some embodiments, the oligonucleotide described herein comprises at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleotide bond, and at least one modified base. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The oligonucleotide comprises at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleotide bond, and at least one modified base.

[0143] In some embodiments, the oligonucleotide described herein comprises at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleotide bond, and at least one modified base. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide comprises at least one modified sugar, a 5'-terminal phosphate group, at least one modified internucleotide bond, and at least one modified base.

[0144] The number of modifications on an oligonucleotide (e.g., RNAi oligonucleotide) and the location of these modifications can affect the properties of the oligonucleotide. For example, the oligonucleotide can be delivered in vivo by conjugating or encapsulating it within a lipid nanoparticle (LNP) or similar carrier. However, when the oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Therefore, in some embodiments, all or substantially all of the nucleotides of the oligonucleotide are modified. In some embodiments, more than half of the nucleotides are modified. In some embodiments, less than half of the nucleotides are modified. In some embodiments, the sugar moiety of all the nucleotides constituting the oligonucleotide is modified at the 2' position. The modification may be reversible or irreversible. In some embodiments, the oligonucleotides disclosed herein have a sufficient number and type of modified nucleotides to produce the desired properties (e.g., protection against enzymatic degradation, the ability to target desired cells after in vivo administration, and / or thermodynamic stability).

[0145] Sugar modification In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise modified sugars. In some embodiments, the modified sugars (also referred to herein as sugar analogs) comprise modified deoxyribose or ribose moieties, wherein, for example, one or more modifications occur at the 2', 3', 4', and / or 5' carbon positions of the sugar. In some embodiments, the modified sugars may also comprise non-natural alternative carbon structures, such as those found in locked nucleic acids (“LNAs”; see, for example, Koshkin et al. (1998)). 54:3607-30), Unidentified Nucleic Acids (“UNA”; see, for example, Snead et al. (2013) 2:e103) and bridging nucleic acids (“BNA”; see, for example, Imanishi & Obika (2002) The structures in 21:1653-59.

[0146] In some embodiments, the nucleotide modification of the sugar includes a 2'-modification. In some embodiments, the 2'-modification can be 2'-O-propargyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F), 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), or 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA). In some embodiments, the modification is 2'-F, 2'-OMe, or 2'-MOE. In some embodiments, the modification of the sugar includes modification of the sugar ring, which may include modification of one or more carbons of the sugar ring. For example, modification of the sugar in a nucleotide may include linking the 2'-oxygen of the sugar to the 1'-carbon or 4'-carbon of the sugar, or linking the 2'-oxygen to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, the modified nucleotide has an acyclic sugar lacking the 2'-carbon to 3'-carbon bond. In some embodiments, the modified nucleotide has a thiol group, for example, at the 4' position of the sugar.

[0147] In some embodiments, the oligonucleotide described herein (e.g., RNAi oligonucleotide) comprises at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 or more). In some embodiments, the sense strand of the oligonucleotide comprises at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35 or more). In some embodiments, the antisense strand of the oligonucleotide comprises at least about one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20 or more).

[0148] In some embodiments, all nucleotides of the sense strand of the oligonucleotide are modified. In some embodiments, all nucleotides of the antisense strand of the oligonucleotide are modified. In some embodiments, all nucleotides of the oligonucleotide (i.e., both the sense and antisense strands) are modified. In some embodiments, the modified nucleotides contain 2'-modifications (e.g., 2'-F or 2'-OMe, 2'-MOE, and 2'-deoxy-2'-fluoro-β-d-arabinose).

[0149] In some embodiments, this disclosure provides oligonucleotides with different modification patterns. In some embodiments, the oligonucleotides herein comprise a sense strand having the modification patterns as illustrated in the examples and sequence listing, and an antisense strand having the modification patterns as illustrated in the examples and sequence listing.

[0150] In some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) comprise an antisense strand having a nucleotide modified with 2'-F. In some embodiments, the oligonucleotides disclosed herein comprise an antisense strand having a nucleotide modified with both 2'-F and 2'-OMe. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand having a nucleotide modified with 2'-F. In some embodiments, the oligonucleotides disclosed herein comprise a sense strand having a nucleotide modified with both 2'-F and 2'-OMe.

[0151] In some embodiments, the oligonucleotides described herein comprise a sense strand, of which about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides contain a 2'-fluorine modification. In some embodiments, about 11% of the nucleotides in the sense strand contain a 2'-fluorine modification. In some embodiments, the oligonucleotides described herein comprise an antisense strand, of which about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides contain a 2'-fluorine modification. In some embodiments, about 32% of the nucleotides in the antisense strand contain a 2'-fluorine modification. In some embodiments, about 15-25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of the nucleotides in the oligonucleotide contain a 2'-fluorine modification. In some embodiments, about 19% of the nucleotides in the oligonucleotide contain a 2'-fluorine modification.

[0152] In some embodiments, one or more positions of the sense strand at positions 8, 9, 10, or 11 are modified with a 2'-F group. In some embodiments, one or more positions of the sense strand at positions 3, 8, 9, 10, 12, 13, and 17 are modified with a 2'-F group. In some embodiments, one or more positions of the antisense strand at positions 2, 3, 4, 5, 7, 10, and 14 are modified with a 2'-F group. In some embodiments, one or more positions of positions 2, 3, 4, 5, 7, 8, 10, 14, 16, and 19 are modified with a 2'-F group. In some embodiments, the sugar moiety at each nucleotide at positions 1-7 and 12-20 of the sense strand is modified with 2'-OMe. In some embodiments, the sugar moiety at each nucleotide at positions 1-7, 12-27, and 31-36 of the sense strand is modified with 2'-OMe. In some implementations, the sugar portion of each nucleotide at positions 6, 9, 11-13, 15, 17, 18, and 20-22 in the sense strand is modified with 2'-OMe.

[0153] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. One or more of positions 8, 9, 10, or 11 of the sense chain are modified by a 2'-F group.

[0154] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. One or more of positions 8, 9, 10, or 11 of the sense chain are modified by a 2'-F group.

[0155] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. One or more of positions 8, 9, 10, or 11 of the sense chain are modified by a 2'-F group.

[0156] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety of each nucleotide at positions 2, 5, and 14 is modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0157] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety of each nucleotide at positions 1, 2, 5, and 14 is modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0158] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety of each nucleotide at positions 2, 4, 5, and 14 is modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0159] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety of each nucleotide at positions 1, 2, 3, 5, 7, and 14 is modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0160] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety of each nucleotide at positions 2, 3, 4, 5, 7, and 14 is modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0161] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety of each nucleotide at positions 1, 2, 3, 5, 10, and 14 is modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0162] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety of each nucleotide at positions 2, 3, 4, 5, 10, and 14 is modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a selection from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0163] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety of each nucleotide at positions 2, 3, 5, 7, 10, and 14 is modified with 2'-F, and the sugar moiety of each of the remaining nucleotides of the antisense strand is modified with a modification selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0164] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety of each nucleotide at positions 2, 3, 4, 5, 7, 10, and 14 is modified with 2'-F, and the sugar moiety of the remaining nucleotides of the antisense strand is modified with a selection from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0165] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety of each nucleotide at positions 2, 3, 4, 5, 7, 8, 10, 14, 16, and 19 is modified with 2'-F, and the sugar moiety of the remaining nucleotides of the antisense strand is modified with a modification selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0166] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21 or position 22 is modified with 2'-F.

[0167] In some embodiments, the oligonucleotides provided herein comprise an antisense strand in which the sugar moiety at position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21 or position 22 is modified with 2'-OMe.

[0168] In some embodiments, the oligonucleotides provided herein comprise an antisense strand whose sugar moiety at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 is modified with a sugar moiety selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0169] In some embodiments, the oligonucleotides provided herein include a sense strand in which the sugar moiety at positions 8-11 is modified with 2'-F. In some embodiments, the oligonucleotides provided herein include a sense strand in which the sugar moiety at positions 3, 8, 9, 10, 12, 13, and 17 is modified with 2'-F. In some embodiments, the oligonucleotides provided herein include a sense strand in which the sugar moiety at positions 1-7 and 12-17 or 12-20 is modified with 2'OMe. In some embodiments, the oligonucleotides provided herein include a sense strand in which the sugar moiety at positions 1-7, 12-27, and 31-36 is modified with 2'OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand in which the sugar moiety of each nucleotide at positions 1-7 and 12-17 or 12-20 is modified with a modification selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA). In some embodiments, the oligonucleotides provided herein comprise a sense strand in which the sugar moiety at positions 1-2, 4-7, 11, 14-16, and 18-20 is modified with 2'OMe. In some embodiments, the oligonucleotides provided herein comprise a sense strand in which the sugar moiety of each nucleotide at positions 1-2, 4-7, 11, 14-16, and 18-20 is modified with a modification selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0170] In some embodiments, the oligonucleotides provided herein comprise a sense strand in which the sugar moiety at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 is modified with 2'-F.

[0171] In some embodiments, the oligonucleotides provided herein comprise a sense strand in which the sugar moiety at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 is modified with 2'-OMe.

[0172] In some embodiments, the oligonucleotides provided herein comprise a sense strand at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 25. The sugar moiety at positions 32, 33, 34, 35, or 36 is modified by a group selected from 2'-O-propynyl, 2'-O-propylamino, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), and 2'-deoxy-2'-fluoro-β-d-arabinose (2'-FANA).

[0173] 5'-terminal phosphate In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise a sense strand and an antisense strand, wherein the antisense strand comprises a 5'-terminal phosphate ester. In some embodiments, the 5'-terminal phosphate ester group of the RNAi oligonucleotide enhances its interaction with Ago2. However, oligonucleotides comprising a 5'-phosphate ester group may be readily degraded via phosphatases or other enzymes, which may limit their performance and / or bioavailability in vivo. In some embodiments, the oligonucleotides described herein comprise 5'-phosphate ester analogs resistant to such degradation. In some embodiments, the phosphate ester analog is an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate, or a combination thereof. In some embodiments, the 5' end of the oligonucleotide chain is attached to a chemical moiety (“phosphate ester analog”) that mimics the electrostatic and steric properties of the native 5'-phosphate ester group. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. The antisense chain contains a 5'-terminal phosphate ester, optionally a 5'-terminal phosphate ester analog.

[0174] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The antisense chain contains a 5'-terminal phosphate ester, optionally a 5'-terminal phosphate ester analog.

[0175] In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) SEQ ID NO: 484 and 725, respectively; (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The antisense chain contains a 5'-terminal phosphate ester, optionally a 5'-terminal phosphate ester analog.

[0176] In some embodiments, the oligonucleotides herein (e.g., RNAi oligonucleotides) have a phosphate ester analog (referred to as a "4'-phosphate ester analog") at the 4'-carbon position of the sugar. See, for example, International Patent Application Publication No. WO 2018 / 045317. In some embodiments, the oligonucleotides herein contain a 4'-phosphate ester analog at the 5'-terminal nucleotide. In some embodiments, the phosphate ester analog is an oxymethylphosphonate, wherein the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4'-carbon position) or an analog thereof. In other embodiments, the 4'-phosphate ester analog is a thiomethylphosphonate or an aminomethylphosphonate, wherein the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bound to the 4'-carbon of the sugar moiety or an analog thereof. In some embodiments, the 4'-phosphate ester analog is an oxymethylphosphonate. In some embodiments, the oxymethylphosphonate is represented by the formula –O–CH2–PO(OH)2, –O–CH2–PO(OR)2, or -O-CH2-POOH(R), wherein R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group. In some embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3. In some embodiments, R is CH3. In some embodiments, the 4'-phosphate analog is a 4'-oxymethylphosphonate.

[0177] In some embodiments, the oligonucleotides provided herein comprise an antisense strand containing a 4'-phosphate analog at the 5'-terminal nucleotide, wherein the 5'-terminal nucleotide comprises the following structure: 4'-O-monomethylphosphonate-2'-O-methyluridine thiophosphate [MePhosphonate-4O-mUs].

[0178] Modified nucleotide interbonding In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise modified internucleotide bonds. In some embodiments, phosphate modification or substitution produces an oligonucleotide comprising at least one (e.g., at least one, at least two, at least three, or at least five) modified internucleotide bonds. In some embodiments, any oligonucleotide disclosed herein comprises about one to about ten (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3, or 1 to 2) modified internucleotide bonds. In some embodiments, any oligonucleotide disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide bonds.

[0179] The modified internucleotide bonds can be dithiophosphate bonds, thiophosphate bonds, phosphate trimer bonds, thioalkylphosphonate bonds, thioalkylphosphonate bonds, phosphoramide bonds, phosphonate bonds, or boranophosphate bonds. In some embodiments, at least one modified internucleotide bond of any of the oligonucleotides disclosed herein is a thiophosphate bond.

[0180] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) have a phosphate thioester bond between one or more of the following: sense strand positions 1 and 2, antisense strand positions 1 and 2, antisense strand positions 2 and 3, antisense strand positions 3 and 4, antisense strand positions 20 and 21, and antisense strand positions 21 and 22. In some embodiments, the oligonucleotides described herein have a phosphate thioester bond between each of the following: sense strand positions 1 and 2, antisense strand positions 1 and 2, antisense strand positions 2 and 3, antisense strand positions 20 and 21, and antisense strand positions 21 and 22. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. The oligonucleotides described therein comprise modified internucleotide bonds.

[0181] In some embodiments, the oligonucleotide described herein has a phosphate thioester bond between each of the sense strand positions 1 and 2, the antisense strand positions 1 and 2, the antisense strand positions 2 and 3, the antisense strand positions 20 and 21, and the antisense strand positions 21 and 22. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The oligonucleotides described therein comprise modified internucleotide bonds.

[0182] In some embodiments, the oligonucleotide described herein has a phosphate thioester bond between each of the sense strand positions 1 and 2, the antisense strand positions 1 and 2, the antisense strand positions 2 and 3, the antisense strand positions 20 and 21, and the antisense strand positions 21 and 22. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The oligonucleotides described therein comprise modified internucleotide bonds.

[0183] Base modification In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise one or more modified nucleobases. In some embodiments, the modified nucleobase (also referred to herein as a base analog) is linked at the 1' position of the nucleotide sugar moiety. In some embodiments, the modified nucleobase is a nitrogenous base. In some embodiments, the modified nucleobase does not contain a nitrogen atom. See, for example, U.S. Patent Application Publication No. 2008 / 0274462. In some embodiments, the modified nucleotide comprises a universal base. In some embodiments, the modified nucleotide does not contain a nucleobase (base-free). In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively.

[0184] The oligonucleotides mentioned therein contain one or more modified nucleobases.

[0185] In some embodiments, the modified nucleotide contains universal bases. In some embodiments, the modified nucleotide does not contain nucleobases (base-free). In some embodiments, the sense and antisense strands of the oligonucleotide contain nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The oligonucleotides mentioned therein contain one or more modified nucleobases.

[0186] In some embodiments, the modified nucleotide contains universal bases. In some embodiments, the modified nucleotide does not contain nucleobases (base-free). In some embodiments, the sense and antisense strands of the oligonucleotide contain nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The oligonucleotides mentioned therein contain one or more modified nucleobases.

[0187] In some embodiments, the universal base is a heterocyclic moiety located at the 1' position of the nucleotide sugar moiety in the modified nucleotide, or at an equivalent position in a nucleotide sugar moiety substitution, such that when present in a duplex, it can be positioned opposite more than one type of base without substantially altering the duplex structure. In some embodiments, compared to the target nucleic acid (e.g., CD274 A reference single-stranded nucleic acid (e.g., an oligonucleotide) that is completely complementary to mRNA, containing universal bases, forms a double strand with the target nucleic acid. The T of this double strand... m The T-stranded nucleic acid is lower than that formed with complementary nucleic acids. In some embodiments, when compared with a reference single-stranded nucleic acid in which universal bases have been replaced to generate a single mismatch, the single-stranded nucleic acid containing universal bases forms a double-stranded nucleic acid with the target nucleic acid, the T-stranded nucleic acid of which... m It is higher than the duplex formed with nucleic acid containing the mismatched base.

[0188] Non-limiting examples of general binding nucleotides include, but are not limited to, inosine, 1-β-D-furanoribosyl-5-nitroindole, and / or 1-β-D-furanoribosyl-3-nitropyrrole (see, U.S. Patent Application Publication No. 2007 / 0254362; Van Aerschot et al. (1995)). . 23:4363-4370; Loakes et al. (1995) Nucleic Acids Res .23:2361-66; and Loakes & Brown (1994) Nucleic Acids Res. 22:4039-43.

[0189] Targeted ligands In some implementations, it is desirable to target the oligonucleotides provided herein (e.g., RNAi oligonucleotides) to one or more cells or cell types, tissues, organs, or anatomical regions or compartments. Such strategies can help avoid adverse effects on the treated organism and / or prevent excessive loss of the oligonucleotide to areas from which its action (e.g., CD274 Cells, tissues, organs, or anatomical regions or compartments that benefit from the inhibition or reduction of expression. Therefore, in some embodiments, the oligonucleotides disclosed herein (e.g., RNAi oligonucleotides) are modified to facilitate targeting and / or delivery to specific cells or cell types, tissues, organs, or anatomical regions or compartments (e.g., facilitating oligonucleotide delivery to tumors). In some embodiments, the oligonucleotide comprises at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, or more nucleotides) conjugated to one or more targeting ligands. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide contains a targeting ligand conjugated to at least one nucleotide.

[0190] In some embodiments, the oligonucleotide comprises at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6 or more nucleotides) conjugated to one or more target ligands. In some embodiments, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The oligonucleotide contains a targeting ligand conjugated to at least one nucleotide.

[0191] In some implementations, the sense and antisense strands of the oligonucleotide comprise the following nucleotide sequences: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide contains a targeting ligand conjugated to at least one nucleotide.

[0192] In some embodiments, the targeting ligand comprises a carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein, or a portion of a protein (e.g., an antibody or antibody fragment), or lipid. In some embodiments, the targeting ligand is a carbohydrate containing at least one GalNAc moiety.

[0193] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide (e.g., RNAi oligonucleotide) provided herein are each conjugated to a separate target ligand (e.g., the GalNAc moiety). In some embodiments, two to four nucleotides of the oligonucleotide are each conjugated to a separate target ligand. In some embodiments, the target ligand is conjugated to two to four nucleotides at either end of the sense or antisense strand (e.g., the target ligand is conjugated to a protruding or extended portion of two to four nucleotides at the 5' or 3' end of the sense or antisense strand), such that the target ligand is analogous to the bristles of a toothbrush, and the oligonucleotide is analogous to the toothbrush. For example, the oligonucleotide may include a stem-loop at the 5' or 3' end of the sense strand, and one, two, three, or four nucleotides of the stem-loop may be conjugated to the target ligand individually. In some embodiments, the oligonucleotides provided in this disclosure (e.g., RNAi oligonucleotides) include a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop comprises a tricyclic or tetracyclic ring, and wherein the three or four nucleotides constituting the tricyclic or tetracyclic ring are individually conjugated to a target ligand. In some embodiments, the oligonucleotides provided in this disclosure (e.g., RNAi oligonucleotides) include a stem-loop at the 3' end of the sense strand, wherein the loop of the stem-loop comprises a tetracyclic ring, and wherein the three nucleotides of the tetracyclic ring are individually conjugated to a target ligand.

[0194] a. GalNAc targeting ligands GalNAc is a high-affinity carbohydrate ligand for the asialic acid glycoprotein receptor (ASGPR), which is primarily expressed on the surface of hepatocytes and plays a major role in the binding, internalization, and subsequent clearance of circulating glycoproteins (asialic acid glycoproteins) containing terminal galactose or GalNAc residues. Conjugation (indirectly or directly) of the GalNAc moiety to the oligonucleotides of this disclosure can be used to target these oligonucleotides to ASGPR expressed on cells. In some embodiments, the oligonucleotides of this disclosure (e.g., RNAi oligonucleotides) are conjugated to at least one or more GalNAc moieties, wherein the GalNAc moieties target the oligonucleotide to ASGPR expressed on human liver cells (e.g., human hepatocytes). In some embodiments, the GalNAc moieties target the oligonucleotide to the liver.

[0195] In some embodiments, the oligonucleotide of this disclosure (e.g., RNAi oligonucleotide) is conjugated directly or indirectly to a monovalent GalNAc moiety. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc moiety (i.e., conjugated to 2, 3, or 4 monovalent GalNAc moieties, and typically to 3 or 4 monovalent GalNAc moieties). In some embodiments, the oligonucleotide is conjugated to one or more divalent, trivalent, or tetravalent GalNAc moieties. In some embodiments, the divalent, trivalent, or tetravalent GalNAc moieties are conjugated to the oligonucleotide via a branched linker. In some embodiments, the monovalent GalNAc moieties are conjugated to a first nucleotide, and the divalent, trivalent, or tetravalent GalNAc moieties are conjugated to a second nucleotide via a branched linker.

[0196] In some embodiments, one (1) or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide described herein (e.g., RNAi oligonucleotides) are each conjugated to a GalNAc moiety. In some embodiments, two (2) to four (4) nucleotides of a tetracyclic ring are each conjugated to a separate GalNAc moiety. In some embodiments, one (1) to three (3) nucleotides of a tricyclic ring are each conjugated to a separate GalNAc moiety. In some embodiments, a targeting ligand is conjugated to two (2) to four (4) nucleotides at either end of the sense or antisense strand (e.g., the ligand is conjugated to a protruding or extended portion of two (2) to four (4) nucleotides at the 5' or 3' end of the sense or antisense strand), such that the GalNAc moiety resembles the bristles of a toothbrush, and the oligonucleotide resembles a toothbrush. In some embodiments, the GalNAc moiety is conjugated to a nucleotide of the sense strand. For example, three (3) or four (4) GalNAc moieties can be conjugated to nucleotides in a tetracycle of sense strand, wherein each GalNAc moiety is conjugated to one (1) nucleotide.

[0197] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise a tetracycle, wherein the tetracycle (L) is any combination of adenine (A) and guanine (G) nucleotides. In some embodiments, the tetracycle (L) comprises a monovalent GalNAc moiety attached to any one or more guanine (G) nucleotides of the tetracycle via any linker described herein, as follows (X = heteroatom): In some embodiments, the tetracycle (L) has a monovalent GalNAc attached to any one or more adenine nucleotides of the tetracycle via any linker described herein, as shown below (X = heteroatom): In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) include a monovalent GalNAc moiety attached to a guanine (G) nucleotide, referred to as [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanine-GalNAc, as follows: In some embodiments, the oligonucleotides described herein include a monovalent GalNAc moiety attached to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc, as shown below: Examples of this type of conjugation are shown below for rings containing the nucleotide sequence GAAA from 5' to 3' (L = linker, X = heteroatom). For example, such a ring can exist at positions 27-30 of the sense strand provided herein. In the chemical formula, Used to describe the attachment point to the oligonucleotide chain.

[0198] Targeting ligands can be linked to nucleotides using appropriate methods or chemistry (e.g., click chemistry). In some embodiments, a click linker is used to conjugate the targeting ligand to a nucleotide of the oligonucleotide (e.g., RNAi oligonucleotide) constituting this document. In some embodiments, an acetal-based linker is used to conjugate the targeting ligand to a nucleotide of any of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO2016 / 100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is stable. An example of a ring from 5' to 3' containing the nucleotide GAAA is shown below, wherein the GalNAc moiety is attached to the nucleotide of the ring using an acetal linker. For example, such a ring can be present at position 27-30 of either sense strand. In the chemical formula, It is the attachment point to the oligonucleotide chain.

[0199] or As mentioned, various suitable methods or chemical synthesis techniques (e.g., click chemistry) can be used to link the target ligand to a nucleotide. In some embodiments, a click linker is used to conjugate the target ligand to a nucleotide. In some embodiments, an acetal-based linker is used to conjugate the target ligand to a nucleotide of any of the oligonucleotides described herein. An acetal-based linker is disclosed, for example, in International Patent Application Publication No. WO 2016 / 100401. In some embodiments, the linker is an unstable linker. However, in other embodiments, the linker is a stable linker.

[0200] In some embodiments, a double-stranded extension (e.g., up to 3, 4, 5, or 6 bp in length) is provided between the targeting ligand (e.g., the GalNAc moiety) and the oligonucleotide. In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) do not have a GalNAc conjugated thereto.

[0201] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide comprises at least one GalNAc moiety conjugated to a nucleotide.

[0202] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The oligonucleotide comprises at least one GalNAc moiety conjugated to a nucleotide.

[0203] b. Lipid-targeting ligands In some embodiments, this disclosure provides an oligonucleotide-ligand conjugate comprising an oligonucleotide containing a nucleotide sequence for inhibiting the expression of a target mRNA (e.g., CD274) expressed in a tumor, and one or more targeting ligands conjugated to the oligonucleotide. In some embodiments, the oligonucleotide-ligand conjugate described herein comprises a nucleotide sequence and one or more targeting ligands, wherein the nucleotide sequence comprises one or more nucleosides (nucleic acids) conjugated to one or more targeting ligands according to formula Ia: ; Ia Or its pharmaceutically acceptable salt. in: B is a nucleobase or hydrogen; R 1 and R 2 Independently hydrogen, halogen, R A -CN, -S(O)R, -S(O)2R, -Si(OR)2R, -Si(OR)R2 or -SiR3; or R on the same carbon 1 and R 2 Together with their intermediary atoms, they form 3-7 member saturated or partially unsaturated rings with 0-3 independent heteroatoms selected from nitrogen, oxygen and sulfur; Each R A Independently, the optionally substituted group is selected from C. 1-6 Aliphatic group, phenyl, 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; Each R is independently hydrogen, a suitable protecting group, or an optionally substituted group, the optionally substituted group being selected from C. 1-6 Aliphatic group, phenyl, having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, comprising a 4-7 membered saturated or partially unsaturated heterocycle, and a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; or Two R groups on the same atom, together with their intervening atoms, form a 4-7 member saturated, partially unsaturated or heteroaryl ring having 0-3 independent heteroatoms selected from nitrogen, oxygen, silicon and sulfur. Each targeting ligand is a lipid conjugate moiety (LC); and each LC is independently composed of saturated or unsaturated straight-chain or branched C. 1-50 The lipid conjugate portion of a hydrocarbon chain, wherein 0-10 methylene units of the hydrocarbon chain are independently replaced by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, -P(S)OR-; Each -Cy- is independently a optionally substituted divalent ring selected from phenylene, 8-10 membered bicyclic arylene, 4-7 membered saturated or partially unsaturated carbocyclic, 4-11 membered saturated or partially unsaturated spirocyclic, 8-10 membered bicyclic saturated or partially unsaturated carbocyclic, 4-7 membered saturated or partially unsaturated heterocyclic with 1-3 heteroatoms independently selected from nitrogen, oxygen and sulfur, 4-11 membered saturated or partially unsaturated spirocyclic with 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, 8-10 membered bicyclic saturated or partially unsaturated heterocyclic with 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, 5-6 membered heterocyclic with 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur, or 8-10 membered bicyclic heterocyclic with 1-5 heteroatoms independently selected from nitrogen, oxygen or sulfur. n is 1-10; L represents a covalent bond or a divalent saturated or unsaturated straight chain or branched chain. 1-50 A hydrocarbon chain wherein 0-10 methylene units of the hydrocarbon chain are independently converted by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O-, -S(O)2-, -P(O)OR-, -P(S)OR-, -V 1 CR 2 W 1 -or replace; m is 1-50; X 1 V 1 and W 1 Independently -C(R)2-, -OR, -O-, -S-, -Se-, or -NR-; Y represents hydrogen, a suitable hydroxyl protecting group, or ; R 3 The group may be hydrogen, a suitable protecting group, a suitable prodrug, or an optionally substituted group, wherein the optionally substituted group is selected from C.1-6 Aliphatic group, phenyl, 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur; X 2 For O, S, or NR; X 3 It can be -O-, -S-, -BH2- or covalent; Y 1 A linker group attached to the 2'- or 3'-terminus of a nucleoside, nucleotide, or oligonucleotide; Y 2 Hydrogen, a suitable protecting group, a phosphoramide analog, an internucleotide linker attached to the 5'-terminus of a nucleoside, nucleotide, or oligonucleotide, or a linker attached to a solid support; and Z can be -O-, -S-, -NR-, or -CR2-.

[0204] In some embodiments, the oligonucleotide-ligand conjugate comprises one or more nucleic acids conjugated to a target ligand, represented by formula II-a: II-a Or its pharmaceutically acceptable salt.

[0205] In some embodiments, the oligonucleotide-ligand conjugate comprises one or more nucleic acids conjugated to a target ligand, represented by formula II-b or II-c: II-b II-c Or its pharmaceutically acceptable salt, wherein: L 1 C is a straight or branched chain that is covalent, monovalent, divalent, saturated, or unsaturated. 1-50 A hydrocarbon chain wherein 0-10 methylene units of the hydrocarbon chain are independently converted by -Cy-, -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O-, -S(O)2-, -P(O)OR-, -P(S)OR- or replace; R 4 For hydrogen, R A Or a suitable amine protecting group; and R 5 It is an adamantyl group, or a saturated or unsaturated straight-chain or branched C group. 1-50A hydrocarbon chain in which 0-10 methylene units are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR- or -P(S)OR.

[0206] In some implementation schemes, R 5 Selected from .

[0207] In some implementation schemes, R 5 Selected from: .

[0208] In some implementation schemes, R 5 for .

[0209] In some implementation schemes, R 5 for .

[0210] In some implementation schemes, R 5 for .

[0211] In some implementation schemes, R 5 for .

[0212] In some implementation schemes, R 5 for .

[0213] In some implementation schemes, R 5 for .

[0214] In some implementation schemes, R 5 for .

[0215] In some implementation schemes, R 5 for .

[0216] In some implementation schemes, R 5 for .

[0217] In some implementation schemes, R 5 for .

[0218] In some implementation schemes, R 5 for .

[0219] In some implementation schemes, R 5 for .

[0220] In some implementation schemes, R 5 for .

[0221] In some implementation schemes, R 5 for .

[0222] In some embodiments, the oligonucleotide-ligand conjugate comprises one or more nucleic acids conjugated to a target ligand, represented by formula II-Ib or II-Ic: II-Ib II-Ic Or its pharmaceutically acceptable salt; wherein B is a nucleobase or hydrogen; m is 1-50; X 1 It can be -O- or -S-; Y represents hydrogen. or ; R 3 Hydrogen or a suitable protecting group; X 2 For O or S; X 3 It can be -O-, -S-, or covalent; Y 1 A linker group attached to the 2'- or 3'-terminus of a nucleoside, nucleotide, or oligonucleotide; Y 2 It is a hydrogen, phosphoramide analogue, an internucleotide linker attached to the 5'-terminus of a nucleoside, nucleotide or oligonucleotide, or a linker attached to a solid support. R 5 It is an adamantyl group, or a saturated or unsaturated straight-chain or branched C group. 1-50 A hydrocarbon chain wherein 0-10 methylene units are independently replaced by -O-, -C(O)NR-, -NR-, -S-, -C(O)-, -C(O)O-, -S(O)-, -S(O)2-, -P(O)OR-, or -P(S)OR-; and R is hydrogen, a suitable protecting group, or an optionally substituted group, wherein the optionally substituted group is selected from C. 1-6Aliphatic group, phenyl, 4-7 membered saturated or partially unsaturated heterocycle having 1-2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen and sulfur.

[0223] In some implementation schemes, R 5 Selected from .

[0224] In some implementation schemes, R 5 for .

[0225] In some embodiments, the oligonucleotide sequence contains 1-10 targeting ligands. In some embodiments, the nucleotide sequence contains 1, 2, or 3 targeting ligands. In some embodiments, the nucleotide sequence contains 1 targeting ligand.

[0226] In some embodiments, the oligonucleotide of the oligonucleotide-ligand conjugate is a double-stranded molecule. In some embodiments, the oligonucleotide is an RNAi molecule. In some embodiments, the double-stranded oligonucleotide includes a stem-loop. In some embodiments, the ligand is conjugated to any nucleotide in the stem-loop. In some embodiments, the ligand is conjugated to the first nucleotide from 5' to 3' in the stem-loop. In some embodiments, the ligand is conjugated to the second nucleotide from 5' to 3' in the stem-loop. In some embodiments, the ligand is conjugated to the third nucleotide from 5' to 3' in the stem-loop. In some embodiments, the ligand is conjugated to the fourth nucleotide from 5' to 3' in the stem-loop. In some embodiments, the ligand is conjugated to one, two, three, or four nucleotides in the stem-loop. In some embodiments, the ligand is conjugated to three nucleotides in the stem-loop.

[0227] In some embodiments, the oligonucleotide-ligand conjugate comprises a 36-nucleotide sense strand, with positions from 5' to 3' numbered 1-36. In some embodiments, the oligonucleotide-ligand conjugate comprises a lipid conjugated to position 1 of the 36-nucleotide sense strand. In some embodiments, the oligonucleotide-ligand conjugate comprises a lipid conjugated to the 5' terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide-ligand conjugate comprises a lipid conjugated to the 5' terminal nucleotide of the 36-nucleotide sense strand. In some embodiments, the oligonucleotide-ligand conjugate comprises a lipid conjugated to position 27 of the 36-nucleotide sense strand. In some embodiments, the oligonucleotide-ligand conjugate comprises a lipid conjugated to position 28 of the 36-nucleotide sense strand. In some embodiments, the oligonucleotide conjugate comprises a lipid conjugated to position 29 of the 36-nucleotide sense strand. In some embodiments, the oligonucleotide conjugate comprises a lipid conjugated to position 30 of the 36-nucleotide sense strand.

[0228] In some embodiments, the oligonucleotide-ligand conjugate comprises a C8-C30 hydrocarbon chain conjugated at position 1 of the sense strand of 36 nucleotides. In some embodiments, the oligonucleotide-ligand conjugate comprises a C22 hydrocarbon chain conjugated at position 1 of the sense strand of 36 nucleotides.

[0229] In some embodiments, the oligonucleotide-ligand conjugate comprises a lipid conjugated to the 5' terminal nucleotide of the sense chain. In some embodiments, the oligonucleotide-ligand conjugate comprises a C8-C30 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense chain. In some embodiments, the oligonucleotide-ligand conjugate comprises a C22 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense chain. In some embodiments, the oligonucleotide-ligand conjugate comprises a C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense chain.

[0230] In some embodiments, the oligonucleotide-ligand conjugate comprises a hydrocarbon chain conjugated to the 2' carbon of the ribosome ring of the 5' terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide-ligand conjugate comprises a C8-C30 hydrocarbon chain conjugated to the 2' carbon of the ribosome ring of the 5' terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide-ligand conjugate comprises a C22 hydrocarbon chain conjugated to the 2' carbon of the ribosome ring of the 5' terminal nucleotide of the sense strand. In some embodiments, the oligonucleotide-ligand conjugate comprises a C18 hydrocarbon chain conjugated to the 2' carbon of the ribosome ring of the 5' terminal nucleotide of the sense strand.

[0231] In some embodiments, the oligonucleotide-ligand conjugate comprises a lipid conjugated to the 5' terminal nucleotide of the sense chain via a linker. In some embodiments, the oligonucleotide-ligand conjugate comprises a C8-C30 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense chain via a linker. In some embodiments, the oligonucleotide-ligand conjugate comprises a C22 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense chain via a linker. In some embodiments, the oligonucleotide-ligand conjugate comprises a C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense chain via a linker.

[0232] In some embodiments, the oligonucleotide-ligand conjugate comprises a hydrocarbon chain conjugated to the 2' carbon of the ribosome ring of the 5' terminal nucleotide of the sense strand via a linker. In some embodiments, the oligonucleotide-ligand conjugate comprises a C8-C30 hydrocarbon chain conjugated to the 2' carbon of the ribosome ring of the 5' terminal nucleotide of the sense strand via a linker. In some embodiments, the oligonucleotide-ligand conjugate comprises a C22 hydrocarbon chain conjugated to the 2' carbon of the ribosome ring of the 5' terminal nucleotide of the sense strand via a linker. In some embodiments, the oligonucleotide-ligand conjugate comprises a C18 hydrocarbon chain conjugated to the 2' carbon of the ribosome ring of the 5' terminal nucleotide of the sense strand via a linker.

[0233] In some embodiments, the oligonucleotide-ligand conjugate comprises an antisense strand of 15 to 30 nucleotides and a sense strand of 15 to 40 nucleotides, wherein the sense strand and the antisense strand form a bistranded region, wherein the antisense strand contains a complementary region complementary to a target sequence expressed in the adrenal gland or adrenal cortex, and wherein the sense strand comprises a stem-loop at its 3' end, the stem-loop comprising a tetracycle containing four nucleotides, wherein one or more of the four nucleotides are represented by formula II-Ib: , Where B is selected from adenine and guanine nucleobases, and where R... 5 It is a hydrocarbon chain. In some embodiments, m is 1, X1 is O, and Y2 is a nucleotide linker attached to the 5' end of the nucleoside. Y by This indicates that Y1 is a linker group attached to the 2' or 3' end of the nucleotide, X2 is O, X3 is O, and R3 is H. In some embodiments, the hydrocarbon chain is a C8-C30 hydrocarbon chain. In some embodiments, the hydrocarbon chain is a C22 hydrocarbon chain. In some embodiments, the C22 hydrocarbon chain is represented by the following formula. .

[0234] In some embodiments, the four nucleotides of the tetracycle are numbered 1-4 from 5' to 3', and position 1 is represented by formula II-Ib. In some embodiments, position 2 is represented by formula II-Ib. In some embodiments, position 3 is represented by formula II-Ib. In some embodiments, position 4 is represented by formula II-Ib. In some embodiments, the sense strand is 36 nucleotides, which are numbered 1-36 from 5' to 3', wherein the stem-loop contains nucleotides at positions 21-36, and wherein one or more nucleotides at positions 27-30 are represented by formula II-Ib. In some embodiments, the antisense strand is 22 nucleotides.

[0235] In some aspects, this disclosure provides oligonucleotide-ligand conjugates for targeting target mRNAs (e.g., target mRNAs that regulate immunosuppression) and inhibiting or reducing the expression of target genes (e.g., via the RNAi pathway), wherein the oligonucleotide-ligand conjugate is a double-stranded (ds) nucleic acid molecule comprising a sense strand (also referred to herein as a guest strand) and an antisense strand (also referred herein as a guide strand). In some embodiments, the sense and antisense strands are separate strands and are not covalently linked. In some embodiments, the sense and antisense strands are covalently linked. In some embodiments, the sense and antisense strands form a double-stranded region, wherein the sense and antisense strands, or portions thereof, bind or anneal to each other in a complementary manner (e.g., via Watson-Crick base pairing).

[0236] In some embodiments, the oligonucleotide-ligand conjugate comprises an antisense strand of 15 to 30 nucleotides and a sense strand of 15 to 40 nucleotides, wherein the sense strand and the antisense strand form a bistranded region, wherein the antisense strand comprises a complementary region complementary to a target sequence expressed in the adrenal gland or adrenal cortex, and wherein the 5' terminal nucleotide of the sense strand comprises a nucleoside represented by formula II-Ib: , Where B is selected from adenine and guanine nucleobases, and where R... 5 It is a hydrocarbon chain. In some embodiments, m is 1, X1 is O, and Y2 is a nucleotide linker attached to the 5' end of the nucleoside. Y by This indicates that Y1 is a linker group attached to the 2' or 3' end of the nucleotide, X2 is O, X3 is O, and R3 is H. In some embodiments, the hydrocarbon chain is a C8-C30 hydrocarbon chain. In some embodiments, the hydrocarbon chain is a C22 hydrocarbon chain. In some embodiments, the C22 hydrocarbon chain is represented by the following formula. .

[0237] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide comprises at least one lipid moiety conjugated to a nucleotide.

[0238] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The oligonucleotide comprises at least one lipid moiety conjugated to a nucleotide.

[0239] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide comprises at least one lipid moiety conjugated to a nucleotide.

[0240] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide comprises at least one hydrocarbon chain conjugated with a nucleotide.

[0241] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The oligonucleotide contains at least one hydrocarbon conjugated with a nucleotide.

[0242] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide contains at least one hydrocarbon conjugated with a nucleotide.

[0243] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide comprises at least one C18 hydrocarbon chain conjugated to a nucleotide.

[0244] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The oligonucleotide contains at least one C18 hydrocarbon conjugated with a nucleotide.

[0245] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide contains at least one C18 hydrocarbon conjugated with a nucleotide.

[0246] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively. The oligonucleotide comprises at least one C22 hydrocarbon chain conjugated with a nucleotide.

[0247] In some implementations, the sense and antisense strands of the oligonucleotide comprise nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively. The oligonucleotide contains at least one C22 hydrocarbon conjugated with a nucleotide.

[0248] Exemplary oligonucleotides for reducing CD274 expression In some implementations, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide (e.g., RNAi oligonucleotide) comprises a sense strand and an antisense strand, wherein all nucleotides constituting the sense strand and the antisense strand are modified, wherein the antisense strand comprises any of SEQ ID NO: 1-2 and 4-241. CD274 The complementary region is complementary to the mRNA target sequence, and the length of the complementary region is at least 15 consecutive nucleotides. In some embodiments, the 5'-terminal nucleotide of the antisense strand comprises 4'-O-monomethylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU], as described herein. In some embodiments, the 5'-terminal nucleotide of the antisense strand comprises a phosphate thioester bond. In some embodiments, the antisense strand and the sense strand comprise one or more nucleotides modified with 2'-fluoro(2'-F) and 2'-O-methyl(2'-OMe) and at least one phosphate thioester bond. In some embodiments, the antisense strand comprises four (4) phosphate thioester bonds, while the sense strand comprises one (1) phosphate thioester bond. In some embodiments, the antisense strand comprises five (5) phosphate thioester bonds, while the sense strand comprises one (1) phosphate thioester bond.

[0249] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a sense strand having a sequence of any one of SEQ ID NO: 483-484 and 486-723 and an antisense strand comprising a complementary sequence selected from SEQ ID NO: 724-725 and 727-964.

[0250] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) comprise a sense strand having a sequence of any one of SEQ ID NO: 965-1000 and an antisense strand comprising a complementary sequence selected from SEQ ID NO: 1001-1036.

[0251] In some implementations, the methods provided herein for reducing CD274 The expressed oligonucleotides (e.g., RNAi oligonucleotides) contain: The sense chain contains nucleotides modified by 2'-F at positions 8-11, nucleotides modified by 2'-OMe at positions 1-7, 12-27 and 31-36, nucleotides conjugated with GalNAc at positions 28, 29 and 30; and phosphate thioester bonds between positions 1 and 2. The antisense strand comprises 2'-F modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14; 2'-OMe at positions 1, 6, 8, 9, 11-13, and 15-22; thiophosphate bonds between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22; and a 5'-terminal nucleotide at position 1 comprising a 4'-phosphate analogue, optionally wherein the 5'-terminal nucleotide comprises a 4'-O-monophosphate bond. Methylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU]; wherein positions 1-20 of the antisense strand and positions 1-20 of the sense strand form a double-stranded region, wherein positions 21-36 of the sense strand form a stem-loop, wherein positions 27-30 constitute the loop of the stem-loop, optionally wherein positions 27-30 contain a tetracycle, wherein positions 21 and 22 of the antisense strand contain overhangs, and wherein the sense strand and antisense strand contain nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 483 and 724, respectively; (b) are SEQ ID NO: 484 and 725, respectively; (c) are SEQ ID NO: 486 and 727, respectively; (d) are SEQ ID NO: 487 and 728, respectively; (e) are SEQ ID NO: 488 and 729, respectively; (f) are SEQ ID NO: 489 and 730, respectively; (g) are SEQ ID NO: 490 and 731, respectively; (h) are SEQ ID NO: 492 and 733, respectively; (i) are SEQ ID NO: 493 and 734, respectively; (j) are SEQ ID NO: 494 and 735, respectively; (k) are SEQ ID NO: 495 and 736, respectively; (l) are SEQ ID NO: 496 and 737, respectively; (m) are SEQ ID NO: 497 and 738, respectively; (n) are SEQ ID NO: 498 and 739, respectively; (o) are SEQ ID NO: 499 and 740, respectively; (p) are SEQ ID NO: 500 and 741, respectively; (q) are SEQ ID NO: 501 and 742, respectively; (r) are SEQ ID NO: 502 and 743, respectively; (s) are SEQ ID NO: 503 and 744, respectively; (t) are SEQ ID NO: 504 and 745, respectively; (u) are SEQ ID NO: 505 and 746, respectively; (v) are SEQ ID NO: 506 and 747, respectively; (w) are SEQ ID NO: 507 and 748, respectively; (x) are SEQ ID NO: 508 and 749, respectively; (y) are SEQ ID NO: 509 and 750, respectively; (z) are SEQ ID NO: 510 and 751, respectively; (aa) are SEQ ID NO: 511 and 752 respectively; (bb) are SEQ ID NO: 512 and 753, respectively; (cc) are SEQ ID NO: 513 and 754 respectively; (dd) are SEQ ID NO: 514 and 755 respectively; (ee) are SEQ ID NO: 515 and 756 respectively; (ff) are SEQ ID NO: 516 and 757 respectively; (gg) are SEQ ID NO: 517 and 758 respectively; (hh) are SEQ ID NO: 518 and 758 respectively; and, (ii) are SEQ ID NO: 491 and 732, respectively.

[0252] In some implementations, it is used to reduce CD274 The expressed oligonucleotides targeting CD274 (e.g., RNAi oligonucleotides) include: The sense chain contains nucleotides modified by 2'-F at positions 8-11, nucleotides modified by 2'-OMe at positions 1-7, 12-27 and 31-36, nucleotides conjugated with GalNAc at positions 28, 29 and 30; and phosphate thioester bonds between positions 1 and 2. The antisense strand comprises 2'-F modified nucleotides at positions 2, 3, 4, 5, 7, 10, and 14; 2'-OMe at positions 1, 6, 8, 9, 11-13, and 15-22; thiophosphate bonds between positions 1 and 2, 2 and 3, 3 and 4, 20 and 21, and 21 and 22; and a 5'-terminal nucleotide at position 1 comprising a 4'-phosphate analogue, optionally wherein the 5'-terminal nucleotide comprises a 4'-O-monophosphate bond. Methylphosphonate-2'-O-methyluridine [MePhosphonate-4O-mU]; wherein positions 1-20 of the antisense strand and positions 1-20 of the sense strand form a double-stranded region, wherein positions 21-36 of the sense strand form a stem-loop, wherein positions 27-30 constitute the loop of the stem-loop, optionally wherein positions 27-30 contain a tetracycle, wherein positions 21 and 22 of the antisense strand contain overhangs, and wherein the sense strand and antisense strand contain nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 486 and 727, respectively; (c) are SEQ ID NO: 487 and 728, respectively; (d) are SEQ ID NO: 488 and 729, respectively; (e) are SEQ ID NO: 489 and 730, respectively; (f) are SEQ ID NO: 491 and 732 respectively; and, (g) are SEQ ID NO: 502 and 743, respectively.

[0253] In some implementations, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 484, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 725. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 486, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 727. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 487, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 728. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 488, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 729. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 489, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 730. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 491, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 732. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 502, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 743.

[0254] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 243; and (ii) a sense strand of 19-50 nucleotides in length, which contains a complementary region complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0255] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274(ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 245; and (ii) a sense strand of 19-50 nucleotides in length, which contains a complementary region complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0256] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 246; and (ii) a sense strand of 19-50 nucleotides in length, which contains a complementary region complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0257] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 247; and (ii) a sense strand of 19-50 nucleotides in length, which contains a complementary region complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0258] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 248; and (ii) a sense strand of 19-50 nucleotides in length, which contains a complementary region complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0259] In some implementations, it is used to reduce CD274The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 250; and (ii) a sense strand of 19-50 nucleotides in length, which contains a complementary region complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0260] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 261; and (ii) a sense strand of 19-50 nucleotides in length, which contains a complementary region complementary to the antisense strand, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0261] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 243; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0262] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274(ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 245; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0263] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 246; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0264] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 247; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0265] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 248; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0266] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 250; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0267] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, wherein the complementary region is as shown in SEQ ID NO: 261; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0268] In some implementations, it is used to reduce CD274The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 243; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand, as shown in SEQ ID NO: 2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with a protruding end consisting of 1-4 nucleotides.

[0269] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 245; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand, as shown in SEQ ID NO: 4, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with a protruding end consisting of 1-4 nucleotides.

[0270] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 246; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand, as shown in SEQ ID NO: 5, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with a protruding end consisting of 1-4 nucleotides.

[0271] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274(ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 247; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand, as shown in SEQ ID NO: 6, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with a protruding end consisting of 1-4 nucleotides.

[0272] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 248; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand, as shown in SEQ ID NO: 7, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with a protruding end consisting of 1-4 nucleotides.

[0273] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 250; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand, as shown in SEQ ID NO: 9, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with a protruding end consisting of 1-4 nucleotides.

[0274] In some implementations, it is used to reduce CD274 The expressed oligonucleotide targeting CD274 (e.g., RNAi oligonucleotide) comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains [a sequence related to CD274]. CD274(ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 261; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand, as shown in SEQ ID NO: 20, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0275] In some embodiments, the CD274-targeting oligonucleotide (e.g., RNAi oligonucleotide) for reducing CD274 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains... CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 243; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is shown in SEQ ID NO: 2, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0276] In some embodiments, the CD274-targeting oligonucleotide (e.g., RNAi oligonucleotide) for reducing CD274 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains... CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 245; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is shown in SEQ ID NO: 4, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0277] In some embodiments, the CD274-targeting oligonucleotide (e.g., RNAi oligonucleotide) for reducing CD274 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains... CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 246; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is shown in SEQ ID NO: 5, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0278] In some embodiments, the CD274-targeting oligonucleotide (e.g., RNAi oligonucleotide) for reducing CD274 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains... CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 247; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is shown in SEQ ID NO: 6, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0279] In some embodiments, the CD274-targeting oligonucleotide (e.g., RNAi oligonucleotide) for reducing CD274 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains... CD274(ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 248; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is shown in SEQ ID NO: 7, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0280] In some embodiments, the CD274-targeting oligonucleotide (e.g., RNAi oligonucleotide) for reducing CD274 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains... CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 250; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is shown in SEQ ID NO: 9, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0281] In some embodiments, the CD274-targeting oligonucleotide (e.g., RNAi oligonucleotide) for reducing CD274 expression comprises (i) an antisense strand of 19-30 nucleotides in length, wherein the antisense strand contains a nucleotide sequence that contains... CD274 (ii) a complementary region complementary to the mRNA target sequence, as shown in SEQ ID NO: 261; and (ii) a sense strand of 19-50 nucleotides in length, comprising a complementary region complementary to the antisense strand and a stem-loop at the 3' end, wherein the complementary region complementary to the antisense strand is shown in SEQ ID NO: 20, wherein the stem-loop is represented as S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand are separate strands that form an asymmetric double-stranded region at the 3' end of the antisense strand with an overhang of 1-4 nucleotides.

[0282] In some implementations, this disclosure provides methods for reducing CD274 Expressed oligonucleotides (e.g., RNAi oligonucleotides), wherein the oligonucleotide comprises a sense strand and an antisense strand according to the following: Significant chain: 5'-mX- S -mX-mX-mX-mX-mX-mX-fX-fX-fX-fX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-[ademX-L]-mX-mX-mX-mX-mX-mX-mX-mX-3'; Hybridization to: Antonym chain: 5'-[MePhosphonate-4O-mX]- S -fX- S -fX-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-mX-mX-mX-mX-mX- S -mX- S -mX-3'; Where mX = 2'- O -Methyl modified nucleotides, fX = 2'-fluoro modified nucleotides, - S- = Phosphophosphate bond, - = Phosphodiester bond, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and AdemX-L = lipid molecule (e.g., C18) attached to the nucleotide.

[0283] In some implementations, this disclosure provides methods for reducing CD274 Expressed oligonucleotides (e.g., RNAi oligonucleotides), wherein the oligonucleotide comprises a sense strand and an antisense strand according to the following: Significant chain: 5'-mX- S -mX-mX-mX-mX-mX-mX-fX-fX-fX-fX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-m X-mX-mX-[ademX-GalNAc]-[ademX-GalNAc]-[ademX-GalNAc]-mX-mX-mX-mX-mX-mX- 3'; Hybridization to: Antonym chain: 5'-[MePhosphonate-4O-mX]- S -fX- S -fX- S-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-mX-mX-mX-mX-mX- S -mX- S -mX-3'; Where mX = 2'- O -Methyl modified nucleotides, fX = 2'-fluoro modified nucleotides, - S- = Phosphophosphate bond, - = Phosphodiester bond, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and ademX-GalNAc = GalNAc attached to the nucleotide.

[0284] In some implementations, this disclosure provides methods for reducing CD274 The expressed RNAi oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence 5'[ademGs-C18][mG][mA][mU][mA][mU][mU][fU][fG][fC][fU][mG][mU][mC][mU][mU][mU][mU][mA][mU][mA][mG][mC][mA][mG][mC][mG][mC][mG][mA][mA][mG][mC][mU][mG][mC]-3' (SEQ ID NO: 1050) and all modifications, and wherein the antisense strand comprises 5' The sequence of [MePhosphonate-4O-mUs][fAs][fUs][fA][fA][mA][fG][mA][mC][fA][mG][mC][mA][fA][mA][mU][mA][mU][mC][mCs][mGs][mG]-3' (SEQ ID NO:1005) and all modifications thereof, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'F ribonucleoside; s = phosphate thioester, and wherein ademA-GalNAc = GalNAc modified adenine nucleotide.

[0285] In some implementations, this disclosure provides methods for reducing CD274The expressed RNAi oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence 5′[mAs][mU][mG][mA][mG][mG][mA][fU][fA][fU][fU][mU][mG][mC][mU][mG][mU][mC][mU][mA][mG][mC][mA][mG][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3′ (SEQ ID NO: 966) and all modifications, and wherein the antisense strand comprises 5′ The sequence of [MePhosphonate-4O-mUs][fAs][fGs][fA][fC][mA][fG][mC][mA][fA][mA][mU][mA][fU][mC][mC][mU][mC][mA][mUs][mGs][mG]3′ (SEQ ID NO: 1002) and all modifications thereof, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'F ribonucleoside; s = phosphate thioester, and wherein ademA-GalNAc = GalNAc modified adenine nucleotide.

[0286] In some implementations, this disclosure provides methods for reducing CD274The expressed RNAi oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence 5′[mAs][mG][mG][mA][mU][mA][mU][fU][fU][fG][fC][mU][mG][mU][mC][mU][mU][mU][mA][mA][mG][mC][mA][mG][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3′ (SEQ ID NO: 968) and all modifications, and wherein the antisense strand comprises 5′ The sequence of [MePhosphonate-4O-mUs][fUs][fAs][fA][fA][mG][fA][mC][mA][fG][mC][mA][mA][fA][mU][mA][mU][mC][mC][mUs][mGs][mG]-3′ (SEQ ID NO: 1004) and all modifications thereof, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'F ribonucleoside; s = phosphate thioester, and wherein ademA-GalNAc = GalNAc modified adenine nucleotide.

[0287] In some implementations, this disclosure provides methods for reducing CD274The expressed RNAi oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence 5′-[mGs][mG][mA][mU][mA][mU][mU][fU][fG][fC][fU][mG][mU][mC][mU][mU][mU][mA][mU][mA][mG][mC][mA][mG][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3′ (SEQ ID NO: 969) and all modifications, and wherein the antisense strand comprises 5′- The sequence of [MePhosphonate-4O-mUs][fAs][fUs][fA][fA][mA][fG][mA][mC][fA][mG][mC][mA][fA][mA][mU][mA][mU][mC][mCs][mGs][mG]-3′ (SEQ ID NO: 1005) and all modifications thereof, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'F ribonucleoside; s = phosphate thioester, and wherein ademA-GalNAc = GalNAc modified adenine nucleotide.

[0288] In some implementations, this disclosure provides methods for reducing CD274The expressed RNAi oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence 5′- [mAs][mU][mA][mU][mU][mU][mG][fC][fU][fG][fU][mC][mU][mU][mU][mA][mU][mA][mU][mA][mG][mC][mA][mG][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3′ (SEQ ID NO: 970) and all modifications, and wherein the antisense strand comprises 5′- The sequence of [MePhosphonate-4O-mUs][fAs][fUs][fA][fU][mA][fA][mA][mG][fA][mC][mA][mG][fC][mA][mA][mA][mU][mUs][mGs][mG]-3′ (SEQ ID NO: 1006) and all modifications thereof, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'F ribonucleoside; s = phosphate thioester, and wherein ademA-GalNAc = GalNAc modified adenine nucleotide.

[0289] In some implementations, this disclosure provides methods for reducing CD274The expressed RNAi oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence 5′-[mAs][mU][mU][mU][mG][mC][mU][fG][fU][fC][fU][mU][mU][mA][mU][mA][mU][mU][mC][mA][mG][mC][mA][mG][mC][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3′ (SEQ ID NO: 971) and all modifications, and wherein the antisense strand comprises 5′- The sequence of [MePhosphonate-4O-mUs][fGs][fAs][fA][fU][mA][fU][mA][mA][fA][mG][mA][mC][fA][mG][mC][mA][mA][mA][mUs][mGs][mG]-3′ (SEQ ID NO: 1007) and all modifications thereof, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'F ribonucleoside; s = phosphate thioester, and wherein ademA-GalNAc = GalNAc modified adenine nucleotide.

[0290] In some implementations, this disclosure provides methods for reducing CD274The expressed RNAi oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence 5′[mGs][mC][mA][mA][mU][mA][mU][fG][fA][fC][fA][mA][mU][mU][mG][mA][mA][mU][mG][mA][mU][mG][mA][mG][mC][mA][mG][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3′ (SEQ ID NO: 973) and all modifications, and wherein the antisense strand comprises 5′- The sequence of [MePhosphonate-4O-mUs][fCs][fAs][fU][fU][mC][fA][mA][mU][fU][mG][mU][mC][fA][mU][mA][mU][mU][mG][mCs][mGs][mG]-3′ (SEQ ID NO: 1009) and all modifications thereof, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'F ribonucleoside; s = phosphate thioester, and wherein ademA-GalNAc = GalNAc modified adenine nucleotide.

[0291] In some implementations, this disclosure provides methods for reducing CD274The expressed RNAi oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand comprises the sequence 5′-[mGs][mA][mU][mA][mA][mG][mA][fA][fC][fA][fU][mU][mA][mU][mU][mC][mA][mA][mU][mA][mG][mC][mA][mG][mC][mG][ademA-GalNAc][ademA-GalNAc][ademA-GalNAc][mG][mG][mC][mU][mG][mC]-3′ (SEQ ID NO: 984) and all modifications, and wherein the antisense strand comprises 5′- The sequence of [MePhosphonate-4O-mUs][fAs][fUs][fU][fG][mA][fA][mU][mA][fA][mU][mG][mU][fU][mC][mU][mU][mA][mU][mCs][mGs][mG]-3′ (SEQ ID NO: 1020) and all modifications thereof, wherein mC, mA, mG, mU = 2'-OMe ribonucleoside; fA, fC, fG, fU = 2'F ribonucleoside; s = phosphate thioester, and wherein ademA-GalNAc = GalNAc modified adenine nucleotide.

[0292] In some implementations, this disclosure provides methods for reducing CD274 Expressed oligonucleotides (e.g., RNAi oligonucleotides), wherein the oligonucleotide comprises a sense strand and an antisense strand according to the following: Sense chain: 5'-[ademXs-L][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][m X][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX]- 3' Hybridization to: Antisense strand: 5'- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX]-3'; Where mX = 2'- O-Methyl modified nucleotide, fX = 2'-fluoromodified nucleotide, s = thiophosphate bond, ][ = phosphodiester bond, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and AdemX-L = lipid molecule attached to the nucleotide (e.g., C18).

[0293] In some implementations, this disclosure provides methods for reducing CD274 Expressed oligonucleotides (e.g., RNAi oligonucleotides), wherein the oligonucleotide comprises a sense strand and an antisense strand according to the following: Sense strand: 5'-[ademXs-C18][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][ mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX]- 3' Hybridization to: Antisense strand: 5'- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX]-3'; Where mX = 2'- O -Methyl modified nucleotide, fX = 2'-fluoromodified nucleotide, s = thiophosphate bond, ][ = phosphodiester bond, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and AdemX-C18 = C18 hydrocarbon chain attached to the nucleotide.

[0294] In some implementations, it is used to reduce CD274 The oligonucleotides expressing mRNA contain sense and antisense strands, which consist of nucleotide sequences selected from the following: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The semantic and antisemantic chains are modified based on the following pattern. Sense strand: 5'-[ademXs-C18][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][ mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX]- 3' Hybridization to: Antisense strand: 5'- [MePhosphonate-4O-mXs][fXs][fXs][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX]-3'; Where mX = 2'- O -Methyl modified nucleotide, fX = 2'-fluoromodified nucleotide, s = thiophosphate bond, ][ = phosphodiester bond, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and AdemX-C18 = C18 hydrocarbon chain attached to the nucleotide.

[0295] In some implementations, this disclosure provides methods for reducing CD274 Expressed oligonucleotides (e.g., RNAi oligonucleotides), wherein the oligonucleotide comprises a sense strand and an antisense strand according to the following: Sense chain: 5'-[ademXs-L][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][m X][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX]- 3' Hybridization to: Antisense strand: 5'- [MePhosphonate-4O-mXs][fXs][fX][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX]-3'; Where mX = 2'- O-Methyl modified nucleotide, fX = 2'-fluoromodified nucleotide, s = thiophosphate bond, ][ = phosphodiester bond, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and AdemX-L = lipid molecule attached to the nucleotide (e.g., C18).

[0296] In some implementations, this disclosure provides methods for reducing CD274 Expressed oligonucleotides (e.g., RNAi oligonucleotides), wherein the oligonucleotide comprises a sense strand and an antisense strand according to the following: Sense strand: 5'-[ademXs-C18][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][ mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX]- 3' Hybridization to: Antisense strand: 5'- [MePhosphonate-4O-mXs][fXs][fX][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX]-3'; Where mX = 2'- O -Methyl modified nucleotide, fX = 2'-fluoromodified nucleotide, s = thiophosphate bond, ][ = phosphodiester bond, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and AdemX-C18 = C18 hydrocarbon chain attached to the nucleotide.

[0297] In some implementations, it is used to reduce CD274 The oligonucleotides expressing mRNA contain sense and antisense strands, which consist of nucleotide sequences selected from the following: (a) are SEQ ID NO: 484 and 725, respectively; (b) are SEQ ID NO: 487 and 728 respectively; and, (c) are SEQ ID NO: 491 and 732, respectively. The semantic and antisemantic chains are modified based on the following pattern. Sense strand: 5'-[ademXs-C18][mX][mX][mX][mX][mX][mX][fX][fX][fX][fX][mX][mX][mX][mX][mX][ mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX][mX]- 3' Hybridization to: Antisense strand: 5'- [MePhosphonate-4O-mXs][fXs][fX][fX][fX][mX][fX][mX][mX][fX][mX][mX][mX][fX][mX][mX][mX][mX][mX][mXs][mXs][mX]-3'; Where mX = 2'- O -Methyl modified nucleotide, fX = 2'-fluoromodified nucleotide, s = thiophosphate bond, ][ = phosphodiester bond, [MePhosphonate-4O-mX] = 4'-O-monomethylphosphonate-2'-O-methyl modified nucleotide, and AdemX-C18 = C18 hydrocarbon chain attached to the nucleotide.

[0298] In some implementations, this disclosure provides methods for reducing CD274 The expressed oligonucleotide (e.g., RNAi oligonucleotide), wherein the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand comprising nucleotide sequences selected from the group consisting of: (a) are SEQ ID NO: 965 and 1001, respectively; (b) are SEQ ID NO: 966 and 1002, respectively; (c) are SEQ ID NO: 968 and 1004, respectively; (d) are SEQ ID NO: 969 and 1005, respectively; (e) are SEQ ID NO: 970 and 1006, respectively; (f) are SEQ ID NO: 971 and 1007, respectively; (g) are SEQ ID NO: 972 and 1008, respectively; (h) are SEQ ID NO: 974 and 1010, respectively; (i) are SEQ ID NO: 975 and 1011, respectively; (j) are SEQ ID NO: 976 and 1012, respectively; (k) are SEQ ID NO: 977 and 1013, respectively; (l) are SEQ ID NO: 978 and 1014, respectively; (m) are SEQ ID NO: 979 and 1015, respectively; (n) are SEQ ID NO: 980 and 1016, respectively; (o) are SEQ ID NO: 981 and 1017 respectively; (p) are SEQ ID NO: 982 and 1018, respectively; (q) are SEQ ID NO: 983 and 1019, respectively; (r) are SEQ ID NO: 984 and 1020, respectively; (s) are SEQ ID NO: 985 and 1021, respectively; (t) are SEQ ID NO: 986 and 1022, respectively; (u) are SEQ ID NO: 987 and 1023, respectively; (v) are SEQ ID NO: 988 and 1024, respectively; (w) are SEQ ID NO: 989 and 1025, respectively; (x) are SEQ ID NO: 990 and 1026, respectively; (y) are SEQ ID NO: 991 and 1027, respectively; (z) are SEQ ID NO: 992 and 1028, respectively; (aa) are SEQ ID NO: 993 and 1029, respectively; (bb) are SEQ ID NO: 994 and 1030 respectively; (cc) are SEQ ID NO: 995 and 1031 respectively; (dd) are SEQ ID NO: 996 and 1032, respectively; (ee) are SEQ ID NO: 997 and 1033 respectively; (ff) are SEQ ID NO: 998 and 1034, respectively; (gg) are SEQ ID NO: 999 and 1035 respectively; (hh) are SEQ ID NO: 1000 and 1036 respectively; and, (ii) are SEQ ID NO: 973 and 1009, respectively.

[0299] In some implementations, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 966, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1002. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 968, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1004. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 969, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1005. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1050, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1005. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 970, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1006. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 971, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1007. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 973, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1009. In some embodiments, the present disclosure provides for reducing CD274 Target of expression CD274The oligonucleotide comprises a sense strand and an antisense strand, the sense strand comprising a nucleotide sequence as shown in SEQ ID NO: 984, and the antisense strand comprising a nucleotide sequence as shown in SEQ ID NO: 1020.

[0300] In some implementations, the present disclosure provides for reducing CD274 Target of expression CD274 The oligonucleotide comprises an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 728, the sense strand comprising the nucleotide sequence of SEQ ID NO: 487, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide inter-bonded.

[0301] In some implementations, the present disclosure provides for reducing CD274 The expressed CD274-targeting oligonucleotide comprises an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 728, the sense strand comprising the nucleotide sequence of SEQ ID NO: 487, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 2' carbon of the ribose ring of the 5' terminal nucleotide of the sense strand via a linker, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide inter-bonded.

[0302] In some implementations, the present disclosure provides for reducing CD274 The expressed oligonucleotide targeting CD274 comprises an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 725, the sense strand comprising the nucleotide sequence of SEQ ID NO: 484, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide inter-bonded.

[0303] In some implementations, the present disclosure provides for reducing CD274 The expressed CD274-targeting oligonucleotide comprises an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 725, the sense strand comprising the nucleotide sequence of SEQ ID NO: 484, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 2' carbon of the ribose ring of the 5' terminal nucleotide of the sense strand via a linker, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide inter-bonded.

[0304] In some implementations, the present disclosure provides for reducing CD274 The expressed CD274-targeting oligonucleotide comprises an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 732, the sense strand comprising the nucleotide sequence of SEQ ID NO: 491, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide inter-bonded.

[0305] In some implementations, the present disclosure provides for reducing CD274 The expressed CD274-targeting oligonucleotide comprises an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of SEQ ID NO: 732, the sense strand comprising the nucleotide sequence of SEQ ID NO: 491, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 2' carbon of the ribose ring of the 5' terminal nucleotide of the sense strand via a linker, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide inter-bonded.

[0306] In some implementations, the present disclosure provides for reducing CD274 The expressed oligonucleotide targeting CD274 comprises an antisense strand and a sense strand, wherein the antisense strand is 20 to 30 nucleotides in length and has a complementary region of 19 to 29 nucleotides complementary to the target sequence of CD274 shown in any of SEQ ID NO: 2, 5 and 9, wherein the sense strand is 28 to 40 nucleotides in length and comprises a stem-loop at its 3' end as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand form a double-stranded region of at least 19 nucleotides in length, and wherein the sense strand comprises a C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand.

[0307] In some implementations, the present disclosure provides for reducing CD274The expressed oligonucleotide targeting CD274 comprises an antisense strand of approximately 20 to 22 nucleotides in length and a sense strand of approximately 28 to 40 nucleotides in length, wherein the antisense strand and the sense strand form an asymmetric duplex region of approximately 20 to 22 base pairs, the asymmetric duplex region comprising a 3' end overhang of at least one nucleotide of the antisense strand, wherein the antisense strand comprises a structure consistent with SEQ ID NO: The complementary region of 19 to 21 nucleotides complementary to the target sequence of CD274 shown in any of 2, 5, and 9, wherein the sense strand comprises: (i) a stem-loop at the 3' end of the sense strand, wherein the stem-loop comprises a nucleotide sequence represented by the following formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, and (ii) at least one C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified nucleotide interbonded.

[0308] preparation Various formulations (e.g., pharmaceutical formulations) have been developed for the application of oligonucleotides. For example, oligonucleotides (e.g., RNAi oligonucleotides) can be delivered to a subject or cellular environment using formulations that minimize degradation, promote delivery and / or uptake, or provide another beneficial property to the oligonucleotides in the formulation. In some embodiments, this document provides methods that include reducing… CD274 Compositions of expressed oligonucleotides (e.g., RNAi oligonucleotides). Such compositions can be suitably formulated so that, when administered to a subject, whether directly to the target cells or systemically, a sufficient fraction of the oligonucleotides enters the cells to reduce [the condition / progression]. CD274 Expression. As disclosed herein, any suitable oligonucleotide formulation of any kind can be used to deliver oligonucleotides to reduce [the risk of adverse reactions]. CD274 In some embodiments, the oligonucleotide is formulated in buffer solutions such as phosphate-buffered saline, liposomes, micelle structures, and shells. Any oligonucleotide described herein may be provided not only as a nucleic acid but also in the form of a pharmaceutically acceptable salt.

[0309] Oligonucleotide formulations containing cationic lipids can be used to promote the transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include oligofectamine, lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche), all of which can be used according to the manufacturer's instructions.

[0310] Therefore, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the excipient comprises liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles, or may otherwise be formulated for administration to the cells, tissues, organs, or bodies of a subject in need (see, for example, Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition, Pharmaceutical Press, 2013).

[0311] In some embodiments, the formulations described herein include excipients. In some embodiments, the excipients impart improved stability, improved absorption, improved solubility, and / or therapeutic enhancement to the composition of the active ingredient. In some embodiments, the excipients are buffers (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or mediators (e.g., buffer solutions, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotide is lyophilized to extend its shelf life and then formulated into a solution prior to use (e.g., administration to a subject). Therefore, the excipient in a composition comprising any of the oligonucleotides described herein may be a lyophilization protectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature regulator (e.g., dextran, Ficoll™, or gelatin).

[0312] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous), oral (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration.

[0313] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (when water-soluble) or dispersions, as well as sterile powders for the immediate preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In many cases, isotonic agents, such as sugars, polyols like mannitol, sorbitol, and sodium chloride, are preferably included in the composition. Sterile injectable solutions can be prepared by introducing the desired amount of oligonucleotides with one or a combination of the components listed above into a selected solvent, followed by sterile filtration.

[0314] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., for reducing...). CD274 (Expressed RNAi oligonucleotides), although the percentage of the active ingredient may be between about 1% and about 80% or more of the total composition by weight or volume. Those skilled in the art who prepare such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore, multiple dosages and treatment regimens may be desirable.

[0315] Cytotoxic T-lymphocyte-associated antigen (CTLA4) inhibitors In some embodiments, this disclosure provides a CTLA4 inhibitor for use in conjunction with the oligonucleotides described herein. In some embodiments, the CTLA4 inhibitor inhibits the association of CTLA4 with its ligands B7.1 or B7.2. In some embodiments, the CTLA4 inhibitor is specific to CTLA4.

[0316] In some embodiments, the CTLA4 inhibitor is an anti-CTLA4 antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an antibody fragment. In some embodiments, the CTLA4 inhibitor is a small molecule.

[0317] In some embodiments, the anti-CTLA4 antibody is ipilimumab (MDX-010). In some embodiments, the anti-CTLA4 antibody is trimemumab.

[0318] In some embodiments, the anti-CTLA4 antibody is any anti-CTLA4 antibody known in the art, including but not limited to Ascierto et al. "Anti-CTLA4 monoclonal antibodies: the past and the future in clinical application" J. of Translational Medicine. 9(196): Anti-CTLA4 antibody disclosed in 2011.

[0319] In some embodiments, the anti-CTLA4 antibody described herein binds to CTLA4 with an affinity of about 30 nM to about 100 nM. In some embodiments, the anti-CTLA4 antibody described herein binds to CTLA4 with an affinity of about 30 nM. In some embodiments, the anti-PD-L1 antibody described herein binds to CTLA4 with an affinity of about 40 nM. In some embodiments, the anti-CTLA4 antibody described herein binds to CTLA4 with an affinity of about 50 nM. In some embodiments, the anti-CTLA4 antibody described herein binds to CTLA4 with an affinity of about 60 nM. In some embodiments, the anti-CTLA4 antibody described herein binds to CTLA4 with an affinity of about 70 nM. In some embodiments, the anti-CTLA4 antibody described herein binds to CTLA4 with an affinity of about 80 nM. In some embodiments, the anti-CTLA4 antibody described herein binds to CTLA4 with an affinity of about 90 nM. In some implementations, the anti-CTLA4 antibody described herein binds to CTLA4 with an affinity of approximately 100 nM.

[0320] In some embodiments, the antibody is generated using a display technology. Display technologies used to generate antibody peptides include any display technology (e.g., display library screening technology). In some embodiments, synthetic antibodies are designed, selected, or optimized by screening target antigens using a display technology (e.g., phage display technology). Phage display libraries can contain millions to billions of phage vectors, each expressing a unique antibody fragment on its viral coat. Such libraries provide a rich and diverse resource for selecting potentially hundreds of antibody fragments with different affinity levels for one or more antigens of interest (McCafferty et al., 1990. Nature. 348:552-4; Edwards, BM et al., 2003. JMB. 334: 103-18; Schofield, D. et al., 2007. Genome Biol. 8, R254; and Pershad, K. et al., 2010. Protein Engineering Design and Selection. 23:279-88; the contents of each of these are incorporated herein by reference in their entirety). Typically, antibody fragments present in such libraries include scFv antibody fragments, which contain V linked by flexible linkers. H and V L Fusion proteins of antibody domains. In some cases, scFvs may contain the same sequence except for the unique sequence encoding the variable loop of the CDR. In some cases, scFvs are expressed as fusion proteins linked to viral capsid proteins (e.g., the N-terminus of viral pellet proteins). The VL chain can be expressed separately for assembly with the VH chain in the periplasm, after which the complex is incorporated into the viral capsid. Library members precipitated from bound phages can be sequenced to obtain cDNA encoding the desired scFv. Antibody variable domains or CDRs from such sequences can be directly incorporated into antibody sequences for the generation of recombinant antibodies, or mutated via in vitro affinity maturation for further optimization.

[0321] In some embodiments, yeast surface display technology is used to generate sequences of peptides to be encoded in the viral genome. In some embodiments, recombinant antibodies are developed by displaying antibody fragments of interest as fusion bodies on a yeast surface, wherein the protein interacts with proteins and small molecules in solution. scFvs with affinity for the desired receptor can be isolated from the yeast surface using magnetic separation and flow cytometry. Several cycles of yeast surface display and isolation can be performed to obtain scFvs with the desired properties through directed evolution.

[0322] Methods for determining the affinity of an antibody for its antigen are known in the art. An exemplary method for determining binding affinity employs surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson, U. et al., (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., i(1991) Biotechniques 11:620-627; Johnson, B. et al., (1995) J. Mol. Recognit. 8:125-131; and Johnson, B. et al., (1991) Anal. Biochem. 198:268-277.

[0323] How to use Reduce CD274 expression In some embodiments, this disclosure provides for contacting or delivering to cells or cell populations an effective amount of the oligonucleotides (e.g., RNAi oligonucleotides) provided herein to reduce CD274 Methods of expression. In some implementations, this is achieved by measuring cells... CD274 The decrease in the amount or level of mRNA, PD-L1 protein, or PD-L1 activity is used to determine CD274 The reduction in expression. These methods include those described herein and those known to those skilled in the art.

[0324] The methods described herein can be used with any suitable cell type. In some implementations, the cells are expression cells. CD274 Any cell (e.g., hepatocytes) to which mRNA is generated. In some embodiments, the cells are primary cells obtained from the subject. In some embodiments, the primary cells have undergone a limited number of passages, such that the cells substantially retain their native phenotypic properties. In some embodiments, the cells to which the oligonucleotides are delivered are ex vivo or in vitro (i.e., can be delivered to cells in culture or the organism in which the cells reside).

[0325] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) are delivered to cells or cell populations using nucleic acid delivery methods known in the art, including but not limited to injecting a solution containing the oligonucleotide, bombarding with particles coated with the oligonucleotide, exposing cells or cell populations to a solution containing the oligonucleotide, or electroporating cell membranes in the presence of the oligonucleotide. Other methods known in the art for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier transport, chemically mediated transport, and cationic liposome transfection such as calcium phosphate.

[0326] In some implementation schemes, CD274 The reduction in expression is achieved through evaluation and CD274 The expression of one or more molecules, properties, or characteristics of the relevant cells or cell populations is determined by assays or techniques, or by evaluation of direct indicators of expression in the cells or cell populations. CD274 Expressed molecules (e.g., CD274 The determination is made by assaying or using techniques to identify mRNA or PD-L1 protein. In some embodiments, this is achieved by measuring the levels of the oligonucleotide in cells or cell populations that have come into contact with it. CD274 The expression was evaluated by comparing it with an appropriate control (e.g., appropriate cells or cell populations that were not exposed to the oligonucleotide or exposed to a control oligonucleotide) to assess the oligonucleotide reduction provided in this paper. CD274 The degree of expression. In some implementations, the level of expression in control cells or cell populations is predetermined. CD274 The predetermined control level or value is presented so that it is not necessary to measure the control level or value every time the assay or technique is performed. The predetermined level or value can take many forms. In some embodiments, the predetermined level or value may be a single cutoff value, such as the median or mean.

[0327] In some embodiments, contacting or delivering the oligonucleotide described herein (e.g., RNAi oligonucleotide) to cells or cell populations results in a decrease in the number of cells or cell populations that were not contacted with the oligonucleotide or were contacted with a control oligonucleotide. CD274 Expression is reduced. In some implementation schemes, CD274 The decrease in expression is relative to CD274 The control levels or amounts expressed are approximately 1% or less, approximately 5% or less, approximately 10% or less, approximately 15% or less, approximately 20% or less, approximately 25% or less, approximately 30% or less, approximately 35% or less, approximately 40% or less, approximately 45% or less, approximately 50% or less, approximately 55% or less, approximately 60% or less, approximately 70% or less, approximately 80% or less, or approximately 90% or less. In some embodiments, CD274 The control level or level of expression is in cells or cell populations that have not yet been exposed to the oligonucleotides described in this article. CD274The amount or level of mRNA and / or PD-L1 protein. In some embodiments, the effect of delivering the oligonucleotides of this invention to cells or cell populations according to the methods herein is evaluated after any limited time period or time quantity (e.g., minutes, hours, days, weeks, months). For example, in some embodiments, the determination is performed in cells or cell populations at least about 4 hours, about 8 hours, about 12 hours, about 18 hours, about 24 hours; or at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, about 56 days, about 63 days, about 70 days, about 77 days, or about 84 days or longer. CD274 Expression. In some embodiments, the expression is determined in cells or cell populations at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months or longer after contacting or delivering the oligonucleotide to cells or cell populations. CD274 Express.

[0328] In some embodiments, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) are delivered in the form of a transgene engineered to express the oligonucleotide or a chain constituting the oligonucleotide (e.g., its sense and antisense strands) in cells. In some embodiments, the oligonucleotides of this document are delivered using a transgene engineered to express any of the oligonucleotides disclosed herein. The transgene can be delivered using a viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or a non-viral vector (e.g., plasmid or synthetic mRNA). In some embodiments, the transgene can be injected directly into a subject.

[0329] In some embodiments, contacting or delivering the oligonucleotide described herein (e.g., RNAi oligonucleotide) to tumor draining lymph nodes results in tumor draining lymph nodes that have not been contacted with the oligonucleotide or have been contacted with a control oligonucleotide. CD274 Expression is reduced. In some implementations, tumor-draining lymph nodes... CD274 The decrease in expression is relative to no expression. CD274 In oligonucleotide-treated tumor-draining lymph nodes CD274 The control levels or amounts expressed are approximately 1% or less, approximately 5% or less, approximately 10% or less, approximately 15% or less, approximately 20% or less, approximately 25% or less, approximately 30% or less, approximately 35% or less, approximately 40% or less, approximately 45% or less, approximately 50% or less, approximately 55% or less, approximately 60% or less, approximately 70% or less, approximately 80% or less, or approximately 90% or less. In some embodiments, CD274The control level or level of expression is in cells or cell populations that have not yet been exposed to the oligonucleotides described in this article. CD274 The amount or level of mRNA and / or PD-L1 protein.

[0330] In some embodiments, contacting or delivering the oligonucleotides described herein (e.g., RNAi oligonucleotides) to the tumor microenvironment results in tumor microenvironments that have not been contacted with the oligonucleotide or have been contacted with a control oligonucleotide showing a decrease in the concentration of the oligonucleotide. CD274 Expression is reduced. In some implementations, this occurs in the tumor microenvironment. CD274 The decrease in expression is relative to no expression. CD274 In tumor microenvironments treated with oligonucleotides CD274 The control levels or amounts expressed are approximately 1% or less, approximately 5% or less, approximately 10% or less, approximately 15% or less, approximately 20% or less, approximately 25% or less, approximately 30% or less, approximately 35% or less, approximately 40% or less, approximately 45% or less, approximately 50% or less, approximately 55% or less, approximately 60% or less, approximately 70% or less, approximately 80% or less, or approximately 90% or less. In some embodiments, CD274 The control level or level of expression is in cells or cell populations that have not yet been exposed to the oligonucleotides described in this article. CD274 The amount or level of mRNA and / or PD-L1 protein.

[0331] Combination of CD274 oligonucleotides and CTLA4 inhibitors In some embodiments, this disclosure provides CD274 oligonucleotides for or suitable for use in treating subjects who have received or are receiving CTLA4 inhibitors (e.g., people with a disease, condition, or symptom associated with CD274 expression).

[0332] In some embodiments, the methods described herein include selecting subjects who have or are susceptible to diseases, conditions, or illnesses associated with CD274 expression and / or CTLA4 expression. In some cases, the methods may include selecting individuals with biomarkers associated with diseases such as cancer or other chronic lymphocytic disorders related to CD274 expression and / or CTLA4 expression.

[0333] Similarly, and as detailed herein, the method may also include steps such as: measuring or obtaining baseline values ​​of markers of CD274 expression and / or CTLA4 expression, and then comparing such values ​​with one or more other baseline values ​​or values ​​obtained after administration of oligonucleotides to assess the effectiveness of treatment.

[0334] In some embodiments, this disclosure provides methods for treating subjects who have, are suspected of having, or are at risk of developing a disease, condition, or symptom with the CD274 oligonucleotide described herein, wherein the subject has received or is receiving a CTLA4 inhibitor. In some embodiments, this disclosure provides methods for treating subjects who have, are suspected of having, or are at risk of developing a disease, condition, or symptom with the CTLA4 inhibitor described herein, wherein the subject has received or is receiving the CD274 oligonucleotide described herein.

[0335] In some aspects, this disclosure provides methods for treating or attenuating the onset or progression of a disease, condition, or symptom associated with CD274 expression using the CD274 oligonucleotide of this invention in combination with a CTLA4 inhibitor. In other aspects, this disclosure provides methods for achieving one or more therapeutic benefits in a subject suffering from a disease, condition, or symptom associated with CD274 expression using the CD274 oligonucleotide of this invention in combination with a CTLA4 inhibitor. In some embodiments of the methods of this invention, the subject is treated by administering a therapeutically effective amount of the CD274 oligonucleotide of this invention in combination with a CTLA4 inhibitor. In some embodiments of the methods of this invention, the subject is treated by administering a therapeutically effective amount of the CD274 oligonucleotide of this invention to a subject who has received or is receiving a CTLA4 inhibitor. In some embodiments of the methods of this invention, the subject is treated by administering a therapeutically effective amount of a CTLA4 inhibitor to a subject who has received or is receiving the CD274 oligonucleotide of this invention. In some embodiments, the subject is given therapeutic treatment. In some embodiments, the subject is given prophylactic treatment.

[0336] In some embodiments of the methods described herein, one or more of the CD274 oligonucleotides described herein, or a pharmaceutical composition comprising one or more CD274 oligonucleotides, are administered to a subject who has received or is receiving a CTLA4 inhibitor and has a disease, condition, or symptom associated with CD274 expression, thereby treating the subject by reducing CD274 expression in the subject. In some embodiments, the subject… CD274 The amount or level of mRNA is reduced. In some embodiments, the amount or level of CD274 and / or protein is reduced in the subject. In some embodiments of the methods described herein, one or more of the CD274 oligonucleotides described herein or a pharmaceutical composition comprising one or more CD274 oligonucleotides is administered to a subject who has received or is receiving a CTLA4 inhibitor and has a disease, condition, or status associated with CD274 expression, thereby treating the subject by reducing CD274 expression and CTLA4 signaling in the subject. In some embodiments, the amount or level of mRNA in the subject is reduced. CD274The amount or level of mRNA and CTLA4 signaling is reduced. In some implementations, the amount or level of CD274 and / or protein is reduced in the subject, and CTLA4 signaling is reduced in the subject.

[0337] In some implementations, a therapeutically effective amount of CD274 oligonucleotide and / or CTLA4 inhibitor is administered to the subject. A therapeutically acceptable amount may be one that can therapeutically treat a disease or condition. The appropriate dose for any subject will depend on several factors, including the subject's body size, body surface area, age, the specific composition to be administered, the active ingredient in the composition, the time and route of administration, general health status, and any other medications administered concurrently.

[0338] In some embodiments, any of the compositions herein are administered to a subject via the enteral (e.g., orally, via a gastric feeding tube, via a duodenal feeding tube, via a gastrostomy, or via the rectum), parenteral (e.g., subcutaneously, intravenously, intra-arterially, intraosseously, intramuscularly, intracerebrally, intraventricularly, or intrathecally), topical (e.g., epidermis, inhalation, via eye drops, or via mucous membranes), or by direct injection into the target organ. Typically, the oligonucleotides herein are administered intravenously or subcutaneously.

[0339] As a set of non-limiting examples, the oligonucleotides described herein are typically administered quarterly (once every three months), every two months (once every two months), monthly, or weekly. For example, the oligonucleotide may be administered weekly or at two- or three-week intervals. Alternatively, the oligonucleotide may be administered daily. In some embodiments, a loading dose of one or more oligonucleotides is administered to the subject, followed by one or more maintenance doses of the oligonucleotide.

[0340] In some implementations, the CTLA4 inhibitor described herein (e.g., an anti-CTLA4 antibody) is administered quarterly (every three months), every two months (every two months), monthly, or weekly. For example, the inhibitor may be administered weekly or at two- or three-week intervals. Alternatively, the inhibitor may be administered daily.

[0341] In some embodiments, the oligonucleotide described herein is administered in combination with a CTLA4 inhibitor. In some embodiments, the oligonucleotide and the inhibitor are administered simultaneously, sequentially (in any order), or intermittently. For example, the oligonucleotide and the inhibitor may be administered simultaneously. Alternatively, the oligonucleotide may be administered, followed by the inhibitor after any time period (e.g., one hour, one day, one week, or one month), or vice versa.

[0342] In some implementations, the subjects to be treated are human or non-human primates or other mammal subjects. Other exemplary subjects include domestic animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

[0343] In some embodiments, this disclosure provides a method of treating a subject's cancer, the method comprising (i) administering an oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length, wherein the antisense strand and the sense strand form a bistranded region, wherein the antisense strand comprises an oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length. CD274 (ii) administration of a CTLA-4 inhibitor.

[0344] In some implementations, this disclosure provides treatment and activation. CD274 Methods for expressing a related disease, symptom, or condition, comprising administering an RNAi oligonucleotide and a CTLA-4 inhibitor to a subject in need, wherein the oligonucleotide comprises an antisense strand of 15 to 30 nucleotides in length and a sense strand of 15 to 40 nucleotides in length, wherein the antisense strand and the sense strand form a double-stranded region, wherein the antisense strand comprises an RNAi oligonucleotide and a CTLA-4 inhibitor. CD274 The complementary region is complementary to the mRNA target sequence, and the length of the complementary region is at least 15 consecutive nucleotides. Combination of CD274 oligonucleotides with cancer therapy In some embodiments, this disclosure provides CD274 oligonucleotides for or suitable for treating subjects who have received or are receiving cancer therapies (e.g., people with a disease, condition, or symptom associated with CD274 expression). In some embodiments, the cancer therapy is chemotherapy, immunotherapy, radiation therapy, surgical resection, targeted therapy, transplantation (solid tissue or stem cells), or a combination thereof.

[0346] In some embodiments, the method described herein includes selecting subjects who have or are susceptible to diseases, conditions, or illnesses associated with CD274 expression. In some embodiments, the method includes selecting individuals who have biomarkers associated with diseases such as cancer that are associated with CD274 expression.

[0347] In some implementations, this disclosure provides methods for treating subjects who have, are suspected of having, or are at risk of developing a disease, condition, or symptom with the CD274 oligonucleotide described herein, wherein the subject has received or is receiving cancer therapy.

[0348] In some embodiments, this disclosure provides methods for treating or attenuating the onset or progression of a disease, condition, or illness associated with CD274 expression using the CD274 oligonucleotides described herein in combination with cancer therapy. In some embodiments, this disclosure provides methods for treating or attenuating the onset or progression of a disease, condition, or illness associated with CD274 expression using one or more of chemotherapy, immunotherapy, radiotherapy, resection, targeted therapy, and transplantation (solid tissue or stem cells). In some embodiments, this disclosure provides methods for achieving one or more therapeutic benefits in a subject suffering from a disease, condition, or illness associated with CD274 expression using the CD274 oligonucleotides described herein in combination with cancer therapy. In some embodiments of the methods described herein, a subject is treated by administering a therapeutically effective amount of the CD274 oligonucleotides described herein in combination with cancer therapy. In some embodiments of the methods described herein, a subject is treated by administering a therapeutically effective amount of the CD274 oligonucleotides described herein to a subject who has received or is receiving cancer therapy. In some embodiments of the methods described herein, a subject is treated by administering a therapeutically effective amount of cancer therapy to a subject who has received or is receiving the CD274 oligonucleotides described herein.

[0349] In some embodiments of the methods described herein, one or more of the CD274 oligonucleotides described herein, or a pharmaceutical composition comprising one or more CD274 oligonucleotides, are administered to a subject who has received or is receiving cancer therapy and has a disease, condition, or symptom associated with CD274 expression, thereby treating the subject by reducing CD274 expression in the subject. In some embodiments, the subject… CD274 The amount or level of mRNA is reduced. In some implementations, the amount or level of CD274 and / or protein is reduced in the subject.

[0350] In some implementations, a therapeutically effective amount of CD274 oligonucleotide and / or cancer therapy is administered to the subject. A therapeutically acceptable amount may be an amount capable of therapeutically treating the d...

Claims

1. An oligonucleotide comprising an antisense strand comprising the nucleotide sequence of SEQ ID NO: 728 and a sense strand comprising the nucleotide sequence of SEQ ID NO: 487, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprises at least one 2 '-modified nucleotide and at least one modified internucleotide linkage.

2. An oligonucleotide comprising an antisense strand comprising the nucleotide sequence of SEQ ID NO: 728 and a sense strand comprising the nucleotide sequence of SEQ ID NO: 487, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 2' carbon of the ribose ring of the 5' terminal nucleotide of the sense strand via a linker, and wherein each of the antisense and sense strands comprises at least one 2 '-modified nucleotide and at least one modified internucleotide linkage.

3. An oligonucleotide comprising an antisense strand comprising the nucleotide sequence of SEQ ID NO: 725 and a sense strand comprising the nucleotide sequence of SEQ ID NO: 484, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprises at least one 2 '-modified nucleotide and at least one modified internucleotide linkage.

4. An oligonucleotide comprising an antisense strand comprising the nucleotide sequence of SEQ ID NO: 725 and a sense strand comprising the nucleotide sequence of SEQ ID NO: 484, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 2' carbon of the ribose ring of the 5' terminal nucleotide of the sense strand via a linker, and wherein each of the antisense and sense strands comprises at least one 2 '-modified nucleotide and at least one modified internucleotide linkage.

5. An oligonucleotide comprising an antisense strand comprising the nucleotide sequence of SEQ ID NO: 732 and a sense strand comprising the nucleotide sequence of SEQ ID NO: 491, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand, and wherein each of the antisense and sense strands comprises at least one 2 '-modified nucleotide and at least one modified internucleotide linkage.

6. An oligonucleotide comprising an antisense strand comprising the nucleotide sequence of SEQ ID NO: 732 and a sense strand comprising the nucleotide sequence of SEQ ID NO: 491, wherein the sense strand comprises a saturated C18 hydrocarbon chain conjugated to the 2' carbon of the ribose ring of the 5' terminal nucleotide of the sense strand via a linker, and wherein each of the antisense and sense strands comprises at least one 2 '-modified nucleotide and at least one modified internucleotide linkage.

7. The oligonucleotide of any one of claims 1-6, wherein the 2’-modified nucleotide comprises a 2’-modification selected from 2’-aminoethyl, 2’-fluoro, 2’-O-methyl, 2’-O- methoxyethyl, and 2’-deoxy-2’-fluoro-beta-d-arabinonucleic acid.

8. The oligonucleotide of any one of claims 1-7, wherein about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2’-fluoro modification.

9. The oligonucleotide of any one of claims 1-8, wherein about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand comprise a 2’-fluoro modification.

10. The oligonucleotide of any one of claims 1-9, wherein about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the oligonucleotide comprise a 2’-fluoro modification.

11. The oligonucleotide of any one of claims 1-10, wherein positions 8-11 of the sense strand each comprise a 2’-fluoro modification.

12. The oligonucleotide of any one of claims 1-11, wherein positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand each comprise a 2’-fluoro modification.

13. The oligonucleotide of any one of claims 1-12, wherein the remaining nucleotides comprise a 2’-O-methyl modification, provided that the 5’ terminal nucleotide of the sense strand conjugated to the saturated C18 hydrocarbon chain does not comprise a 2’-O-methyl modification.

14. The oligonucleotide of any one of claims 1-13, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

15. The oligonucleotide of claim 14, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2 of the sense strand.

16. The oligonucleotide of claim 14, wherein the sense strand comprises phosphorothioate linkages between positions 1 and 2, 2 and 3, and 3 and 4 of the sense strand.

17. The oligonucleotide of any one of claims 1-16, wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22.

18. The oligonucleotide of any one of claims 1-17, wherein the 4’-carbon of the sugar of the 5’-nucleotide of the antisense strand comprises a phosphate analog.

19. The oligonucleotide of claim 18, wherein the phosphate analog is an oxy-methylphosphonate, a vinylphosphonate, or a malonylphosphonate.

20. An oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand is 20 to 30 nucleotides in length and has a region of complementarity of 19 to 29 nucleotides that is complementary to a target sequence of CD274 set forth in any one of SEQ ID NOs: 2, 5, and 9, wherein the sense strand is 28 to 40 nucleotides in length and comprises a stem-loop at its 3' end as set forth below: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, wherein the antisense strand and the sense strand form a duplex region that is at least 19 nucleotides in length, and wherein the sense strand comprises a C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand.

21. An oligonucleotide comprising an antisense strand that is about 20 to 22 nucleotides in length and a sense strand that is about 28 to 40 nucleotides in length, wherein the antisense strand and the sense strand form an asymmetric duplex region of about 20 to 22 base pairs that comprises a 3' terminal overhang of at least 1 nucleotide of the antisense strand, wherein the antisense strand comprises a region of complementarity of 19 to 21 nucleotides that is complementary to a target sequence of CD274 set forth in any one of SEQ ID NOs: 2, 5, and 9, wherein the sense strand comprises: (i) a stem-loop at the 3' end of the sense strand, wherein the stem-loop comprises a sequence of nucleotides represented by the following formula: 5'-S1-L-S2-3', wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, and (ii) at least one C18 hydrocarbon chain conjugated to the 5' terminal nucleotide of the sense strand, and wherein each of the antisense strand and the sense strand comprises at least one 2'-modified nucleotide and at least one modified internucleotide linkage.

22. The oligonucleotide of any one of claims 20-21, wherein the antisense strand comprises a sequence as set forth in any one of SEQ ID NOs: 725, 728, and 732.

23. The oligonucleotide of any one of claims 20-22, wherein the sense strand comprises a sequence as set forth in any one of SEQ ID NOs: 966, 969, and 973.

24. The oligonucleotide of any one of claims 20-23, wherein L is a tetraloop.

25. The oligonucleotide of any one of claims 20-24, wherein L is 4 nucleotides in length.

26. The oligonucleotide of any one of claims 20-25, wherein L comprises a sequence represented by GAAA.

27. The oligonucleotide of any one of claims 20 and 22-26, wherein the antisense strand comprises a 3' terminal overhang of one or more nucleotides in length.

28. The oligonucleotide of any one of claims 21-27, wherein the 3' terminal overhang is 2 nucleotides in length, optionally wherein the 3' terminal overhang sequence is GG.

29. The oligonucleotide of any one of claims 20 and 22-29, wherein the oligonucleotide comprises at least one modified nucleotide.

30. The oligonucleotide of claim 29, wherein the modified nucleotide is a 2’-modified nucleotide.

31. The oligonucleotide of claim 30, wherein the 2’-modified nucleotide comprises a 2’- modification selected from 2’-aminoethyl, 2’-fluoro, 2’-O-methyl, 2’-O-methoxyethyl, and 2’-deoxy-2’-fluoro-beta-d-arabinonucleic acid.

32. The oligonucleotide of any one of claims 21-31, wherein about 10-15%, 10%, 11%, 12%, 13%, 14%, or 15% of the nucleotides of the sense strand comprise a 2’-fluoro modification.

33. The oligonucleotide of any one of claims 21-32, wherein about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the antisense strand comprise a 2’-fluoro modification.

34. The oligonucleotide of any one of claims 21-33, wherein about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% of the nucleotides of the oligonucleotide comprise a 2’-fluoro modification.

35. The oligonucleotide of any one of claims 21-34, wherein the sense strand comprises 36 nucleotides from 5’ to 3’ at positions 1-36, wherein positions 8-11 comprise a 2’-fluoro modification.

36. The oligonucleotide of any one of claims 21-35, wherein the antisense strand comprises 22 nucleotides from 3’ to 5’ at positions 1-22, and wherein positions 2, 3, 4, 5, 7, 10, and 14 comprise a 2’-fluoro modification.

37. The oligonucleotide of any one of claims 32-36, wherein the remaining nucleotides comprise a 2’-O-methyl modification, provided that the 5’ terminal nucleotide of the sense strand conjugated to the saturated C18 hydrocarbon chain does not comprise a 2’-O-methyl modification.

38. The oligonucleotide of any one of claims 20 and 22-37, wherein the oligonucleotide comprises at least one modified internucleotide linkage.

39. The oligonucleotide of any one of claims 21-28, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

40. The oligonucleotide of claim 39, wherein the sense strand comprises a phosphorothioate linkage between positions 1 and 2 of the sense strand.

41. The oligonucleotide of claim 39, wherein the sense strand comprises phosphorothioate linkages between positions 1 and 2, 2 and 3, and 3 and 4 of the sense strand.

42. The oligonucleotide of any one of claims 39-41, wherein the antisense strand comprises 22 nucleotides from 3’ to 5’ at positions 1-22, wherein the antisense strand comprises phosphorothioate linkages between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22.

43. The oligonucleotide of any one of claims 20-42, wherein the 4’-carbon of the sugar of the 5’-nucleotide of the antisense strand comprises a phosphate analog.

44. The oligonucleotide of claim 43, wherein the phosphate analog is oxy-methylphosphonate, vinylphosphonate, or malonylphosphonate.

45. An oligonucleotide comprising a sense strand comprising a nucleotide sequence as set forth in SEQ ID NO: 1050 and an antisense strand comprising a nucleotide sequence as set forth in SEQ ID NO: 1005.

46. A pharmaceutical composition comprising an oligonucleotide according to any one of claims 1-45 and a pharmaceutically acceptable carrier, delivery agent, or excipient.

47. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an oligonucleotide according to any one of claims 1-45 or a pharmaceutical composition according to claim 46.

48. A method of treating a disease, disorder, or condition associated with activation of CD274 expression, comprising administering to a subject in need thereof an oligonucleotide of any one of claims 1-45 or a pharmaceutical composition of claim 46.

49. A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an oligonucleotide according to any one of claims 1-45 or a pharmaceutical composition according to claim 46 in combination with a CTLA4 inhibitor.

50. A method of treating a disease, disorder, or condition associated with activation of CD274 a CTLA4 inhibitor.

51. The method of claim 48 or 50, wherein the disease, disorder, or condition associated with activation of CD274 The disease, disorder, or condition associated with expression is cancer.

52. The method of any one of claims 47, 49, and 51, wherein the cancer is selected from the group consisting of carcinoma, sarcoma, melanoma, lymphoma and leukemia, prostate cancer, breast cancer, hepatocellular carcinoma (HCC), colorectal cancer, pancreatic cancer, and glioblastoma.

53. The method of any one of claims 47, 49, and 51-52, wherein the cancer comprises an immunosuppressive tumor microenvironment.

54. The method of any one of claims 47, 49, and 51-52, wherein the cancer comprises an inflamed tumor microenvironment.

55. The method of claim 54, wherein the inflamed tumor microenvironment comprises infiltrating T cells.

56. The method of any one of claims 49-55, wherein the CTLA-4 inhibitor is an antibody.

57. The method of claim 56, wherein the antibody is an anti-CTLA-4 antibody.

58. The method of claim 57, wherein the anti-CTLA-4 antibody is selected from the group consisting of ipilimumab or tremelimumab.

59. A method of delivering an oligonucleotide targeted CD274 to a lymph node of a subject, comprising administering an oligonucleotide according to any one of claims 1-45.

60. The method of claim 59, wherein the lymph node is a tumor-draining lymph node.

61. Use of an oligonucleotide of any one of claims 1-45 or a pharmaceutical composition of claim 46 in the manufacture of a medicament for the treatment of a disease, disorder, or condition associated with CD274 expression, optionally for the treatment of cancer.

62. The oligonucleotide of any one of claims 1-45 or the pharmaceutical composition of claim 46, for or suitable for use in treating a disease, disorder, or condition associated with CD274 expression, optionally for use in treating a cancer.

63. A kit comprising the oligonucleotide of any one of claims 1-45, optionally a pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder, or condition associated with expression of CD274 the oligonucleotide of any one of claims 1-45, optionally a pharmaceutically acceptable carrier, and a package insert comprising instructions for administration to a subject having a disease, disorder, or condition associated with expression of 64. The use according to claim 61, the oligonucleotide or pharmaceutical composition for use or suitable for use according to claim 62, or the kit according to claim 63, wherein the oligonucleotide or pharmaceutical composition is suitable for use or suitable for use. CD274 The related disease, symptom, or condition is cancer.

65. The oligonucleotide of any one of claims 1-45 or the pharmaceutical composition of claim 46 for use in the manufacture of a medicament for treating a disease, disorder, or condition associated with CD274 expression in combination with a CTLA4 inhibitor.

66. The oligonucleotide of any one of claims 1-45 or the pharmaceutical composition of claim 46, for use or suitable for use in combination therapy with a CTLA4 inhibitor in the treatment of a disease, disorder, or condition associated with expression of CD274 a disease, disorder, or condition associated with expression.

67. A kit comprising the oligonucleotide of any one of claims 1-45, optionally a pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the RNAi oligonucleotide in combination with a CTLA4 inhibitor to a subject having a disease, disorder, or condition associated with expression of CD274 a disease, disorder, or condition associated with expression of the target gene.

68. The use according to claim 65, the oligonucleotide or pharmaceutical composition for use or suitable for use according to claim 66, or the kit according to claim 67, wherein the oligonucleotide or pharmaceutical composition is suitable for use or suitable for use. CD274 The related disease, symptom, or condition is cancer.

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