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COMPOSITIONS AND METHODS FOR INHIBITING HMGB1 EXPRESSION

20220072024 · 2022-03-10

    Inventors

    Cpc classification

    International classification

    Abstract

    This disclosure relates to oligonucleotides, compositions and methods useful for reducing HMGB1 expression, particularly in hepatocytes. Disclosed oligonucleotides for the reduction of HMGB1 expression may be either double-stranded or single-stranded and may be modified for improved characteristics such as stronger resistance to nucleases and lower immunogenicity. Disclosed oligonucleotides for the reduction of HMGB1 expression may also be designed to include targeting ligands to target a particular cell or organ, such as the hepatocytes of the liver, and may be used to treat liver fibrosis and related conditions.

    Claims

    1. An oligonucleotide for reducing expression of HMGB1, the oligonucleotide comprising a sense strand of 15 to 50 nucleotides in length and an antisense strand of 15 to 30 nucleotides in length, wherein the sense strand forms a duplex region with the antisense strand, wherein the sense strand comprises a sequence as set forth in any one of SEQ ID NOs.: 1-13 and wherein the antisense strand comprises a complementary sequence selected from SEQ ID NOs: 14-26.

    2. The oligonucleotide of claim 1, wherein the sense strand sequence comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 27-39.

    3. The oligonucleotide of claim 1 or claim A2, wherein the antisense strand sequence consists of a sequence as set forth in any one of SEQ ID NOs: 14-26.

    4. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 27 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 14.

    5. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 28 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 15.

    6. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 29 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 16.

    7. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 30 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 17.

    8. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 31 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 18.

    9. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 32 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 19.

    10. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 33 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 20.

    11. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 34 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 21.

    12. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 35 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 22.

    13. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 36 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 23.

    14. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 37 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 24.

    15. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 38 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 25.

    16. The oligonucleotide of claim 1, wherein the sense strand sequence comprises a sequence as set forth in SEQ ID NO: 39 and the antisense strand sequence comprises a sequence as set forth in SEQ ID NO: 26.

    17. The oligonucleotide of any one of claims 1-16, wherein the oligonucleotide comprises at least one modified nucleotide.

    18. The oligonucleotide of claim 17, wherein all the nucleotides of the oligonucleotide are modified.

    19. The oligonucleotide of claim 17 or claim 18, wherein the modified nucleotide comprises a 2′-modification.

    20. The oligonucleotide of claim 19, wherein the 2′-modification is a 2′-fluoro or 2′-O-methyl.

    21. The oligonucleotide of any one of claims 17-20, wherein one or more of positions 1, 2, 4, 6, 7, 12, 14, 16, 18-27, and 31-36 of the sense strand, and/or one or more of positions 1, 4, 6, 8, 9, 11, 13, 15, 18, and 20-22 of the antisense strand are modified with a 2′-O-methyl.

    22. The oligonucleotide of claim 21, wherein all of positions 1, 2, 4, 6, 7, 12, 14, 16, 18-27, and 31-36 of the sense strand, and all of positions 1, 4, 6, 8, 9, 11, 13, 15, 18, and 20-22 of the antisense strand are modified with a 2′-O-methyl.

    23. The oligonucleotide of any one of claims 17-22, wherein one or more of positions 3, 5, 8-11, 13, 15, and 17 of the sense strand, and/or one or more of positions 2, 3, 5, 7, 10, 12, 14, 16, 17, and 19 of the antisense strand are modified with a 2′-fluoro.

    24. The oligonucleotide of claim 23, wherein all of positions 3, 5, 8-11, 13, 15, and 17 of the sense strand, and all of positions 2, 3, 5, 7, 10, 12, 14, 16, 17, and 19 of the antisense strand are modified with a 2′-fluoro.

    25. The oligonucleotide of any one of claims 17-20, wherein one or more of positions 1-7, 12-27, and 31-36 of the sense strand, and/or one or more of positions 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with a 2′-O-methyl.

    26. The oligonucleotide of claim 25, wherein all of positions 1-7, 12-27, and 31-36 of the sense strand, and all of positions 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with a 2′-O-methyl.

    27. The oligonucleotide of any one of claims 17-20, 25 and 26, wherein one or more of positions 8-11 of the sense strand, and/or one or more of positions 2, 3, 5, 7, 10, and 14 of the antisense strand are modified with a 2′-fluoro.

    28. The oligonucleotide of claim 27, wherein all of positions 8-11 of the sense strand, and all of positions 2, 3, 5, 7, 10, and 14 of the antisense strand are modified with a 2′-fluoro.

    29. The oligonucleotide of any one of claims 17-20, wherein one or more of positions 1, 2, 4-7, 9, 11, 14-16, 18-27, and 31-36 of the sense strand, and/or one or more of positions 1, 6, 8, 9, 11, 13, 15, 18, and 20-22 of the antisense strand are modified with a 2′-O-methyl.

    30. The oligonucleotide of claim 29, wherein all of positions 1, 2, 4-7, 9, 11, 14-16, 18-27, and 31-36 of the sense strand, and all of positions 1, 6, 8, 9, 11, 13, 15, 18, and 20-22 of the antisense strand are modified with a 2′-O-methyl.

    31. The oligonucleotide of any one of claims 17-20, 29, and 30, wherein one or more of positions 3, 8, 10, 12, 13, and 17 of the sense strand, and/or one or more of positions 2-5, 7, 10, 12, 14, 16, 17, and 19 of the antisense strand are modified with a 2′-fluoro.

    32. The oligonucleotide of claim 31, wherein all of positions 3, 8, 10, 12, 13, and 17 of the sense strand, and all of positions 2-5, 7, 10, 12, 14, 16, 17, and 19 of the antisense strand are modified with a 2′-fluoro.

    33. The oligonucleotide of any one of claims 1-32, wherein the oligonucleotide comprises at least one modified internucleotide linkage.

    34. The oligonucleotide of claim 33, wherein the at least one modified internucleotide linkage is a phosphorothioate linkage.

    35. The oligonucleotide of claim 33 or claim 34, wherein the oligonucleotide has a phosphorothioate linkage between one or more of: positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.

    36. The oligonucleotide of claim 35, wherein the oligonucleotide has a phosphorothioate linkage between each of: positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.

    37. The oligonucleotide of any one of claims 1-36, wherein the uridine at the first position of the antisense strand comprises a phosphate analog.

    38. The oligonucleotide of claim 37, comprising the following structure at position 1 of the antisense strand: ##STR00011##

    39. The oligonucleotide of any one claims 1-38, wherein one or more of the nucleotides of the -AAA- sequence at positions 28-30 on the sense strand is conjugated to a monovalent GalNAc moiety.

    40. The oligonucleotide of claim 39, wherein each of the nucleotides of the -AAA- sequence at positions 28-30 on the sense strand is conjugated to a monovalent GalNAc moiety.

    41. The oligonucleotide of claim 40, wherein the -AAA- motif at positions 28-30 on the sense strand comprises the structure: ##STR00012## wherein: L represents a bond, click chemistry handle, or a linker of 1 to 20, inclusive, consecutive, covalently bonded atoms in length, selected from the group consisting of substituted and unsubstituted alkylene, substituted and unsubstituted alkenylene, substituted and unsubstituted alkynylene, substituted and unsubstituted heteroalkylene, substituted and unsubstituted heteroalkenylene, substituted and unsubstituted heteroalkynylene, and combinations thereof; and X is O, S, or N.

    42. The oligonucleotide of claim 41, wherein L is an acetal linker.

    43. The oligonucleotide of claim 41 or claim 42, wherein X is O.

    44. The oligonucleotide of any one of claims 41-43, wherein the -AAA- sequence at positions 28-30 on the sense strand comprises the structure: ##STR00013##

    45. An oligonucleotide for reducing expression of HMGB1, the oligonucleotide comprising an antisense strand of 15 to 30 nucleotides in length, wherein the antisense strand has a region of complementarity to HMGB1 that is complementary to at least 15 contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-13.

    46. The oligonucleotide of claim 45, wherein the antisense strand is 19 to 27 nucleotides in length.

    47. The oligonucleotide of claim 45, wherein the antisense strand is 22 nucleotides in length.

    48. The oligonucleotide of any one of claims 45 to 47, further comprising a sense strand of 15 to 50 nucleotides in length, wherein the sense strand forms a duplex region with the antisense strand.

    49. The oligonucleotide of claim 48, wherein the sense strand is 19 to 50 nucleotides in length.

    50. The oligonucleotide of claim 48 or 49, wherein the duplex region is 20 nucleotides in length.

    51. An oligonucleotide for reducing expression of HMGB1 comprising a sense strand and an antisense strand, wherein: (a) the sense strand comprises a sequence as set forth in SEQ ID NO: 788 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 814; (b) the sense strand comprises a sequence as set forth in SEQ ID NO: 789 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 815; (c) the sense strand comprises a sequence as set forth in SEQ ID NO: 790 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 816; (d) the sense strand comprises a sequence as set forth in SEQ ID NO: 791 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 817; (e) the sense strand comprises a sequence as set forth in SEQ ID NO: 792 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 818; (f) the sense strand comprises a sequence as set forth in SEQ ID NO: 793 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 819; (g) the sense strand comprises a sequence as set forth in SEQ ID NO: 794 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 820; (h) the sense strand comprises a sequence as set forth in SEQ ID NO: 795 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 821; (i) the sense strand comprises a sequence as set forth in SEQ ID NO: 796 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 822; (j) the sense strand comprises a sequence as set forth in SEQ ID NO: 797 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 823; (k) the sense strand comprises a sequence as set forth in SEQ ID NO: 798 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 824; (l) the sense strand comprises a sequence as set forth in SEQ ID NO: 799 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 825; (m) the sense strand comprises a sequence as set forth in SEQ ID NO: 800 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 826; (n) the sense strand comprises a sequence as set forth in SEQ ID NO: 801 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 827; (o) the sense strand comprises a sequence as set forth in SEQ ID NO: 802 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 828; (p) the sense strand comprises a sequence as set forth in SEQ ID NO: 803 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 829; (q) the sense strand comprises a sequence as set forth in SEQ ID NO: 804 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 830; (r) the sense strand comprises a sequence as set forth in SEQ ID NO: 805 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 831; (s) the sense strand comprises a sequence as set forth in SEQ ID NO: 806 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 832; (t) the sense strand comprises a sequence as set forth in SEQ ID NO: 807 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 833; (u) the sense strand comprises a sequence as set forth in SEQ ID NO: 808 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 834; (v) the sense strand comprises a sequence as set forth in SEQ ID NO: 809 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 835; (w) the sense strand comprises a sequence as set forth in SEQ ID NO: 810 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 836; (x) the sense strand comprises a sequence as set forth in SEQ ID NO: 811 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 837; (y) the sense strand comprises a sequence as set forth in SEQ ID NO:812 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 838; or (z) the sense strand comprises a sequence as set forth in SEQ ID NO: 813 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 839.

    52. A composition comprising the oligonucleotide of any one of claims 1-51 and an excipient.

    53. A method of delivering an oligonucleotide to a subject, the method comprising administering the composition of claim 52 to the subject.

    54. The method of claim 53, wherein the subject has or is at risk of having liver fibrosis.

    55. The method of claim 54, wherein the subject has cholestatic or autoimmune liver disease.

    56. The method of any one of claims 53-55, wherein expression of HMGB1 protein is reduced by administering to the subject the oligonucleotide.

    57. A method of treating a subject having or at risk of having liver fibrosis, the method comprising administering to the subject an oligonucleotide of any one of claims 1-51.

    58. The method of claim 57, wherein the subject has cholestatic or autoimmune liver disease.

    59. The method of claim 58, wherein the subject has nonalcoholic steatohepatitis (NASH).

    60. The method of any one of claims 57-59, wherein the oligonucleotide is administered prior to exposure of the subject to a hepatotoxic agent.

    61. The method of any one of claims 57-59, wherein the oligonucleotide is administered subsequent to exposure of the subject to a hepatotoxic agent.

    62. The method of any one of claims 57-59, wherein the oligonucleotide is administered simultaneously with the subject's exposure to a hepatotoxic agent.

    63. The method of any one of claims 57-62, wherein the administration results in a reduction in liver HMGB1 levels.

    64. The method of any one of claims 57-63, wherein the administration results in a reduction in serum HMGB1 levels.

    65. Use of an oligonucleotide of any one of claims 1-51 for treating a subject having or at risk of having liver fibrosis.

    66. The use of claim 65, wherein the subject has cholestatic or autoimmune liver disease.

    67. The use of claim 65, wherein the subject has nonalcoholic steatohepatitis (NASH).

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0067] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain embodiments, and together with the written description, serve to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein.

    [0068] FIG. 1A and FIG. 1B: In vivo activity evaluation of 3 GalNAc-conjugated HMGB1 oligonucleotides (FIG. 1A) with 3 different modification patterns (FIG. 1B). NM_002128.5 location numbers were used on x-axis.

    [0069] FIG. 2: In vivo activity evaluation of 3 GalNAc-conjugated HMGB1 oligonucleotides at three different concentrations. NM_002128.5 location numbers were used on x-axis.

    [0070] FIG. 3: In vivo activity evaluation of three GalNAc-conjugated HMGB1 oligonucleotides at 4-different time points. NM_002128.5 location numbers were used on x-axis.

    [0071] FIGS. 4A-4F: Screening of 288 triple commons HMGB1 RNAi oligonucleotides in Huh-7 (Human liver) cells. The nucleotide position in NM_002128.5 that corresponds to the 3′ end of the sense strand of each siRNA is indicated on the x axis. The percent mRNA remaining is shown for each of the 5′ assay (red) and the 3′ assay (blue).

    [0072] FIG. 5: In vivo activity evaluation of 22 HMGB1 RNAi oligonucleotides identified in the screen of FIGS. 4A-4F. The 22 HMGB1 oligonucleotides are GalNAc-conjugated with 2 different modification patterns (M2 and M3, see FIG. 1B for modification patterns). NM_002128.5 location numbers were used on x-axis.

    [0073] FIG. 6: In vivo activity evaluation of lead GalNAc-conjugated HMGB1 oligonucleotides 21 days after administration. NM_002128.5 location numbers were used on x-axis

    [0074] FIG. 7: GalNAc-conjugated HMGB1 oligonucleotides that were tested in primary monkey/human hepatocytes. Arrow indicates that the oligonucleotide is also selected to be tested in non-human primate screen.

    [0075] FIGS. 8A-8D: Activity of the 6 GalNAc-conjugates HMGB1 oligonucleotides in primary monkey (cynomolgous) and human hepatocytes shown by IC50 curve. (FIG. 8A) Cyno hepatocyte #1. (FIG. 8B) Cyno hepatocyte #2. (FIG. 8C) Human hepatocyte #1. (FIG. 8D) Positive control GalNAc-conjugated LDHA oligonucleotide.

    [0076] FIGS. 9A-9B: In vivo activity evaluation of GalNAc-conjugated HMGB1 oligonucleotides in non-human primates. (FIG. 9A) 4 mg/kg, one dose. (FIG. 9B) 2 mg/kg dosing, 4 repeat doses.

    [0077] FIGS. 10A-10F: Screening of 6 HMGB1 RNAi oligonucleotides at 3 different concentrations (0.03 nM, 0.1 nM and 1 nM) in mouse, monkey, and human cell lines. (FIG. 10A) Mouse cell line, 5′ assay. (FIG. 10B) Mouse cell line, 3′ assay. (FIG. 10C) Monkey cell line, 5′ assay. (FIG. 10D) Monkey cell line, 3′ assay. (FIG. 10E) Human cell line, 5′ assay. (FIG. 10F) Human cell line, 3′ assay.

    [0078] FIGS. 11A-11C: Activity of 4 GalNAc-conjugate HMGB1 oligonucleotides in Huh-7 cells by IC50 curve. IC50 curves of HMGB1 (FIG. 11A), HMGB2 (FIG. 11B) and HMGB3 (FIG. 11C), normalized to mock.

    DETAILED DESCRIPTION OF THE INVENTION

    [0079] According to some aspects, the disclosure provides oligonucleotides targeting HMGB1 mRNA that are effective for reducing HMGB1 expression in cells, particularly liver cells (e.g., hepatocytes) for the treatment of liver fibrosis. Accordingly, in related aspects, the disclosure provides methods of treating fibrosis that involve selectively reducing HMGB1 gene expression in liver. In certain embodiments, HMGB1 targeting oligonucleotides provided herein are designed for delivery to selected cells of target tissues (e.g., liver hepatocytes) to treat fibrosis in those tissues. RNAi oligonucleotides having particular modification patterns are disclosed herein (as outlined in Table 2) that are particularly useful for knocking down HMGB1 mRNA in vivo.

    [0080] Further aspects of the disclosure, including a description of defined terms, are provided below.

    I. Definitions

    [0081] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

    [0082] Administering: As used herein, the terms “administering” or “administration” means to provide a substance (e.g., an oligonucleotide) to a subject in a manner that is pharmacologically useful (e.g., to treat a condition in the subject).

    [0083] Asialoglycoprotein receptor (ASGPR): As used herein, the term “Asialoglycoprotein receptor” or “ASGPR” refers to a bipartite C-type lectin formed by a major 48 kDa (ASGPR-1) and minor 40 kDa subunit (ASGPR-2). ASGPR is primarily expressed on the sinusoidal surface of hepatocyte cells, and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins).

    [0084] Attenuates: As used herein, the term “attenuates” means reduces or effectively halts. As a non-limiting example, one or more of the treatments provided herein may reduce or effectively halt the onset or progression of liver fibrosis or liver inflammation in a subject. This attenuation may be exemplified by, for example, a decrease in one or more aspects (e.g., symptoms, tissue characteristics, and cellular, inflammatory or immunological activity, etc.) of liver fibrosis or liver inflammation, no detectable progression (worsening) of one or more aspects of liver fibrosis or liver inflammation, or no detectable aspects of liver fibrosis or liver inflammation in a subject when they might otherwise be expected.

    [0085] Complementary: As used herein, the term “complementary” refers to a structural relationship between two nucleotides (e.g., on two opposing nucleic acids or on opposing regions of a single nucleic acid strand) that permits the two nucleotides to form base pairs with one another. For example, a purine nucleotide of one nucleic acid that is complementary to a pyrimidine nucleotide of an opposing nucleic acid may base pair together by forming hydrogen bonds with one another. In some embodiments, complementary nucleotides can base pair in the Watson-Crick manner or in any other manner that allows for the formation of stable duplexes. In some embodiments, two nucleic acids may have regions of multiple nucleotides that are complementary with each other so as to form regions of complementarity, as described herein.

    [0086] Deoxyribonucleotide: As used herein, the term “deoxyribonucleotide” refers to a nucleotide having a hydrogen in place of a hydroxyl at the 2′ position of its pentose sugar as compared with a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than at the 2′ position, including modifications or substitutions in or of the sugar, phosphate group or base.

    [0087] Double-stranded oligonucleotide: As used herein, the term “double-stranded oligonucleotide” refers to an oligonucleotide that is substantially in a duplex form. In some embodiments, the complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed between of antiparallel sequences of nucleotides of covalently separate nucleic acid strands. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed between antiparallel sequences of nucleotides of nucleic acid strands that are covalently linked. In some embodiments, complementary base-pairing of duplex region(s) of a double-stranded oligonucleotide is formed from single nucleic acid strand that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that base pair together. In some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are fully duplexed with one another. However, in some embodiments, a double-stranded oligonucleotide comprises two covalently separate nucleic acid strands that are partially duplexed, e.g., having overhangs at one or both ends. In some embodiments, a double-stranded oligonucleotide comprises antiparallel sequence of nucleotides that are partially complementary, and thus, may have one or more mismatches, which may include internal mismatches or end mismatches.

    [0088] Duplex: As used herein, the term “duplex,” in reference to nucleic acids (e.g., oligonucleotides), refers to a structure formed through complementary base pairing of two antiparallel sequences of nucleotides.

    [0089] Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect.

    [0090] Hepatocyte: As used herein, the term “hepatocyte” or “hepatocytes” refers to cells of the parenchymal tissues of the liver. These cells make up approximately 70-85% of the liver's mass and manufacture serum albumin, fibrinogen, and the prothrombin group of clotting factors (except for Factors 3 and 4). Markers for hepatocyte lineage cells may include, but are not limited to: transthyretin (Ttr), glutamine synthetase (Glu1), hepatocyte nuclear factor 1a (Hnf1a), and hepatocyte nuclear factor 4a (Hnf4a). Markers for mature hepatocytes may include, but are not limited to: cytochrome P450 (Cyp3a11), fumarylacetoacetate hydrolase (Fah), glucose 6-phosphate (G6p), albumin (Alb), and OC2-2F8 (see, e.g., Huch et al., Nature, 2013, 494(7436):247-250, the contents of which relating to hepatocyte markers is incorporated herein by reference.

    [0091] Hepatotoxic agent: As used herein, a “hepatotoxic agent” is a chemical compound, virus, or other substance that is itself toxic to the liver or can be processed to form a metabolite that is toxic to the liver. Hepatotoxic agents may include, but are not limited to, carbon tetrachloride (CC14), acetaminophen (paracetamol), vinyl chloride, arsenic, chloroform, and nonsteroidal anti-inflammatory drugs (such as aspirin and phenylbutazone).

    [0092] Liver inflammation: As used herein, the term “liver inflammation” or “hepatitis” refers to a physical condition in which the liver becomes swollen, dysfunctional, and/or painful, especially as a result of injury or infection, as may be caused by exposure to a hepatotoxic agent. Symptoms may include jaundice (yellowing of the skin or eyes), fatigue, weakness, nausea, vomiting, appetite reduction, and weight loss. Liver inflammation, if left untreated, may progress to fibrosis, cirrhosis, liver failure, or liver cancer.

    [0093] Liver fibrosis: As used herein, the term “liver fibrosis” or “fibrosis of the liver” refers to an excessive accumulation in the liver of extracellular matrix proteins, which could include collagens (I, III, and IV), fibronectin, undulin, elastin, laminin, hyaluronan, and proteoglycans resulting from inflammation and liver cell death. Liver fibrosis, if left untreated, may progress to cirrhosis, liver failure, or liver cancer.

    [0094] Loop: As used herein the term, “loop” refers to a unpaired region of a nucleic acid (e.g., oligonucleotide) that is flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to one another, such that under appropriate hybridization conditions (e.g., in a phosphate buffer, in a cell), the two antiparallel regions, which flank the unpaired region, hybridize to form a duplex (referred to as a “stem”).

    [0095] Modified Internucleotide Linkage: As used herein, the term “modified internucleotide linkage” refers to an internucleotide linkage having one or more chemical modifications compared with a reference internucleotide linkage comprising a phosphodiester bond. In some embodiments, a modified nucleotide is a non-naturally occurring linkage. Typically, a modified internucleotide linkage confers one or more desirable properties to a nucleic acid in which the modified internucleotide linkage is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.

    [0096] Modified Nucleotide: As used herein, the term “modified nucleotide” refers to a nucleotide having one or more chemical modifications compared with a corresponding reference nucleotide selected from: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide and thymidine deoxyribonucleotide. In some embodiments, a modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, a modified nucleotide has one or more chemical modification in its sugar, nucleobase, and/or phosphate group. In some embodiments, a modified nucleotide has one or more chemical moieties conjugated to a corresponding reference nucleotide. Typically, a modified nucleotide confers one or more desirable properties to a nucleic acid in which the modified nucleotide is present. For example, a modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, bioactivity, reduced immunogenicity, etc.

    [0097] Nicked Tetraloop Structure: A “nicked tetraloop structure” is a structure of a RNAi oligonucleotide characterized by the presence of separate sense (passenger) and antisense (guide) strands, in which the sense strand has a region of complementarity with the antisense strand, and in which at least one of the strands, generally the sense strand, has a tetraloop configured to stabilize an adjacent stem region formed within the at least one strand.

    [0098] Oligonucleotide: As used herein, the term “oligonucleotide” refers to a short nucleic acid, e.g., of less than 100 nucleotides in length. An oligonucleotide may be single-stranded or double-stranded. An oligonucleotide may or may not have duplex regions. As a set of non-limiting examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), dicer substrate interfering RNA (dsiRNA), antisense oligonucleotide, short siRNA, or single-stranded siRNA. In some embodiments, a double-stranded oligonucleotide is an RNAi oligonucleotide.

    [0099] Overhang: As used herein, the term “overhang” refers to terminal non-base pairing nucleotide(s) resulting from one strand or region extending beyond the terminus of a complementary strand with which the one strand or region forms a duplex. In some embodiments, an overhang comprises one or more unpaired nucleotides extending from a duplex region at the 5′ terminus or 3′ terminus of a double-stranded oligonucleotide. In certain embodiments, the overhang is a 3′ or 5′ overhang on the antisense strand or sense strand of a double-stranded oligonucleotides.

    [0100] Phosphate analog: As used herein, the term “phosphate analog” refers to a chemical moiety that mimics the electrostatic and/or steric properties of a phosphate group. In some embodiments, a phosphate analog is positioned at the 5′ terminal nucleotide of an oligonucleotide in place of a 5′-phosphate, which is often susceptible to enzymatic removal. In some embodiments, a 5′ phosphate analogs contain a phosphatase-resistant linkage Examples of phosphate analogs include 5′ phosphonates, such as 5′ methylenephosphonate (5′-MP) and 5′-(E)-vinylphosphonate (5′-VP). In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”) at a 5′-terminal nucleotide. An example of a 4′-phosphate analog is oxymethylphosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof (see, e.g., International patent publication WO/2018/045317, the contents of which relating to phosphate analogs is incorporated herein by reference. Other modifications have been developed for the 5′ end of oligonucleotides (see, e.g., WO 2011/133871; U.S. Pat. No. 8,927,513; and Prakash et al., Nucleic Acids Res., 2015, 43(6):2993-3011, the contents of each of which relating to phosphate analogs are incorporated herein by reference).

    [0101] Reduced expression: As used herein, the term “reduced expression” of a gene refers to a decrease in the amount of RNA transcript or protein encoded by the gene and/or a decrease in the amount of activity of the gene in a cell or subject, as compared to an appropriate reference cell or subject. For example, the act of treating a cell with a double-stranded oligonucleotide (e.g., one having an antisense strand that is complementary to HMGB1 mRNA sequence) may result in a decrease in the amount of RNA transcript, protein and/or activity (e.g., encoded by the HMGB1 gene) compared to a cell that is not treated with the double-stranded oligonucleotide. Similarly, “reducing expression” as used herein refers to an act that results in reduced expression of a gene (e.g., HMGB1).

    [0102] Region of Complementarity: As used herein, the term “region of complementary” refers to a sequence of nucleotides of a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel sequence of nucleotides to permit hybridization between the two sequences of nucleotides under appropriate hybridization conditions, e.g., in a phosphate buffer, in a cell, etc.

    [0103] Ribonucleotide: As used herein, the term “ribonucleotide” refers to a nucleotide having a ribose as its pentose sugar, which contains a hydroxyl group at its 2′ position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of atoms other than at the 2′ position, including modifications or substitutions in or of the ribose, phosphate group or base.

    [0104] RNAi Oligonucleotide: As used herein, the term “RNAi oligonucleotide” refers to either (a) a double stranded oligonucleotide having a sense strand (passenger) and antisense strand (guide), in which the antisense strand or part of the antisense strand is used by the Argonaute 2 (Ago2) endonuclease in the cleavage of a target mRNA or (b) a single stranded oligonucleotide having a single antisense strand, where that antisense strand (or part of that antisense strand) is used by the Ago2 endonuclease in the cleavage of a target mRNA.

    [0105] Strand: As used herein, the term “strand” refers to a single contiguous sequence of nucleotides linked together through internucleotide linkages (e.g., phosphodiester linkages, phosphorothioate linkages). In some embodiments, a strand has two free ends, e.g., a 5′-end and a 3′-end.

    [0106] Subject: As used herein, the term “subject” means any mammal, including mice, rabbits, and humans. In some embodiments, the subject is a human or non-human primate. The terms “individual” or “patient” may be used interchangeably with “subject.”

    [0107] Synthetic: As used herein, the term “synthetic” refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or that is otherwise not derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

    [0108] Targeting ligand: As used herein, the term “targeting ligand” refers to a molecule (e.g., a carbohydrate, amino sugar, cholesterol, polypeptide or lipid) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and that is conjugatable to another substance for purposes of targeting the other substance to the tissue or cell of interest. For example, in some embodiments, a targeting ligand may be conjugated to an oligonucleotide for purposes of targeting the oligonucleotide to a specific tissue or cell of interest. In some embodiments, a targeting ligand selectively binds to a cell surface receptor. Accordingly, in some embodiments, a targeting ligand when conjugated to an oligonucleotide facilitates delivery of the oligonucleotide into a particular cell through selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of the complex comprising the oligonucleotide, targeting ligand and receptor. In some embodiments, a targeting ligand is conjugated to an oligonucleotide via a linker that is cleaved following or during cellular internalization such that the oligonucleotide is released from the targeting ligand in the cell.

    [0109] Tetraloop: As used herein, the term “tetraloop” refers to a loop that increases stability of an adjacent duplex formed by hybridization of flanking sequences of nucleotides. The increase in stability is detectable as an increase in melting temperature (Tm) of an adjacent stem duplex that is higher than the Tm of the adjacent stem duplex expected, on average, from a set of loops of comparable length consisting of randomly selected sequences of nucleotides. For example, a tetraloop can confer a melting temperature of at least 50° C., at least 55° C., at least 56° C., at least 58° C., at least 60° C., at least 65° C. or at least 75° C. in 10 mM NaHPO.sub.4 to a hairpin comprising a duplex of at least 2 base pairs in length. In some embodiments, a tetraloop may stabilize a base pair in an adjacent stem duplex by stacking interactions. In addition, interactions among the nucleotides in a tetraloop include but are not limited to non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature, 1990, 346(6285):680-2; Heus and Pardi, Science, 1991, 253(5016):191-4). In some embodiments, a tetraloop comprises or consists of 3 to 6 nucleotides, and is typically 4 to 5 nucleotides. In certain embodiments, a tetraloop comprises or consists of three, four, five, or six nucleotides, which may or may not be modified (e.g., which may or may not be conjugated to a targeting moiety). In one embodiment, a tetraloop consists of four nucleotides. Any nucleotide may be used in the tetraloop and standard IUPAC-IUB symbols for such nucleotides may be used, as described in Cornish-Bowden, Nucleic Acids Res., 1985, 13:3021-3030. For example, the letter “N” may be used to mean that any base may be in that position, the letter “R” may be used to show that A (adenine) or G (guanine) may be in that position, and “B” may be used to show that C (cytosine), G (guanine), or T (thymine) may be in that position. Examples of tetraloops include the UNCG family of tetraloops (e.g., UUCG), the GNRA family of tetraloops (e.g., GAAA), and the CUUG tetraloop (Woese et al., Proc Natl Acad Sci USA., 1990, 87(21):8467-71; Antao et al., Nucleic Acids Res., 1991, 19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, for example, Nakano et al., Biochemistry, 2002, 41(48):14281-14292; Shinji et al., Nippon Kagakkai Koen Yokoshu, 2000, 78(2):731; which are incorporated by reference herein for their relevant disclosures. In some embodiments, the tetraloop is contained within a nicked tetraloop structure.

    [0110] Treat: As used herein, the term “treat” refers to the act of providing care to a subject in need thereof, e.g., through the administration a therapeutic agent (e.g., an oligonucleotide) to the subject, for purposes of improving the health and/or well-being of the subject with respect to an existing condition (e.g., a disease, disorder) or to prevent or decrease the likelihood of the occurrence of a condition. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom or contributing factor of a condition (e.g., disease, disorder) experienced by a subject.

    II. Oligonucleotide-Based Inhibitors of HMGB1 Expression

    [0111] i. HMGB1 Target Sequences

    [0112] In some embodiments, oligonucleotide-based inhibitors of HMGB1 expression are provided herein that can be used to achieve a therapeutic benefit. Through examination of the HMGB1 mRNA, including mRNAs of multiple different species (human, rhesus monkey, and mouse (see, e.g., Example 1) and in vitro and in vivo testing, it has been discovered that certain sequences of HMGB1 mRNA are useful as targeting sequences because they are more amenable than others to oligonucleotide-based inhibition. In some embodiments, a HMGB1 target sequence comprises, or consists of, a sequence as forth in any one of SEQ ID NOs: 1-13. These regions of HMGB1 mRNA may be targeted using oligonucleotides as discussed herein for purposes of inhibiting HMGB1 mRNA expression.

    [0113] Accordingly, in some embodiments, oligonucleotides provided herein are designed so as to have regions of complementarity to HMGB1 mRNA (e.g., within a target sequence of HMGB1 mRNA) for purposes of targeting the mRNA in cells and inhibiting its expression. The region of complementary is generally of a suitable length and base content to enable annealing of the oligonucleotide (or a strand thereof) to HMGB1 mRNA for purposes of inhibiting its expression. In some embodiments, the region of complementarity is at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 nucleotides in length. In some embodiments, an oligonucleotide provided herein has a region of complementarity to HMGB1 that is in the range of 12 to 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 length. In some embodiments, an oligonucleotide provided herein has a region of complementarity to HMGB1 that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

    [0114] In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is at least partially complementary to a sequence as set forth in any one of SEQ ID NOs: 1-13. In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is fully complementary to a sequence as set forth in any one of SEQ ID NOs: 1-13. In some embodiments, a region of complementarity of an oligonucleotide (e.g., on an antisense strand of a double-stranded oligonucleotide) is complementary to a contiguous sequence of nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-13 that is in the range of 12 to 20 nucleotides (e.g., 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 20, 14 to 18, 14 to 16, 16 to 20, 16 to 18, or 18 to 20) in length. In some embodiments, a region of complementarity of an oligonucleotide (e.g., on an antisense strand of a double-stranded oligonucleotide) is complementary to a contiguous sequence of nucleotides of a sequence as set forth in any one of SEQ ID NOs: 11-13 that is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides in length.

    [0115] In some embodiments, a region of complementarity of an oligonucleotide that is complementary to contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-13 spans the entire length of an antisense strand. In some embodiments, a region of complementarity of an oligonucleotide that is complementary to contiguous nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-13 spans a portion of the entire length of an antisense strand. In some embodiments, an oligonucleotide disclosed herein comprises a region of complementarity (e.g., on an antisense strand of a double-stranded oligonucleotide) that is at least partially (e.g., fully) complementary to a contiguous stretch of nucleotides spanning nucleotides 1-20 of a sequence as set forth in any one of SEQ ID NOs: 1-13.

    [0116] In some embodiments, a region of complementarity to HMGB1 may have one or more mismatches compared with a corresponding sequence of HMGB1 mRNA. A region of complementarity on an oligonucleotide may have up to 1, up to 2, up to 3, up to 4, up to 5, etc. mismatches provided that it maintains the ability to form complementary base pairs with HMGB1 mRNA under appropriate hybridization conditions. Alternatively, a region of complementarity on an oligonucleotide may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches provided that it maintains the ability to form complementary base pairs with HMGB1 mRNA under appropriate hybridization conditions. In some embodiments, if there are more than one mismatches in a region of complementarity, they may be positioned consecutively (e.g., 2, 3, 4, or more in a row), or interspersed throughout the region of complementarity provided that the oligonucleotide maintains the ability to form complementary base pairs with HMGB1 mRNA under appropriate hybridization conditions.

    [0117] ii. Types of Oligonucleotides

    [0118] There are a variety of structures of oligonucleotides that are useful for targeting HMGB1 in the methods of the present disclosure, including RNAi, antisense, miRNA, etc. Any of the structures described herein or elsewhere may be used as a framework to incorporate or target a sequence described herein (e.g., a hotpot sequence of HMBG1 such as those illustrated in SEQ ID NOs: 1-13).

    [0119] In some embodiments, oligonucleotides for reducing the expression of HMGB1 expression engage RNA interference (RNAi) pathways upstream or downstream of dicer involvement. For example, RNAi oligonucleotides have been developed with each strand having sizes of 19-25 nucleotides with at least one 3′ overhang of 1 to 5 nucleotides (see, e.g., U.S. Pat. No. 8,372,968). Longer oligonucleotides have also been developed that are processed by Dicer to generate active RNAi products (see, e.g., U.S. Pat. No. 8,883,996). Further work produced extended double-stranded oligonucleotides where at least one end of at least one strand is extended beyond a duplex targeting region, including structures where one of the strands includes a thermodynamically-stabilizing tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, as well as International patent publication WO2010033225, which are incorporated by reference herein for their disclosure of the structures and form of these oligonucleotides). Such structures may include single-stranded extensions (on one or both sides of the molecule) as well as double-stranded extensions.

    [0120] In some embodiments, oligonucleotides provided herein are designed to engage in the RNA interference pathway downstream of the involvement of dicer (e.g., dicer cleavage). Such oligonucleotides may have an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3′ end of the sense strand. Such oligonucleotides (e.g., siRNAs) may comprise a 21 nucleotide guide strand that is antisense to a target RNA and a complementary passenger strand, in which both strands anneal to form a 19-bp duplex and 2 nucleotide overhangs at either or both 3′ ends. Longer oligonucleotide designs are also available including oligonucleotides having a guide strand of 23 nucleotides and a passenger strand of 21 nucleotides, where there is a blunt end on the right side of the molecule (3′-end of passenger strand/5′-end of guide strand) and a two nucleotide 3′-guide strand overhang on the left side of the molecule (5′-end of the passenger strand/3′-end of the guide strand). In such molecules, there is a 21 base pair duplex region. See, for example, U.S. Pat. Nos. 9,012,138; 9,012,621; and 9,193,753, each of which are incorporated herein for their relevant disclosures.

    [0121] In some embodiments, oligonucleotides as disclosed herein may comprise sense and antisense strands that are both in the range of 17 to 26 (e.g., 17 to 26, 20 to 25, or 21-23) nucleotides in length. In some embodiments, an oligonucleotide as disclosed herein comprises a sense and antisense strand that are both in the range of 19-22 nucleotide in length. In some embodiments, the sense and antisense strands are of equal length. In some embodiments, an oligonucleotide comprises sense and antisense strands, such that there is a 3′-overhang on either the sense strand or the antisense strand, or both the sense and antisense strand. In some embodiments, for oligonucleotides that have sense and antisense strands that are both in the range of 21-23 nucleotides in length, a 3′ overhang on the sense, antisense, or both sense and antisense strands is 1 or 2 nucleotides in length. In some embodiments, the oligonucleotide has a guide strand of 22 nucleotides and a passenger strand of 20 nucleotides, where there is a blunt end on the right side of the molecule (3′-end of passenger strand/5′-end of guide strand) and a two nucleotide 3′-guide strand overhang on the left side of the molecule (5′-end of the passenger strand/3′-end of the guide strand). In such molecules, there is a 20 base pair duplex region.

    [0122] Other oligonucleotides designs for use with the compositions and methods disclosed herein include: 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology, Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., having 19 bp or shorter stems; see, e.g., Moore et al. Methods Mol. Biol., 2010, 629:141-158), blunt siRNAs (e.g., of 19 bps in length; see, e.g., Kraynack and Baker, R N A, 2006, 12:163-176), asymmetrical siRNAs (aiRNA; see, e.g., Sun et al., Nat. Biotechnol., 2008, 26:1379-1382), asymmetric shorter-duplex siRNA (see, e.g., Chang et al., Mol Ther., 2009, 17(4):725-32), fork siRNAs (see, e.g., Hohjoh, FEBS Letters, 2004, 557(1-3):193-198), single-stranded siRNAs (Elsner, Nature Biotechnology, 2012, 30:1063), dumbbell-shaped circular siRNAs (see, e.g., Abe et al., J Am Chem Soc., 2007, 129:15108-15109), and small internally segmented interfering RNA (siRNA; see, e.g., Bramsen et al., Nucleic Acids Res., 2007, 35(17):5886-5897). Each of the foregoing references is incorporated by reference in its entirety for the related disclosures therein. Further non-limiting examples of oligonucleotide structures that may be used in some embodiments to reduce or inhibit the expression of HMGB1 are microRNA (miRNA), short hairpin RNA (shRNA), and short siRNA (see, e.g., Hamilton et al., EMBO J., 2002, 21(17):4671-4679; see also U.S. patent publication no. 20090099115).

    [0123] Still, in some embodiments, an oligonucleotide for reducing HMGB1 expression as described herein is single-stranded. Such structures may include, but are not limited to single-stranded RNAi molecules. Recent efforts have demonstrated the activity of single-stranded RNAi molecules (see, e.g., Matsui et al., Molecular Therapy, 2016, 24(5):946-955). However, in some embodiments, oligonucleotides provided herein are antisense oligonucleotides (ASOs). An antisense oligonucleotide is a single-stranded oligonucleotide that has a nucleobase sequence which, when written in the 5′ to 3′ direction, comprises the reverse complement of a targeted segment of a particular nucleic acid and is suitably modified (e.g., as a gapmer) so as to induce RNaseH mediated cleavage of its target RNA in cells or (e.g., as a mixmer) so as to inhibit translation of the target mRNA in cells. Antisense oligonucleotides for use in the instant disclosure may be modified in any suitable manner known in the art including, for example, as shown in U.S. Pat. No. 9,567,587, which is incorporated by reference herein for its disclosure regarding modification of antisense oligonucleotides (including, e.g., length, sugar moieties of the nucleobase (pyrimidine, purine), and alterations of the heterocyclic portion of the nucleobase). Further, antisense molecules have been used for decades to reduce expression of specific target genes (see, e.g., Bennett et al., “Pharmacology of Antisense Drugs,” Ann Rev Pharmacol Toxicol., 2017, 57:81-105).

    [0124] iii. Double-Stranded Oligonucleotides

    [0125] Double-stranded oligonucleotides for targeting HMGB1 expression (e.g., via the RNAi pathway) generally have a sense strand and an antisense strand that form a duplex with one another. In some embodiments, the sense and antisense strands are not covalently linked. However, in some embodiments, the sense and antisense strands are covalently linked. In some embodiments, a duplex formed between a sense and antisense strand is at least 15 (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 length. In some embodiments, a duplex formed between a sense and antisense strand is in the range of 15-30 nucleotides in length (e.g., 15 to 30, 15 to 27, 15 to 22, 18 to 22, 18 to 25, 18 to 27, 18 to 30, or 21 to 30 nucleotides in length). In some embodiments, a duplex formed between a sense and antisense strand is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the duplex region is 20 nucleotides in length. In some embodiments a duplex formed between a sense and antisense strand does not span the entire length of the sense strand and/or antisense strand. In some embodiments, a duplex between a sense and antisense strand spans the entire length of either the sense or antisense strands. In certain embodiments, a duplex between a sense and antisense strand spans the entire length of both the sense strand and the antisense strand.

    [0126] In some embodiments, an oligonucleotide provided herein comprises a sense strand having a sequence as set forth in any one of SEQ ID NOs: 1-13 and an antisense strand comprising a complementary sequence selected from SEQ ID NOs: 14-26, as is arranged Table 1.

    [0127] In some embodiments, the sense strand comprising a sequence as set forth in SEQ ID NO: 1 and the antisense strand comprising a sequence as set forth in SEQ ID NO: 14. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 2 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 15. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 3 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 16. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 4 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 17. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 5 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 18. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 6 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 19. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 7 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 20. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 8 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 21. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 9 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 22. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 10 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 23. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 11 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 24. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 12 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 25. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 13 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 26.

    [0128] In some embodiments, an oligonucleotide provided herein comprises a sense strand comprising a sequence as set forth in any one of SEQ ID NOs: 27-39 and an antisense strand comprising a complementary sequence selected from SEQ ID NOs: 14-26, as is also arranged in Table 2, including modifications to the sense sequence and antisense sequences. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 27 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 14. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 28 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 15. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 29 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 16. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 30 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 17. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 31 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 18. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 32 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 19. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 33 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 20. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 34 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 21. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 35 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 22. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 36 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 23. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 37 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 24. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 38 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 25. In some embodiments, the sense strand comprises a sequence as set forth in SEQ ID NO: 39 and the antisense strand comprises a sequence as set forth in SEQ ID NO: 26.

    [0129] It should be appreciated that, in some embodiments, sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and/or one or more modified nucleotides and/or one or more modified internucleotide linkages and/or one or more other modification compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

    [0130] In some embodiments, a double-stranded oligonucleotide comprises a 25-nucleotide sense strand and a 27-nucleotide antisense strand that when acted upon by a dicer enzyme results in an antisense strand that is incorporated into the mature RISC. In some embodiments, a sense strand of an oligonucleotide is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides). In some embodiments, a sense strand of an oligonucleotide is longer than 25 nucleotides (e.g., 26, 27, 28, 29 or 30 nucleotides).

    [0131] In some embodiments, the length of a duplex formed between a sense and antisense strand of an oligonucleotide may be 12 to 30 nucleotides (e.g., 12 to 30, 12 to 27, 15 to 25, 18 to 30 or 19 to 30 nucleotides) in length. In some embodiments, the length of a duplex formed between a sense and antisense strand of an oligonucleotide is at least 12 nucleotides long (e.g., at least 12, at least 15, at least 20, or at least 25 nucleotides long). In some embodiments, the length of a duplex formed between a sense and antisense strand of an oligonucleotide is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

    [0132] In some embodiments, oligonucleotides provided herein have one 5′end that is thermodynamically less stable compared to the other 5′ end. In some embodiments, an asymmetry oligonucleotide is provided that includes a blunt end at the 3′ end of a sense strand and an overhang at the 3′ end of an antisense strand. In some embodiments, a 3′ overhang on an antisense strand is 1-8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides in length). Typically, an oligonucleotide for RNAi has a two-nucleotide overhang on the 3′ end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, an overhang is a 3′ overhang comprising a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides. However, in some embodiments, the overhang is a 5′ overhang comprising a length of between one and six nucleotides, optionally one to five, one to four, one to three, one to two, two to six, two to five, two to four, two to three, three to six, three to five, three to four, four to six, four to five, five to six nucleotides, or one, two, three, four, five or six nucleotides.

    [0133] In some embodiments, two terminal nucleotides on the 3′ end of an antisense strand are modified. In some embodiments, the two terminal nucleotides on the 3′ end of the antisense strand are complementary with the target. In some embodiments, the two terminal nucleotides on the 3′ end of the antisense strand are not complementary with the target. In some embodiments, two terminal nucleotides on each 3′ end of an oligonucleotide in the nicked tetraloop structure are GG. Typically, one or both of the two terminal GG nucleotides on each 3′ end of an oligonucleotide is not complementary with the target.

    [0134] In some embodiments, there is one or more (e.g., 1, 2, 3, 4, 5) mismatches between a sense and antisense strand. If there is more than one mismatch between a sense and antisense strand, they may be positioned consecutively (e.g., 2, 3 or more in a row), or interspersed throughout the region of complementarity. In some embodiments, the 3′-terminus of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated at the 3′ terminus of the sense strand. In some embodiments, base mismatches or destabilization of segments at the 3′-end of the sense strand of the oligonucleotide improved the potency of synthetic duplexes in RNAi, possibly through facilitating processing by Dicer.

    [0135] a. Antisense Strands

    [0136] In some embodiments, an antisense strand of an oligonucleotide may be referred to as a “guide strand.” For example, if an antisense strand can engage with RNA-induced silencing complex (RISC) and bind to an Argonaut protein, or engage with or bind to one or more similar factors, and direct silencing of a target gene, it may be referred to as a guide strand. In some embodiments a sense strand complementary with a guide strand may be referred to as a “passenger strand.”

    [0137] In some embodiments, an oligonucleotide provided herein comprises an antisense strand that is up to 50 nucleotides in length (e.g., up to 30, up to 27, up to 25, up to 21, or up to 19 nucleotides in length). In some embodiments, an oligonucleotide provided herein comprises an antisense strand is at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, or at least 27 nucleotides in length). In some embodiments, an antisense strand of an oligonucleotide disclosed herein is in the range of 12 to 50 or 12 to 30 (e.g., 12 to 30, 11 to 27, 11 to 25, 15 to 21, 15 to 27, 17 to 21, 17 to 25, 19 to 27, or 19 to 30) nucleotides in length. In some embodiments, an antisense strand of any one 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

    [0138] In some embodiments an oligonucleotide disclosed herein comprises an antisense strand comprising a sequence as set forth in any one of SEQ ID NOs: 14-26. In some embodiments, an oligonucleotide comprises an antisense strand comprising a contiguous sequence of nucleotides that is in the range of 12 to 20 nucleotides (e.g., 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 20, 14 to 18, 14 to 16, 16 to 20, 16 to 18, or 18 to 20 nucleotides) in length of any of the sequences as set forth in any one of SEQ ID NOs: 14-26. In some embodiments, an oligonucleotide comprises an antisense strand comprising a contiguous sequence of nucleotides of a sequence as set forth in any one of SEQ ID NOs: 14-26 that is 12, 13, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides in length. In some embodiments, an oligonucleotide comprises an antisense strand that consists of a sequence as set forth in any one of SEQ ID NOs: 14-26.

    [0139] b. Sense Strands

    [0140] In some embodiments, a double-stranded oligonucleotide may have a sense strand of up to 40 nucleotides in length (e.g., 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, an oligonucleotide may have a sense strand of at least 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 35, or at least 38 nucleotides in length). In some embodiments, an oligonucleotide may have a sense strand in a range of 12 to 50 (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 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40) nucleotides in length. In some embodiments, an oligonucleotide may have 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, or 40 nucleotides in length. In some embodiments, a sense strand of an oligonucleotide is longer than 27 nucleotides (e.g., 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides). In some embodiments, a sense strand of an oligonucleotide is longer than 25 nucleotides (e.g., 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides). In some embodiments, the sense strand is 20 nucleotides in length. In some embodiments, the sense strand is 36 nucleotides in length.

    [0141] In some embodiments, an oligonucleotide disclosed herein comprises a sense strand sequence as set forth in in any one of SEQ ID NOs: 1-13 and 27-39. In some embodiments, an oligonucleotide has a sense strand that comprises at least 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) contiguous nucleotides of a sequence as set forth in in any one of SEQ ID NOs: 1-13 and 27-39. In some embodiments, an oligonucleotide has a sense strand that comprises a contiguous sequence of nucleotides that is in the range of 7 to 36 nucleotides (e.g., 12 to 30, 12 to 27, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19-27, 20-36, or 15 to 36 nucleotides) in length of any of the sequences as set forth in any one of SEQ ID NOs: 1-13 and 27-39. In some embodiments, an oligonucleotide has a sense strand that comprises a contiguous sequence of nucleotides of a sequence as set forth in any one of SEQ ID NOs: 1-13 and 27-39 that 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, or 36 nucleotides in length. In some embodiments, an oligonucleotide has a sense strand that consists of a sequence as set forth in any one of SEQ ID NOs: 1-13 and 27-39.

    [0142] In some embodiments, a sense strand comprises a stem-loop at its 3′-end. In some embodiments, a sense strand comprises a stem-loop at its 5′-end. In some embodiments, a strand comprising a stem loop is in the range of 2 to 66 nucleotides long (e.g., 2 to 66, 10 to 52, 14 to 40, 2 to 30, 4 to 26, 8 to 22, 12 to 18, 10 to 22, 14 to 26, or 14 to 30 nucleotides long). In some embodiments, a strand comprising a stem loop is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, a stem comprises a duplex of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. In some embodiments, a stem-loop provides the molecule better protection against degradation (e.g., enzymatic degradation) and facilitates targeting characteristics for delivery to a target cell. For example, in some embodiments, a loop provides added nucleotides on which modification can be made without substantially affecting the gene expression inhibition activity of an oligonucleotide. In certain embodiments, an oligonucleotide is provided herein in which the sense strand comprises (e.g., at its 3′-end) a stem-loop set forth as: S.sub.1-L-S.sub.2, in which S.sub.1 is complementary to S.sub.2, and in which L forms a loop between S.sub.1 and S.sub.2 of up to 10 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length).

    [0143] In some embodiments, a loop (L) of a stem-loop is a tetraloop (e.g., within a nicked tetraloop structure). A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetraloop has 4 to 5 nucleotides. However, in some embodiments, a tetraloop comprises or consists of 3 to 6 nucleotides, and typically consists of 4 to 5 nucleotides. In certain embodiments, a tetraloop comprises or consists of three, four, five, or six nucleotides.

    [0144] iv. Oligonucleotide Modifications

    [0145] Oligonucleotides may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, base-paring properties, RNA distribution and cellular uptake and other features relevant to therapeutic or research use (see, e.g., Bramsen et al., Nucleic Acids Res., 2009, 37:2867-2881; Bramsen and Kjems, Frontiers in Genetics, 2012, 3:1-22). Accordingly, in some embodiments, oligonucleotides of the present disclosure may include one or more suitable modifications. In some embodiments, a modified nucleotide has a modification in its base (or nucleobase), the sugar (e.g., ribose, deoxyribose), or the phosphate group.

    [0146] The number of modifications on an oligonucleotide and the positions of those nucleotide modifications may influence the properties of an oligonucleotide. For example, oligonucleotides maybe be delivered in vivo by conjugating them to or encompassing them in a lipid nanoparticle (LNP) or similar carrier. However, when an oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Accordingly, in certain embodiments of any of the oligonucleotides provided herein, all or substantially all the nucleotides of an oligonucleotide are modified. In certain embodiments, more than half of the nucleotides are modified. In certain embodiments, less than half of the nucleotides are modified. Typically, with naked delivery, every sugar is modified at the 2′-position. These modifications may be reversible or irreversible. In some embodiments, an oligonucleotide as disclosed herein has a number and type of modified nucleotides sufficient to cause the desired characteristic (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and/or thermodynamic stability).

    [0147] a. Sugar Modifications

    [0148] In some embodiments, a modified sugar (also referred herein to a sugar analog) includes a modified deoxyribose or ribose moiety, e.g., in which one or more modifications occur at the 2′, 3′, 4′, and/or 5′ carbon position of the sugar. In some embodiments, a modified sugar may also include non-natural alternative carbon structures such as those present in locked nucleic acids (“LNA”) (see, e.g., Koshkin et al., Tetrahedron, 1998, 54:3607-3630), unlocked nucleic acids (“UNA”) (see, e.g., Snead et al., Molecular Therapy—Nucleic Acids, 2013, 2, e103), and bridged nucleic acids (“BNA”) (see, e.g., Imanishi and Obika, The Royal Society of Chemistry, Chem. Commun., 2002, 16:1653-1659). Koshkin et al., Snead et al., and Imanishi and Obika are incorporated by reference herein for their disclosures relating to sugar modifications.

    [0149] In some embodiments, a nucleotide modification in a sugar comprises a 2′-modification. A 2′-modification may be 2′-aminoethyl, 2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl, and 2′-deoxy-2′-fluoro-β-d-arabinonucleic acid. Typically, the modification is 2′-fluoro, 2′-O-methyl, or 2′-O-methoxyethyl. In some embodiments a modification in a sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring. For example, a modification of a sugar of a nucleotide may comprise a 2′-oxygen of a sugar is linked to a 1′-carbon or 4′-carbon of the sugar, or a 2′-oxygen is linked to the 1′-carbon or 4′-carbon via an ethylene or methylene bridge. In some embodiments, a modified nucleotide has an acyclic sugar that lacks a 2′-carbon to 3′-carbon bond. In some embodiments, a modified nucleotide has a thiol group, e.g., in the 4′ position of the sugar.

    [0150] In some embodiments, the oligonucleotide described herein comprises at least 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 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 one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).

    [0151] In some embodiments, all the nucleotides of the sense strand of the oligonucleotide are modified. In some embodiments, all the nucleotides of the antisense strand of the oligonucleotide are modified. In some embodiments, all the nucleotides of the oligonucleotide (i.e., both the sense strand and the antisense strand) are modified. In some embodiments, the modified nucleotide comprises a 2′-modification (e.g., a 2′-fluoro or 2′-O-methyl).

    [0152] The present disclosure provides oligonucleotides having different modification patterns. In some embodiments, the modified oligonucleotides comprise a sense strand sequence having a sequence as set forth in any one of SEQ ID NOs: 27-39, and an antisense strand sequence having a sequence as set forth in any one of SEQ ID NOs: 14-26. In some embodiments, for these oligonucleotides, one or more of positions 1, 2, 4, 6, 7, 12, 14, 16, 18-27, and 31-36 of the sense strand, and/or one or more of positions 1, 4, 6, 8, 9, 11, 13, 15, 18, and 20-22 of the antisense strand are modified with a 2′-O-methyl. In some embodiments, all of positions 1, 2, 4, 6, 7, 12, 14, 16, 18-27, and 31-36 of the sense strand, and all of positions 1, 4, 6, 8, 9, 11, 13, 15, 18, and 20-22 of the antisense strand are modified with a 2′-O-methyl. In some embodiments, one or more of positions 3, 5, 8-11, 13, 15, and 17 of the sense strand, and/or one or more of positions 2, 3, 5, 7, 10, 12, 14, 16, 17, and 19 of the antisense strand are modified with a 2′-fluoro. In some embodiments, all of positions 3, 5, 8-11, 13, 15, and 17 of the sense strand, and all of positions 2, 3, 5, 7, 10, 12, 14, 16, 17, and 19 of the antisense strand are modified with a 2′-fluoro. In some embodiments, all of positions 1, 2, 4, 6, 7, 12, 14, 16, 18-27, and 31-36 of the sense strand, all of positions 1, 4, 6, 8, 9, 11, 13, 15, 18, and 20-22 of the antisense strand are modified with a 2′-O-methyl, all of positions 3, 5, 8-11, 13, 15, and 17 of the sense strand, and all of positions 2, 3, 5, 7, 10, 12, 14, 16, 17, and 19 of the antisense strand are modified with a 2′-fluoro.

    [0153] In some embodiments, for oligonucleotides comprising a sense strand having a sequence as set forth in any one of SEQ ID NOs: 27-39, and an antisense strand having a sequence as set forth in any one of SEQ ID NOs: 14-26, one or more of positions 1-7, 12-27, and 31-36 of the sense strand, and/or one or more of positions 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with a 2′-O-methyl. In some embodiments, all of positions 1-7, 12-27, and 31-36 of the sense strand, and all of positions 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with a 2′-O-methyl. In some embodiments, one or more of positions 8-11 of the sense strand, and/or one or more of positions 2, 3, 5, 7, 10, and 14 of the antisense strand are modified with a 2′-fluoro. In some embodiments, all of positions 8-11 of the sense strand, and all of positions 2, 3, 5, 7, 10, and 14 of the antisense strand are modified with a 2′-fluoro. In some embodiments, all of positions 1-7, 12-27, and 31-36 of the sense strand, all of positions 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with a 2′-O-methyl, all of positions 8-11 of the sense strand, and all of positions 2, 3, 5, 7, 10, and 14 of the antisense strand are modified with a 2′-fluoro.

    [0154] In some embodiments, for oligonucleotides comprising a sense strand having a sequence as set forth in any one of SEQ ID NOs: 27-39, and an antisense strand having a sequence as set forth in any one of SEQ ID NOs: 14-26, one or more of positions 1, 2, 4-7, 9, 11, 14-16, 18-27, and 31-36 of the sense strand, and/or one or more of positions 1, 6, 8, 9, 11, 13, 15, 18, and 20-22 of the antisense strand are modified with a 2′-O-methyl. In some embodiments, all of positions 1, 2, 4-7, 9, 11, 14-16, 18-27, and 31-36 of the sense strand, and all of positions 1, 6, 8, 9, 11, 13, 15, 18, and 20-22 of the antisense strand are modified with a 2′-O-methyl. In some embodiments, one or more of positions 3, 8, 10, 12, 13, and 17 of the sense strand, and/or one or more of positions 2-5, 7, 10, 12, 14, 16, 17, and 19 of the antisense strand are modified with a 2′-fluoro. In some embodiments, all of positions 3, 8, 10, 12, 13, and 17 of the sense strand, and all of positions 2-5, 7, 10, 12, 14, 16, 17, and 19 of the antisense strand are modified with a 2′-fluoro.

    [0155] In some embodiments, the terminal 3′-end group (e.g., a 3′-hydroxyl) with a phosphate group or other group, which can be used, for example, to attach linkers, adapters or labels or for the direct ligation of an oligonucleotide to another nucleic acid.

    [0156] b. 5′ Terminal Phosphates

    [0157] In some embodiments, 5′-terminal phosphate groups of oligonucleotides enhance the interaction with Argonaute 2. However, oligonucleotides comprising a 5′-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, oligonucleotides include analogs of 5′ phosphates that are resistant to such degradation. In some embodiments, a phosphate analog may be oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. In certain embodiments, the 5′ end of an oligonucleotide strand is attached to chemical moiety that mimics the electrostatic and steric properties of a natural 5′-phosphate group (“phosphate mimic”) (see, e.g., Prakash et al., Nucleic Acids Res., 2015, 43(6):2993-3011, the contents of which relating to phosphate analogs are incorporated herein by reference). Many phosphate mimics have been developed that can be attached to the 5′ end (see, e.g., U.S. Pat. No. 8,927,513, the contents of which relating to phosphate analogs are incorporated herein by reference). Other modifications have been developed for the 5′ end of oligonucleotides (see, e.g., International patent publication WO2011133871, the contents of which relating to phosphate analogs are incorporated herein by reference). In certain embodiments, a hydroxyl group is attached to the 5′ end of the oligonucleotide.

    [0158] In some embodiments, an oligonucleotide has a phosphate analog at a 4′-carbon position of the sugar (referred to as a “4′-phosphate analog”) (see, e.g., International patent publication WO2018045317, entitled 4′-Phosphate Analogs and Oligonucleotides Comprising the Same, which content relating to phosphate analogs is incorporated herein by reference). In some embodiments, an oligonucleotide provided herein comprise a 4′-phosphate analog at a 5′-terminal nucleotide. In some embodiments, a phosphate analog is an oxymethylphosphonate in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4′-carbon) or analog thereof. In other embodiments, a 4′-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate in which 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 analog thereof. In certain embodiments, a 4′-phosphate analog is an oxymethylphosphonate. In some embodiments, an oxymethylphosphonate is represented by the formula —O—CH.sub.2—PO(OH).sub.2 or —O—CH.sub.2—PO(OR).sub.2, in which R is independently selected from H, CH.sub.3, an alkyl group, CH.sub.2CH.sub.2CN, CH.sub.2OCOC(CH.sub.3).sub.3, CH.sub.2OCH.sub.2CH.sub.2Si(CH.sub.3).sub.3, or a protecting group. In certain embodiments, the alkyl group is CH.sub.2CH.sub.3. More typically, R is independently selected from H, CH.sub.3, or CH.sub.2CH.sub.3.

    [0159] In certain embodiments, a phosphate analog attached to the oligonucleotide is a methoxy phosphonate (MOP). In certain embodiments, a phosphate analog attached to the oligonucleotide is a 5′ mono-methyl protected MOP. In some embodiments, the following uridine nucleotide comprising a phosphate analog may be used, e.g., at the first position of a guide (antisense) strand:

    ##STR00004##

    which modified nucleotide is referred to as [MePhosphonate-4O-mU] or 5′-Methoxy, Phosphonate-4′oxy-2′-O-methyluridine.

    [0160] c. Modified Intranucleotide Linkages

    [0161] In some embodiments, phosphate modifications or substitutions may result in an oligonucleotide comprises at least one (e.g., at least 1, at least 2, at least 3 or at least 5) comprising a modified internucleotide linkage. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1 to 10 (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 linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages.

    [0162] A modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage. In some embodiments, at least one modified internucleotide linkage of any one of the oligonucleotides as disclosed herein is a phosphorothioate linkage.

    [0163] In some embodiments, the oligonucleotide described herein has a phosphorothioate linkage between one or more of: positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. In some embodiments, the oligonucleotide described herein has a phosphorothioate linkage between each of: positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.

    [0164] d. Base Modifications

    [0165] In some embodiments, oligonucleotides provided herein have one or more modified nucleobases. In some embodiments, modified nucleobases (also referred to herein as base analogs) are linked at the 1′ position of a nucleotide sugar moiety. In certain embodiments, a modified nucleobase is a nitrogenous base. In certain embodiments, a modified nucleobase does not contain nitrogen atom (see, e.g., U.S. patent publication 20080274462). In some embodiments, a modified nucleotide comprises a universal base. However, in certain embodiments, a modified nucleotide does not contain a nucleobase (abasic).

    [0166] In some embodiments a universal base is a heterocyclic moiety located at the 1′ position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a duplex, can be positioned opposite more than one type of base without substantially altering structure of the duplex. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., oligonucleotide) that is fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a lower T.sub.m than a duplex formed with the complementary nucleic acid. However, in some embodiments, compared to a reference single-stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher T.sub.m than a duplex formed with the nucleic acid comprising the mismatched base.

    [0167] Non-limiting examples of universal-binding nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3-nitropyrrole (U.S. patent publication no. 20070254362; Van Aerschot et al., Nucleic Acids Res. 1995, 23(21):4363-70; Loakes et al., Nucleic Acids Res. 1995, 23(13):2361-6; Loakes and Brown, Nucleic Acids Res., 1994, 22(20):4039-43. Each of the foregoing is incorporated by reference herein for their disclosures relating to base modifications).

    [0168] e. Reversible Modifications

    [0169] While certain modifications to protect an oligonucleotide from the in vivo environment before reaching target cells can be made, they can reduce the potency or activity of the oligonucleotide once it reaches the cytosol of the target cell. Reversible modifications can be made such that the molecule retains desirable properties outside of the cell, which are then removed upon entering the cytosolic environment of the cell. Reversible modification can be removed, for example, by the action of an intracellular enzyme or by the chemical conditions inside of a cell (e.g., through reduction by intracellular glutathione).

    [0170] In some embodiments, a reversibly modified nucleotide comprises a glutathione-sensitive moiety. Typically, nucleic acid molecules have been chemically modified with cyclic disulfide moieties to mask the negative charge created by the internucleotide diphosphate linkages and improve cellular uptake and nuclease resistance (see, e.g., U.S. patent publication 20110294869, International patent publication WO2015188197; Meade et al., Nature Biotechnology, 2014, 32:1256-1263; International patent publication WO2014088920; each of which are incorporated by reference for their disclosures of such modifications). This reversible modification of the internucleotide diphosphate linkages is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g., glutathione). Earlier examples include neutralizing phosphotriester modifications that were reported to be cleavable inside cells (Dellinger et al., J. Am. Chem. Soc., 2003, 125:940-950).

    [0171] In some embodiments, such a reversible modification allows protection during in vivo administration (e.g., transit through the blood and/or lysosomal/endosomal compartments of a cell) where the oligonucleotide will be exposed to nucleases and other harsh environmental conditions (e.g., pH). When released into the cytosol of a cell where the levels of glutathione are higher compared to extracellular space, the modification is reversed and the result is a cleaved oligonucleotide. Using reversible, glutathione sensitive moieties, it is possible to introduce sterically larger chemical groups into the oligonucleotide of interest as compared to the options available using irreversible chemical modifications. This is because these larger chemical groups will be removed in the cytosol and, therefore, should not interfere with the biological activity of the oligonucleotides inside the cytosol of a cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the structure of the glutathione-sensitive moiety can be engineered to modify the kinetics of its release.

    [0172] In some embodiments, a glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, a glutathione-sensitive moiety is attached to the 2′carbon of the sugar of a modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5′-carbon of a sugar, particularly when the modified nucleotide is the 5′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3′-carbon of sugar, particularly when the modified nucleotide is the 3′-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group (see, e.g., International patent publication WO2018039364, the contents of which are incorporated by reference herein for its relevant disclosures).

    [0173] v. Targeting Ligands

    [0174] In some embodiments, it may be desirable to target the oligonucleotides of the disclosure to one or more cells or one or more organs. Such a strategy may help to avoid undesirable effects in other organs or may avoid undue loss of the oligonucleotide to cells, tissue or organs that would not benefit for the oligonucleotide. Accordingly, in some embodiments, oligonucleotides disclosed herein may be modified to facilitate targeting of a particular tissue, cell or organ, e.g., to facilitate delivery of the oligonucleotide to the liver. In certain embodiments, oligonucleotides disclosed herein may be modified to facilitate delivery of the oligonucleotide to the hepatocytes of the liver. In some embodiments, an oligonucleotide comprises a nucleotide that is conjugated to one or more targeting ligand.

    [0175] A targeting ligand may comprise a carbohydrate, amino sugar, cholesterol, peptide, polypeptide, protein or part of a protein (e.g., an antibody or antibody fragment) or lipid. In some embodiments, a targeting ligand is an aptamer. For example, a targeting ligand may be an RGD peptide that is used to target tumor vasculature or glioma cells, CREKA peptide to target tumor vasculature or stoma, transferring, lactoferrin, or an aptamer to target transferrin receptors expressed on CNS vasculature, or an anti-EGFR antibody to target EGFR on glioma cells. In certain embodiments, the targeting ligand is one or more GalNAc moieties.

    [0176] In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of an oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5′ or 3′ end of the sense or antisense strand) such that the targeting ligands resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5′ or 3′ end of the sense strand and 1, 2, 3 or 4 nucleotides of the loop of the stem may be individually conjugated to a targeting ligand.

    [0177] In some embodiments, it is desirable to target an oligonucleotide that reduces the expression of HMGB1 to the hepatocytes of the liver of the subject. Any suitable hepatocyte targeting moiety may be used for this purpose.

    [0178] GalNAc is a high affinity ligand for asialoglycoprotein receptor (ASGPR), which is primarily expressed on the sinusoidal surface of hepatocyte cells and has a major role in binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to oligonucleotides of the instant disclosure may be used to target these oligonucleotides to the ASGPR expressed on these hepatocyte cells.

    [0179] In some embodiments, an oligonucleotide of the instant disclosure is conjugated directly or indirectly to a monovalent GalNAc. In some embodiments, the oligonucleotide is conjugated directly or indirectly to more than one monovalent GalNAc (e.g., is conjugated to 2, 3, or 4 monovalent GalNAc moieties, and is typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, an oligonucleotide of the instant disclosure is conjugated to a one or more bivalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties.

    [0180] In some embodiments, 1 or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of an oligonucleotide are each conjugated to a GalNAc moiety. In some embodiments, 2 to 4 nucleotides of the loop (L) of the stem-loop are each conjugated to a separate GalNAc. In some embodiments, targeting ligands are conjugated to 2 to 4 nucleotides at either ends of the sense or antisense strand (e.g., ligands are conjugated to a 2 to 4 nucleotide overhang or extension on the 5′ or 3′ end of the sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush and the oligonucleotide resembles a toothbrush. For example, an oligonucleotide may comprise a stem-loop at either the 5′ or 3′ end of the sense strand and 1, 2, 3 or 4 nucleotides of the loop of the stem may be individually conjugated to a GalNAc moiety. In some embodiments, GalNAc moieties are conjugated to a nucleotide of the sense strand. For example, four GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand where each GalNAc moiety is conjugated to one nucleotide.

    [0181] In some embodiments, an oligonucleotide herein comprises a monovalent GalNAc attached to a Guanidine nucleotide, referred to as [ademG-GalNAc] or 2′-aminodiethoxymethanol-Guanidine-GalNAc, as depicted below:

    ##STR00005##

    [0182] In some embodiments, an oligonucleotide herein comprises a monovalent GalNAc attached to an adenine nucleotide, referred to as [ademA-GalNAc] or 2′-aminodiethoxymethanol-Adenine-GalNAc, as depicted below.

    ##STR00006##

    [0183] An example of such conjugation is shown below for a loop comprising from 5′ to 3′ the nucleotide sequence GAAA (L=linker, X=heteroatom) stem attachment points are shown. Such a loop may be present, for example, at positions 27-30 of the molecules shown in FIG. 1B. In the chemical formula,

    ##STR00007##

    is used to describe an attachment point to the oligonucleotide strand.

    ##STR00008##

    [0184] Appropriate methods or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International patent publication WO2016100401, the contents of which relating to such linkers are incorporated herein by reference. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. A “labile linker” refers to a linker that can be cleaved, e.g., by acidic pH. A “stable linker” refers to a linker that cannot be cleaved.

    [0185] An example is shown below for a loop comprising from 5′ to 3′ the nucleotides GAAA, in which GalNAc moieties are attached to nucleotides of the loop using an acetal linker. Such a loop may be present, for example, at positions 27-30 of the molecules described in FIG. 10. In the chemical formula,

    ##STR00009##

    is an attachment point to the oligonucleotide strand.

    ##STR00010##

    [0186] Any appropriate method or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International patent publication WO2016100401, the contents of which relating to such linkers are incorporated herein by reference. In some embodiments, the linker is a labile linker. In other embodiments, the linker is stable.

    [0187] In some embodiments, a duplex extension (e.g., of up to 3, 4, 5, or 6 base pairs in length) is provided between a targeting ligand (e.g., a GalNAc moiety) and a double-stranded oligonucleotide.

    III. Formulations

    [0188] Various formulations have been developed to facilitate oligonucleotide use. For example, oligonucleotides can be delivered to a subject or a cellular environment using a formulation that minimizes degradation, facilitates delivery and/or uptake, or provides another beneficial property to the oligonucleotides in the formulation. In some embodiments, provided herein are compositions comprising oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) to reduce the expression of HMGB1. Such compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient portion of the oligonucleotides enter the cell to reduce HMGB1 expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for the reduction of HMGB1 as disclosed herein. In some embodiments, an oligonucleotide is formulated in buffer solutions such as phosphate buffered saline solutions, liposomes, micellar structures, and capsids.

    [0189] Formulations of oligonucleotides with cationic lipids can be used to facilitate transfection of the 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.

    [0190] Accordingly, in some embodiments, a formulation comprises a lipid nanoparticle. In some embodiments, an excipient comprises a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, or a nanoparticle, or may be otherwise formulated for administration to the cells, tissues, organs, or body of a subject in need thereof (see, e.g., Remington: The Science and Practice of Pharmacy, 22nd Ed., Pharmaceutical Press, 2013).

    [0191] In some embodiments, formulations as disclosed herein comprise an excipient. In some embodiments, an excipient confers to a composition improved stability, improved absorption, improved solubility and/or therapeutic enhancement of the active ingredient. In some embodiments, an excipient is a buffering agent (e.g., sodium citrate, sodium phosphate, a tris base, or sodium hydroxide) or a vehicle (e.g., a buffered solution, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, an oligonucleotide is lyophilized for extending its shelf-life and then made into a solution before use (e.g., administration to a subject). Accordingly, an excipient in a composition comprising any one of the oligonucleotides described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinyl pyrolidone), or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

    [0192] In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

    [0193] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J., USA) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the oligonucleotides in a required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

    [0194] In some embodiments, a composition may contain at least about 0.1% of the therapeutic agent (e.g., an oligonucleotide for reducing HMGB1 expression) or more, although the percentage of the active ingredient(s) may be about 1% to about 80% or more of the weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

    [0195] Even though a number of embodiments are directed to liver-targeted delivery of any of the oligonucleotides disclosed herein, targeting of other tissues is also contemplated.

    IV. Methods of Use

    [0196] i. Reducing HMGB1 Expression in Cells

    [0197] In some embodiments, methods are provided for delivering to a cell an effective amount any one of oligonucleotides disclosed herein for purposes of reducing expression of HMGB1 in the cell. Methods provided herein are useful in any appropriate cell type. In some embodiments, a cell is any cell that expresses HMGB1 (e.g., hepatocytes, macrophages, monocyte-derived cells, prostate cancer cells, cells of the brain, endocrine tissue, bone marrow, lymph nodes, lung, gall bladder, liver, duodenum, small intestine, pancreas, kidney, gastrointestinal tract, bladder, adipose and soft tissue and skin). In some embodiments, the cell is a primary cell that has been obtained from a subject and that may have undergone a limited number of a passages, such that the cell substantially maintains is natural phenotypic properties. In some embodiments, a cell to which the oligonucleotide is delivered is ex vivo or in vitro (e.g., can be delivered to a cell in culture or to an organism in which the cell resides). In specific embodiments, methods are provided for delivering to a cell an effective amount any one of oligonucleotides disclosed herein for purposes of reducing expression of HMGB1 solely in hepatocytes.

    [0198] In some embodiments, oligonucleotides disclosed herein can be introduced using appropriate nucleic acid delivery methods including injection of a solution containing the oligonucleotides, bombardment by particles covered by the oligonucleotides, exposing the cell or organism to a solution containing the oligonucleotides, or electroporation of cell membranes in the presence of the oligonucleotides. Other appropriate methods for delivering oligonucleotides to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and others.

    [0199] The consequences of inhibition can be confirmed by an appropriate assay to evaluate one or more properties of a cell or subject, or by biochemical techniques that evaluate molecules indicative of HMGB1 expression (e.g., RNA, protein). In some embodiments, the extent to which an oligonucleotide provided herein reduces levels of expression of HMGB1 is evaluated by comparing expression levels (e.g., mRNA or protein levels of HMGB1 to an appropriate control (e.g., a level of HMGB1 expression in a cell or population of cells to which an oligonucleotide has not been delivered or to which a negative control has been delivered). In some embodiments, an appropriate control level of HMGB1 expression may be a predetermined level or value, such that a control level need not be measured every time. The predetermined level or value can take a variety of forms. In some embodiments, a predetermined level or value can be single cut-off value, such as a median or mean.

    [0200] In some embodiments, administration of an oligonucleotide as described herein results in a reduction in the level of HMGB1 expression in a cell. In some embodiments, the reduction in levels of HMGB1 expression may be a reduction to 1% or lower, 5% or lower, 10% or lower, 15% or lower, 20% or lower, 25% or lower, 30% or lower, 35% or lower, 40% or lower, 45% or lower, 50% or lower, 55% or lower, 60% or lower, 70% or lower, 80% or lower, or 90% or lower compared with an appropriate control level of HMGB1. The appropriate control level may be a level of HMGB1 expression in a cell or population of cells that has not been contacted with an oligonucleotide as described herein. In some embodiments, the effect of delivery of an oligonucleotide to a cell according to a method disclosed herein is assessed after a finite period of time. For example, levels of HMGB1 may be analyzed in a cell at least 8 hours, 12 hours, 18 hours, 24 hours; or at least one, two, three, four, five, six, seven, or fourteen days after introduction of the oligonucleotide into the cell.

    [0201] In some embodiments, an oligonucleotide is delivered in the form of a transgene that is engineered to express in a cell the oligonucleotides (e.g., its sense and antisense strands). In some embodiments, an oligonucleotide is delivered using a transgene that is engineered to express any oligonucleotide disclosed herein. Transgenes may be delivered using viral vectors (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus or herpes simplex virus) or non-viral vectors (e.g., plasmids or synthetic mRNAs). In some embodiments, transgenes can be injected directly to a subject.

    [0202] ii. Treatment Methods

    [0203] Aspects of the disclosure relate to methods for reducing HMGB1 expression in for attenuating the onset or progression of liver fibrosis in a subject. In some embodiments, the methods may comprise administering to a subject in need thereof an effective amount of any one of the oligonucleotides disclosed herein. Such treatments could be used, for example, to slow or halt any type of liver fibrosis. The present disclosure provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disease or disorder associated with liver fibrosis and/or liver inflammation.

    [0204] The compounds of the invention selectively target HMGB1 mRNA exclusively in the liver. Compared to other NASH therapies, this selectivity is expected to increase the potency of the HMGB1 inhibitors of the inventions while decreasing off-target interactions and potential side effects.

    [0205] In certain aspects, the disclosure provides a method for preventing in a subject, a disease or disorder as described herein by administering to the subject a therapeutic agent (e.g., an oligonucleotide or vector or transgene encoding same). In some embodiments, the subject to be treated is a subject who will benefit therapeutically from a reduction in the amount of HMGB1 protein, e.g., in the liver. Subjects at risk for the disease or disorder can be identified by, for example, one or a combination of diagnostic or prognostic assays known in the art (e.g., identification of liver fibrosis and/or liver inflammation). Administration of a prophylactic agent can occur prior to the detection of or the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.

    [0206] In some embodiments, the disclosure provides methods for using RNAi oligonucleotides of the invention for treating subjects having or suspected of having liver conditions such as, for example, cholestatic liver disease, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH).

    [0207] In some embodiments, the disclosure provides RNAi oligonucleotides described herein for use in treating subjects having or suspected of having liver conditions such as, for example, cholestatic liver disease, NAFLD and NASH.

    [0208] In some embodiments, the disclosure provides RNAi for the preparation of a medicament for treatment of subjects having or suspected of having liver conditions such as, for example, cholestatic liver disease, NAFLD and nonalcoholic steatohepatitis NASH.

    [0209] Methods described herein are typically involve administering to a subject in an effective amount of an oligonucleotide, that is, an amount capable of producing a desirable therapeutic result. A therapeutically acceptable amount may be an amount that is capable of treating a disease or disorder. The appropriate dosage for any one subject will depend on certain factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.

    [0210] In some embodiments, a subject is administered any one of the compositions disclosed herein either enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intracerebroventricular injection, intrathecal), topically (e.g., epicutaneous, inhalational, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the liver of a subject). Typically, oligonucleotides disclosed herein are administered intravenously or subcutaneously.

    [0211] As a non-limiting set of examples, the oligonucleotides of the instant disclosure would typically be administered quarterly (once every three months), bi-monthly (once every two months), monthly, or weekly. For example, the oligonucleotides may be administered every week or at intervals of two, or three weeks. In some embodiments, the oligonucleotides may be administered daily.

    [0212] In some embodiments, the subject to be treated is a human or non-human primate or other mammalian subject. Other exemplary subjects include domesticated 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.

    EXAMPLES

    Example 1: In Vivo Activity of GalNAc-Conjugated HMGB1 Oligonucleotides

    [0213] HMGB1 oligonucleotides used in this example were designed to bind to conserved sequences identified by the algorithm in the human, monkey (both rhesus), and mouse sequences (“triple common” sequences). In this study, three triple-common oligonucleotide sequences (S31-AS18, S35-AS22, and S36-AS23) were tested in 3 different modification patterns (M1, M2 and M3, see FIGS. 1A and 1B). Oligonucleotides were subcutaneously administered to CD-1 mice at 1 mg/kg and mice were euthanized on day 5 following administration. Liver samples were obtained and RNA was extracted to evaluate HMGB1 mRNA levels by qPCR (normalized to HPRT1-F576 (Housekeeping gene). The levels of remaining HMGB1 mRNA were interrogated using TAQMAN®-based qPCR assays.

    [0214] The qPCR was performed using two different primers specific to different regions in the HMGB1 mRNA. The qPCR performed using the primer at the 5′ end relative to the other primer was designated “5′ qPCR.” Similarly, the qPCR performed using the primer at the 3′ end relative to the other primer was designated “3′ qPCR.” In this experiment, a 3′ qPCR assay (using primer MmHMGB1-F1541) was used. The data showed that all tested HMGB1 oligonucleotides were potent in knockdown HMGB1 5 days after administration, as indicated by the reduced amount of HMGB1 mRNA remaining in mice liver (normalized to a PBS control treatment) (FIG. 1A).

    [0215] Two GalNAc-conjugated HMGB1 oligonucleotides with different modification patterns (S31-AS18-M3 and S35-AS22-M2) were further tested in a dose response analysis, using the tool compound S36-AS23-M2 as control. The three oligonucleotides were subcutaneously administered to CD-1 mice at three different dosages (0.5 mg/kg, 1 mg/kg, and 2 mg/kg). Mice were euthanized on day 5 following administration. Liver samples were obtained, and RNA was extracted to evaluate HMGB1 mRNA levels by qPCR (normalized to HPRT1-F576 (Housekeeping gene). The levels of remaining HMGB1 mRNA were interrogated using TAQMAN®-based qPCR assays (a 3′assay as described above was used). The results show that the two tested GalNAc-conjugated HMGB1 oligonucleotides, in both modification patterns, reduced liver HMGB1 mRNA level in mice liver (FIG. 2). All oligonucleotides showed ED.sub.50s of ˜0.5-1.0 mg/kg, especially if non-hepatocyte ‘floor’ is considered.

    [0216] The two GalNAc-conjugated HMGB1 oligonucleotides with different modification patterns (S31-AS18-M3 and S35-AS22-M2) were then tested in vivo in a duration study to evaluate their activity in inhibiting HMGB1 expression in mice. PBS was used as negative control, and the tool compound S36-AS23-M2 was used as positive control in this experiment. Oligonucleotides were subcutaneously administered to CD-1 mice at 1 mg/kg and mice were euthanized on days 7, 14, 21, and 28 following administration. Liver samples were obtained and RNA was extracted to evaluate HMGB1 mRNA levels by qPCR (normalized to HPRT1-F576 housekeeping gene). A 3′assay as described above was used. The data showed that all tested HMGB1 oligonucleotides were potent in knockdown HMGB1 3 weeks after injection, even with 1 mg/kg single dose, as indicated by the reduced amount of HMGB1 mRNA remaining in mice liver at days 7, 14, 21, and 28 (normalized to a PBS control treatment) (FIG. 3).

    Example 2: Additional In Vitro Screening of New HMGB1 RNAi Oligonucleotides Sequences to Identify Additional RNAi Oligonucleotides that Inhibit HMGB1 Expression

    [0217] Additional 288 triple commons HMGB1 RNAi oligonucleotides (see Table 4 for sequences) were screened in an in vitro activity assay to identify additional RNAi oligonucleotides sequences that are effective at inhibiting HMGB1 expression (FIGS. 4A-4F). In this assay, Huh-7 cells were transfected with the indicated oligonucleotides. Cells were maintained for 24 h following transfection, RNAs were isolated using the iScript R2′-qPCR sample preparation buffer. HMGB1 mRNA were interrogated using TAQMAN®-based qPCR assays. Two qPCR assays, a 5′ assay and a 3′ assay, were used to determine mRNA levels as measured by HEX (housekeeping gene—HPR2′-F576/SFRS9-F594) and FAM probes, respectively.

    [0218] The percent mRNA remaining is shown for each of the 5′ assay (red) and the 3′ assay (blue). Oligonucleotides with the lowest percentage of mRNA remaining compared to mock transfection controls were considered hits. Oligonucleotides with low complementarity to the human genome were used as negative controls.

    [0219] Twenty-two (22) new sequences identified in the screen were made into GalNAc-conjugated tetraloop oligonucleotides (see Table 2 and Table 5 for sequences) with two different modification patterns (M2 and M3, respectively) and their in vivo activities in reduce liver HMGB1 mRNA level were tested. PBS was used as negative control, and the tool compound in two different modification patterns (S36-AS23-M2 and S36-AS23-M3) were used as positive control in this experiment. Oligonucleotides were subcutaneously administered to CD-1 mice at 1 mg/kg, and mice were euthanized on day 5 following administration. Liver samples were obtained and RNA was extracted to evaluate HMGB1 mRNA levels by qPCR (normalized to HPRT1-F576 (Housekeeping gene). The levels of remaining HMGB1 mRNA were interrogated using TAQMAN®-based qPCR assays (3′assay was used). The results show that the new GalNAc-conjugated oligonucleotides had different levels of activity in inhibiting liver HMGB1 and 7 new GalNAc-conjugated HMGB1 oligonucleotides (as indicated by the arrow, S27-AS14, S28-AS15, S29-AS16, S30-AS17, S32-AS19, S33-AS20, and S34-AS21) were selected to be included in knockdown duration study, if at least 50% suppression of HMGB1 mRNA is achieved 3 weeks after a single dose of 3 to 5 mg/kg in mice (FIG. 5).

    [0220] For the knockdown duration assay, the 7 new GalNAc-conjugated HMGB1 oligonucleotides (S27-AS14, S28-AS15, S29-AS16, S30-AS17, S32-AS19, S33-AS20, and S34-AS21) with different modification patterns (M2 or M3) were tested and compared with two previously identified GalNAc-conjugated HMGB1 oligonucleotides (S31-AS18-M3 and S35-AS22-M2). PBS was used as negative control, and the tool compound S36-AS23-M2 was used as positive control. Oligonucleotides were subcutaneously administered to CD-1 mice at 4 mg/kg and mice were euthanized on day 21 following administration. Liver samples were obtained and RNA was extracted to evaluate HMGB1 mRNA levels by qPCR (normalized to HPRT1-F576 (Housekeeping gene). The levels of remaining HMGB1 mRNA were interrogated using TAQMAN®-based qPCR assays (3′ assay was used). The percent remaining HMGB1 mRNA in the liver 21 days after the administration of the oligonucleotides, normalized to PBS control treatment, is shown in FIG. 6. All tested HMGB1 oligonucleotides were potent in knockdown HMGB1 3 weeks after injection.

    Example 3: Testing GalNAc-Conjugated HMGB1 Oligonucleotides in Primary Monkey or Human Hepatocytes

    [0221] The GalNAc-conjugated HMGB1 oligonucleotides tested in this experiment were shown in FIG. 7. Also shown in FIG. 7 is a GalNAc-conjugated LDHA oligonucleotide for using as positive control. In this assay, GalNAc-conjugates HMGB1 oligonucleotides were delivered by ASGPR receptor-mediated uptake into the monkey primary hepatocyte (FIGS. 8A and 8B) or the human hepatocyte (FIG. 8C) cells.

    [0222] Cells were maintained for 24 h following oligonucleotides delivery. RNA was extracted to evaluate HMGB1 mRNA levels by qPCR (normalized to RhPPIB (Housekeeping gene) for monkey hepatocytes and HPRT1-F576 (Housekeeping gene) for human hepatocytes). The levels of remaining HMGB1 mRNA were interrogated using TAQMAN®-based qPCR assays. RhMHGB1-F457 qPCR assay (FIGS. 8A and 8B) and HsHMGB1-F81 assay (FIG. 8C) were used. GalNAc-conjugate LDHA-1360 oligonucleotide was used as assay control (FIG. 8D) and the level of remaining LDHA mRNA was measured by RhLDHA-F887 qPCR assay. IC50 curves of RhHMGB1, normalized to Mock treatment, are shown.

    [0223] Four exemplary GalNAc-conjugated HMGB1 oligonucleotides (S27-AS14-M2, S33-AS20-M2, S35-AS22-M2, and S37-AS24-M2) were further tested in vivo in non-human primates (monkeys) for their activity in HMGB1 knockdown. PBS was used as negative control, and the tool compound S36-AS23-M2 was used as positive control in this experiment. The structures of S27-AS14-M2, S33-AS20-M2, S35-AS22-M2, and S36-AS23-M2 are depicted in FIG. 7.

    [0224] Oligonucleotides were subcutaneously administered to non-human primate at 4 mg/kg, one single dose, or 2 mg/kg, 4 repeat doses. Monkey liver biopsies were taken each time point following administration. Liver samples were obtained and RNA was extracted to evaluate HMGB1 mRNA levels by qPCR. The percentages of remaining HMGB1 mRNA in monkey liver 7, 14, 25, 54, 81 and 112 days after the administration, normalized to PBS control treatment, are shown (FIGS. 9A-9B). The results show that all oligonucleotides tested significantly reduced liver HMGB1 mRNA level at 25 days post administration. With 4 repeated 2 mg/kg doses, the HMGB1 knockdown effect lasted through 112 days (FIG. 9B).

    Example 4: Comparing HMGB1 RNAi Oligonucleotides in Primary Monkey/Human Hepatocytes

    [0225] The activity of five HMGB1 double strand RNAi oligonucleotides (5866-AS887, S869-AS878, 5116-AS490, 5117-AS491, 5871-AS880, and 5872-AS881 in Table 4) were compared in mouse, monkey, and human cell lines. PBS was used as negative control, and the tool compound S36-AS23-M2 was used as positive control in this experiment. In this assay, mouse cells (Hepa1-6 cells, FIGS. 10A and 10B), monkey cells (LLC-MK2 cells, FIGS. 10C and 10D), and human cells (Huh-7, FIGS. 10E and 10F) were transfected with the indicated oligonucleotides, respectively. Cells were maintained for 24 h following transfection, RNAs were isolated using the iScript R2′-qPCR sample preparation buffer. HMGB1 mRNA were interrogated using TAQMAN®-based qPCR assays. Two qPCR assays, a 5′ assay (FIGS. 10A, 10C, and 10E) and a 3′ assay (FIGS. 10B, 10D, and 10F), were used to determine mRNA levels as measured by HEX (housekeeping gene—HPR2′-F576) and FAM probes, respectively. Oligonucleotides 210, 840, 852, 853 show more than 80% knockdown at 0.1 nM and 1 nM concentrations in both assays in mouse cell line (FIGS. 10A and 10B). All RNAi oligonucleotides display more than 80% KD at 0.1 nM and 1 nM concentrations (FIGS. 10C and 10D). Oligonucleotides 210, 840, 852, 853, 932 show better potency with more than −90% KD at 0.1 nM and 1 nM concentrations in human cell line (FIGS. 10E and 10F).

    Example 5: Testing GalXC-HMGB1 for HMGB1 Selectivity

    [0226] The selectivity of GalNAc-conjugated HMGB1 oligonucleotides S27-AS14-M2, S33-AS20-M2, S35-AS22-M2, and S36-AS23-M2 against HMGB1, HMGB2, and HMGB3 were tested in human hepatocytes. The structures of S27-AS14-M2, S33-AS20-M2, S35-AS22-M2, and S36-AS23-M2 are depicted in FIG. 7. A GalNAc-conjugated LDHA oligonucleotide for using as positive control. Huh-7 cells were transfected with the indicated oligonucleotides with 8 different concentration (4-fold dilution with 8-points, highest concentration is 1 nM). Cells were maintained for 24 h following transfection, RNAs were isolated using the iScript R2′-qPCR sample preparation buffer. HMGB1 mRNA were interrogated using TAQMAN®-based qPCR assays. 5′ qPCR assay used to determine mRNA levels as measured by HEX (housekeeping gene—SFRS9) and FAM probes, respectively. IC50 curves of HMGB1 (FIG. 11A), HMGB2 (FIG. 11B) and HMGB3 (FIG. 11C), normalized to mock treatment, are shown. All four tested conjugates had similar IC50 value for HMGB1 (FIG. 11A). None of the conjugates (including the LDHA control oligonucleotide) had any effect of HMGB2 mRNA level (FIG. 11B) or HMGB3 mRNA level (FIG. 11C).

    Example 6: Materials and Methods

    Transfection

    [0227] For the first screen, Lipofectamine RNAiMAX™ was used to complex the oligonucleotides for efficient transfection. Oligonucleotides, RNAiMAX and Opti-MEM were added to a plate and incubated at room temperature for 20 minutes prior to transfection. Media was aspirated from a flask of actively passaging cells and the cells are incubated at 37° C. in the presence of trypsin for 3-5 minutes. After cells no longer adhered to the flask, cell growth media (lacking penicillin and streptomycin) was added to neutralize the trypsin and to suspend the cells. A 10 μL aliquot was removed and counted with a hemocytometer to quantify the cells on a per millimeter basis. For HeLa cells, 20,000 cells were seeded per well in 100 μL of media. The suspension was diluted with the known cell concentration to obtain the total volume required for the number of cells to be transfected. The diluted cell suspension was added to the 96 well transfection plates, which already contained the oligonucleotides in Opti-MEM. The transfection plates were then incubated for 24 hours at 37° C. After 24 hours of incubation, media was aspirated from each well. Cells were lysed using the lysis buffer from the Promega RNA Isolation kit. The lysis buffer was added to each well. The lysed cells were then transferred to the Corbett XtractorGENE (QIAxtractor) for RNA isolation or stored at -80° C.

    [0228] For subsequent screens and experiments, e.g., the secondary screen, Lipofectamine RNAiMAx was used to complex the oligonucleotides for reverse transfection. The complexes were made by mixing RNAiMAX and siRNAs in OptiMEM medium for 15 minutes. The transfection mixture was transferred to multi-well plates and cell suspension was added to the wells. After 24 hours incubation the cells were washed once with PBS and then lysed using lysis buffer from the Promega SV96 kit. The RNA was purified using the SV96 plates in a vacuum manifold. Four microliters of the purified RNA was then heated at 65° C. for 5 minutes and cooled to 4° C. The RNA was then used for reverse transcription using the High Capacity Reverse Transcription kit (Life Technologies) in a 10-microliter reaction. The cDNA was then diluted to 50 μL with nuclease free water and used for quantitative PCR with multiplexed 5′-endonuclease assays and SSoFast qPCR mastermix (Bio-Rad laboratories).

    cDNA Synthesis

    [0229] RNA was isolated from mammalian cells in tissue culture using the Corbett X-tractor Gene™ (QIAxtractor). A modified SuperScript II protocol was used to synthesize cDNA from the isolated RNA. Isolated RNA (approximately 5 ng/μL) was heated to 65° C. for five minutes and incubated with dNPs, random hexamers, oligo dTs and water. The mixture was cooled for 15 seconds. An “enzyme mix,” consisting of water, 5× first strand buffer, DTT, SUPERase⋅In™ (an RNA inhibitor), and SuperScript II RTase was added to the mixture. The contents were heated to 42° C. for one hour, then to 70° C. for 15 minutes, and then cooled to 4° C. using a thermocycler. The resulting cDNA was then subjected to SYBR®-based qPCR. The qPCR reactions were multiplexed, containing two 5′ endonuclease assays per reaction.

    qPCR Assays

    [0230] Primer sets were initially screened using SYBR®-based qPCR. Assay specificity was verified by assessing melt curves as well as “minus RT” controls. Dilutions of cDNA template (10-fold serial dilutions from 20 ng and to 0.02 ng per reaction) from HeLa and Hepa1-6 cells are used to test human (Hs) and mouse (Mm) assays, respectively. qPCR assays were set up in 384-well plates, covered with MicroAmp film, and run on the 7900HT from Applied Biosystems. Reagent concentrations and cycling conditions included the following: 2×SYBR mix, 10 μM forward primer, 10 μM reverse primer, DD H.sub.2O, and cDNA template up to a total volume of 10 μL.

    [0231] In some cases, as noted, qPCR was performed using TAQMAN®-based qPCR assays. TAQMAN® probes target two different positions (5′ and 3′ to one another) within the coding region of the target mRNA (e.g., HMGB1) were generally used to provide additional confirmation of mRNA levels in the analysis.

    Cloning

    [0232] PCR amplicons that displayed a single melt-curve were ligated into the pGEM®-T Easy vector kit from Promega according to the manufacturer's instructions. Following the manufacturer's protocol, JM109 High Efficiency cells were transformed with the newly ligated vectors. The cells were then plated on LB plates containing ampicillin and incubated at 37° C. overnight for colony growth.

    PCR Screening and Plasmid Mini-Prep

    [0233] PCR was used to identify colonies of E. coli that had been transformed with a vector containing the ligated amplicon of interest. Vector-specific primers that flank the insert were used in the PCR reaction. All PCR products were then run on a 1% agarose gel and imaged by a transilluminator following staining Gels were assessed qualitatively to determine which plasmids appeared to contain a ligated amplicon of the expected size (approximately 300 bp, including the amplicon and the flanking vector sequences specific to the primers used).

    [0234] The colonies that were confirmed transformants by PCR screening were then incubated overnight in cultures consisting of 2 mL LB broth with ampicillin at 37° C. with shaking. E. coli cells were then lysed, and the plasmids of interest were isolated using Promega's Mini-Prep kit. Plasmid concentration was determined by UV absorbance at 260 nm.

    Plasmid Sequencing and Quantification

    [0235] Purified plasmids were sequenced using the BigDye® Terminator sequencing kit. The vector-specific primer, T7, was used to give read lengths that span the insert. The following reagents were used in the sequencing reactions: water, 5× sequencing buffer, BigDye terminator mix, T7 primer, and plasmid (100 ng/μL) to a volume of 10 μL. The mixture was held at 96° C. for one minute, then subjected to 15 cycles of 96° C. for 10 seconds, 50° C. for 5 seconds, 60° C. for 1 minute, 15 seconds; 5 cycles of 96° C. for 10 seconds, 50° C. for 5 seconds, 60° C. for 1 minute, 30 seconds; and 5 cycles of 96° C. for 10 seconds, 50° C. for 5 seconds, and 60° C. for 2 minutes. Dye termination reactions were then sequenced using Applied Biosystems' capillary electrophoresis sequencers.

    [0236] Sequence-verified plasmids were then quantified. They were linearized using a single cutting restriction endonuclease. Linearity was confirmed using agarose gel electrophoresis. All plasmid dilutions were made in TE buffer (pH 7.5) with 100 μg of tRNA per mL buffer to reduce non-specific binding of plasmid to the polypropylene vials.

    [0237] The linearized plasmids were then serially diluted from 1,000,000 to 01 copies per μL and subjected to qPCR. Assay efficiency was calculated, and the assays were deemed acceptable if the efficiency was in the range of 90-110%.

    Multi-Plexing Assays

    [0238] For each target, mRNA levels were quantified by two 5′ nuclease assays. In general, several assays are screened for each target. The two assays selected displayed a combination of good efficiency, low limit of detection, and broad 5′43′ coverage of the gene of interest (GOI). Both assays against one GOI could be combined in one reaction when different fluorophores were used on the respective probes. Thus, the final step in assay validation was to determine the efficiency of the selected assays when they were combined in the same qPCR or “multi-plexed”.

    [0239] Linearized plasmids for both assays in 10-fold dilutions were combined and qPCR was performed. The efficiency of each assay was determined as described above. The accepted efficiency rate was 90-110%.

    [0240] While validating multi-plexed reactions using linearized plasmid standards, C.sub.q values for the target of interest were also assessed using cDNA as the template. For human or mouse targets, HeLa and Hepa1-6 cDNA were used, respectively. The cDNA, in this case, was derived from RNA isolated on the Corbett (˜5 ng/μL in water) from untransfected cells. In this way, the observed C.sub.q values from this sample cDNA were representative of the expected C.sub.q values from a 96-well plate transfection. In cases where C.sub.q values were greater than 30, other cell lines were sought that exhibit higher expression levels of the gene of interest. A library of total RNA isolated from via high-throughput methods on the Corbett from each human and mouse line was generated and used to screen for acceptable levels of target expression.

    Description of Oligonucleotide Nomenclature

    [0241] All oligonucleotides described herein are designated either SN.sub.1-ASN.sub.2-MN.sub.3. The following designations apply: [0242] N.sub.1: sequence identifier number of the sense strand sequence [0243] N.sub.2: sequence identifier number of the antisense strand sequence [0244] N.sub.3: reference number of modification pattern, in which each number represents a pattern of modified nucleotides in the oligonucleotide.

    [0245] For example, S1-AS14-M1 represents an oligonucleotide with a sense sequence that is set forth by SEQ ID NO: 1, an antisense sequence that is set forth by SEQ ID NO: 14, and which is adapted to modification pattern number 1.

    TABLE-US-00001 TABLE 1 Lead HMGB1 RNAi Oligonucleotide Sequences Sense (S)- S AS Antisense (AS) SEQ SEQ Designation Sense Sequence/mRNA Sequence ID NO: Antisense Sequence ID NO: S1-AS14 UGGGCAAAGGAGAUCCUAAA  1 UUUAGGAUCUCCUUUGCCCAGG 14 S2-AS15 AAAGAGAAAUGAAAACCUAA  2 UUAGGUUUUCAUUUCUCUUUGG 15 S3-AS16 AAGAAGAUGAUGAUGAUGAA  3 UUCAUCAUCAUCAUCUUCUUGG 16 S4-AS17 AUGAUGAUGAUGAAUAAGUA  4 UACUUAUUCAUCAUCAUCAUGG 17 S5-AS18 GAUGAUGAAUAAGUUGGUUC  5 GAACCAACUUAUUCAUCAUCGG 18 S6-AS19 UGAAUAAGUUGGUUCUAGCA  6 UGCUAGAACCAACUUAUUCAGG 19 S7-AS20 AAUAAGUUGGUUCUAGCGCA  7 UGCGCUAGAACCAACUUAUUGG 20 S8-AS21 AUAAGUUGGUUCUAGCGCAA  8 UUGCGCUAGAACCAACUUAUGG 21 S9-AS22 AGAAAAAAAUUGAAAUGUAA  9 UUACAUUUCAAUUUUUUUCUGG 22 S10-AS23 UUGUUGUUCUGUUAACUGAA 10 UUCAGUUAACAGAACAACAAGG 23 S11-AS24 UUCUGAAUGCUUCUAAGUAA 11 UUACUUAGAAGCAUUCAGAAGG 24 S12-AS25 CUGAAUGCUUCUAAGUAAAA 12 UUUUACUUAGAAGCAUUCAGGG 25 S13-AS26 GAAUGCUUCUAAGUAAAUAA 13 UUAUUUACUUAGAAGCAUUCGG 26

    TABLE-US-00002 TABLE 2 Lead GalNAc-conjugated HMGB1 oligonucleotide sequences with modifications GalNA Sense Antisense conjugated sequence sequence Antisense sequence oligonucleotides (unmodified) Sense Sequence (modified) (unmodified) (modified) S27-AS14-M2 UGGGCAAAGGAGA [mUs][mG][mG][mG][mC][mA] UUUAGGAUCUCC [MePhosphonate-4O- UCCUAAAGCAGCC [mA][fA][fG][fG][fA][mG] UUUGCCCAGG [mUs][fUs][fU][mA] GAAAGGCUGC [mA][mU][mC][mC][mU][mA] (SEQ ID [fG][mG][fA][mU][mC] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 14) [FR][mC][mC][mU][fU] NO: 27) [mC][mC][mG][ademA- [mU][mG][mC][mC][mC] GalNAc][ademA-GalNAc] [mAs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 814) [mU][mG][mC] (SEQ ID NO: 788) S27-AS14-M3 UGGGCAAAGGAGA [mUs][mG][fG][mG][mC][mA] UUUAGGAUCUCC [MePhosphonate-4O- UCCUAAAGCAGCC [mA][fA][mG][fG][mA][fG] UUUGCCCAGG [mUs][fUs][fU][fA] GAAAGGCUGC [fA][mU][mC][mC][fU][mA] (SEQ ID [fG][mG][fA][mU][mC] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 14) [fU][mC][fC][mU][fU] NO: 27) [mC][mC][mG][ademA- [mU][fG][fC][mC][fC] GalNAc][ademA-GalNAc] [mAs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 815) [mU][mG][mC] (SEQ ID NO: 789) S28-AS15-M2 AAAGAGAAAUGAA [mAs][mA][mA][mG][mA][mG] UUAGGUUUUCAU [MePhosphonate-4O- AACCUAAGCAGCC [mA][fA][fA][fU][fG][mA] UUCUCUUUGG [mUs][fUs][fA][mG] GAAAGGCUGC [mA][mA][mA][mC][mC][mU] (SEQ ID [fG][mU][fU][mU][mU] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 15) [fC][mA][mU][mU][fU] NO: 28) [mC][mC][mG][ademA- [mC][mU][mC][mU][mU] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 816) [mU][mG][mC] (SEQ ID NO: 790) S28-AS15-M3 AAAGAGAAAUGAA [mAs][mA][fA][mG][mA][mG] UUAGGUUUUCAU [MePhosphonate-4O- AACCUAAGCAGCC [mA][fA][mA][fU][mG][fA] UUCUCUUUGG [mUs][fUs][fA][fG] GAAAGGCUGC [fA][mA][mA][mC][fC][mU] (SEQ ID [fG][mU][fU][mU][mU] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 15) [fC][mA][fU][mU][fU] NO: 28) [mC][mC][mG][ademA- [mC][fU][fC][mU][fU] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQID NO: 817) [mU][mG][mC] (SEQ ID NO: 791) S29-AS16-M2 AAGAAGAUGAUGA [mAs][mA][mG][mA][mA][mG] UUCAUCAUCAUC [MePhosphonate-4O- UGAUGAAGCAGCC [mA][fU][fG][fA][fU][mG] AUCUUCUUGG [mUs][fUs][fC][mA] GAAAGGCUGC [mA][mU][mG][mA][mU][mG] (SEQ ID [fU][mC][fA][mU][mC] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 16) [fA][mU][mC][mA][fU] NO: 29) [mC][mC][mG][ademA- [mC][mU][mU][mC][mU] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 818) [mU][mG][mC] (SEQ ID NO: 792) S29-AS16-M3 AAGAAGAUGAUGA [mAs][mA][fG][mA][mA][mG] UUCAUCAUCAUC [MePhosphonate-4O- UGAUGAAGCAGCC [mA][fU][mG][fA][mU][fG] AUCUUCUUGG [mUs][fUs][fC][fA] GAAAGGCUGC [fA][mU][mG][mA][fU][mG] (SEQ ID [fU][mC][fA][mU][mC] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 16) [fA][mU][fC][mA][fU] NO: 29) [mC][mC][mG][ademA- [mC][fU][fU][mC][fU] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 819) [mU][mG][mC] (SEQ ID NO: 793) S30-AS17-M2 AUGAUGAUGAUGA [mAs][mU][mG][mA][mU][mG] UACUUAUUCAUC [MePhosphonate-4O- AUAAGUAGCAGCC [mA][fU][fG][fA][fU][mG] AUCAUCAUGG [mUs][FAS][fC][mU] GAAAGGCUGC [mA][mA][mU][mA][mA][mG] (SEQ ID [fU][mA][fU][mU][mC] (SEQ ID [mU][mA][mG][mC][mA][mG] NO: 17) [fA][mU][mC][mA][fU] NO: 30) [mC][mC][mG][ademA- [mC][mA][mU][mC][mA] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 820) [mU][mG][mC] (SEQ ID NO: 794) S30-AS17-M3 AUGAUGAUGAUGA [mAs][mU][fG][mA][mU][mG] UACUUAUUCAUC [MePhosphonate-4O- AUAAGUAGCAGCC [mA][fU][mG][fA][mU][fG] AUCAUCAUGG [mUs][FAS][fC][fU] GAAAGGCUGC [fA][mA][mU][mA][fA][mG] (SEQ ID [fU][mA][fU][mU][mC] (SEQ ID [mU][mA][mG][mC][mA][mG] NO: 17) [fA][mU][fC][mA][fU] NO: 30) [mC][mC][mG][ademA- [mC][fA][fU][mC][fA] GalNAc][ademA-GalNAc] [mUs][FGS][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 821) [mU][mG][mC] (SEQ ID NO: 795) S31-AS18-M1 GAUGAUGAAUAAG [mGs][mA][fU][mG][fA][mU] GAACCAACUUAU [MePhosphonate-4O- UUGGUUAGCAGCC [mG][fA][fA][fU][fA][mA] UCAUCAUCGG [mUs][FAS][fA][mC] GAAAGGCUGC [fG][mU][fU][mG][fG][mU] (SEQ ID [fC][mA][fA][mC][mU] (SEQ ID [mU][mA][mG][mC][mA][mG] NO: 18) [fU][mA][fU][mU][fC] NO: 31) [mC][mC][mG][ademA- [mA][fU][fC][mA][fU] GalNAc][ademA-GalNAc] [mCs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 822) [mU][mG][mC] (SEQ ID NO: 796) S31-AS18-M2 GAUGAUGAAUAAG [mGs][mA][mU][mG][mA][mU] GAACCAACUUAU [MePhosphonate-4O- UUGGUUAGCAGCC [mG][fA][fA][fU][fA][mA] UCAUCAUCGG [mUs][FAS][fA][mC] GAAAGGCUGC [mG][mU][mU][mG][mG][mU] (SEQ ID [fC][mA][fA][mC][mU] (SEQ ID [mU][mA][mG][mC][mA][mG] NO: 18) [fU][mA][mU][mU][fC] NO: 31) [mC][mC][mG][ademA- [mA][mU][mC][mA][mU] GalNAc][ademA-GalNAc] [mCs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 823) [mU][mG][mC] (SEQ ID NO: 797) S31-AS18-M3 GAUGAUGAAUAAG [mGs][mA][fU][mG][mA][mU] GAACCAACUUAU [MePhosphonate-4O- UUGGUUAGCAGCC [mG][fA][mA][fU][mA][fA] UCAUCAUCGG [mUs][FAS][fA][fC] GAAAGGCUGC [fG][mU][mU][mG][fG][mU] (SEQ ID [fC][mA][fA][mC][mU] (SEQ ID [mU][mA][mG][mC][mA][mG] NO: 18) [fU][mA][fU][mU][fC] NO: 31) [mC][mC][mG][ademA- [mA][fU][fC][mA][fU] GalNAc][ademA-GalNAc] [mCs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 824) [mU][mG][mC] (SEQ ID NO: 798) S32-AS19-M2 UGAAUAAGUUGGU [mUs][mG][mA][mA][mU][mA] UGCUAGAACCAA [MePhosphonate-4O- UCUAGCAGCAGCC [mA][fG][fU][fU][fG][mG] CUUAUUCAGG [mUs][FGS][fC][mU] GAAAGGCUGC [mU][mU][mC][mU][mA][mG] (SEQ ID [fA][mG][fA][mA][mC] (SEQ ID [mC][mA][mG][mC][mA][mG] NO: 19) [fC][mA][mA][mC][fU] NO: 32) [mC][mC][mG][ademA- [mU][mA][mU][mU][mC] GalNAc][ademA-GalNAc] [mAs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 825) [mU][mG][mC] (SEQ ID NO: 799) S32-AS19-M3 UGAAUAAGUUGGU [mUs][mG][fA][mA][mU][mA] UGCUAGAACCAA [MePhosphonate-4O- UCUAGCAGCAGCC [mA][fG][mU][fU][mG][fG] CUUAUUCAGG [mUs][FGS][fC][fU] GAAAGGCUGC [fU][mU][mC][mU][fA][mG] (SEQ ID [fA][mG][fA][mA][mC] (SEQ ID [mC][mA][mG][mC][mA][mG] NO: 19) [fC][mA][fA][mC][fU] NO: 32) [mC][mC][mG][ademA- [mU][fA][fU][mU][fC] GalNAc][ademA-GalNAc] [mAs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 826) [mU][mG][mC] (SEQ ID NO: 800) S33-AS20-M2 AAUAAGUUGGUUC [mUs][mG][fA][mA][mU][mA] UGCGCUAGAACC [MePhosphonate-4O- UAGCGCAGCAGCC [mA][fG][mU][fU][mG][fG] AACUUAUUGG [mUs][FGS][fC][mG] GAAAGGCUGC [fU][mU][mC][mU][fA][mG] (SEQ ID [fC][mU][fA][mG][mA] (SEQ ID [mC][mA][mG][mC][mA][mG] NO: 20) [fA][mC][mC][mA][fA] NO: 33) [mC][mC][mG][ademA- [mC][mU][mU][mA][mU] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 827) [mU][mG][mC] (SEQ ID NO: 801) S33-AS20-M3 AAUAAGUUGGUUC [mAs][mA][fU][mA][mA][mG] UGCGCUAGAACC [MePhosphonate-4O- UAGCGCAGCAGCC [mU][fU][mG][fG][mU][fU] AACUUAUUGG [mUs][FGS][fC][fG] GAAAGGCUGC [fC][mU][mA][mG][fC][mG] (SEQ ID [fC][mU][fA][mG][mA] (SEQ ID [mC][mA][mG][mC][mA][mG] NO: 20) [fA][mC][fC][mA][fA] NO: 33) [mC][mC][mG][ademA- [mC][fU][fU][mA][fU] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 828) [mU][mG][mC] (SEQ ID NO: 802) S34-AS21-M2 AUAAGUUGGUUCU [mAs][mU][mA][mA][mG][mU] UUGCGCUAGAAC [MePhosphonate-4O- AGCGCAAGCAGCC [mU][fG][fG][fU][fU][mC] CAACUUAUGG [mUs][fUs][fG][mC] GAAAGGCUGC [mU][mA][mG][mC][mG][mC] (SEQ ID [fG][mC][fU][mA][mG] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 21) [fA][mA][mC][mC][fA] NO: 34) [mC][mC][mG][ademA- [mA][mC][mU][mU][mA] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 829) [mU][mG][mC] (SEQ ID NO: 803) S34-AS21-M3 AUAAGUUGGUUCU [mAs][mU][fA][mA][mG][mU] UUGCGCUAGAAC [MePhosphonate-4O- AGCGCAAGCAGCC [mU][fG][mG][fU][mU][fC] CAACUUAUGG [mUs][fUs][fG][fC] GAAAGGCUGC [fU][mA][mG][mC][fG][mC] (SEQ ID [fG][mC][fU][mA][mG] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 21) [fA][mA][fC][mC][fA] NO: 34) [mC][mC][mG][ademA- [mA][fC][fU][mU][fA] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 830) [mU][mG][mC] (SEQ ID NO: 804) S35-AS22-M1 AGAAAAAAAUUGA [mAs][mG][fA][mA][fA][mA] UUACAUUUCAAU [MePhosphonate-4O- AAUGUAAGCAGCC [mA][fA][fA][fU][fU][mG] UUUUUUCUGG [mUs][fUs][fA][mC] GAAAGGCUGC [fA][mA][fA][mU][fG][mU] (SEQ ID [fA][mU][fU][mU][mC] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 22) [fA][mA][fU][mU][fU] NO: 35) [mC][mC][mG][ademA- [mU][fU][fU][mU][fC] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 831) [mU][mG][mC] (SEQ ID NO: 805) S35-AS22-M2 AGAAAAAAAUUGA [mAs][mG][mA][mA][mA][mA] UUACAUUUCAAU [MePhosphonate-4O- AAUGUAAGCAGCC [mA][fA][fA][fU][fU][mG] UUUUUUCUGG [mUs][fUs][fA][mC] GAAAGGCUGC [mA][mA][mA][mU][mG][mU] (SEQ ID [fA][mU][fU][mU][mC] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 22) [fA][mA][mU][mU][fU] NO: 35) [mC][mC][mG][ademA- [mU][mU][mU][mU][mC] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 832) [mU][mG][mC] (SEQ ID NO: 806) S35-AS22-M3 AGAAAAAAAUUGA [mAs][mG][fA][mA][mA][mA] UUACAUUUCAAU [MePhosphonate-4O- AAUGUAAGCAGCC [mA][fA][mA][fU][mU][fG] UUUUUUCUGG [mUs][fUs][fA][fC] GAAAGGCUGC [fA][mA][mA][mU][fG][mU] (SEQ ID [fA][mU][fU][mU][mC] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 22) [fA][mA][fU][mU][fU] NO: 35) [mC][mC][mG][ademA- [mU][fU][fU][mU][fC] GalNAc][ademA-GalNAc] [mUs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 833) [mU][mG][mC] (SEQ ID NO: 807) S36-AS23-M1 UUGUUGUUCUGUU [mUs][mU][fG][mU][fU][mG] UUCAGUUAACAG [MePhosphonate-4O- AACUGAAGCAGCC [mU][fU][fC][fU][fG][mU] AACAACAAGG [mUs][fUs][fC][mA] GAAAGGCUGC [fU][mA][fA][mC][fU][mG] (SEQ ID [fG][mU][fU][mA][mA] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 23) [fC][mA][fG][mA][fA] NO: 36) [mC][mC][mG][ademA- [mC][fA][fA][mC][fA] GalNAc][ademA-GalNAc] [mAs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 834) [mU][mG][mC] (SEQ ID NO: 808) S36-AS23-M2 UUGUUGUUCUGUU [mUs][mU][mG][mU][mU][mG] UUCAGUUAACAG [MePhosphonate-4O- AACUGAAGCAGCC [mU][fU][fC][fU][fG][mU] AACAACAAGG [mUs][fUs][fC][mA] GAAAGGCUGC [mU][mA][mA][mC][mU][mG] (SEQ ID [fG][mU][fU][mA][mA] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 23) [fC][mA][mG][mA][fA] NO: 36) [mC][mC][mG][ademA- [mC][mA][mA][mC][mA] GalNAc][ademA-GalNAc] [mAs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 835) [mU][mG][mC] (SEQ ID NO: 809) S36-AS23-M3 UUGUUGUUCUGUU [mUs][mU][fG][mU][mU][mG] UUCAGUUAACAG [MePhosphonate-4O- AACUGAAGCAGCC [mU][fU][mC][fU][mG][fU] AACAACAAGG [mUs][fUs][fC][fA] GAAAGGCUGC [fU][mA][mA][mC][fU][mG] (SEQ ID [fG][mU][fU][mA][mA] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 23) [fC][mA][fG][mA][fA] NO: 36) [mC][mC][mG][ademA- [mC][fA][fA][mC][fA] GalNAc][ademA-GalNAc] [mAs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 836) [mU][mG][mC] (SEQ ID NO: 810) S37-AS24-M2 UUCUGAAUGCUUC [mUs][mU][mC][mU][mG][mA] UUACUUAGAAGC [MePhosphonate-4O- UAAGUAAGCAGCC [mA][fU][fG][fC][fU][mU] AUUCAGAAGG [mUs][fUs][fA][mC] GAAAGGCUGC [mC][mU][mA][mA][mG][mU] (SEQ ID [fU][mU][fA][mG][mA] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 24) [fA][mG][mC][mA][fU] NO: 37) [mC][mC][mG][ademA- [mU][mC][mA][mG][mA] GalNAc][ademA-GalNAc] [mAs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 837) [mU][mG][mC] (SEQ ID NO: 811) S38-AS25-M2 CUGAAUGCUUCUA [mCs][mU][mG][mA][mA][mU] UUUUACUUAGAA [MePhosphonate-4O- AGUAAAAGCAGCC [mG][fC][fU][fU][fC][mU] GCAUUCAGGG [mUs][fUs][fU][mU] GAAAGGCUGC [mA][mA][mG][mU][mA][mA] (SEQ ID [fA][mC][fU][mU][mA] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 25) [fG][mA][mA][mG][fC] NO: 38) [mC][mC][mG][ademA- [mA][mU][mU][mC][mA] GalNAc][ademA-GalNAc] [mGs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 838) [mU][mG][mC] (SEQ ID NO: 812) S39-AS26-M2 GAAUGCUUCUAAG [mGs][mA][mA][mU][mG][mC] UUAUUUACUUAG [MePhosphonate-4O- UAAAUAAGCAGCC [mU][fU][fC][fU][fA][mA] AAGCAUUCGG [mUs][fUs][fA][mU] GAAAGGCUGC [mG][mU][mA][mA][mA][mU] (SEQ ID [fU][mU][fA][mC][mU] (SEQ ID [mA][mA][mG][mC][mA][mG] NO: 26) [fU][mA][mG][mA][fA] NO: 39) [mC][mC][mG][ademA- [mG][mC][mA][mU][mU] GalNAc][ademA-GalNAc] [mCs][mGs][mG] [ademA-GalNAc][mG][mG][mC] (SEQ ID NO: 839) [mU][mG][mC] (SEQ ID NO: 813)

    TABLE-US-00003 TABLE 3 Modification Key for RNAi Oligonucleotides Symbol Modification Description ademA-GalNAc 2′-aminodiethoxymethanol-GalNAc adenosine fA 2′-fluoro-deoxyadenosine fAs 2′-fluoro-deoxyadenosine followed by a phosphorothioate linkage fC 2′-fluoro-deoxycytosine fG 2′-fluoro-deoxyguanosine fGs 2′-fluoro-deoxyguanosine followed by a phosphorothioate linkage fU 2′-fluoro-uridine fUs 2′-fluoro-uridine followed by a phosphorothioate linkage mA 2′-O-methyl adenosine mAs 2′-O-methyl adenosine followed by a phosphorothioate linkage mC 2′-O-methyl cytosine mCs 2′-O-methyl cytosine followed by a phosphorothioate linkage MePhosphonate-4O-mU Methyl-4′-O-methylphosphonate-2′-O- methyl uridine MePhosphonate-4O-mUs Methyl-4′-O-methylphosphonate-2′-O- methyl uridine followed by a phosphorothioate linkage mG 2′-O-methyl guanosine mGs 2′-O-methyl guanosine followed by a phosphorothioate linkage mU 2′-O-methyl uridine mUs 2′-O-methyl uridine followed by a phosphorothioate linkage s Phosphorothioate linkage

    TABLE-US-00004 TABLE 4 HMGB1 RNAi Oligonucleotide Sequences Included in Screen S AS RNAi Sense Sequence/ SEQ SEQ oligonucleotides mRNA seq ID NO Antisense ID NO S40-AS414 AACUAAACAUGGGCAAA  40 GGAUCUCCUUUGCCCAUGU 414 GGAGAUCC UUAGUUAU S41-AS415 ACUAAACAUGGGCAAAG  41 AGGAUUUCCUUUGCCCAUG 415 GAAAUCCT UUUAGUUA S42-AS416 CUAAACAUGGGCAAAGG  42 UAGGAUCUCCUUUGCCCAU 416 AGAUCCTA GUUUAGUU S43-AS417 UAAACAUGGGCAAAGGA  43 UUAGGUUCUCCUUUGCCCA 417 GAACCUAA UGUUUAGU S44-AS418 AAACAUGGGCAAAGGAG  44 CUUAGUAUCUCCUUUGCCC 418 AUACUAAG AUGUUUAG S45-AS419 AACAUGGGCAAAGGAGA  45 UCUUAUGAUCUCCUUUGCC 419 UCAUAAGA CAUGUUUA S46-AS420 ACAUGGGCAAAGGAGAU  46 UUCUUUGGAUCUCCUUUGC 420 CCAAAGAA CCAUGUUU S47-AS421 CAUGGGCAAAGGAGAUC  47 CUUCUUAGGAUCUCCUUUG 421 CUAAGAAG CCCAUGUU S48-AS422 AUGGGCAAAGGAGAUCC  48 GCUUCUUAGGAUCUCCUUU 422 UAAGAAGC GCCCAUGU S49-AS423 AAGCCGAGAGGCAAAAU  49 AUGAUUACAUUUUGCCUCU 423 GUAAUCAT CGGCUUCU S50-AS424 AGCCGAGAGGCAAAAUG  50 UAUGAUGACAUUUUGCCUC 424 UCAUCATA UCGGCUUC S51-AS425 AAAUGUCAUCAUAUGCA  51 ACAAAUAAUGCAUAUGAUG 425 UUAUUUGT ACAUUUUG S52-AS426 CAUCAUAUGCAUUUUUU  52 GUUUGUACAAAAAAUGCAU 426 GUACAAAC AUGAUGAC S53-AS427 AUCAUAUGCAUUUUUUG  53 AGUUUUCACAAAAAAUGCA 427 UGAAAACT UAUGAUGA S54-AS428 AUAUGCAUUUUUUGUGC  54 ACAAGUUUGCACAAAAAAU 428 AAACUUGT GCAUAUGA S55-AS429 GUCAACUUCUCAGAGUU  55 UCUUAUAAAACUCUGAGAA 429 UUAUAAGA GUUGACUG S56-AS430 AAGAAGUGCUCAGAGAG  56 UCUUCUACCUCUCUGAGCA 430 GUAGAAGA CUUCUUAG S57-AS431 AGAAGUGCUCAGAGAGG  57 GUCUUUCACCUCUCUGAGC 431 UGAAAGAC ACUUCUUA S58-AS432 GAAGUGCUCAGAGAGGU  58 GGUCUUCCACCUCUCUGAG 432 GGAAGACC CACUUCUU S59-AS433 AAGUGCUCAGAGAGGUG  59 UGGUCUUCCACCUCUCUGA 433 GAAGACCA GCACUUCU S60-AS434 AGUGCUCAGAGAGGUGG  60 AUGGUUUUCCACCUCUCUG 434 AAAACCAT AGCACUUC S61-AS435 GUGCUCAGAGAGGUGGA  61 CAUGGUCUUCCACCUCUCU 435 AGACCATG GAGCACUU S62-AS436 UGCUCAGAGAGGUGGAA  62 ACAUGUUCUUCCACCUCUC 436 GAACAUGT UGAGCACU S63-AS437 GCUCAGAGAGGUGGAAG  63 GACAUUGUCUUCCACCUCU 437 ACAAUGTC CUGAGCAC S64-AS438 CUCAGAGAGGUGGAAGA  64 AGACAUGGUCUUCCACCUC 438 CCAUGUCT UCUGAGCA S65-AS439 UCAGAGAGGUGGAAGAC  65 CAGACUUGGUCUUCCACCU 439 CAAGUCTG CUCUGAGC S66-AS440 CAGAGAGGUGGAAGACC  66 GCAGAUAUGGUCUUCCACC 440 AUAUCUGC UCUCUGAG S67-AS441 AGAGAGGUGGAAGACCA  67 AGCAGUCAUGGUCUUCCAC 441 UGACUGCT CUCUCUGA S68-AS442 GAGAGGUGGAAGACCAU  68 UAGCAUACAUGGUCUUCCA 442 GUAUGCTA CCUCUCUG S69-AS443 AGAGGUGGAAGACCAUG  69 UUAGCUGACAUGGUCUUCC 443 UCAGCUAA ACCUCUCU S70-AS444 GAGGUGGAAGACCAUGU  70 UUUAGUAGACAUGGUCUUC 444 CUACUAAA CACCUCUC S71-AS445 AGGUGGAAGACCAUGUC  71 CUUUAUCAGACAUGGUCUU 445 UGAUAAAG CCACCUCU S72-AS446 GGUGGAAGACCAUGUCU  72 UCUUUUGCAGACAUGGUCU 446 GCAAAAGA UCCACCUC S73-AS447 GUGGAAGACCAUGUCUG  73 CUCUUUAGCAGACAUGGUC 447 CUAAAGAG UUCCACCU S74-AS448 UGGAAGACCAUGUCUGC  74 UCUCUUUAGCAGACAUGGU 448 UAAAGAGA CUUCCACC S75-AS449 GGAAGACCAUGUCUGCU  75 UUCUCUUUAGCAGACAUGG 449 AAAGAGAA UCUUCCAC S76-AS450 GAAGACCAUGUCUGCUA  76 UUUCUUUUUAGCAGACAUG 450 AAAAGAAA GUCUUCCA S77-AS451 AAGACCAUGUCUGCUAA  77 CUUUCUCUUUAGCAGACAU 451 AGAGAAAG GGUCUUCC S78-AS452 AGACCAUGUCUGCUAAA  78 CCUUUUUCUUUAGCAGACA 452 GAAAAAGG UGGUCUUC S79-AS453 AAAUUUGAAGAUAUGGC  79 CCGCUUUUGCCAUAUCUUC 453 AAAAGCGG AAAUUUUC S80-AS454 AAUUUGAAGAUAUGGCA  80 UCCGCUUUUGCCAUAUCUU 454 AAAGCGGA CAAAUUUU S81-AS455 GAAAGAGAAAUGAAAAC  81 GGAUAUAGGUUUUCAUUUC 455 CUAUAUCC UCUUUCAU S82-AS456 AAAAAAGAAGUUCAAGG  82 AUUGGUAUCCUUGAACUUC 456 AUACCAAT UUUUUUGU S83-AS457 CCCAAUGCACCCAAGAG  83 AAGGAUGCCUCUUGGGUGC 457 GCAUCCTT AUUGGGAU S84-AS458 CCAAUGCACCCAAGAGG  84 GAAGGUGGCCUCUUGGGUG 458 CCACCUTC CAUUGGGA S85-AS459 CAAUGCACCCAAGAGGC  85 CGAAGUAGGCCUCUUGGGU 459 CUACUUCG GCAUUGGG S86-AS460 AAUGCACCCAAGAGGCC  86 CCGAAUGAGGCCUCUUGGG 460 UCAUUCGG UGCAUUGG S87-AS461 AUGCACCCAAGAGGCCU  87 GCCGAUGGAGGCCUCUUGG 461 CCAUCGGC GUGCAUUG S88-AS462 UGCACCCAAGAGGCCUC  88 GGCCGUAGGAGGCCUCUUG 462 CUACGGCC GGUGCAUU S89-AS463 GCACCCAAGAGGCCUCC  89 AGGCCUAAGGAGGCCUCUU 463 UUAGGCCT GGGUGCAU S90-AS464 CACCCAAGAGGCCUCCU  90 AAGGCUGAAGGAGGCCUCU 464 UCAGCCTT UGGGUGCA S91-AS465 ACCCAAGAGGCCUCCUU  91 GAAGGUCGAAGGAGGCCUC 465 CGACCUTC UUGGGUGC S92-AS466 CCCAAGAGGCCUCCUUC  92 AGAAGUCCGAAGGAGGCCU 466 GGACUUCT CUUGGGUG S93-AS467 CCAAGAGGCCUCCUUCG  93 AAGAAUGCCGAAGGAGGCC 467 GCAUUCTT UCUUGGGU S94-AS468 CAAGAGGCCUCCUUCGG  94 GAAGAUGGCCGAAGGAGGC 468 CCAUCUTC CUCUUGGG S95-AS469 AAGAGGCCUCCUUCGGC  95 GGAAGUAGGCCGAAGGAGG 469 CUACUUCC CCUCUUGG S96-AS470 AGAGGCCUCCUUCGGCC  96 AGGAAUAAGGCCGAAGGAG 470 UUAUUCCT GCCUCUUG S97-AS471 GAGGCCUCCUUCGGCCU  97 GAGGAUGAAGGCCGAAGGA 471 UCAUCCTC GGCCUCUU S98-AS472 AGGCCUCCUUCGGCCUU  98 AGAGGUAGAAGGCCGAAGG 472 CUACCUCT AGGCCUCU S99-AS473 GGCCUCCUUCGGCCUUC  99 AAGAGUAAGAAGGCCGAAG 473 UUACUCTT GAGGCCUC S100-AS474 GCCUCCUUCGGCCUUCU 100 GAAGAUGAAGAAGGCCGAA 474 UCAUCUTC GGAGGCCU S101-AS475 GGAAUAACACUGCUGCA 101 UUGUCUUCUGCAGCAGUGU 475 GAAGACAA UAUUCCAC S102-AS476 GAUGACAAGCAGCCUUA 102 UCUUUUCAUAAGGCUGCUU 476 UGAAAAGA GUCAUCUG S103-AS477 GGAUAUUGCUGCAUAUC 103 UUUAGUUCGAUAUGCAGCA 477 GAACUAAA AUAUCCUU S104-AS478 AGCAAGAAAAAGAAGGA 104 CCUCCUCUUCCUUCUUUUU 478 AGAGGAGG CUUGCUUU S105-AS479 GCAAGAAAAAGAAGGAA 105 UCCUCUUCUUCCUUCUUUU 479 GAAGAGGA UCUUGCUU S106-AS480 CAAGAAAAAGAAGGAAG 106 UUCCUUCUCUUCCUUCUUU 480 AGAAGGAA UUCUUGCU S107-AS481 AAGAAAAAGAAGGAAGA 107 CUUCCUCCUCUUCCUUCUU 481 GGAGGAAG UUUCUUGC S108-AS482 AGAAAAAGAAGGAAGAG 108 UCUUCUUCCUCUUCCUUCU 482 GAAGAAGA UUUUCUUG S109-AS483 GAAGAAGAUGAUGAUGA 109 CUUAUUCAUCAUCAUCAUC 483 UGAAUAAG UUCUUCUU S110-AS484 GAUGAUGAUGAUGAAUA 110 AACCAUCUUAUUCAUCAUC 484 AGAUGGTT AUCAUCUU S111-AS485 GAUGAUGAUGAAUAAGU 111 UAGAAUCAACUUAUUCAUC 485 UGAUUCTA AUCAUCAU S112-AS486 AUGAUGAUGAAUAAGUU 112 CUAGAUCCAACUUAUUCAU 486 GGAUCUAG CAUCAUCA S113-AS487 GAUGAAUAAGUUGGUUC 113 CUGCGUUAGAACCAACUUA 487 UAACGCAG UUCAUCAU S114-AS488 UGAAUAAGUUGGUUCUA 114 AACUGUGCUAGAACCAACU 488 GCACAGTT UAUUCAUC S115-AS489 GAAUAAGUUGGUUCUAG 115 AAACUUCGCUAGAACCAAC 489 CGAAGUTT UUAUUCAU S116-AS490 AAUAAGUUGGUUCUAGC 116 AAAACUGCGCUAGAACCAA 490 GCAGUUTT CUUAUUCA S117-AS491 AUAAGUUGGUUCUAGCG 117 AAAAAUUGCGCUAGAACCA 491 CAAUUUTT ACUUAUUC S118-AS492 UAAGUUGGUUCUAGCGC 118 AAAAAUCUGCGCUAGAACC 492 AGAUUUTT AACUUAUU S119-AS493 AAGUUGGUUCUAGCGCA 119 AAAAAUACUGCGCUAGAAC 493 GUAUUUTT CAACUUAU S120-AS494 UUUUUCUUGUCUAUAAA 120 UUAAAUGCUUUAUAGACAA 494 GCAUUUAA GAAAAAAA S121-AS495 UUUUCUUGUCUAUAAAG 121 GUUAAUUGCUUUAUAGACA 495 CAAUUAAC AGAAAAAA S122-AS496 UUUCUUGUCUAUAAAGC 122 GGUUAUAUGCUUUAUAGAC 496 AUAUAACC AAGAAAAA S123-AS497 UUCUUGUCUAUAAAGCA 123 GGGUUUAAUGCUUUAUAGA 497 UUAAACCC CAAGAAAA S124-AS498 UCUUGUCUAUAAAGCAU 124 GGGGUUAAAUGCUUUAUAG 498 UUAACCCC ACAAGAAA S125-AS499 CAACUCACUCCUUUUAA 125 UUUUUUCUUUAAAAGGAGU 499 AGAAAAAA GAGUUGUG S126-AS500 AACUCACUCCUUUUAAA 126 UUUUUUUCUUUAAAAGGAG 500 GAAAAAAA UGAGUUGU S127-AS501 ACUCACUCCUUUUAAAG 127 AUUUUUUUCUUUAAAAGGA 501 AAAAAAAT GUGAGUUG S128-AS502 CUCACUCCUUUUAAAGA 128 AAUUUUUUUCUUUAAAAGG 502 AAAAAATT AGUGAGUU S129-AS503 UCACUCCUUUUAAAGAA 129 CAAUUUUUUUCUUUAAAAG 503 AAAAAUTG GAGUGAGU S130-AS504 CACUCCUUUUAAAGAAA 130 UCAAUUUUUUUCUUUAAAA 504 AAAAUUGA GGAGUGAG S131-AS505 ACUCCUUUUAAAGAAAA 131 UUCAAUUUUUUUCUUUAAA 505 AAAUUGAA AGGAGUGA S132-AS506 CUCCUUUUAAAGAAAAA 132 UUUCAUUUUUUUUCUUUAA 506 AAAUGAAA AAGGAGUG S133-AS507 UCCUUUUAAAGAAAAAA 133 AUUUCUAUUUUUUUCUUUA 507 AUAGAAAT AAAGGAGU S134-AS508 CCUUUUAAAGAAAAAAA 134 CAUUUUAAUUUUUUUCUUU 508 UUAAAATG AAAAGGAG S135-AS509 CUUUUAAAGAAAAAAAU 135 ACAUUUCAAUUUUUUUCUU 509 UGAAAUGT UAAAAGGA S136-AS510 UUUUAAAGAAAAAAAUU 136 UACAUUUCAAUUUUUUUCU 510 GAAAUGTA UUAAAAGG S137-AS511 UUUAAAGAAAAAAAUUG 137 UUACAUUUCAAUUUUUUUC 511 AAAUGUAA UUUAAAAG S138-AS512 UUAAAGAAAAAAAUUGA 138 CUUACUUUUCAAUUUUUUU 512 AAAGUAAG CUUUAAAA S139-AS513 UAAAGAAAAAAAUUGAA 139 CCUUAUAUUUCAAUUUUUU 513 AUAUAAGG UCUUUAAA S140-AS514 AAAGAAAAAAAUUGAAA 140 GCCUUUCAUUUCAAUUUUU 514 UGAAAGGC UUCUUUAA S141-AS515 GAAAAAAAUUGAAAUGU 141 ACAGCUUUACAUUUCAAUU 515 AAAGCUGT UUUUUCUU S142-AS516 AAAAAAAUUGAAAUGUA 142 CACAGUCUUACAUUUCAAU 516 AGACUGTG UUUUUUCU S143-AS517 GUAAGAUUUGUUUUUAA 143 UGUACUGUUUAAAAACAAA 517 ACAGUACA UCUUACAC S144-AS518 UAAGAUUUGUUUUUAAA 144 CUGUAUAGUUUAAAAACAA 518 CUAUACAG AUCUUACA S145-AS519 AAGAUUUGUUUUUAAAC 145 ACUGUUCAGUUUAAAAACA 519 UGAACAGT AAUCUUAC S146-AS520 AUUUGUUUUUAAACUGU 146 GACACUGUACAGUUUAAAA 520 ACAGUGTC ACAAAUCU S147-AS521 UUGUUUUUAAACUGUAC 147 AAGACUCUGUACAGUUUAA 521 AGAGUCTT AAACAAAU S148-AS522 UGUUUUUAAACUGUACA 148 AAAGAUACUGUACAGUUUA 522 GUAUCUTT AAAACAAA S149-AS523 GUUUUUAAACUGUACAG 149 AAAAGUCACUGUACAGUUU 523 UGACUUTT AAAAACAA S150-AS524 UUUUUAAACUGUACAGU 150 AAAAAUACACUGUACAGUU 524 GUAUUUTT UAAAAACA S151-AS525 UUUUAAACUGUACAGUG 151 AAAAAUGACACUGUACAGU 525 UCAUUUTT UUAAAAAC S152-AS526 UUUAAACUGUACAGUGU 152 AAAAAUAGACACUGUACAG 526 CUAUUUTT UUUAAAAA S153-AS527 UUAAACUGUACAGUGUC 153 CAAAAUAAGACACUGUACA 527 UUAUUUTG GUUUAAAA S154-AS528 UAAACUGUACAGUGUCU 154 ACAAAUAAAGACACUGUAC 528 UUAUUUGT AGUUUAAA S155-AS529 AAACUGUACAGUGUCUU 155 UACAAUAAAAGACACUGUA 529 UUAUUGTA CAGUUUAA S156-AS530 AACUGUACAGUGUCUUU 156 AUACAUAAAAAGACACUGU 530 UUAUGUAT ACAGUUUA S157-AS531 ACUGUACAGUGUCUUUU 157 UAUACUAAAAAAGACACUG 531 UUAGUATA UACAGUUU S158-AS532 CUGUACAGUGUCUUUUU 158 CUAUAUAAAAAAAGACACU 532 UUAUAUAG GUACAGUU S159-AS533 UGUACAGUGUCUUUUUU 159 ACUAUUCAAAAAAAGACAC 533 UGAAUAGT UGUACAGU S160-AS534 UACAGUGUCUUUUUUUG 160 UAACUUUACAAAAAAAGAC 534 UAAAGUTA ACUGUACA S161-AS535 CAGUGUCUUUUUUUGUA 161 GUUAAUUAUACAAAAAAAG 535 UAAUUAAC ACACUGUA S162-AS536 GUGGUAUUUUCAAUAGC 162 GGUUAUUGGCUAUUGAAAA 536 CAAUAACC UACCACCA S163-AS537 UGGUAUUUUCAAUAGCC 163 AGGUUUGUGGCUAUUGAAA 537 ACAAACCT AUACCACC S164-AS538 GGUAUUUUCAAUAGCCA 164 AAGGUUAGUGGCUAUUGAA 538 CUAACCTT AAUACCAC S165-AS539 UAUUUUCAAUAGCCACU 165 GCAAGUUUAGUGGCUAUUG 539 AAACUUGC AAAAUACC S166-AS540 AUUUUCAAUAGCCACUA 166 GGCAAUGUUAGUGGCUAUU 540 ACAUUGCC GAAAAUAC S167-AS541 UUUUCAAUAGCCACUAA 167 AGGCAUGGUUAGUGGCUAU 541 CCAUGCCT UGAAAAUA S168-AS542 UUUCAAUAGCCACUAAC 168 CAGGCUAGGUUAGUGGCUA 542 CUAGCCTG UUGAAAAU S169-AS543 UUCAAUAGCCACUAACC 169 CCAGGUAAGGUUAGUGGCU 543 UUACCUGG AUUGAAAA S170-AS544 UCAAUAGCCACUAACCU 170 ACCAGUCAAGGUUAGUGGC 544 UGACUGGT UAUUGAAA S171-AS545 CAAUAGCCACUAACCUU 171 UACCAUGCAAGGUUAGUGG 545 GCAUGGTA CUAUUGAA S172-AS546 AAUAGCCACUAACCUUG 172 GUACCUGGCAAGGUUAGUG 546 CCAGGUAC GCUAUUGA S173-AS547 AUAGCCACUAACCUUGC 173 UGUACUAGGCAAGGUUAGU 547 CUAGUACA GGCUAUUG S174-AS548 UAGCCACUAACCUUGCC 174 CUGUAUCAGGCAAGGUUAG 548 UGAUACAG UGGCUAUU S175-AS549 AGCCACUAACCUUGCCU 175 ACUGUUCCAGGCAAGGUUA 549 GGAACAGT GUGGCUAU S176-AS550 GCCACUAACCUUGCCUG 176 UACUGUACCAGGCAAGGUU 550 GUACAGTA AGUGGCUA S177-AS551 CCACUAACCUUGCCUGG 177 AUACUUUACCAGGCAAGGU 551 UAAAGUAT UAGUGGCU S178-AS552 CACUAACCUUGCCUGGU 178 CAUACUGUACCAGGCAAGG 552 ACAGUATG UUAGUGGC S179-AS553 ACUAACCUUGCCUGGUA 179 CCAUAUUGUACCAGGCAAG 553 CAAUAUGG GUUAGUGG S180-AS554 GGGUUGUAAAUUGGCAU 180 AAAUUUCCAUGCCAAUUUA 554 GGAAAUTT CAACCCCC S181-AS555 GGUUGUAAAUUGGCAUG 181 UAAAUUUCCAUGCCAAUUU 555 GAAAUUTA ACAACCCC S182-AS556 GUUGUAAAUUGGCAUGG 182 UUAAAUUUCCAUGCCAAUU 556 AAAUUUAA UACAACCC S183-AS557 UUGUAAAUUGGCAUGGA 183 UUUAAUUUUCCAUGCCAAU 557 AAAUUAAA UUACAACC S184-AS558 UGUAAAUUGGCAUGGAA 184 CUUUAUAUUUCCAUGCCAA 558 AUAUAAAG UUUACAAC S185-AS559 GUAAAUUGGCAUGGAAA 185 GCUUUUAAUUUCCAUGCCA 559 UUAAAAGC AUUUACAA S186-AS560 UAAAUUGGCAUGGAAAU 186 UGCUUUAAAUUUCCAUGCC 560 UUAAAGCA AAUUUACA S187-AS561 AAAUUGGCAUGGAAAUU 187 CUGCUUUAAAUUUCCAUGC 561 UAAAGCAG CAAUUUAC S188-AS562 AAUUGGCAUGGAAAUUU 188 CCUGCUUUAAAUUUCCAUG 562 AAAGCAGG CCAAUUUA S189-AS563 AUUGGCAUGGAAAUUUA 189 ACCUGUUUUAAAUUUCCAU 563 AAACAGGT GCCAAUUU S190-AS564 UUGGCAUGGAAAUUUAA 190 AACCUUCUUUAAAUUUCCA 564 AGAAGGTT UGCCAAUU S191-AS565 UGGCAUGGAAAUUUAAA 191 GAACCUGCUUUAAAUUUCC 565 GCAGGUTC AUGCCAAU S192-AS566 GGCAUGGAAAUUUAAAG 192 AGAACUUGCUUUAAAUUUC 566 CAAGUUCT CAUGCCAA S193-AS567 GCAUGGAAAUUUAAAGC 193 AAGAAUCUGCUUUAAAUUU 567 AGAUUCTT CCAUGCCA S194-AS568 CAUGGAAAUUUAAAGCA 194 CAAGAUCCUGCUUUAAAUU 568 GGAUCUTG UCCAUGCC S195-AS569 AUGGAAAUUUAAAGCAG 195 ACAAGUACCUGCUUUAAAU 569 GUACUUGT UUCCAUGC S196-AS570 UGGAAAUUUAAAGCAGG 196 AACAAUAACCUGCUUUAAA 570 UUAUUGTT UUUCCAUG S197-AS571 GGAAAUUUAAAGCAGGU 197 CAACAUGAACCUGCUUUAA 571 UCAUGUTG AUUUCCAU S198-AS572 GAAAUUUAAAGCAGGUU 198 CCAACUAGAACCUGCUUUA 572 CUAGUUGG AAUUUCCA S199-AS573 AAAUUUAAAGCAGGUUC 199 ACCAAUAAGAACCUGCUUU 573 UUAUUGGT AAAUUUCC S200-AS574 AAUUUAAAGCAGGUUCU 200 CACCAUCAAGAACCUGCUU 574 UGAUGGTG UAAAUUUC S201-AS575 AUUUAAAGCAGGUUCUU 201 GCACCUACAAGAACCUGCU 575 GUAGGUGC UUAAAUUU S202-AS576 UUUAAAGCAGGUUCUUG 202 UGCACUAACAAGAACCUGC 576 UUAGUGCA UUUAAAUU S203-AS577 UUAAAGCAGGUUCUUGU 203 GUGCAUCAACAAGAACCUG 577 UGAUGCAC CUUUAAAU S204-AS578 UAAAGCAGGUUCUUGUU 204 UGUGCUCCAACAAGAACCU 578 GGAGCACA GCUUUAAA S205-AS579 AAAGCAGGUUCUUGUUG 205 CUGUGUACCAACAAGAACC 579 GUACACAG UGCUUUAA S206-AS580 AAGCAGGUUCUUGUUGG 206 GCUGUUCACCAACAAGAAC 580 UGAACAGC CUGCUUUA S207-AS581 AGCAGGUUCUUGUUGGU 207 UGCUGUGCACCAACAAGAA 581 GCACAGCA CCUGCUUU S208-AS582 GCAGGUUCUUGUUGGUG 208 GUGCUUUGCACCAACAAGA 582 CAAAGCAC ACCUGCUU S209-AS583 CAGGUUCUUGUUGGUGC 209 UGUGCUGUGCACCAACAAG 583 ACAGCACA AACCUGCU S210-AS584 AGGUUCUUGUUGGUGCA 210 UUGUGUUGUGCACCAACAA 584 CAACACAA GAACCUGC S211-AS585 GGUUCUUGUUGGUGCAC 211 UUUGUUCUGUGCACCAACA 585 AGAACAAA AGAACCUG S212-AS586 GUUCUUGUUGGUGCACA 212 AUUUGUGCUGUGCACCAAC 586 GCACAAAT AAGAACCU S213-AS587 UUCUUGUUGGUGCACAG 213 AAUUUUUGCUGUGCACCAA 587 CAAAAATT CAAGAACC S214-AS588 UCUUGUUGGUGCACAGC 214 UAAUUUGUGCUGUGCACCA 588 ACAAAUTA ACAAGAAC S215-AS589 CUUGUUGGUGCACAGCA 215 CUAAUUUGUGCUGUGCACC 589 CAAAUUAG AACAAGAA S216-AS590 UUGUUGGUGCACAGCAC 216 ACUAAUUUGUGCUGUGCAC 590 AAAUUAGT CAACAAGA S217-AS591 UGUUGGUGCACAGCACA 217 AACUAUUUUGUGCUGUGCA 591 AAAUAGTT CCAACAAG S218-AS592 GUUGGUGCACAGCACAA 218 UAACUUAUUUGUGCUGUGC 592 AUAAGUTA ACCAACAA S219-AS593 UUGGUGCACAGCACAAA 219 AUAACUAAUUUGUGCUGUG 593 UUAGUUAT CACCAACA S220-AS594 UGGUGCACAGCACAAAU 220 UAUAAUUAAUUUGUGCUGU 594 UAAUUATA GCACCAAC S221-AS595 GGUGCACAGCACAAAUU 221 AUAUAUCUAAUUUGUGCUG 595 AGAUAUAT UGCACCAA S222-AS596 UUUUUUCAUCUUCAGUU 222 UCAGAUACAACUGAAGAUG 596 GUAUCUGA AAAAAACU S223-AS597 UUUUUCAUCUUCAGUUG 223 AUCAGUGACAACUGAAGAU 597 UCACUGAT GAAAAAAC S224-AS598 UUUUCAUCUUCAGUUGU 224 CAUCAUAGACAACUGAAGA 598 CUAUGATG UGAAAAAA S225-AS599 UUUCAUCUUCAGUUGUC 225 GCAUCUGAGACAACUGAAG 599 UCAGAUGC AUGAAAAA S226-AS600 UUCAUCUUCAGUUGUCU 226 UGCAUUAGAGACAACUGAA 600 CUAAUGCA GAUGAAAA S227-AS601 UCAUCUUCAGUUGUCUC 227 CUGCAUCAGAGACAACUGA 601 UGAUGCAG AGAUGAAA S228-AS602 CAUCUUCAGUUGUCUCU 228 GCUGCUUCAGAGACAACUG 602 GAAGCAGC AAGAUGAA S229-AS603 AUCUUCAGUUGUCUCUG 229 AGCUGUAUCAGAGACAACU 603 AUACAGCT GAAGAUGA S230-AS604 UCUGAUGCAGCUUAUAC 230 AUUAUUUCGUAUAAGCUGC 604 GAAAUAAT AUCAGAGA S231-AS605 CUGAUGCAGCUUAUACG 231 AAUUAUUUCGUAUAAGCUG 605 AAAUAATT CAUCAGAG S232-AS606 CAGCUUAUACGAAAUAA 232 AACAAUAAUUAUUUCGUAU 606 UUAUUGTT AAGCUGCA S233-AS607 AGCUUAUACGAAAUAAU 233 GAACAUCAAUUAUUUCGUA 607 UGAUGUTC UAAGCUGC S234-AS608 GCUUAUACGAAAUAAUU 234 AGAACUACAAUUAUUUCGU 608 GUAGUUCT AUAAGCUG S235-AS609 CUUAUACGAAAUAAUUG 235 CAGAAUAACAAUUAUUUCG 609 UUAUUCTG UAUAAGCU S236-AS610 UAUACGAAAUAAUUGUU 236 AACAGUACAACAAUUAUUU 610 GUACUGTT CGUAUAAG S237-AS611 AUACGAAAUAAUUGUUG 237 UAACAUAACAACAAUUAUU 611 UUAUGUTA UCGUAUAA S238-AS612 UACGAAAUAAUUGUUGU 238 UUAACUGAACAACAAUUAU 612 UCAGUUAA UUCGUAUA S239-AS613 ACGAAAUAAUUGUUGUU 239 GUUAAUAGAACAACAAUUA 613 CUAUUAAC UUUCGUAU S240-AS614 CGAAAUAAUUGUUGUUC 240 AGUUAUCAGAACAACAAUU 614 UGAUAACT AUUUCGUA S241-AS615 GAAAUAAUUGUUGUUCU 241 CAGUUUACAGAACAACAAU 615 GUAAACTG UAUUUCGU S242-AS616 AAAUAAUUGUUGUUCUG 242 UCAGUUAACAGAACAACAA 616 UUAACUGA UUAUUUCG S243-AS617 AAUAAUUGUUGUUCUGU 243 UUCAGUUAACAGAACAACA 617 UAACUGAA AUUAUUUC S244-AS618 AAUUGUUGUUCUGUUAA 244 GUAUUUAGUUAACAGAACA 618 CUAAAUAC ACAAUUAU S245-AS619 AUUGUUGUUCUGUUAAC 245 GGUAUUCAGUUAACAGAAC 619 UGAAUACC AACAAUUA S246-AS620 UGUUGUUCUGUUAACUG 246 GUGGUUUUCAGUUAACAGA 620 AAAACCAC ACAACAAU S247-AS621 GUUGUUCUGUUAACUGA 247 AGUGGUAUUCAGUUAACAG 621 AUACCACT AACAACAA S248-AS622 UGUUCUGUUAACUGAAU 248 AGAGUUGUAUUCAGUUAAC 622 ACAACUCT AGAACAAC S249-AS623 UCUGUUAACUGAAUACC 249 UACAGUGUGGUAUUCAGUU 623 ACACUGTA AACAGAAC S250-AS624 CUGUUAACUGAAUACCA 250 UUACAUAGUGGUAUUCAGU 624 CUAUGUAA UAACAGAA S251-AS625 GUUAACUGAAUACCACU 251 AAUUAUAGAGUGGUAUUCA 625 CUAUAATT GUUAACAG S252-AS626 AACUGAAUACCACUCUG 252 UGCAAUUACAGAGUGGUAU 626 UAAUUGCA UCAGUUAA S253-AS627 ACUGAAUACCACUCUGU 253 UUGCAUUUACAGAGUGGUA 627 AAAUGCAA UUCAGUUA S254-AS628 CUGAAUACCACUCUGUA 254 UUUGCUAUUACAGAGUGGU 628 AUAGCAAA AUUCAGUU S255-AS629 UGAAUACCACUCUGUAA 255 UUUUGUAAUUACAGAGUGG 629 UUACAAAA UAUUCAGU S256-AS630 GAAUACCACUCUGUAAU 256 UUUUUUCAAUUACAGAGUG 630 UGAAAAAA GUAUUCAG S257-AS631 AAUACCACUCUGUAAUU 257 UUUUUUGCAAUUACAGAGU 631 GCAAAAAA GGUAUUCA S258-AS632 AAAAAGUUGCAGCUGUU 258 GUCAAUAAAACAGCUGCAA 632 UUAUUGAC CUUUUUUU S259-AS633 AAAGUUGCAGCUGUUUU 259 AUGUCUACAAAACAGCUGC 633 GUAGACAT AACUUUUU S260-AS634 AAGUUGCAGCUGUUUUG 260 AAUGUUAACAAAACAGCUG 634 UUAACATT CAACUUUU S261-AS635 AGUUGCAGCUGUUUUGU 261 GAAUGUCAACAAAACAGCU 635 UGACAUTC GCAACUUU S262-AS636 GUUGCAGCUGUUUUGUU 262 AGAAUUUCAACAAAACAGC 636 GAAAUUCT UGCAACUU S263-AS637 UGCAGCUGUUUUGUUGA 263 UCAGAUUGUCAACAAAACA 637 CAAUCUGA GCUGCAAC S264-AS638 GCAGCUGUUUUGUUGAC 264 UUCAGUAUGUCAACAAAAC 638 AUACUGAA AGCUGCAA S265-AS639 CAGCUGUUUUGUUGACA 265 AUUCAUAAUGUCAACAAAA 639 UUAUGAAT CAGCUGCA S266-AS640 AGCUGUUUUGUUGACAU 266 CAUUCUGAAUGUCAACAAA 640 UCAGAATG ACAGCUGC S267-AS641 GCUGUUUUGUUGACAUU 267 GCAUUUAGAAUGUCAACAA 641 CUAAAUGC AACAGCUG S268-AS642 UGUUUUGUUGACAUUCU 268 AAGCAUUCAGAAUGUCAAC 642 GAAUGCTT AAAACAGC S269-AS643 UUUGUUGACAUUCUGAA 269 UAGAAUCAUUCAGAAUGUC 643 UGAUUCTA AACAAAAC S270-AS644 UUGUUGACAUUCUGAAU 270 UUAGAUGCAUUCAGAAUGU 644 GCAUCUAA CAACAAAA S271-AS645 UGUUGACAUUCUGAAUG 271 CUUAGUAGCAUUCAGAAUG 645 CUACUAAG UCAACAAA S272-AS646 GUUGACAUUCUGAAUGC 272 ACUUAUAAGCAUUCAGAAU 646 UUAUAAGT GUCAACAA S273-AS647 UUGACAUUCUGAAUGCU 273 UACUUUGAAGCAUUCAGAA 647 UCAAAGTA UGUCAACA S274-AS648 UGACAUUCUGAAUGCUU 274 UUACUUAGAAGCAUUCAGA 648 CUAAGUAA AUGUCAAC S275-AS649 GACAUUCUGAAUGCUUC 275 UUUACUUAGAAGCAUUCAG 649 UAAGUAAA AAUGUCAA S276-AS650 CAUUCUGAAUGCUUCUA 276 UAUUUUCUUAGAAGCAUUC 650 AGAAAATA AGAAUGUC S277-AS651 AUUCUGAAUGCUUCUAA 277 GUAUUUACUUAGAAGCAUU 651 GUAAAUAC CAGAAUGU S278-AS652 UCUGAAUGCUUCUAAGU 278 UUGUAUUUACUUAGAAGCA 652 AAAUACAA UUCAGAAU S279-AS653 UGAAUGCUUCUAAGUAA 279 AAUUGUAUUUACUUAGAAG 653 AUACAATT CAUUCAGA S280-AS654 AAUGCUUCUAAGUAAAU 280 AAAAUUGUAUUUACUUAGA 654 ACAAUUTT AGCAUUCA S281-AS655 AUGCUUCUAAGUAAAUA 281 AAAAAUUGUAUUUACUUAG 655 CAAUUUTT AAGCAUUC S282-AS656 GCUUCUAAGUAAAUACA 282 AAAAAUAUUGUAUUUACUU 656 AUAUUUTT AGAAGCAU S283-AS657 UUUUUAUUAGUAUUGUU 283 AAAAGUACAACAAUACUAA 657 GUACUUTT UAAAAAAA S284-AS658 UUUUAUUAGUAUUGUUG 284 GAAAAUGACAACAAUACUA 658 UCAUUUTC AUAAAAAA S285-AS659 UUUAUUAGUAUUGUUGU 285 UGAAAUGGACAACAAUACU 659 CCAUUUCA AAUAAAAA S286-AS660 UUAUUAGUAUUGUUGUC 286 AUGAAUAGGACAACAAUAC 660 CUAUUCAT UAAUAAAA S287-AS661 UAUUAGUAUUGUUGUCC 287 UAUGAUAAGGACAACAAUA 661 UUAUCATA CUAAUAAA S288-AS662 AUUAGUAUUGUUGUCCU 288 CUAUGUAAAGGACAACAAU 662 UUACAUAG ACUAAUAA S289-AS663 UUAGUAUUGUUGUCCUU 289 CCUAUUAAAAGGACAACAA 663 UUAAUAGG UACUAAUA S290-AS664 UAGUAUUGUUGUCCUUU 290 ACCUAUGAAAAGGACAACA 664 UCAUAGGT AUACUAAU S291-AS665 GUAUUGUUGUCCUUUUC 291 AGACCUAUGAAAAGGACAA 665 AUAGGUCT CAAUACUA S292-AS666 UUGUUGUCCUUUUCAUA 292 UUCAGUCCUAUGAAAAGGA 666 GGACUGAA CAACAAUA S293-AS667 UGUUGUCCUUUUCAUAG 293 UUUCAUACCUAUGAAAAGG 667 GUAUGAAA ACAACAAU S294-AS668 UUGUCCUUUUCAUAGGU 294 AAUUUUAGACCUAUGAAAA 668 CUAAAATT GGACAACA S295-AS669 UGUCCUUUUCAUAGGUC 295 AAAUUUCAGACCUAUGAAA 669 UGAAAUTT AGGACAAC S296-AS670 GUCCUUUUCAUAGGUCU 296 AAAAUUUCAGACCUAUGAA 670 GAAAUUTT AAGGACAA S297-AS671 UCCUUUUCAUAGGUCUG 297 AAAAAUUUCAGACCUAUGA 671 AAAUUUTT AAAGGACA S298-AS672 CCUUUUCAUAGGUCUGA 298 GAAAAUUUUCAGACCUAUG 672 AAAUUUTC AAAAGGAC S299-AS673 AUAGGUCUGAAAUUUUU 299 UCAAGUAGAAAAAUUUCAG 673 CUACUUGA ACCUAUGA S300-AS674 AGGGGAAGCUAGUCUUU 300 CAAAAUCAAAAGACUAGCU 674 UGAUUUTG UCCCCUCA S301-AS675 GGGGAAGCUAGUCUUUU 301 GCAAAUGCAAAAGACUAGC 675 GCAUUUGC UUCCCCUC S302-AS676 GGGAAGCUAGUCUUUUG 302 GGCAAUAGCAAAAGACUAG 676 CUAUUGCC CUUCCCCU S303-AS677 GGAAGCUAGUCUUUUGC 303 GGGCAUAAGCAAAAGACUA 677 UUAUGCCC GCUUCCCC S304-AS678 GAAGCUAGUCUUUUGCU 304 UGGGCUAAAGCAAAAGACU 678 UUAGCCCA AGCUUCCC S305-AS679 CAGUGUUUAUCCUUUCA 305 UAACUUUAUGAAAGGAUAA 679 UAAAGUTA ACACUGUA S306-AS680 GUGUUUAUCCUUUCAUA 306 GCUAAUUAUAUGAAAGGAU 680 UAAUUAGC AAACACUG S307-AS681 UUAUCCUUUCAUAUAGU 307 AUUAGUUAACUAUAUGAAA 681 UAACUAAT GGAUAAAC S308-AS682 GCUAAUAAAAAGCUUUU 308 GUGUAUACAAAAGCUUUUU 682 GUAUACAC AUUAGCUA S309-AS683 GUAAAGUUAAGUUGAGA 309 GAAAAUUAUCUCAACUUAA 683 UAAUUUTC CUUUACCC S310-AS684 AAAGUUAAGUUGAGAUA 310 AUGAAUACUAUCUCAACUU 684 GUAUUCAT AACUUUAC S311-AS685 GUUAAGUUGAGAUAGUU 311 UGGAUUAAAACUAUCUCAA 685 UUAAUCCA CUUAACUU S312-AS686 UAAGUUGAGAUAGUUUU 312 UAUGGUUGAAAACUAUCUC 686 CAACCATA AACUUAAC S313-AS687 AAGUUGAGAUAGUUUUC 313 UUAUGUAUGAAAACUAUCU 687 AUACAUAA CAACUUAA S314-AS688 AGUUGAGAUAGUUUUCA 314 GUUAUUGAUGAAAACUAUC 688 UCAAUAAC UCAACUUA S315-AS689 GUUGAGAUAGUUUUCAU 315 AGUUAUGGAUGAAAACUAU 689 CCAUAACT CUCAACUU S316-AS690 UGAGAUAGUUUUCAUCC 316 UCAGUUAUGGAUGAAAACU 690 AUAACUGA AUCUCAAC S317-AS691 GAGAUAGUUUUCAUCCA 317 UUCAGUUAUGGAUGAAAAC 691 UAACUGAA UAUCUCAA S318-AS692 AGAUAGUUUUCAUCCAU 318 GUUCAUUUAUGGAUGAAAA 692 AAAUGAAC CUAUCUCA S319-AS693 GUUUUCAUCCAUAACUG 319 UGGAUUUUCAGUUAUGGAU 693 AAAAUCCA GAAAACUA S320-AS694 UUCAUCCAUAACUGAAC 320 UUUUGUAUGUUCAGUUAUG 694 AUACAAAA GAUGAAAA S321-AS695 UUGAUCAGUUAAGAAAU 321 UAUGUUAAAUUUCUUAACU 695 UUAACATA GAUCAAGA S322-AS696 GAUCAGUUAAGAAAUUU 322 GCUAUUUGAAAUUUCUUAA 696 CAAAUAGC CUGAUCAA S323-AS697 CAUUUACAAACUGAAGA 323 UUGAUUACUCUUCAGUUUG 697 GUAAUCAA UAAAUGUA S324-AS698 AUUUACAAACUGAAGAG 324 AUUGAUUACUCUUCAGUUU 698 UAAUCAAT GUAAAUGU S325-AS699 ACAAACUGAAGAGUAAU 325 GUAGAUUGAUUACUCUUCA 699 CAAUCUAC GUUUGUAA S326-AS700 AAACAUUUUGAAAGUCU 326 UCAAGUACAGACUUUCAAA 700 GUACUUGA AUGUUUGA S327-AS701 AAGGACUAAUAGAAAAG 327 AGAACUUACUUUUCUAUUA 701 UAAGUUCT GUCCUUCA S328-AS702 ACUAAUAGAAAAGUAUG 328 GGUUAUAACAUACUUUUCU 702 UUAUAACC AUUAGUCC S329-AS703 AGAAAAGUAUGUUCUAA 329 UGUAAUGGUUAGAACAUAC 703 CCAUUACA UUUUCUAU S330-AS704 GAAAAGUAUGUUCUAAC 330 AUGUAUAGGUUAGAACAUA 704 CUAUACAT CUUUUCUA S331-AS705 AAGUAUGUUCUAACCUU 331 CUCAUUUAAAGGUUAGAAC 705 UAAAUGAG AUACUUUU S332-AS706 GUAAUGGCAGUUAUAUU 332 AACUGUAAAAUAUAACUGC 706 UUACAGTT CAUUACAU S333-AS707 UAAUGGCAGUUAUAUUU 333 GAACUUCAAAAUAUAACUG 707 UGAAGUTC CCAUUACA S334-AS708 UAAAGAAGACCUGAGAA 334 GGGAUUCAUUCUCAGGUCU 708 UGAAUCCC UCUUUAAU S335-AS709 AAAGAAGACCUGAGAAU 335 GGGGAUACAUUCUCAGGUC 709 GUAUCCCC UUCUUUAA S336-AS710 GAAGACCUGAGAAUGUA 336 UUUGGUGAUACAUUCUCAG 710 UCACCAAA GUCUUCUU S337-AS711 AAGACCUGAGAAUGUAU 337 UUUUGUGGAUACAUUCUCA 711 CCACAAAA GGUCUUCU S338-AS712 AGACCUGAGAAUGUAUC 338 CUUUUUGGGAUACAUUCUC 712 CCAAAAAG AGGUCUUC S339-AS713 GACCUGAGAAUGUAUCC 339 GCUUUUGGGGAUACAUUCU 713 CCAAAAGC CAGGUCUU S340-AS715 ACCUGAGAAUGUAUCCC 340 CGCUUUUGGGGAUACAUUC 714 CAAAAGCG UCAGGUCU S341-AS715 CCUGAGAAUGUAUCCCC 341 ACGCUUUUGGGGAUACAUU 715 AAAAGCGT CUCAGGUC S342-AS716 CUGAGAAUGUAUCCCCA 342 CACGCUUUUGGGGAUACAU 716 AAAGCGTG UCUCAGGU S343-AS717 UGAGAAUGUAUCCCCAA 343 UCACGUUUUUGGGGAUACA 717 AAACGUGA UUCUCAGG S344-AS718 GAGAAUGUAUCCCCAAA 344 CUCACUCUUUUGGGGAUAC 718 AGAGUGAG AUUCUCAG S345-AS719 GCCAUAUUAAAUUUUUU 345 AUGUCUACAAAAAAUUUAA 719 GUAGACAT UAUGGCAG S346-AS720 CCAUAUUAAAUUUUUUG 346 AAUGUUAACAAAAAAUUUA 720 UUAACATT AUAUGGCA S347-AS721 CAUAUUAAAUUUUUUGU 347 UAAUGUCAACAAAAAAUUU 721 UGACAUTA AAUAUGGC S348-AS722 AUAUUAAAUUUUUUGUU 348 CUAAUUUCAACAAAAAAUU 722 GAAAUUAG UAAUAUGG S349-AS723 AUUAAAUUUUUUGUUGA 349 GACUAUUGUCAACAAAAAA 723 CAAUAGTC UUUAAUAU S350-AS724 AAAUUUUUUGUUGACAU 350 UGAGAUUAAUGUCAACAAA 724 UAAUCUCA AAAUUUAA S351-AS725 AAUUUUUUGUUGACAUU 351 CUGAGUCUAAUGUCAACAA 725 AGACUCAG AAAAUUUA S352-AS726 AUUUUUUGUUGACAUUA 352 ACUGAUACUAAUGUCAACA 726 GUAUCAGT AAAAAUUU S353-AS727 GAAGACUAUGAAAAUGC 353 UAUAGUCAGCAUUUUCAUA 727 UGACUATA GUCUUCAC S354-AS728 AGACUUUCCAUUACAAG 354 UAAAAUUACUUGUAAUGGA 728 UAAUUUTA AAGUCUCG S355-AS729 ACUUUGCAUCUCAGUAU 355 AAUAAUUCAUACUGAGAUG 729 GAAUUATT CAAAGUUU S356-AS730 CUUUGCAUCUCAGUAUG 356 GAAUAUUUCAUACUGAGAU 730 AAAUAUTC GCAAAGUU S357-AS731 UUGCAUCUCAGUAUGAA 357 UUGAAUAAUUCAUACUGAG 731 UUAUUCAA AUGCAAAG S358-AS732 GCAUCUCAGUAUGAAUU 358 AAUUGUAUAAUUCAUACUG 732 AUACAATT AGAUGCAA S359-AS733 CAUCUCAGUAUGAAUUA 359 AAAUUUAAUAAUUCAUACU 733 UUAAAUTT GAGAUGCA S360-AS734 GAAUGAUUUUUCUUUAC 360 UUUGUUUUGUAAAGAAAAA 734 AAAACAAA UCAUUCAA S361-AS735 AGUUUAGGGAACAAUUU 361 AAAUUUCCAAAUUGUUCCC 735 GGAAAUTT UAAACUCC S362-AS736 GUUUAGGGAACAAUUUG 362 AAAAUUGCCAAAUUGUUCC 736 GCAAUUTT CUAAACUC S363-AS737 UUUAGGGAACAAUUUGG 363 CAAAAUUGCCAAAUUGUUC 737 CAAUUUTG CCUAAACU S364-AS738 UUAGGGAACAAUUUGGC 364 ACAAAUUUGCCAAAUUGUU 738 AAAUUUGT CCCUAAAC S365-AS739 UAGGGAACAAUUUGGCA 365 CACAAUAUUGCCAAAUUGU 739 AUAUUGTG UCCCUAAA S366-AS740 AGGGAACAAUUUGGCAA 366 CCACAUAAUUGCCAAAUUG 740 UUAUGUGG UUCCCUAA S367-AS741 GGGAACAAUUUGGCAAU 367 ACCACUAAAUUGCCAAAUU 741 UUAGUGGT GUUCCCUA S368-AS742 GGAACAAUUUGGCAAUU 368 AACCAUAAAAUUGCCAAAU 742 UUAUGGTT UGUUCCCU S369-AS743 GAACAAUUUGGCAAUUU 369 AAACCUCAAAAUUGCCAAA 743 UGAGGUTT UUGUUCCC S370-AS744 AACAAUUUGGCAAUUUU 370 AAAACUACAAAAUUGCCAA 744 GUAGUUTT AUUGUUCC S371-AS745 ACAAUUUGGCAAUUUUG 371 GAAAAUCACAAAAUUGCCA 745 UGAUUUTC AAUUGUUC S372-AS746 CAAUUUGGCAAUUUUGU 372 CGAAAUCCACAAAAUUGCC 746 GGAUUUCG AAAUUGUU S373-AS747 AAAUAGCGUUCUUGUAA 373 GUGUAUAAUUACAAGAACG 747 UUAUACAC CUAUUUUA S374-AS748 AAUAGCGUUCUUGUAAU 374 CGUGUUAAAUUACAAGAAC 748 UUAACACG GCUAUUUU S375-AS749 GCGUUCUUGUAAUUUUA 375 AAAGCUUGUAAAAUUACAA 749 CAAGCUTT GAACGCUA S376-AS750 UAAUUUUACACGCUUUU 376 UCCAUUACAAAAGCGUGUA 750 GUAAUGGA AAAUUACA S377-AS751 CGCUUUUGUGAUGGAGU 377 AAAACUGCACUCCAUCACA 751 GCAGUUTT AAAGCGUG S378-AS752 GCUUUUGUGAUGGAGUG 378 CAAAAUAGCACUCCAUCAC 752 CUAUUUTG AAAAGCGU S379-AS753 CUUUUGUGAUGGAGUGC 379 ACAAAUCAGCACUCCAUCA 753 UGAUUUGT CAAAAGCG S380-AS754 UUUUGUGAUGGAGUGCU 380 AACAAUACAGCACUCCAUC 754 GUAUUGTT ACAAAAGC S381-AS755 UUUGUGAUGGAGUGCUG 381 UAACAUAACAGCACUCCAU 755 UUAUGUTA CACAAAAG S382-AS756 UGUGAUGGAGUGCUGUU 382 UAUAAUAAAACAGCACUCC 756 UUAUUATA AUCACAAA S383-AS757 UGAUGGAGUGCUGUUUU 383 UAUAUUACAAAACAGCACU 757 GUAAUATA CCAUCACA S384-AS758 GAUGGAGUGCUGUUUUG 384 UUAUAUAACAAAACAGCAC 758 UUAUAUAA UCCAUCAC S385-AS759 AUGGAGUGCUGUUUUGU 385 AUUAUUUAACAAAACAGCA 759 UAAAUAAT CUCCAUCA S386-AS760 UGGAGUGCUGUUUUGUU 386 AAUUAUAUAACAAAACAGC 760 AUAUAATT ACUCCAUC S387-AS761 GGAGUGCUGUUUUGUUA 387 AAAUUUUAUAACAAAACAG 761 UAAAAUTT CACUCCAU S388-AS762 GAGUGCUGUUUUGUUAU 388 UAAAUUAUAUAACAAAACA 762 AUAAUUTA GCACUCCA S389-AS763 AGUGCUGUUUUGUUAUA 389 CUAAAUUAUAUAACAAAAC 763 UAAUUUAG AGCACUCC S390-AS764 GUGCUGUUUUGUUAUAU 390 UCUAAUUUAUAUAACAAAA 764 AAAUUAGA CAGCACUC S391-AS765 UGCUGUUUUGUUAUAUA 391 GUCUAUAUUAUAUAACAAA 765 AUAUAGAC ACAGCACU S392-AS766 GCUGUUUUGUUAUAUAA 392 AGUCUUAAUUAUAUAACAA 766 UUAAGACT AACAGCAC S393-AS767 CUGUUUUGUUAUAUAAU 393 AAGUCUAAAUUAUAUAACA 767 UUAGACTT AAACAGCA S394-AS768 AUUUGCAUUUGUUUAUG 394 UGAAAUUACAUAAACAAAU 768 UAAUUUCA GCAAAUGG S395-AS769 GUUUAUGUAAUUUCAGG 395 GUAUUUCUCCUGAAAUUAC 769 AGAAAUAC AUAAACAA S396-AS770 AUGUAAUUUCAGGAGGA 396 UUCAGUAUUCCUCCUGAAA 770 AUACUGAA UUACAUAA S397-AS771 GAAUACUGAACAUCUGA 397 UCCAGUACUCAGAUGUUCA 771 GUACUGGA GUAUUCCU S398-AS772 CAUCUGAGUCCUGGAUG 398 AUUAGUAUCAUCCAGGACU 772 AUACUAAT CAGAUGUU S399-AS773 AUCUGAGUCCUGGAUGA 399 UAUUAUUAUCAUCCAGGAC 773 UAAUAATA UCAGAUGU S400-AS774 UGAGUCCUGGAUGAUAC 400 GUUUAUUAGUAUCAUCCAG 774 UAAUAAAC GACUCAGA S401-AS775 AGUCCUGGAUGAUACUA 401 UAGUUUAUUAGUAUCAUCC 775 AUAAACTA AGGACUCA S402-AS776 GUCCUGGAUGAUACUAA 402 UUAGUUUAUUAGUAUCAUC 776 UAAACUAA CAGGACUC S403-AS777 UCCUGGAUGAUACUAAU 403 AUUAGUUUAUUAGUAUCAU 777 AAACUAAT CCAGGACU S404-AS778 CCUGGAUGAUACUAAUA 404 UAUUAUUUUAUUAGUAUCA 778 AAAUAATA UCCAGGAC S405-AS779 CUGGAUGAUACUAAUAA 405 UUAUUUGUUUAUUAGUAUC 779 ACAAAUAA AUCCAGGA S406-AS780 UGGAUGAUACUAAUAAA 406 AUUAUUAGUUUAUUAGUAU 780 CUAAUAAT CAUCCAGG S407-AS781 GGAUGAUACUAAUAAAC 407 AAUUAUUAGUUUAUUAGUA 781 UAAUAATT UCAUCCAG S408-AS782 GAUGAUACUAAUAAACU 408 CAAUUUUUAGUUUAUUAGU 782 AAAAAUTG AUCAUCCA S409-AS783 AUGAUACUAAUAAACUA 409 GCAAUUAUUAGUUUAUUAG 783 AUAAUUGC UAUCAUCC S410-AS784 UGAUACUAAUAAACUAA 410 UGCAAUUAUUAGUUUAUUA 784 UAAUUGCA GUAUCAUC S411-AS785 GAUACUAAUAAACUAAU 411 CUGCAUUUAUUAGUUUAUU 785 AAAUGCAG AGUAUCAU S412-AS786 AUACUAAUAAACUAAUA 412 UCUGCUAUUAUUAGUUUAU 786 AUAGCAGA UAGUAUCA S413-AS787 UACUAAUAAACUAAUAA 413 CUCUGUAAUUAUUAGUUUA 787 UUACAGAG UUAGUAUC S866-AS887 UGGGCAAAGGAGAUCCU 866 GGCUUUUUAGGAUCUCCUU 887 AAAAAGCC UGCCCAUG S867-AS876 AAAGAGAAAUGAAAACC 867 GGGAUUUAGGUUUUCAUUU 876 UAAAUCCC CUCUUUCA S868-AS877 AAGAAGAUGAUGAUGAU 868 ACUUAUUCAUCAUCAUCAU 877 GAAUAAGT CUUCUUCU S869-AS878 AUGAUGAUGAUGAAUAA 869 GAACCUACUUAUUCAUCAU 878 GUAGGUTC CAUCAUCU S870-AS879 GAUGAUGAAUAAGUUGG 870 CGCUAUAACCAACUUAUUC 879 UUAUAGCG AUCAUCAU S871-AS880 AGAAAAAAAUUGAAAUG 871 CAGCCUUACAUUUCAAUUU 880 UAAGGCTG UUUUCUUU S872-AS881 UUGUUGUUCUGUUAACU 872 UGGUAUUCAGUUAACAGAA 881 GAAUACCA CAACAAUU S873-AS882 UUCUGAAUGCUUCUAAG 873 UGUAUUUACUUAGAAGCAU 882 UAAAUACA UCAGAAUG S874-AS883 CUGAAUGCUUCUAAGUA 874 AUUGUUUUUACUUAGAAGC 883 AAAACAAT AUUCAGAA S875-AS884 GAAUGCUUCUAAGUAAA 875 AAAUUUUAUUUACUUAGAA 884 UAAAAUTT GCAUUCAG

    TABLE-US-00005 TABLE 5 Additinoal GalNAc-conjugated HMGB1 oligonucleotide sequences with modifications (tested in FIG. 5). GalNAc- conjugated Sense Antisense oligo- sequence Sense sequence SEQ sequence Antisense sequence SEQ nucleotides (unmodified) (modified) ID NO (unmodified) (unmodified) ID NO S840- CUAAACAUGGG [mCs][mU][mA][mA][mA] 840 UCUCCUUUGCCC [MePhosphonate-4O- 853 AS853-M2 CAAAGGAGAGC [mC][mA][fU][fG][fG] AUGUUUAGGG [mUs][FCS][fU][mC] AGCCGAAGGCU [mC][mA][mA][mA][mG] [fC][mU][fU][mU] GC [mG][mA][mG][mA][mG] [mG][fC][mC][mC] [mC][mA][mG][mC][mC] [mA][fU][mG][mU] [mG][ademA-GalNAc] [mU][mU][mA][mGs] [ademA-GalNAc][mG] [mGs][mG] [mG][mC][mU][mG][mC] S840- CUAAACAUGGG [mCs][mU][fA][mA][mA] 840 UCUCCUUUGCCC [MePhosphonate-4O- 853 AS853-M3 CAAAGGAGAGC [mC][mA][fU][mG][fG] AUGUUUAGGG [mUs][FCS][fU][fC] AGCCGAAAGGC [mG][fC][fA][mA][mA] [GC][mU][fU][mU] UGC [mG][fG][mA][mG][mA] [mG][fC][mC][fC] [mG][mC][mA][mG][mC] [mA][fU][mG][fU] [mC][mG][ademA- [fU][mU][fA][mGs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S841- UAAACAUGGGC [mUs][mA][mA][mA][mC] 841 UUCUCCUUUGCC [MePhosphonate-4O- 854 AS854-M2 AAAGGAGAAGC [mA][mU][fG][fG][fG] CAUGUUUAGG [mUs][fUs][fC][mU] AGCCGAAAGGC [fC][mA][mA][mA][mG] [fC][mC][fU][mU] UGC [mG][mA][mG][mA][mA] [mU][fG][mC][mC] [mG][mC][mA][mG][mC] [mC][fA][mU][mG] [mC][mG][ademA- [mU][mU][mU][mAs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S841- UAAACAUGGGC [mUs][mA][fA][mA][mC] 841 UUCUCCUUUGCC [MePhosphonate-4O- 854 AS854-M3 AAAGGAGAAGC [mA][mU][fG][mG][fG] CAUGUUUAGG [mUs][fUs][fC][fU] AGCCGAAAGGC [mC][fA][fA][mA][mG] (SEQ ID [fC][mC][fU][mU] UGC [mG][fA][mG][mA][mA] NO: 854) [mU][fG][mC][fC] [mG][mC][mA][mG][mC] [mC][fA][mU][fG] [mC][mG][ademA- [fU][mU][fU][mAs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S842- AGCCGAGAGGC [mAs][mG][mC][mC][mG] 842 UGACAUUUUGCC [MePhosphonate-4O- 855 AS855-M2 AAAAUGUCAGC [mA][mG][fA][fG][fG] UCUCGGCUGG [mUs][FGS][fA][mC] AGCCGAAAGGC [fC][mA][mA][mA][mA] [fA][mU][fU][mU] UGC [mU][mG][mU][mC][mA] [mU][fG][mC][mC] [mG][mC][mA][mG][mC] [mU][fC][mU][mC] [mC][mG][ademA- [mG][mG][mC][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S842- AGCCGAGAGGC [mAs][mG][fC][mC][mG] 842 UGACAUUUUGCC [MePhosphonate-4O- 855 AS855-M3 AAAAUGUCAGC [mA][mG][fA][mG][fG] UCUCGGCUGG [mUs][FGS][fA][fC] AGCCGAAAGG [mC][fA][fA][mA][mA] [fA][mU][fU][mU] [mU][fG][mU][mC][mA] [mU][fG][mC][fC] [mG][mC][mA][mG][mC] [mU][fC][mU][fC] [mC][mG][ademA- [fG][mG][fC][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S843- AAGAAGUGCUC [mAs][mA][mG][mA][mA] 843 UACCUCUCUGAG [MePhosphonate-4O- 856 AS856-M2 AGAGAGGUAGC [mG][mU][fG][fC][fU] CACUUCUUGG [mUs][FAS][fC][mC] AGCCGAAAGGC [fC][mA][mG][mA][mG] [fU][mC][fU][mC] UGC [mA][mG][mG][mU][mA] [mU][fG][mA][mG] [mG][mC][mA][mG][mC] [mC][fA][mC][mU] [mC][mG][ademA- [mU][mC][mU][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S843- AAGAAGUGCUC [mAs][mA][fG][mA][mA] 843 UACCUCUCUGAG [MePhosphonate-4O- 856 AS856-M3 AGAGAGGUAGC [mG][mU][fG][mC][fU] CACUUCUUGG [mUs][FAS][fC][fC] AGCCGAAAGGC [mC][fA][fG][mA][mG] [fU][mC][fU][mC] UGC [mA][fG][mG][mU][mA] [mU][fG][mA][fG] [mG][mC][mA][mG][mC] [mC][fA][mC][fU] [mC][mG][ademA- [fU][mC][fU][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S844- CUCUGAGAGGU [mCs][mU][mC][mA][mG] 844 UGGUCUUCCACC [MePhosphonate-4O- 857 AS857-M2 GGAAGACCAGC [mA][mG][fA][fG][fG] UCUCUGAGGG [mUs][FGS][fG][mU] AGCCGAAAGGC [fU][mG][mG][mA][mA] [fC][mU][fU][mC] UGC [mG][mA][mC][mC][mA] [mC][fA][mC][mC] [mG][mC][mA][mG][mC] [mU][fC][mU][mC] [mC][mG][ademA- [mU][mG][mA][mGs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S844- CUCUGAGAGGU [mCs][mU][fC][mA][mG] 844 [MePhosphonate-4O- 857 AS857-M3 GGAAGACCAGC [mA][mG][fA][mG][fG] [mUs][FGS][fG][fU] AGCCGAAAGGC [mU][fG][fG][mA][mA] [fC][mU][fU][mC] UGC [mG][fA][mC][mC][mA] [mC][fA][mC][fC] [mG][mC][mA][mG][mC] [mU][fC][mU][fC] [mC][mG][ademA- [fU][mG][fA][mGs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S845- AGGUGGAAGAC [mCs][mU][fC][mA][mG] 845 UCAGACAUGGUC [MePhosphonate-4O- 858 AS858-M2 CAUGUCUGAGC [mA][mG][fA][mG][fG] UUCCACCUGG [mUs][FCS][fA][mG] AGCCGAAAGGC [mU][fG][fG][mA][mA] [fA][mC][fA][mU] UGC [mG][fA][mC][mC][mA] [mG][fG][mU][mC] [mG][mC][mA][mG][mC] [mU][fU][mC][mC] [mC][mG][ademA- [mA][mC][mC][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S845- AGGUGGAAGAC [mAs][mG][fG][mU][mG] 845 UCAGACAUGGUC [MePhosphonate-4O- 858 AS858- CAUGUCUGAGC [mG][mA][fA][mG][fA] UUCCACCUGG [mUs][FCS][fA][fG] AGCCGAAAGGC [mC][fC][fA][mU][mG] [fA][mC][fA][mU] UGC [mU][fC][mU][mG][mA] [mG][fG][mU][fC] [mG][mC][mA][mG][mC] [mU][fU][mC][fC] [mC][mG]demA- [fA][mC][fC][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S885- AAGACCAUGUC [mAs][mA][mG][mA][mC] 885 UCUUUAGCAGAC [MePhosphonate-4O- 886 AS886-M2 UGCUAAAGAGC [mC][mA][fU][fG][fU] AUGGUCUUGG [mUs][FCS][fU][mU] AGCCGAAAGGC [fC][mU][mG][mC][mU] [fU][mA][fG][mC] UGC [mA][mA][mA][mG][mA] [mA][fG][mA][mC] [mG][mC][mA][mG][mC] [mA][fU][mG][mG] [mC][mG][ademA- [mU][mC][mU][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S885- AAGACCAUGUC [mAs][mA][fG][mA][mC] UCUUUAGCAGAC [MePhosphonate-4O- 886 AS886-M3 UGCUAAAGAGC [mC][mA][fU][mG][fU] AUGGUCUUGG [mUs][FCS][fU][fU] AGCCGAAAGGC [mC][fU][fG][mC][mU] [fU][mA][fG][mC] UGC [mA][fA][mA][mG][mA] [mA][fG][mA][fC] [mG][mC][mA][mG][mC] [mA][fU][mG][fG] [mC][mG][ademA- [fU][mC][fU][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S846- AAAUUUGAAGA [mAs][mA][mA][mU][mU] 846 UUUGCCAUAUCU [MePhosphonate-4O- 859 AS859-M2 UAUGGCAAAGC [mU][mG][fA][fA][fG] UCAAAUCCGG [mUs][fUs][fU][mG] AGCCGAAAGGC [fA][mU][mA][mU][mG] [fC][mC][fA][mU] UGC [mG][mC][mA][mA][mA] [mA][fU][mC][mU] [mG][mC][mA][mG][mC] [mU][fC][mA][mA] [mC][mG][ademA- [mA][mU][mU][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S846- AAAUUUGAAGA [mAs][mA][fA][mU][mU] 846 UUUGCCAUAUCU [MePhosphonate-4O- 859 AS859-M3 UAUGGCAAAGC [mU][mG][fA][mA][fG] UCAAAUUUGG [mUs][fUs][fU][fG] AGCCGAAAGGC [mA][fU][fA][mU][mG] [fC][mC][fA][mU] UGC [mG][fC][mA][mA][mA] [mA][fU][mC][fU] [mG][mC][mA][mG][mC] [mU][fC][mA][fA] [mC][mG][ademA- [fA][mU][fU][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S847- AGCAAGAAAAA [mAs][mG][mC][mA][mA] 847 UCUUCCUUCUUU [MePhosphonate-4O- 860 AS860-M2 GAAGGAAGAGC [mG][mA][fA][fA][fA] UUCUUGCUGG [mUs][FCS][fU][mU] AGCCGAAAGGC [fA][mG][mA][mA][mG] [fC][mC][fU][mU] UGC [mG][mA][mA][mG][mA] [mC][fU][mU][mU] [mG][mC][mA][mG][mC] [mU][fU][mC][mU] [mC][mG][ademA- [mU][mG][mC][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S847- AGCAAGAAAAA [mAs][mG][fC][mA][mA] 847 UCUUCCUUCUUU [MePhosphonate-4O- 860 AS860-M3 GAAGGAAGAGC [mG][mA][fA][mA][fA] UUCUUGCUGG [mUs][FCS][fU][fU] AGCCGAAAGGC [mA][fG][fA][mA][mG] [fC][mC][fU][mU] UGC [mG][fA][mA][mG][mA] [mC][fU][mU][fU] [mG][mC][mA][mG][mC] [mU][fU][mC][fU] [mC][mG][ademA- [fU][mG][fC][mUs] GalNAc][ademA-GalNAc] [MGS[mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S848- CAAGAAAAAGA [mCs][mA][mA][mG][mA] 848 UCUCUUCCUUCU [MePhosphonate-4O- 861 AS861-M2 AGGAAGAGAGC [mA][mA][fA][fA][fG] UUUUCUUGGG [mUs][FCS][fU][mC] AGCCGAAAGGC [fA][mA][mG][mG][mA] [fU][mU][fC][mC] UGC [mA][mG][mA][mG][mA] [mU][fU][mC][mU] [mG][mC][mA][mG][mC] [mU][fU][mU][mU] [mC][mG][ademA- [mC][mU][mU][mGs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S848- CAAGAAAAAGA [mCs][mA][fA][mG][mA] 848 UCUCUUCCUUCU [MePhosphonate-4O- 861 AS861-M3 AGGAAGAGAGC [mA][mA][fA][mA][fG] UUUUCUUGGG [mUs][FCS][fU][fC] AGCCGAAAGGC [mA][fA][fG][mG][mA] [fU][mU][fC][mC] UGC [mA][fG][mA][mG][mA] [mU][fU][mC][fU] [mG][mC][mA][mG][mC] [mU][fU][mU][fU] [mC][mG][ademA- [fC][mU][fU][mGs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S849- GAUGAUGAUGA [mGs][mA][mU][mG][mA] 849 UCAACUUAUUCA [MePhosphonate-4O- 862 AS862-M2 AUAAGUUGAGC [mU][mG][fA][fU][fG] UCAUCAUCGG [mUs][FCS][fA][mA] AGCCGAAAGGC [fA][mA][mU][mA][mA] [fC][mU][fU][mA] UGC [mG][mU][mU][mG][mA] [mU][fU][mC][mA] [mG][mC][mA][mG][mC] [mU][fC][mA][mU] [mC][mG][ademA- [mC][mA][mU][mCs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S849- GAUGAUGAUGA [mGs][mA][fU][mG][mA] 849 UCAACUUAUUCA [MePhosphonate-4O- 862 AS862-M3 AUAAGUUGAGC [mU][mG][fA][mU][fG] UCAUCAUCGG [mUs][FCS][fA][fA] AGCCGAAAGGC [mA][fA][fU][mA][mA] [fC][mU][fU][mA] UGC [mG][fU][mU][mG][mA] [mU][fU][mC][fA] [mG][mC][mA][mG][mC] [mU][fC][mA][fU] [mC][mG][ademA- [fC][mA][fU][mCs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S850- AUGAUGAUGAA [mAs][mU][mG][mA][mU] 850 UCCAACUUAUUC [MePhosphonate-4O- 863 AS863-M2 UAAGUUGGAGC [mG][mA][fU][fG][fA] AUCAUCAUGG [mUs][FCS][fC][mA] AGCCGAAAGGC [fA][mU][mA][mA][mG] [fA][mC][fU][mU] UGS [mU][mU][mG][mG][mA] [mA][fU][mU][mC] [mG][mC][mA][mG][mC] [mA][fU][mC][mA] [mC][mG][ademA- [mU][mC][mA][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S850- AUGAUGAUGAA [mAs][mU][fG][mA][mU] 850 UCCAACUUAUUC [MePhosphonate-4O- 863 AS863-M3 UAAGUUGGAGC [mG][mA][fU][mG][fA] AUCAUCAUGG [mUs][FCS][fC][fA] AGCCGAAAGGC [mA][fU][fA][mA][mG] [fA][mC][fU][mU] UGC [mU][fU][mG][mG][mA] [mA][fU][mU][fC] (SEQ ID [mG][mC][mA][mG][mC] [mA][fU][mC][fA] NO: 850) [mC][mG][ademA- [fU][mC][fA][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S851- AAGUUGGUUCU [mAs][mA][mG][mU][mU] 851 UACUGCGCUAGA [MePhosphonate-4O- 864 AS864-M2 AGCGCAGUAGC [mG][mG][fU][fU][fC] ACCAACUUGG [mUs][FAS][fC][mU] AGCCGAAAGGC [fU][mA][mG][mC][mG] [fG][mC][fG][mC] UGC [mC][mA][mG][mU][mA] [mU][fA][mG][mA] [mG][mC][mA][mG][mC] [mA][fC][mC][mA] [mC][mG][ademA- [mA][mC][mU][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S851- AAGUUGGUUCU [mAs][mA][fG][mU][mU] 851 UACUGCGCUAGA [MePhosphonate-4O- 864 AS864-M3 AGCGCAGUAGC [mG][mG][fU][mU][fC] ACCAACUUGG [mUs][FAS][fC][fU] AGCCGAAAGGC [mU][fA][fG][mC][mG] [fG][mC][fG][mC] UGC [mC][fA][mG][mU][mA] [mU][fA][mG][fA] [mG][mC][mA][mG][mC] [mA][fC][mC][fA] [mC][mG][ademA- [fA][mC][fU][mUs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S852- UUAUUAGUAUU [mUs][mU][mA][mU][mU] 852 UAGGACAACAAU [MePhosphonate-4O- 865 AS865-M2 GUUGUCCUAGC [mA][mG][fU][fA][fU] ACUAAUAAGG [mUs][FAS][fG][mG] AGCCGAAAGGC [fU][mG][mU][mU][mG] [fA][mC][fA][mA] UGC [mU][mC][mC][mU][mA] [mC][fA][mA][mU] [mG][mC][mA][mG][mC] [mA][fC][mU][mA] [mC][mG][ademA- [mA][mU][mA][mAs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC] S852- UUAUUAGUAUU [mUs][mU][fA][mU][mU] 852 UAGGACAACAAU [MePhosphonate-4O- 865 AS865-M3 GUUGUCCUA [mA][mG][fU][mA][fU] ACUAAUAAGG [mUs][FAS][fG][fG] [mU][fG][fU][mU][mG] [fA][mC][fA][mA] [mU][fC][mC][mU][mA] [mC][fA][mA][fU] [mG][mC][mA][mG][mC] [mA][fC][mU][fA] [mC][mG][ademA- [fA][mU][fA][mAs] GalNAc][ademA-GalNAc] [mGs][mG] [ademAGalNAc][mG][mG] [mC][mU][mG][mC]

    [0246] The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the description and the appended claims.

    [0247] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

    [0248] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

    [0249] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

    [0250] The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.