Compositions and methods for inhibiting expression of complement component 3

Abstract

Described herein are oligonucleotides (e.g., RNAi oligonucleotides) that comprise sense and antisense strands for targeting complement component 3 (C3) mRNA. The RNAi oligonucleotides can be used to inhibit C3 expression, levels, and / or activity in cells. Also described are methods of using the oligonucleotides (e.g., RNAi oligonucleotides) for the prevention or treatment of diseases, disorders, or conditions mediated by complement pathway activation or dysregulation.

Description

[Technical field]

[0001] Sequence Listing This application has been filed together with a Sequence Listing in electronic format. The Sequence Listing is provided in a file entitled 50694-093WO3_Sequence_Listing_4_18_22_ST25_FINAL, created on April 20, 2021, and is 77,827 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0002] The complement system plays a central role in the clearance of immune complexes and immune responses against infectious agents, foreign antigens, virus-infected cells, and tumor cells. Complement consists of a group of more than 50 proteins that form part of the innate immune system. The complement system functions to arm the body against microbial infections and maintain tissue hemostasis. Complement is a tightly regulated enzyme cascade that can be activated by one of three pathways: the classical pathway, in which antibody complexes trigger activation; the alternative pathway, which is constitutively activated at low levels by a process called "tickover" and can be amplified by bacterial pathogens or damaged tissue surfaces; or the lectin pathway, which is initiated by mannose residues present on certain microorganisms, such as certain bacteria, fungi, and viruses. Uncontrolled activation or inadequate regulation of the complement pathway can lead to systemic inflammation, cell injury, and tissue damage. Thus, the complement pathway is involved in the pathogenesis of many diverse diseases. Inhibition or modulation of complement pathway activity is recognized as a promising therapeutic strategy. There are a limited number of treatment options available for these diseases. Therefore, there is a significant unmet need to develop innovative strategies to treat diseases associated with activation or dysregulation of the complement pathway.

[0003] Regardless of which complement pathway initiates the process, complement activation converges on complement component 3 (C3) within the cascade. The C3 protein plays a central role in driving several important biological processes, including complement activation, opsonization and clearance of pathogens, immune complexes and damaged cells, and regulation of humoral and T cell adaptive immune responses.

[0004] C3 is an essential protein in the complement system that helps initiate the complement pathway cascade. C3 activation via the classical, alternative, or lectin pathways leads to cleavage of C3 resulting in the breakdown products C3a and C3b. C3a is a potent anaphylatoxin and chemoattractant for neutrophils, eosinophils, and mast cells. C3b participates in the formation of C3 convertase in the alternative pathway, and C5 convertase is involved in all three complement pathways, which drives the complement cascade leading to further activation of downstream terminal complement. C5 cleavage leads to the formation of C5a, which is also a potent chemotactic driver and anaphylatoxin, and C5b, which rapidly assembles with complement proteins C6, 7, 8, and 9 to build the pore-forming complex C5b-9 on pathogen or tissue surfaces. As a result, C3 may be an ideal target for inhibition or silencing to selectively inhibit the complement pathway as a way to treat diseases associated with activation or dysregulation of the complement pathway. Summary of the Invention [Means for solving the problem]

[0005] Described herein are oligonucleotides (e.g., RNAi oligonucleotides, such as sense and antisense strand oligonucleotides) that target complement component (C3), known to play a role in complement pathway activation. The RNAi oligonucleotides, or pharma- ceutically acceptable salts thereof (e.g., sodium salts thereof), can be used to treat patients with diseases associated with complement pathway activation or dysregulation.

[0006] In one aspect, the present disclosure provides an RNAi oligonucleotide for reducing complement component 3 (C3) expression, comprising a sense strand and an antisense strand, or a pharma- ceutically acceptable salt thereof, wherein the sense strand and the antisense strand form a double helical region. The antisense strand comprises a region of complementarity to a C3 mRNA target sequence of SEQ ID NO: 13 or 14, and the region of complementarity is at least 15 contiguous nucleotides in length. In some embodiments, the sense strand is 15-50 nucleotides in length (e.g., 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, and 50 nucleotides in length). In some embodiments, the sense strand is 18-36 nucleotides in length (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36 nucleotides in length). In some embodiments, the antisense strand is 15-30 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides in length). In some embodiments, the antisense strand is 22 nucleotides in length, and the antisense strand and the sense strand form a double helical region at least 19 nucleotides in length, optionally at least 20 nucleotides in length. In some embodiments, the sense strand is 36 nucleotides in length, and the antisense strand and the sense strand form a double helical region at least 19 nucleotides in length, optionally at least 20 nucleotides in length. In some embodiments, the region of complementarity is at least 19 contiguous nucleotides in length, optionally at least 20 nucleotides in length.

[0007] In some embodiments, the 3' end of the sense strand comprises a stem-loop described as S1-L-S2, where S1 is complementary to S2 and L forms a loop between S1 and S2 that is 3-5 nucleotides long. In some embodiments, L is a tri-loop or tetra-loop. In some embodiments, L is a tetra-loop. In some embodiments, the tetra-loop comprises a nucleic acid sequence of SEQ ID NO:8. In some embodiments, S1 and S2 are 1-10 nucleotides in length, where optionally, S1 and S2 have the same length. In some embodiments, S1 and S2 are 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, or 10 nucleotides in length. In some embodiments, S1 and S2 are 6 nucleotides in length. In some embodiments, the stem-loop region comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO:7. In some embodiments, the stem-loop region comprises a nucleic acid sequence having at least 95% identity to SEQ ID NO: 7 (e.g., at least 95%, 96%, 97%, 98%, 99%, and 100% identity to SEQ ID NO: 7). In some embodiments, the stem-loop region comprises SEQ ID NO: 7. In some embodiments, the stem-loop comprises a nucleic acid having up to 1, 2, or 3 substitutions, insertions, or deletions relative to SEQ ID NO: 7.

[0008] In some embodiments, the antisense strand comprises a 3' overhang sequence that is 1 nucleotide or more in length.In some embodiments, the antisense strand comprises a 3' overhang of at least two linked nucleotides.In some embodiments, the 3' overhang sequence is 2 nucleotides in length, and in this case, optionally, the 3' overhang sequence is GG.

[0009] In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:4. In some embodiments, the antisense strand comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:6. In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of (a) SEQ ID NO:1 and 3, respectively, and (b) SEQ ID NO:4 and 6, respectively. In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:1, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:4, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:6. In some embodiments, as shown in Compound A, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:37, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:38. In some embodiments, as shown in Compound B, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:39, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:40. In some embodiments, as shown in Compound C, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:41, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:42. In some embodiments, as shown in Compound D, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 43, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 44. In some embodiments, as shown in Compound E, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 45, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 46. In some embodiments, as shown in Compound F, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 47, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 48. In some embodiments, as shown in Compound G, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 49, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 50. In some embodiments, as shown in Compound H, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 51, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 52.In some embodiments, as shown in Compound I, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:53 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:54.

[0010] Provided herein is an RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprising a sense strand and an antisense strand, wherein the sense strand has a nucleic acid sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to either SEQ ID NO:1 or SEQ ID NO:4, and the antisense strand has a nucleic acid sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to either SEQ ID NO:3 or SEQ ID NO:6. In some embodiments, the sense strand has at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequence identity to either SEQ ID NO:1 or SEQ ID NO:4, and the antisense strand has at least 95% (e.g., at least 96%, 97%, 98%, or 99%) sequence identity to at least one of SEQ ID NO:3 or SEQ ID NO:6. In some embodiments, the sense strand has the nucleic acid sequence of SEQ ID NO:1 or 4, and the antisense strand has the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:6. In some embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises SEQ ID NO:4 and SEQ ID NO:6. In other embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises SEQ ID NO:1 and SEQ ID NO:3.

[0011] In some embodiments, the antisense strand has at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to either SEQ ID NO:3 or SEQ ID NO:6.

[0012] In one embodiment, the sense strand comprises a stem-loop region that is not complementary to the antisense strand and a double helix region that is substantially complementary to the antisense strand, hi another embodiment, the double helix region comprises a length of 20-22 nucleosides.

[0013] In other embodiments, the stem-loop region comprises a nucleic acid sequence having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) identity to SEQ ID NO:7. In some embodiments, the stem-loop region has at least 95% (e.g., at least 96%, 97%, 98%, and 99%) identity to SEQ ID NO:7. In some embodiments, the stem-loop region comprises SEQ ID NO:7.

[0014] In some embodiments, the 4'-carbon of the sugar of the 5' nucleotide of the antisense strand comprises a phosphate analog. In some embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises a uridine at position 1 of the 5' end of the antisense strand. In some embodiments, the uridine comprises a phosphate analog. In some embodiments, the phosphate analog is 4'-O-monomethylphosphonate. In some embodiments, the uridine comprising a phosphate analog has the following structure: [ka] Includes.

[0015] In some embodiments, the oligonucleotide comprises at least one (e.g., at least 2, 5, 10, 15, 20, 30, and 40) modified nucleotide. In some embodiments, the oligonucleotide comprises 20-50 modified nucleotides (e.g., 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, and 50 modified oligonucleotides). In some embodiments, the oligonucleotide comprises 20-40 (e.g., 25-40, 30-40, 35-40, 30-35, 25-35, 20-25, 21-30, and 31-40) modified nucleotides. In some embodiments, all of the nucleotides of the oligonucleotide are modified. In some embodiments, at least one (e.g., at least 2, 5, 10, 15, 20, 30, and 40) modified nucleotides comprises a 2'-modification. In some embodiments, the 2'-modification is 2'-fluoro or 2'-O-methyl, where optionally the 2'-fluoro modification is a 2'-fluoro deoxyribonucleoside and / or the 2'-O-methyl modification is a 2'-O-methyl ribonucleoside.

[0016] In one embodiment, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises between 40 and 50 (e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50) 2'-O-methyl modifications, where optionally the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises between 40 and 50 (e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50) 2'-O-methyl ribonucleosides. In one embodiment, at least one (e.g., at least two, at least five, at least 10, at least 20, and at least 30) of nucleotides 1-7, 11-27, and 31-36 of the sense strand and one or more or all of nucleotides 1, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with 2'-O-methyl, e.g., 2'-O-methyl ribonucleosides. In one embodiment, 10-30 (e.g., 12-28, 12-24, 12-20, 12-16, 16-30, 20-30, and 24-30) of nucleotides 1-7, 11-27, and 31-36 of the sense strand and one or more or all of nucleotides 1, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with 2'-O-methyl, e.g., 2'-O-methyl ribonucleosides. In one embodiment, all of nucleotides 1-7, 12-27, and 31-36 of the sense strand and one or more or all of nucleotides 1, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with 2'-O-methyl, e.g., 2'-O-methyl ribonucleosides. In some embodiments, all of nucleotides 1, 2, 4-7, 11, 14-16, 18-27, and 31-36 of the sense strand and one or more or all of nucleotides 1, 6, 9, 11, 13, 15, 17, 18, and 20-22 of the antisense strand are modified with 2'-O-methyl, e.g., 2'-O-methyl ribonucleosides.

[0017] In another embodiment, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises 5-15 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15) 2'-fluoro modifications, such as 2'-fluoro deoxyribonucleosides. In some embodiments, at least one (e.g., at least 2, 3, 4, 5, 6, or 7) of nucleotides 3, 8, 9, 10, 11, 12, 13, and 17 of the sense strand, and one or more or all of nucleotides 2, 3, 4, 5, 7, 8, 10, 12, 14, 16, and 19 of the antisense strand are modified with 2'-fluoro, such as 2'-fluoro deoxyribonucleosides. In another embodiment, two to four of nucleotides 3, 8, 9, 10, 11, 12, 13, and 17 of the sense strand and one or more or all of nucleotides 2, 3, 4, 5, 7, 8, 10, 12, 14, 16, and 19 of the antisense strand are modified with 2'-fluoro, e.g., 2'-fluoro deoxyribonucleosides. In another embodiment, all of nucleotides 8, 9, 10, and 11 of the sense strand and one or more or all of nucleotides 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with 2'-fluoro, e.g., 2'-fluoro deoxyribonucleosides.

[0018] In some embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises at least one (e.g., at least two, at least five, at least ten, at least twenty, and at least thirty) modified internucleotide linkage. In some embodiments, at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, has a phosphorothioate linkage between nucleotides 1 and 2 of the sense strand and between nucleotides 1 and 2, 2 and 3, 20 and 21, and 21 and 22 of the antisense strand.

[0019] In one embodiment, there are no internucleotide linkages between the sense and antisense strands.

[0020] In some embodiments, at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands. In some embodiments, each targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid. In some embodiments, each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety. In some embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises 1-5 2'-ON-acetylgalactosamine (GalNAc) moieties conjugated to the sense strand. In some embodiments, up to 4 nucleotides of L of the stem loop are conjugated to a monovalent GalNAc moiety. In some embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises 1-5 (e.g., 2, 3, 4, and 5) GalNAc moieties conjugated to the sense strand. In some embodiments, at least one (e.g., at least two, or at least three) GalNAc moieties are conjugated to the loop region of the sense strand (SEQ ID NO:8). In some embodiments, one or more of the nucleotides at nucleotide positions 28-30 of the sense strand are conjugated to a monovalent GalNAc moiety. In some embodiments, each of the nucleotides at positions 28-30 of the sense strand is conjugated to a monovalent GalNAc moiety.

[0021] In some embodiments, nucleotides 28-30 of the sense strand have the following structure: [ka] wherein Z represents a bond, a click chemistry handle, or a linker of 1 to 20 contiguous covalently bonded atoms in length (inclusive) 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. In some embodiments, Z is an acetal linker. In some embodiments, X is O. In some embodiments, the nucleotides at positions 28-30 of the sense strand comprise the following structures: In some embodiments, nucleotides 28-30 of the sense strand have the following structure: [ka] Includes.

[0022] In one embodiment, the antisense strand is 13 to 27 (e.g., 13 to 25, 13 to 22, 13 to 20, 13 to 18, 13 to 15, 15 to 27, 18 to 27, 20 to 27, 22 to 27, and 25 to 27) nucleotides in length. In one embodiment, the antisense strand is 22 nucleotides in length.

[0023] In another embodiment, the sense strand is 20 to 50 (e.g., 22 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 20 to 22) nucleotides in length. In one embodiment, the sense strand is 30 to 40 (e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40) nucleotides in length.

[0024] In some embodiments, the sense strand forms a double helix with the antisense strand. In some embodiments, the double helix structure comprises a double helix between all or a portion of the sense strand and all or a portion of the antisense strand. In some embodiments, the region of complementarity is 20-30 (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30) nucleotides in length. In some embodiments, the antisense strand and / or the sense strand comprises a 3' overhang of at least two (e.g., at least three, at least four, or at least five) linked nucleotides. In some embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, is a double-stranded ribonucleic acid (dsRNA). In some embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, is a single-stranded ribonucleic acid.

[0025] In some embodiments, the RNA oligonucleotide comprises a pharma- ceutically acceptable salt. In some embodiments, the pharma- ceutically acceptable salt is a sodium salt.

[0026] In another aspect, the disclosure provides a pharmaceutical composition comprising any one of the oligonucleotides described herein (e.g., any of the RNAi oligonucleotides, or a pharma- ceutically acceptable salt thereof) and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0027] In another aspect, the disclosure provides a vector encoding at least one strand of any one of the RNAi oligonucleotides described herein, or a pharma- ceutically acceptable salt thereof.

[0028] In another aspect, the present disclosure provides a vector encoding at least one strand of an RNAi oligonucleotide described herein having a DNA sequence of any one of SEQ ID NOs: 33-36, or a pharma- ceutically acceptable salt thereof.

[0029] In another aspect, the disclosure provides a cell comprising any one of the vectors described herein or the RNAi oligonucleotides described herein, or a pharma- ceutically acceptable salt thereof.

[0030] In another aspect, the disclosure provides a cell comprising any of the vectors described herein or any of the oligonucleotides described herein (e.g., any of the RNAi oligonucleotides, or a pharma- ceutically acceptable salts thereof).

[0031] In another aspect, the disclosure provides a method of treating a disease mediated by complement pathway activation or dysregulation, comprising contacting a cell of a subject with any of the oligonucleotides described herein (e.g., RNAi oligonucleotides), pharmaceutical compositions described herein, vectors described herein, or cells described herein. In some embodiments, the cell is contacted for a time sufficient to achieve degradation of C3 mRNA transcripts. In some embodiments, expression of C3 in the cell is reduced. In some embodiments, transcription of C3 in the cell is reduced. In some embodiments, the level and / or activity of C3 in the cell is reduced. In some embodiments, the level and / or activity of C3 is reduced by 10% to 100% (e.g., reduced by 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, and 90% to 100%) compared to the level and / or activity of C3 in cells of a subject that has not been administered any one of the RNAi oligonucleotides, or pharma- ceutically acceptable salts thereof, pharmaceutical compositions, vectors, or cells described herein. In some embodiments, the level and / or activity of C3 is reduced by 50% to 99% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 99%, 70% to 99%, 80% to 99%, and 90% to 99%) compared to the level and / or activity of C3 in cells of a subject that has not been administered any one of the RNAi oligonucleotides, or pharma- ceutically acceptable salts, pharmaceutical compositions, vectors, or cells described herein. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0032] In another aspect, the disclosure provides a method for reducing C3 expression in a cell, a population of cells, or a subject, comprising: i) contacting the cell or population of cells with any one of the RNAi oligonucleotides, or pharma- ceutically acceptable salts, pharmaceutical compositions, or vectors described herein; or ii) administering to the subject any one of the RNAi oligonucleotides, or pharma- ceutically acceptable salts, pharmaceutical compositions, or vectors described herein. In some embodiments, reducing C3 expression comprises decreasing the amount or level of C3 mRNA, the amount or level of C3 protein, or both. In some embodiments, the level of C3 mRNA, the level of C3 protein, or both is reduced by 10% to 100% (e.g., reduced by 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, and 90% to 100%) compared to the level of C3 mRNA, the level of C3 protein, or both in cells of a subject that has not been administered any one of the RNAi oligonucleotides, or pharma- ceutically acceptable salts thereof, pharmaceutical compositions, vectors, or cells described herein.

[0033] In some embodiments, the level of C3 mRNA, the level of C3 protein, or both, is reduced by 50% to 99% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 99%, 70% to 99%, 80% to 99%, and 90% to 99%) compared to the level of C3 mRNA, the level of C3 protein, or both, in cells of a subject that has not been administered any one of the RNAi oligonucleotides, or pharma- ceutically acceptable salts, pharmaceutical compositions, vectors, or cells described herein.

[0034] In some embodiments, the subject is identified as having a disease mediated by or associated with complement pathway activation or dysregulation (e.g., dysregulation of the alternative complement pathway, the classical complement pathway, and / or the lectin pathway). In some embodiments, the disease mediated by or associated with complement pathway activation or dysregulation is as follows: paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), IgA nephropathy, lupus nephritis, C3 glomerulopathy (C3G), dermatomyositis / autoimmune myositis, systemic sclerosis, demyelinating polyneuropathy, pemphigus, membranous nephropathy, focal segmental glomerulosclerosis (FSGS), bullous pemphigoid, epidermolysis bullosa acquisita (EBA), mucous membrane pemphigoid, ANCA vasculitis, low complement urticarial vasculitis, immune complex small vasculitis, cutaneous small vasculitis, autoimmune necrotizing myopathy, transplanted organ rejection such as kidney, liver, heart or lung transplant rejection, antibody-mediated rejection (AMR) such as chronic AMR (cAMR), antiphospholipid (aPL) syndrome, glomerulonephritis, asthma, dense deposit disease (DDD), age-related macular degeneration (AMD), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), severe refractory RA, Felty's syndrome, multiple sclerosis (MS), traumatic brain injury (TBI) ), spinal cord injury, ischemia-reperfusion injury, preeclampsia, post-transplant dysfunction in acute kidney injury (DGF-AKI), cardiopulmonary bypass-associated acute kidney injury, hypoxic-ischemic encephalopathy, dialysis-induced thrombosis, Takayasu's arteritis, relapsing polychondritis, acute / prophylactic graft-versus-host disease, chronic graft-versus-host disease, beta-thalassemia, stem cell transplant-associated thrombotic microangiopathy, biliary atresia, inflammatory liver disease, Behçet's disease, ischemic stroke, intracerebral hemorrhage, scleroderma, scleroderma renal crisis, scleroderma-associated interstitial lung disease (SSc-ILD), sickle cell disease, autosomal ADPKD, chemotherapy-induced peripheral neuropathy (CIPN), diabetic neuropathy, amyotrophic lateral sclerosis (ALS), diabetic nephropathy, diabetic retinopathy, geographic atrophy, pulmonary arterial hypertension, refractory severe asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF), chronic lung allograft dysfunction, pulmonary disease state in cystic fibrosis, hidradenitis suppurativa, nonalcoholic fatty liver disease (NASH), ankylosing spondylitis, hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) (prevention), coronary artery disease,Atherosclerosis, osteoporosis (prevention), osteoarthritis, high-risk drusen, inflammatory bowel disease, ulcerative colitis, interstitial cystitis, dialysis-induced complement activation, pyoderma gangrenosum, chronic heart failure, autoimmune myocarditis, nasal polyposis, acute and chronic pancreatitis, atherosclerosis, eosinophilic esophagitis, eosinophilic granulomatosis, hypereosinophilic syndrome, wound healing, and thrombotic thrombocytopenic purpura (TTP). In some embodiments, the subject is identified as having antibody-mediated rejection (AMR), such as chronic AMR.

[0035] In some embodiments, the present disclosure provides a method of treating antibody-mediated rejection (AMR), such as chronic AMR (cAMR), comprising contacting a cell of a subject with any one of the RNAi oligonucleotides, or pharma- ceutically acceptable salts thereof, pharmaceutical compositions, vectors, or cells described herein. In some embodiments, the RNAi oligonucleotides, or any one of the pharma- ceutically acceptable salts thereof, pharmaceutical compositions, vectors, or cells described herein are intended for use in the prevention or treatment of antibody-mediated rejection (AMR), such as chronic AMR (cAMR), in a subject in need thereof.

[0036] In some embodiments, the RNAi oligonucleotide, or its pharmaceutically acceptable salt, pharmaceutical composition, vector, or cell is formulated for daily, weekly, monthly, or yearly administration.In some embodiments, the RNAi oligonucleotide, or its pharmaceutically acceptable salt, pharmaceutical composition, vector, or cell is formulated for intravenous, subcutaneous, intramuscular, oral, intranasal, sublingual, intrathecal, and intradermal administration.In some embodiments, the RNAi oligonucleotide, or its pharmaceutically acceptable salt, pharmaceutical composition, vector, or cell is formulated for subcutaneous administration.

[0037] In one embodiment, the oligonucleotide (e.g., the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof), or composition thereof, is formulated for daily, weekly, monthly, or yearly administration. In one embodiment, the oligonucleotide is formulated for subcutaneous, intravenous, intramuscular, oral, nasal, sublingual, intrathecal, and intradermal administration. In one embodiment, the oligonucleotide is formulated for subcutaneous administration. In one embodiment, the oligonucleotide is formulated for administration at a dose of about 0.1 mg / kg to about 150 mg / kg (e.g., 0.1 mg / kg to 125 mg / kg, 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 75 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 25 mg / kg, 0.1 mg / kg to 15 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 5 mg / kg, 5 mg / kg to 150 mg / kg, 25 mg / kg to 150 mg / kg, and 50 mg / kg to 150 mg / kg). In one embodiment, the oligonucleotide is formulated for administration at a dose of about 0.5 mg / kg to about 15 mg / kg (e.g., 0.5 mg / kg to 13 mg / kg, 0.5 mg / kg to 10 mg / kg, 0.5 mg / kg to 5 mg / kg, 0.5 mg / kg to 1 mg / kg, 1 mg / kg to 15 mg / kg, 5 mg / kg to 15 mg / kg, and 10 mg / kg to 15 mg / kg).

[0038] In some embodiments, the oligonucleotide is formulated for administration in combination with one or more additional therapeutic agents.

[0039] In another aspect, the disclosure provides a kit comprising an oligonucleotide described herein (e.g., an RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof), a pharmaceutical composition described herein, a vector described herein, or a cell described herein.

[0040] In another aspect, the disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof) described herein, a pharmaceutical composition described herein, a vector described herein, or a cell described herein for use in the prophylaxis or treatment of a disease mediated by or associated with activation or dysregulation of the complement pathway (e.g., activation or dysregulation of the alternative, classical, and / or lectin pathways).

[0041] In another aspect, the disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof), pharmaceutical composition, composition, vector, or cell, as described herein, wherein the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, pharmaceutical composition, composition, vector, or cell is administered or formulated for subcutaneous administration. [Brief description of the drawings]

[0042] [Figure 1-1] The chemical structure of the antisense strand of compound A is shown. [Figure 1-2] 1 shows the chemical structure of the sense strand of compound A. [Figure 1-3] 1 shows the chemical structure of the RNAi oligonucleotide of compound A. [Figure 1-4] 1 shows the chemical structure of the RNAi oligonucleotide of compound A. [Figure 1-5] The nucleic acid sequences of the sense and antisense strands of compound A are shown. [Figure 1-6] 1 shows a schematic diagram of a double-stranded oligonucleotide of Compound A. [Figure 2-1] 1 shows the chemical structures of the sense and antisense strands of Compound B. [Figure 2-2] 1 shows the chemical structures of the sense and antisense strands of Compound B. [Figure 2-3] The nucleic acid sequences of the sense and antisense strands of compound B are shown. [Diagram 3](FIG. 3A) Graph showing the results of an in vitro screen completed in HepG2 cells measuring the percent of C3 mRNA remaining after treating cells with various oligonucleotides at 1 nM (FIG. 3B) Graph showing the results of an in vitro screen completed in HepG2 cells measuring the percent of C3 mRNA remaining after treating cells with various oligonucleotides at 0.1 nM and 1 nM. [Figure 4-1] (FIG. 4A) Schematic diagram of the RNAi oligonucleotides of compounds AI. [Figure 4-2] (FIG. 4B) Graph showing the results of an in vivo screen of compounds A, B, and C in CD-1 mice expressing human C3 cDNA after hydrodynamic injection. RT-qPCR measurement of the percentage of human C3 mRNA in the liver 4 days after administration of a single subcutaneous dose of 1 mg / kg of compounds A, B, and C compared to the phosphate buffered saline (PBS) control group. (FIG. 4C) Graph showing the results of an in vivo screen of compounds A, D, E, F, G, H, and I in CD-1 mice expressing human C3 cDNA after hydrodynamic injection. RT-qPCR measurement of the percentage of human C3 mRNA in the liver 4 days after administration of a single subcutaneous dose of 0.5 mg / kg of compounds A, D, E, F, G, H, and I compared to the PBS control group. [Diagram 5] FIG. 1 is a graph showing the measurement of the percent of C3 mRNA in the liver of cynomolgus macaques before administration, 28 days and 56 days after treatment with a single dose of 4 mg / kg of Compound A, Compound B, or any one of Compounds C-I, compared to PBS administered as a control. [Figure 6](FIG. 6A) Graph showing measurement of percent C3 mRNA in liver of cynomolgus macaques after treatment with 1 mg / kg or 2 mg / kg of Compound A or Compound B on days 0, 28, 56, and 84, compared to PBS administered as a control. (FIG. 6B) Graph showing measurement of percent C3 in serum of cynomolgus macaques after treatment with 1 mg / kg or 2 mg / kg of Compound A or Compound B, compared to PBS administered as a control. [Figure 7] 1 is a graph showing the estimated ED50 of Compound A and Compound B measured as C3 mRNA in liver of cynomolgus macaques 28 days after administration of a single dose of 2 mg / kg Compound A or Compound B. [Figure 8] Graph showing percent complement activity (AP) in serum of cynomolgus monkeys after treatment with 2 mg / kg Compound A or Compound B on days 0, 28, 56, and 84, as measured by a WIESLAB® ELISA-based functional assay. PBS was administered in the same multiple-dose regimen as the control group. [Figure 9] Graph showing percent lysis from serum of cynomolgus monkeys after treatment with 1 mg / kg or 2 mg / kg of Compound A on days 0, 28, 56 and 84, as measured by rabbit erythrocyte hemolysis. PBS was administered in the same multiple dose regimen as the control group. [Figure 10] (FIG. 10A) RT-qPCR measurement of C3 mRNA (%) in the liver of CD-1 mice after administration of a single subcutaneous dose of 0.5 mg / kg, 1 mg / kg, and 6 mg / kg of Compound J compared to PBS administered as a control. The level of liver knockdown was followed for 70 days, with 5 mice sacrificed at each time point for measurement. (FIG. 10B) ELISA assay measurement of C3 circulating protein (%) in the serum of CD-1 mice over 70 days after administration of a single subcutaneous dose of 0.5 mg / kg, 1 mg / kg, and 6 mg / kg of Compound J compared to PBS administered as a control. [Figure 11] Graph showing stem-loop-qPCR measurement of siRNA exposure in plasma, liver, kidney, and spleen tissues of CD-1 mice administered a single subcutaneous dose of 6 mg / kg Compound J over a period of 672 hours. Five mice were sacrificed at each time point for measurement. [Figure 12] (FIG. 12A) RT-qPCR measurement of percent C3 mRNA in liver of CD-1 mice over a 70-day period after administration of four doses of 1 mg / kg or 6 mg / kg of Compound J on days 0, 14, 28, and 42, compared to PBS administered as a control. (FIG. 12B) ELISA assay measurement of C3 serum protein in CD-1 mice over a 70-day period after administration of four doses of 1 mg / kg or 6 mg / kg of Compound J on days 0, 14, 28, and 42. C3 levels were calculated as a percentage of C3 serum levels measured from the PBS control group (n=5 / time point). [Figure 13] (FIG. 13A) Stem loop-qPCR measurement of Compound J concentration in liver tissue of CD-1 mice administered 4 doses of 1 mg / kg Compound J on days 0, 14, 28, and 42. (FIG. 13B) Stem loop-qPCR measurement of Compound J concentration in plasma of CD-1 mice administered 4 doses of 1 mg / kg Compound J on days 0, 14, 28, and 42. [Figure 14] A set of images showing in situ hybridization of fluorescent tags to C3 and properdin to monitor glomerular complement deposition in the kidneys of NZB / W F1 mice treated monthly with 0.5 mg / kg, 3 mg / kg, or 6 mg / kg of Compound J for 18 weeks from 21 to 37 weeks of age, compared to naive C57BL / 6 mice and age-matched PBS-treated NZB / W F1 mice as controls. [Figure 15](FIG. 15A) Graph showing percentage of C3 mRNA in liver of NZB / W F1 mice after monthly subcutaneous doses of 0.5 mg / kg, 3 mg / kg, or 6 mg / kg Compound J at 21 weeks of age and termination at 29 weeks of age (n=10 / time point). (FIG. 15B) Graph showing percentage of C3 serum protein in NZB / W F1 mice after monthly subcutaneous doses of 0.5 mg / kg, 3 mg / kg, or 6 mg / kg Compound J at 21 weeks of age and termination at 29 weeks of age (n=10 / time point). [Figure 16] (FIG. 16A) Graph showing absorbance measured at 450 nm measuring circulating immune complexes by IgG capture in 29-week-old NZB / W F1 mice subcutaneously administered 0.5 mg / kg, 3 mg / kg, or 6 mg / kg subcutaneous doses of Compound J from 21 weeks of age. (FIG. 16B) Graph showing absorbance measured at 450 nm measuring circulating immune complexes by C1q capture in 37-week-old NZB / W F1 mice monthly administered 0.5 mg / kg, 3 mg / kg, or 6 mg / kg of Compound J from 21 weeks of age. [Figure 17] A series of images showing in situ hybridization of fluorescent tags to C3 and properdin to monitor complement deposition in glomeruli of MRL / Ipr mice treated with a subcutaneous dose of 6 mg / kg Compound J every 2 weeks from 8-16 weeks of age compared to a PBS control group. [Figure 18] This is a series of images showing in situ hybridization of fluorescent tags to C3 and properdin to monitor glomerular complement deposition in kidneys of CFH- / - mice treated monthly for 4 months with 0.5 mg / kg, 3 mg / kg, or 6 mg / kg of Compound J, starting at 4-8 months of age, compared to a PBS control group. Kidneys were harvested and imaged 4 weeks after the last dose. [Figure 19] FIG. 13 is a graph showing RT-qPCR measurement of percent C3 mRNA in livers of CFH− / − mice treated monthly with 0.5 mg / kg, 3 mg / kg, or 6 mg / kg of Compound J for 4 months, starting at 4-8 months of age, compared to CFH− / − mice receiving PBS as a control group. [Figure 20] (FIG. 20A) Graph showing clinical scores of hind paws from a collagen antibody-induced arthritis model in which arthritis was induced on day 0 and treated prophylactically with an LPS booster on day 3, followed by three doses of Compound J at 1 mg / kg or 6 mg / kg doses on days -7, 0, and 7. PBS-treated CAIA animals served as a control group. (FIG. 20B) Graph showing clinical scores of hind paws from a collagen antibody-induced arthritis model in which arthritis was induced on day 0 and treated prophylactically with an LPS booster on day 3, followed by a single dose of Compound J at 1 mg / kg or 6 mg / kg doses on day 5 after disease induction. PBS-treated CAIA animals served as a control group. [Figure 21] (FIG. 21A) A series of images of hind paw inflammation on day 11 in a CAIA mouse model in which arthritis was induced by collagen antibody administered on day 0 and an LPS booster on day 3, followed by prophylactic treatment with three doses of Compound J at a 6 mg / kg dose on days -7, 0, and 7. PBS-treated CAIA animals served as the control group. (FIG. 21B) A series of images of hind paw inflammation on day 13 in a CAIA mouse model in which arthritis was induced by collagen antibody administered on day 0 and an LPS booster on day 3, followed by therapeutic treatment with a single 6 mg / kg dose of Compound J on day 5 after disease induction. PBS-treated CAIA animals served as the control group. [Figure 22] (FIG. 22A) A series of images of H&E staining demonstrating a reduction in mononuclear cell infiltration into the hind paws after prophylactic treatment with three doses of 6 mg / kg dose of Compound J on days -7, 0 and 7. Naive and PBS-treated CAIA animals were used as negative and positive controls for inflammation, respectively. (FIG. 22B) A series of images of H&E staining demonstrating a reduction in mononuclear cell infiltration into the hind paws after therapeutic treatment with a single 6 mg / kg dose of Compound J in a CAIA-induced arthritis mouse model 5 days after disease induction. Naive and PBS-treated CAIA animals were used as negative and positive controls for inflammation, respectively. [Diagram 23]1 is a series of images of Safranin O staining demonstrating prevention of cartilage erosion and pannus formation, and H&E staining demonstrating reduction of mononuclear cell infiltration, in knee joints of a CAIA-induced arthritis model after animals were prophylactically treated with three doses of 6 mg / kg Compound J on days -7, 0, and 7. Naïve and PBS-treated CAIA animals were used as negative and positive controls, respectively. [Figure 24] (FIG. 24A) A series of H&E staining images demonstrating a reduction in mononuclear cell infiltration in the knee joints of the CAIA-induced arthritis model after therapeutic treatment of animals with a single dose of 6 mg / kg of compound J on day 5 after disease induction. Naive and PBS-treated CAIA animals were used as negative and positive controls, respectively. (FIG. 24B) A series of Safranin O staining images demonstrating the prevention of cartilage erosion and pannus formation in the knee joints of the CAIA-induced arthritis model after therapeutic treatment of animals with a single dose of 6 mg / kg of compound J on day 5 after disease induction. Naive and PBS-treated CAIA animals were used as negative and positive controls, respectively. [Diagram 25] Figure 1 is a series of images of lymphocyte (CD45+) staining in the hind paws of CAIA-induced arthritic animals demonstrating a reduction in immune cell infiltration following therapeutic treatment with a single dose of 6 mg / kg Compound J on day 5 after disease induction. Naive and PBS-treated CAIA animals were used as negative and positive controls, respectively. [Figure 26] Figure 1 is a series of images of neutrophil and macrophage (CD11b+) staining of hind paws of CAIA-induced arthritic animals demonstrating a reduction in immune cell infiltration following therapeutic treatment with a single dose of 6 mg / kg Compound J on day 5 after disease induction. Naive and PBS-treated CAIA animals were used as negative and positive controls, respectively. [Figure 27] Figure 1 is a series of images of macrophage (F4 / 80+) staining of the hind paws of CAIA-induced arthritic animals demonstrating a reduction in immune cell infiltration following therapeutic treatment with a single dose of 6 mg / kg Compound J on day 5 after disease induction. Naive and PBS-treated CAIA animals were used as negative and positive controls, respectively. [Figure 28]13 is a series of images of in situ hybridization of fluorescent tags to C3 mRNA (red) to monitor local complement expression and CD45+ cell (green-lymphocyte) infiltration into the hind paws of CAIA-induced animals following therapeutic treatment with a single 6 mg / kg dose of Compound J on day 5 after disease induction. [Figure 29] 1 is a graph showing the mean clinical scores from two experiments with MOG-induced experimental autoimmune encephalomyelitis (EAE) mice in which disease was induced on day 0, received two doses of pertussis toxin on days 0 and 1, and then were therapeutically treated with five weekly doses of Compound J at a 6 mg / kg dose starting on day 7 after disease induction. PBS-treated EAE animals were used as disease positive controls, and C3-deficient (C3- / -) were used as a negative control for C3 expression. [Diagram 30] A representative series of images of luxol fast blue spinal cord staining of MOG-induced EAE mice after receiving five weekly doses of 6 mg / kg Compound J compared to naïve PBS-treated EAE mice (disease control), and C3-deficient mice MOG-induced EAE. [Diagram 31] (FIG. 31A) Graph showing the amount of liver C3 mRNA in MOG-induced EAE mice after receiving 5 weekly doses of 6 mg / kg Compound J compared to naive PBS-treated EAE mice (positive control), naive C3-deficient mice (C3- / -), and C3-deficient mice MOG-induced EAE (negative control). (FIG. 31B) Graph showing the amount of serum C3 in MOG-induced EAE mice after receiving 5 weekly doses of 6 mg / kg Compound J compared to naive PBS-treated EAE mice (disease positive control), naive C3-deficient mice (C3- / -), and C3-deficient mice MOG-induced EAE (C3 expression negative control). [Diagram 32] (FIG. 32A) is a graph showing the mean concentration of Compound A versus time (hours) in plasma of cynomolgus macaques after administration of a single IV or SC dose of 3 mg / kg of Compound A. (FIG. 32B) is a graph showing the mean concentration of Compound A versus time (hours) in liver of cynomolgus macaques after administration of a single IV or SC dose of 3 mg / kg of Compound A. [Diagram 33] FIG. 1 is a graph showing the mean percent (±SD) of C3 mRNA expression in liver of cynomolgus macaques following a single dose of 3 mg / kg Compound A administered by SC or IV injection compared to saline (control). [Diagram 34] FIG. 1 is a graph showing the mean expression (±SD) of C3 protein in serum of cynomolgus macaques following administration of a single IV or SC dose of 3 mg / kg Compound A compared to saline (control). [Diagram 35] FIG. 1 is a graph showing complement C3 classical pathway activity in cynomolgus macaques following administration of a single IV or SC dose of 3 mg / kg Compound A compared to saline (control). [Diagram 36] FIG. 1 is a graph showing complement C3 lectin pathway activity in cynomolgus macaques following administration of a single IV or SC dose of 3 mg / kg Compound A compared to saline (control). [Figure 37] FIG. 1 is a graph showing complement C3 alternative pathway activity in cynomolgus macaques following administration of a single IV or SC dose of 3 mg / kg Compound A compared to saline control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] definition As used herein, the terms "about" and "approximately" refer to an amount that is ±10% of the recited value, optionally ±5% of the recited value, or more optionally, ±2% of the recited value.

[0044] As used herein, "administering" and "administration" refer to any method of providing a pharmaceutical formulation to a subject. The oligonucleotides described herein can be administered by any method known to those skilled in the art. Suitable methods for administering oligonucleotides can include, for example, oral, injection (e.g., intravenous, intraperitoneal, intramuscular, intravitreal, and subcutaneous), infusion formulations, and the like. The method of administering oligonucleotides can include subcutaneous administration. The oligonucleotides prepared as described herein can be administered in a variety of forms, depending on the disorder to be treated and the age, condition, and weight of the subject, as known in the art. The formulations can be administered prophylactically; i.e., administered to reduce the likelihood of developing a disease or condition.

[0045] As used herein, "an agent that reduces the level and / or activity of C3" refers to any oligonucleotide (e.g., an RNAi oligonucleotide) disclosed herein that can be used (e.g., administered) to reduce the level or expression of C3 in a cell or subject, e.g., in the cells or serum of a subject. "Reducing the level of C3," "reducing the expression of C3," and "reducing the transcription of C3" refer to reducing the level, reducing the expression, or reducing the transcription of C3 mRNA and / or C3 protein in a cell or subject, e.g., by administering an RNAi oligonucleotide (such as those described herein) to the cell or subject. The level of C3 mRNA and / or C3 protein can be measured using any method known in the art (e.g., by measuring the level of C3 mRNA or the level of C3 protein in a cell or subject). The decrease can be greater than or equal to about 5% (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100%) of C3 mRNA or protein levels compared to before treatment, or compared to levels of C3 mRNA or C3 protein in an untreated subject (e.g., a subject having a disease or disorder associated with complement activation or dysregulation (e.g., activation or dysregulation of C3)), or compared to a control subject (e.g., a healthy subject (e.g., a subject not having a disease or disorder associated with complement activation or dysregulation (e.g., activation or dysregulation of C3)). It can be a reduction in the level, expression, transcription of mRNA and / or C3 protein. The C3 can be any C3 (e.g., mouse C3, rat C3, monkey C3, or human C3), as well as variants or mutants of C3. Thus, the C3 can be wild-type C3, mutant C3, or transgenic C3 in the context of genetically engineered cells, cell populations, or organisms. "Reducing the activity of C3" also means reducing the level of activity associated with C3 (e.g., by reducing complement pathway activation associated with diseases mediated by complement pathway activation or dysregulation).The activity of C3 may be reduced by about 5% or more (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100%). The activity level of C3 may be measured using any method known in the art. The reduction may be a decrease in the level, expression, or transcription of C3 mRNA and / or C3 protein by at least about 5% or more (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% or more relative to a cell or subject not treated with an RNAi oligonucleotide disclosed herein). The reduction in C3 mRNA and / or C3 protein level, expression, or transcription can be over a period of at least one day or more (e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or more days). The reduction can be a reduction in the amount of C3 protein in the blood of a treated subject (e.g., a human subject) of at least 75-175 mg / dL (e.g., 75-100 mg / dL, 75-125 mg / L, 75-150 mg / dL, 150 mg / dL-175 mg / dL, 125-175 mg / dL, and 100-175 mg / dL).

[0046] The term "alternative nucleoside" or "alternative nucleotide" refers to a nucleoside having an alternative sugar or alternative nucleobase, such as those described herein. Alternative nucleosides can include nucleosides in which the nucleobase portion has been modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or pyrimidine, such as an "alternative nucleobase" selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uridine, 5-bromouridine, 5-thiazolo-uridine, 2-thio-uridine, pseudouridine, 1-methylpseudouridine, 5-methoxyuridine, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine. Alternative nucleosides can also include nucleosides where the sugar moiety has been modified; for example, 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O-methyl cytosine, 2'-O-methyl uridine, 2-fluoro-deoxy adenosine, 2-fluoro-deoxy guanosine, 2-fluoro-deoxy cytidine, 2-fluoro-deoxy uridine.

[0047] Exemplary nucleobases with substituted uracil include the following: pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, i.e., having the nucleic acid base deoxythymine), 1-methyl-pseudouridine (m 1 ψ), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 S 4 ψ), 4-thio-1-methylpseudouridine, 3-methyl-pseudouridine (m 3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-uridine (ψm), 2-thio-2'-O-methyluridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

[0048] Exemplary nucleobases having an alternative cytosine include the following: 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetylcytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k 2 C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N4-acetyl-2'-O-methyl-cytidine (ac 4 Cm), N4,2'-O-dimethyl-cytidine (m 4 Cm), 5-formyl-2'-O-methyl-cytidine (f 5 Cm), N4,N4,2'-O-trimethyl-cytidine (m 4 2 cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0049] Exemplary nucleobases with an alternative adenine include the following: 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m 2 A), N6-methyl-adenosine (m 6 A), 2-methylthio-N6-methyl-adenosine (ms 2 m 6 A), N6-isopentenyl-adenosine (i 6 A), 2-methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A), N6-(cis-hydroxyisopentenyl)adenosine (io 6 A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A), N6-glycinylcarbamoyl-adenosine (g 6 A), N6-threonylcarbamoyl-adenosine (t 6 A), N6-methyl-N6-threonylcarbamoyl-adenosine (m 6 t 6 A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms 2 g 6 A), N6,N6-dimethyladenosine (m 6 2 A), N6-hydroxynorvalylcarbamoyl-adenosine (hn 6 A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms 2 hn 6 A), N6-acetyl-adenosine (ac 6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyladenosine (m 6 Am), N6,N6,2'-O-trimethyladenosine (m 6 2 Am), 1,2'-O-dimethyladenosine (m 1 Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0050] Exemplary nucleobases with substituted guanine include the following: inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxywyosine (o 2 yW), hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ 0 ), 7-aminomethyl-7-deaza-guanosine (preQ 1 ), Archaeosin (G + ), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methylguanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m 1 G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2,7 G), N2,N2,7-dimethyl-guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methylguanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2 Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Im), 2'-O-ribosylguanosine(phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.

[0051] Nucleobase moieties may also be represented by the letter code of each corresponding nucleobase, e.g., A, T, G, C, or U, where each letter can optionally include alternative nucleobases of equivalent function.

[0052] As used herein, the term "antisense" refers to an oligonucleotide that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA (e.g., the sequence of C3 (e.g., SEQ ID NO: 12)) so as to disrupt expression of an endogenous gene (e.g., C3).

[0053] The terms "antisense strand" and "guide strand" refer to the strand of an RNAi oligonucleotide (eg, dsRNA) that includes a region that is substantially complementary to a target sequence, eg, C3 mRNA (eg, SEQ ID NO: 12).

[0054] The term "at least" preceding a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically encompassed, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 10 nucleotides of a 21-nucleotide nucleic acid molecule" means that a range of 10 to 21 nucleotides, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides, have the indicated property. When "at least" is present before a series or range of numbers, it is understood that "at least" can modify each of the numbers in the series or range.

[0055] As used herein, the term "attenuate" means to reduce or substantially stop. As a non-limiting example, one or more of the treatments provided herein may inhibit or substantially stop the onset or progression of a disease mediated by complement pathway activation or dysregulation (e.g., C3 activation or dysregulation) in a subject. This attenuation may be exemplified, for example, by a reduction in one or more aspects (e.g., symptoms, tissue characteristics, and cellular, inflammatory, or immunological activity, etc.) of a disease associated with complement pathway activation or dysregulation, for example, one or more of the diseases associated with complement pathway activation or dysregulation disclosed herein.

[0056] The term "cDNA" refers to a nucleic acid sequence that is the DNA equivalent of an mRNA sequence (i.e., has uridines substituted for thymidines). Generally, those skilled in the art will understand that a cDNA sequence is the same as an mRNA sequence, except that uridines are read as thymidines, and therefore the terms cDNA and mRNA may be used interchangeably with respect to a particular gene (e.g., the C3 gene).

[0057] As used herein, the terms "C3" and "complementary component 3" refer to a protein or gene encoding complementary component 3. The term "C3" refers to naturally occurring variants of wild-type C3 protein, such as a protein having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more identity) to the amino acid sequence of wild-type human C3 as set forth in NCBI Reference No. NP_000055.2 or SEQ ID NO:11. The term "C3" also refers to naturally occurring variants of wild-type C3 polynucleotides, e.g., polynucleotides having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more identity) to the nucleic acid sequence of wild-type human C3 set forth in NCBI Reference No. NM_000064.4 or SEQ ID NO:12.

[0058] As used herein, "combination therapy" or "administered in combination with" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen specifies the dose and periodicity of administration of each agent such that the effects of the individual agents on the subject overlap. In some embodiments, the delivery of two or more agents is simultaneous or concurrent, and the agents may be co-formulated. In some embodiments, the two or more agents are not co-formulated and are administered sequentially as part of a prescribed regimen. In some embodiments, the administration of two or more agents or treatments in combination results in a reduction in symptoms or other parameters associated with the disorder that is greater than would be observed with one agent or treatment alone or in the absence of the other. The effect of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). The sequential or substantially simultaneous administration of each therapeutic agent can be effected by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination can be administered by intravenous injection and a second therapeutic agent of the combination can be administered orally.

[0059] As used herein, the term "complement pathway activation or dysregulation" refers to any abnormality in the ability of the complement pathway, including the classical, alternative, and lectin pathways, to confer host defense against pathogens, to eliminate immune complexes and damaged cells, and to regulate immunity. Complement pathway activation or dysregulation can occur in the fluid phase and on cell surfaces and can lead to excessive complement activation or insufficient regulation, either of which can cause tissue damage.

[0060] As used herein, "complementary," when used to describe a first nucleotide or nucleoside sequence in relation to a second nucleotide or nucleoside sequence, refers to the ability of an oligonucleotide containing the first nucleotide or nucleoside sequence to hybridize and form a double helix structure with an oligonucleotide containing the second nucleotide sequence under specified conditions, as will be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPE pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions may be applied, such as physiologically relevant conditions such as those that may be encountered inside an organism. Those skilled in the art will be able to determine the set of conditions most suitable for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides or nucleosides. Also, as used herein, "complementary" sequences may include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and alternative nucleotides or nucleosides, so long as the above requirements for ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogsteen base pairs. As described herein, a complementary sequence within an oligonucleotide (e.g., an RNAi oligonucleotide) or between an oligonucleotide and a target sequence includes base pairing between an oligonucleotide containing a first nucleotide or nucleoside sequence and an oligonucleotide containing a second nucleotide or nucleoside sequence over the entire length of one or both nucleotide or nucleoside sequences. Such sequences may be referred to herein as "fully complementary" to one another.When a first sequence is referred to as "substantially complementary" with respect to a second sequence, the two sequences may be fully complementary or both may form one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs, while retaining the ability to hybridize under conditions most relevant to their ultimate application (e.g., reducing expression via the RISC pathway) upon hybridization for a duplex of up to 30 base pairs. "Substantially complementary" may also refer to an oligonucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding C3). For example, an oligonucleotide is complementary to at least a portion of C3 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding C3. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered as mismatches for purposes of determining complementarity. For example, oligonucleotides (e.g., RNAi oligonucleotides) comprising one oligonucleotide 22 linked nucleosides in length and another oligonucleotide 20 nucleosides in length can be referred to as "fully complementary" for purposes described herein, even though they have different lengths.

[0061] As used herein, a "complementary oligonucleotide" is one that is capable of base pairing according to standard Watson-Crick complementarity rules. Specifically, purines base pair with pyrimidines to form combinations of guanine paired with cytosine (G:C), and adenine paired with thymine (A:T) in the case of DNA, and adenine paired with uracil (A:U) in the case of RNA. It is understood that two oligonucleotides can hybridize to each other even if they are not completely complementary to each other, so long as each has at least one region that is substantially complementary to the other.

[0062] The phrase "contacting a cell with an oligonucleotide" as used herein includes contacting a cell with an oligonucleotide, e.g., a single-stranded oligonucleotide or a double-stranded oligonucleotide (e.g., a single-stranded RNA or a double-stranded RNA that forms a double helix), by methods known in the art. Contacting a cell with an oligonucleotide includes contacting a cell with an oligonucleotide in vitro or contacting a cell with an oligonucleotide in vivo. Contacting can be performed either directly or indirectly. Thus, for example, the oligonucleotide can be brought into physical contact with the cell by the individual performing the method, or the RNAi oligonucleotide can be placed in a situation that allows or will cause it to subsequently contact the cell. Contacting a cell in vitro can be performed, for example, by incubating the cell with the oligonucleotide. Contacting a cell in vivo can be performed, for example, by injecting the oligonucleotide into or near the tissue in which the cell is located, or by injecting the RNAi oligonucleotide into another area, e.g., the bloodstream or subcutaneous space, so that the agent subsequently reaches the tissue in which the cell to be contacted is located. For example, the oligonucleotide may contain and / or be linked to a ligand that directs the oligonucleotide to the desired site, or may be incorporated into a vector (e.g., a viral vector) that delivers the oligonucleotide to the desired site. A combination of in vitro and in vivo contact methods is also possible. For example, cells may be contacted with oligonucleotides in vitro and then transplanted into a subject.

[0063] The term "contiguous nucleobase region" refers to a region of an oligonucleotide that is complementary to a target nucleic acid (e.g., the antisense strand of an RNAi oligonucleotide). This term may be used interchangeably herein with the term "contiguous nucleotide sequence" or "contiguous nucleotide base sequence". In some embodiments, all of the nucleotides of an oligonucleotide are present in a contiguous nucleotide or nucleoside region. In some embodiments, an oligonucleotide comprises a contiguous nucleotide region and may optionally further comprise one or more nucleotides or one or more nucleosides. The nucleotide linker region may or may not be complementary to the target nucleic acid. The internucleoside linkages present between the nucleotides of the contiguous nucleotide region may include phosphorothioate internucleoside linkages. In addition, the contiguous nucleotide region may include one or more sugar-modified nucleosides.

[0064] As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen instead of a hydroxyl at the 2' position of its pentose sugar compared to a ribonucleotide. Modified deoxyribonucleotides are deoxyribonucleotides that have one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions of the sugar, phosphate group, or base.

[0065] As used herein, the term "disease" refers to the interruption, cessation, or disorder of a bodily function, system, or organ. Diseases or disorders of interest include those that would benefit from treatment with the oligonucleotides described herein (e.g., single-stranded or double-stranded RNA constructs that form a duplex as described herein) that target C3, such as by the therapeutic methods described herein. Non-limiting examples of diseases or disorders mediated by or associated with complement pathway activation or dysregulation that can be treated using the compositions and methods described herein include, for example, skin disorders, neurological disorders, nephrological disorders, acute care, rheumatic disorders, pulmonary disorders, dermatological disorders, hematological disorders, and ophthalmological disorders, such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), IgA nephropathy, and the like. , lupus nephritis, C3 glomerulopathy (C3G), dermatomyositis / autoimmune myositis, systemic sclerosis, demyelinating polyneuropathy, pemphigus, membranous nephropathy, focal segmental glomerulosclerosis (FSGS), bullous pemphigoid, epidermolysis bullosa acquisita (EBA), mucous membrane pemphigoid, ANCA vasculitis, low complement urticarial vasculitis, immune complex small vasculitis, cutaneous small vasculitis, autoimmune necrotizing myopathy, rejection of transplanted organs such as kidney, liver, heart or lung transplant rejection, e.g., chronic AMR (cAMR), Antibody-mediated rejection (AMR) such as antiphospholipid (aPL) syndrome, glomerulonephritis, asthma, dense deposit disease (DDD), age-related macular degeneration (AMD), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), severe refractory RA, Felty syndrome, multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, ischemia-reperfusion injury, preeclampsia, post-transplant dysfunction in acute kidney injury (DGF-AKI), cardiopulmonary bypass-associated acute kidney injury, hypoxic Ischemic encephalopathy, dialysis-induced thrombosis, Takayasu's arteritis, relapsing polychondritis, acute / prophylactic graft-versus-host disease, chronic graft-versus-host disease, beta-thalassemia, stem cell transplant-associated thrombotic microangiopathy, biliary atresia, inflammatory liver disease, Behçet's disease, ischemic stroke, intracerebral hemorrhage, scleroderma, scleroderma renal crisis, scleroderma-associated interstitial lung disease (SSc-ILD), sickle cell disease, autosomal dominant polycystic kidney disease (ADPKD), chemotherapy-induced peripheral neuropathy (CIPN), diabetic neuropathy,Amyotrophic lateral sclerosis (ALS), diabetic nephropathy, diabetic retinopathy, geographic atrophy, pulmonary arterial hypertension, severe intractable asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF), chronic pulmonary allograft dysfunction, pulmonary disease in cystic fibrosis, hidradenitis suppurativa, nonalcoholic fatty liver disease (NASH), ankylosing spondylitis, hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) (prevention), coronary artery disease Disease, atherosclerosis, osteoporosis (prevention), osteoarthritis, high-risk drusen, inflammatory bowel disease, ulcerative colitis, interstitial cystitis, dialysis-induced complement activation, pyoderma gangrenosum, chronic heart failure, autoimmune myocarditis, nasal polyposis, acute and chronic pancreatitis, atherosclerosis, eosinophilic esophagitis, eosinophilic granulomatosis, hypereosinophilic syndrome, wound healing, and thrombotic thrombocytopenic purpura (TTP).

[0066] As used herein, the term "double helix," with respect to nucleic acids (eg, oligonucleotides), refers to the structure formed by complementary base pairing of antiparallel sequences of two nucleotides.

[0067] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of an agent (e.g., an RNAi oligonucleotide described herein) that reduces the level and / or activity of C3 (e.g., in a cell or subject) refer to an amount sufficient to produce a beneficial or desired result, including a clinical result, when administered to a subject, such as a human, and thus "effective amount" or its synonyms vary depending on the context in which it is applied. For example, in the context of treating a disease associated with activation or dysregulation of the complement pathway, it is the amount of an agent that reduces the level and / or activity of C3 sufficient to achieve a therapeutic response compared to the response obtained without administration of the agent that reduces the level and / or activity of C3. The amount of a given agent that reduces the level and / or activity of C3 described herein that corresponds to such an amount may vary depending on a variety of factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, subject identity (e.g., age, sex, and / or weight), or host being treated, but can still be routinely determined by one of skill in the art. Also, as used herein, a "therapeutically effective amount" of an agent that reduces the level and / or activity of C3 of the present disclosure is an amount that produces a beneficial or desired result in a subject compared to a control. As defined herein, a therapeutically effective amount of an agent that reduces the level and / or activity of C3 of the present disclosure can be readily determined by one of ordinary skill in the art by routine methods known in the art. Dosage regimens can be adjusted to provide an optimal therapeutic response.

[0068] As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a composition to, for example, impart or contribute a desired consistency or stabilizing effect.

[0069] "G", "C", "A", "T" and "U" each generally represent nucleotides containing guanine, cytosine, adenine, thymidine and uracil as bases, respectively, but may contain alternative sugar moieties other than ribose and deoxyribose. It is also understood that the term "nucleotide" may refer to alternative nucleotides or surrogate replacement moieties, as further detailed below. Those skilled in the art will appreciate that guanine, cytosine, adenine and uracil may be substituted by other moieties without substantially altering the base pairing properties of an oligonucleotide containing a nucleotide bearing such a replacement moiety. For example, and without limitation, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine may be substituted by a nucleotide containing, for example, inosine in the nucleotide sequence of an oligonucleotide specifically featured in the present disclosure. In another example, any adenine and cytosine in the oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair that pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods specifically featured in this disclosure.

[0070] As used herein, the term "inhibitor" refers to any agent that reduces the level and / or activity of a protein (e.g., C3). Non-limiting examples of inhibitors include oligonucleotides (e.g., RNAi oligonucleotides, e.g., dsRNA, siRNA, or shRNA). As used herein, the term "reduce" is used interchangeably with "silencing," "downregulation," "suppression," and other similar terms, and includes any level reduction of 5% or more (e.g., 10%, 15%, 25%, 35%, 50%, 75%, and 100%). Typical levels of C3 protein found in the serum of healthy humans are about 75-175 mg / dL (e.g., 75-100 mg / dL, 75-125 mg / L, 75-150 mg / dL, 150 mg / dL-175 mg / dL, 125-175 mg / dL, and 100-175 mg / dL).

[0071] By "level" is meant the level or activity of a protein, or of an mRNA encoding a protein (e.g., C3), optionally compared to a reference. The reference may be any useful standard, as defined herein. A "decreased level" or "increased level" of a protein refers to a decrease or increase, respectively, in the protein level compared to the reference (e.g., a decrease or increase of, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or more, compared to the reference; e.g., a decrease or increase of, e.g., about 10%, about 15%, about By "a decrease or increase" is meant a decrease or increase of more than 20%, about 50%, about 75%, about 100%, or about 200%; e.g., a decrease or increase of less than about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold, or less compared to the reference; e.g., a decrease or increase of more than about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 1000-fold, or more compared to the reference. Protein or mRNA levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, ng / mL) or as a percentage of the total protein or mRNA in the sample.

[0072] As used herein, the term "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) flanked by two antiparallel regions of nucleic acid that are sufficiently complementary to each other such that under appropriate hybridization conditions (e.g., in a phosphate buffer or inside a cell), the two antiparallel regions flanking the unpaired region hybridize to form a double helix (called the "stem").

[0073] As used herein, the term "modified internucleotide bond" refers to an internucleotide bond that has one or more chemical modifications compared to a reference internucleotide bond that includes a phosphodiester bond. In some embodiments, the modified nucleotide is a non-naturally occurring linkage. Typically, the modified internucleotide bond confers one or more desirable properties to the nucleic acid in which the modified internucleotide bond is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0074] As used herein, the term "modified nucleotide" refers to a nucleotide that has one or more chemical modifications compared to a corresponding reference nucleotide selected from the following: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. In some embodiments, the modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, the modified nucleotide has one or more chemical modifications in its sugar, nucleobase, and / or phosphate group. In some embodiments, the modified nucleotide has one or more chemical moieties conjugated to the corresponding reference nucleotide. Typically, the modified nucleotide confers one or more desirable properties to the nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0075] A "nick tetraloop structure" is a structure of an RNAi oligonucleotide characterized by the presence of separate sense (passenger) and antisense (guide) strands, where the sense strand has a region of complementarity with the antisense strand, and where at least one of the strands, typically the sense strand, has a tetraloop configured to stabilize the adjacent stem region formed in at least one strand. The nick tetraloop structure creates a single break in the nucleotides of the sense and antisense strands, thereby preventing the strands from being covalently linked at that site.

[0076] The terms "nucleobase" and "base" include purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds during nucleic acid hybridization. In the context of the present disclosure, the term nucleobase also encompasses alternative nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as alternative nucleobases. Such variants are described, for example, in Hirao et al. (Accounts of Chemical Research, vol. 45: page 2055, 2012) and Bergstrom (Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1, 2009).

[0077] The term "nucleoside" refers to a monomeric unit of a nucleobase and a sugar moiety or an oligonucleotide having a nucleobase and a sugar moiety. Nucleosides can include naturally occurring nucleosides as well as alternative nucleosides such as those described herein. The nucleobase of a nucleoside can be a naturally occurring nucleobase or an alternative nucleobase. Similarly, the sugar moiety of a nucleoside can be a naturally occurring sugar or an alternative sugar.

[0078] As used herein, a "nucleotide" refers to a monomeric unit of an oligonucleotide that includes a nucleoside and an internucleoside linkage. The internucleoside linkage may or may not include a phosphate linkage. Similarly, a "linked nucleoside" may or may not be linked by a phosphate linkage. Many "alternative internucleoside linkages" are known in the art, including, but not limited to, phosphate, phosphorothioate, and boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNAs) (e.g., locked nucleosides (LNAs) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNAs), phosphotriesters, phosphorothioates, phosphoramidates, and other variants of the phosphate backbone of natural nucleosides, including those described herein.

[0079] As used herein, the term "oligonucleotide" refers to a short nucleic acid, e.g., a nucleic acid less than 100 nucleotides in length. An oligonucleotide may be either single-stranded or double-stranded. An oligonucleotide may or may not have a double helix region. As a non-limiting set of examples, an oligonucleotide may be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide, a short siRNA, or a single-stranded siRNA. In some embodiments, an oligonucleotide is an RNAi oligonucleotide.

[0080] As used herein, the term "overhang" refers to terminal non-base-paired nucleotides resulting from one strand or region that extends beyond the end of the complementary strand that forms a double helix with the strand or region. In some embodiments, the overhang comprises one or more unpaired nucleotides that extend from the double helix region at the 5'-end or 3'-end of the oligonucleotide (e.g., RNAi oligonucleotide). In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand of the oligonucleotide (e.g., RNAi oligonucleotide).

[0081] As used herein, the term "patient in need thereof" or "subject in need thereof" refers to the identification of a subject based on the need for treatment of a disease or disorder, e.g., a disease mediated by complement dysregulation (e.g., dysregulation associated with C3, dysregulation of one or all of the complement pathways (e.g., alternative, classical, and / or lectin pathways), etc.). A subject can be identified, for example, as having a need for treatment of a disease or disorder (e.g., a disease or disorder associated with complement pathway activation or dysregulation disclosed herein) based, for example, on early diagnosis by a skilled artisan (e.g., a physician).

[0082] "Percent sequence identity" to a reference oligonucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference oligonucleotide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways that are within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or to a given nucleic acid or amino acid sequence, B (alternatively, it can be expressed as a given nucleic acid or amino acid sequence, A, having a particular percent identity to, with, or to a given nucleic acid or amino acid sequence, B) is calculated as follows: (Fraction X / Y)×100 (where X is the number of nucleotides or amino acids that are scored as identical matches by a sequence alignment program (such as BLAST) in its alignment of A and B, and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0083] As used herein, "pharmaceutical acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle that can suspend or dissolve active compounds), and has the property of being substantially non-toxic and non-inflammatory in patients. Excipients include, for example, the following: anti-adhesive agents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (coloring agents), emollients, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, flow agents (glidants), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, the following: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0084] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt of any of the compounds described herein. For example, pharmaceutically acceptable salts of any of the compounds described herein include those that are within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid. The compounds described herein may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts, including inorganic or organic acids, or the salts may be prepared from inorganic or organic bases in the case of the acidic form of the compounds described herein. Often, compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases and methods for preparing suitable salts are well known in the art. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases.Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogensulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, and the like. Representative salts include phosphate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, such as, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0085] The term "pharmaceutical composition" as used herein refers to a composition containing a compound (e.g., an RNAi oligonucleotide) described herein, which is formulated with a pharmaceutical acceptable excipient and is optionally manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal. The pharmaceutical composition can be formulated, for example, for subcutaneous administration, for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); for intrathecal injection; for intracerebroventricular injection; for intraparenchymal injection; for oral administration in unit dosage form (e.g., tablet, capsule, caplet, gel cap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment; or any other pharmaceutical acceptable formulation.

[0086] 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, the phosphate analog is located at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. In some embodiments, the 5' phosphate analog contains a phosphatase-resistant linkage. Examples of phosphate analogs include 5' phosphonates, such as 5' methylene phosphonate (5'-MP) and 5'-(E)-vinyl phosphonate (5'-VP). In some embodiments, an oligonucleotide has a phosphate analog at the 4'-carbon position of the sugar at the 5'-terminal nucleotide (referred to as a "4'-phosphate analog"). One example of a 4'-phosphate analog is an oxymethyl phosphonate, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., at its 4'-carbon), or an analog thereof. See, for example, US Patent Publication No. 2019 / 0177729 (each of which is incorporated herein by reference for its content regarding phosphate analogs). Other modifications have been developed for the 5' end of oligonucleotides (see, for example, WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015), Nucleic Acids Res., 43(6):2993-3011; each of which is incorporated herein by reference for its content regarding phosphate analogs).

[0087] The term "probe" as used herein refers to any molecule that can selectively bind to a specific sequence, for example, a nucleic acid molecule such as mRNA. Probes can be synthesized using methods known and routine in the art, or derived from appropriate biological preparations. Probes may be specially designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0088] As used herein, the term "reduced expression" of a gene refers to a reduction in the amount of RNA transcripts or protein encoded by the gene and / or a reduction in the amount of activity of the gene in a cell or subject, as compared to a suitable reference cell or subject. For example, the act of treating a cell with an RNAi oligonucleotide (e.g., one having an antisense strand complementary to a C3 mRNA sequence) can result in a reduction in the amount of RNA transcripts, protein and / or activity (e.g., encoded by the C3 gene) as compared to a cell that has not been treated with the RNAi oligonucleotide. Similarly, "reducing expression" as used herein refers to an act that results in a decrease in expression of a gene (e.g., C3). Reduction in expression can be assessed by a reduction in serum concentration of C3, as described herein (e.g., for cells that have not been contacted with an oligonucleotide as described herein). Alternatively, decreased expression can be assessed by, for example, a reduction in the levels of C3 mRNA transcription and / or translation (e.g., at least a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, or 60% or more decrease, e.g., a decrease in the range of 1% to 60% or more) compared to a cell not contacted with an oligonucleotide described herein.

[0089] "Reference" means any useful standard used to compare protein or mRNA levels or activity. A reference can be any sample, standard, standard curve, or level used for comparison purposes. A reference can be a normal reference sample or a reference standard or level. A "reference sample" can be, for example, a control, e.g., a predefined negative control value such as a "normal control," or a previous sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (e.g., cell or tissue) from a subject without a disease; a sample from a subject diagnosed with a disease but not yet treated with a compound described herein; a sample from a subject treated with a compound described herein; or a sample of a known normal concentration of a purified oligonucleotide or protein (e.g., any described herein). "Reference standard or level" means a value or numerical value obtained from a reference sample. A "normal control value" is a predetermined value indicative of a non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range ("between X and Y"), a high threshold ("below X"), or a low threshold ("above X"). A subject with a measurement within the normal control value for a particular biomarker is typically considered to be "within the normal range" for that biomarker. A normal reference standard or level can be a value or value obtained from a normal subject without a disease or disorder (e.g., a disease or disorder associated with activation or dysregulation of the complement pathway); a subject treated with a compound described herein. In a preferred embodiment, the reference sample, standard, or level is matched to the subject sample by at least one of the following criteria: age, weight, sex, disease stage, and general health. A standard curve of purified oligonucleotide or protein levels, for example any of those described herein, within the normal reference range can also be used as a reference.

[0090] As used herein, the term "region of complementarity" refers to a region on the antisense strand of an oligonucleotide that is substantially complementary to all or part of a gene, primary transcript, sequence (e.g., a target sequence, e.g., a C3 nucleotide sequence), or an mRNA (e.g., C3) that is processed to disrupt the expression of an endogenous gene. If the region of complementarity is not completely complementary to the target sequence, mismatches may occur in the internal or terminal regions of the molecule. In general, mismatches are most tolerated in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the oligonucleotide (e.g., an RNAi oligonucleotide).

[0091] As used herein, the term "ribonucleotide" refers to a nucleotide having 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 an atom other than the 2' position, including modifications or substitutions of the ribose, phosphate group, or base.

[0092] As used herein, the term "RNAi oligonucleotide" refers to either (a) a double-stranded oligonucleotide having a sense strand (passenger) and an antisense strand (guide), where the antisense strand or a portion of the antisense strand is used by Argonaute 2 (Ago2) endonuclease in cleaving a target mRNA, or (b) a single-stranded oligonucleotide having a single antisense strand, where the antisense strand (or a portion of the antisense strand) is used by Ago2 endonuclease in cleaving a target mRNA. In some embodiments, the RNAi oligonucleotide comprises a loop region, such as a stem-loop, which comprises nucleosides as that term is defined herein. RNAi oligonucleotides include, for example, dsRNA, siRNA, and shRNA, which mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. RNAi oligonucleotides direct sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). RNAi oligonucleotides reduce expression of C3 in cells, e.g., cells in a subject, such as a mammalian subject. Generally, the majority of nucleosides in an RNAi oligonucleotide are ribonucleosides, but as described in detail herein, each or both strands may also include one or more non-ribonucleosides, such as deoxyribonucleosides and / or alternative nucleosides. The RNAi oligonucleotide is substantially in a double helical form. In some embodiments, the complementary base pairing of the double helical region of the RNAi oligonucleotide is formed between antiparallel sequences of nucleotides of covalently separate nucleic acid strands. In some embodiments, the complementary base pairing of the double helical region of the RNAi oligonucleotide is formed between antiparallel sequences of nucleotides of covalently linked nucleic acid strands. In some embodiments, the complementary base pairing of the double helical region of the RNAi oligonucleotide is formed from a single nucleic acid strand that is folded back (e.g., via a hairpin) to provide a complementary antiparallel sequence of nucleotides that base pair together. In some embodiments, the RNAi oligonucleotide comprises two covalently separate nucleic acid strands that are completely double helical with each other.However, in some embodiments, the RNAi oligonucleotide is partially double-stranded, e.g., comprises two covalently separate nucleic acid strands with overhangs at one or both ends. In some embodiments, the RNAi oligonucleotide comprises an antiparallel sequence of partially complementary nucleotides, and thus may have one or more mismatches, which may include internal or terminal mismatches.

[0093] As used herein, the terms "sense strand" and "passenger strand" refer to a strand of an RNAi oligonucleotide that includes a region that is substantially complementary to a region of the antisense strand. The region of the sense strand that is complementary to a region of the antisense strand is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%) identical to a portion of a target gene (e.g., C3 gene). For example, the sense strand may have a region that is at least 85% identical to a portion of SEQ ID NO: 12, e.g., over at least 10-36 nucleotides, e.g., over 10-31 nucleotides, 10-26 nucleotides, 10-20 nucleotides, or 10-15 nucleotides.

[0094] The terms "siRNA" and "short interfering RNA," also known as "small interfering RNA," refer to an RNA agent, optionally an RNAi agent, of about 10-50 nucleotides in length, whose strands have overhangs containing, for example, one, two, or three overhanging linked nucleosides, which can direct or mediate RNA interference. Naturally occurring siRNAs are produced from longer dsRNA molecules (e.g., >25 linked nucleosides in length) by the cellular RNAi machinery (e.g., Dicer or its homologs).

[0095] As used herein, the term "strand" refers to a single continuous sequence of nucleotides linked together via internucleotide bonds (e.g., phosphodiester bonds, phosphorothioate bonds). In some embodiments, the strand has two free ends, e.g., a 5' end and a 3' end.

[0096] As used herein, the term "subject" refers to any organism to which a composition according to the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans). A subject may be a human or animal that is seeking or in need of treatment, is in need of treatment, is undergoing treatment, will be undergoing treatment in the future, or is under the care of a professional trained in a particular disease or condition.

[0097] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring. Sugar also includes "sugar substitutes," defined as structures that can replace the furanose ring of a nucleoside. In certain embodiments, sugar substitutes are non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures may include simple variations on the natural furanose ring, such as six-membered rings, or may be more complex, as in the case of the acyclic systems used in peptide nucleic acids. Sugar substitutes may also include sugar substitutes in which the furanose ring is replaced with another ring system, for example, a morpholino or hexitol ring system. Sugar moieties useful in preparing oligonucleotides having a motif include, but are not limited to, β-D-ribose, β-D-2'-deoxyribose, substituted sugars (e.g., 2', 5' and bis substituted sugars), 4'-S-sugars (e.g., 4'-S-ribose, 4'-S-2'-deoxyribose and 4'-S-2' substituted ribose), bicyclic sugar surrogates (e.g., 2'-O-CH 2 -4' or 2'-O-(CH 2 ) 2 -4'-bridged ribose derived bicyclic sugars and sugar surrogates (e.g., where the ribose ring is replaced with a morpholino or hexitol ring system). The type of heterocyclic base and internucleoside linkage used at each position varies and is not a determining factor of the motif. In most nucleosides with surrogate sugar moieties, the heterocyclic nucleobase is generally maintained to allow hybridization.

[0098] As used herein, the term "stem loop" refers to a region of an oligonucleotide in which two regions have complementary nucleotide sequences when one is read in the 5' to 3' direction and the other is read in the 3' to 5' direction, and the nucleotides between the two regions form an unpaired loop. A stem loop region may also be referred to as a hairpin or hairpin loop.

[0099] As used herein, the term "strand" refers to an oligonucleotide comprising a chain of linked nucleosides. A "strand comprising a nucleobase sequence" refers to an oligonucleotide comprising a chain of linked nucleosides represented by a sequence designated using standard nucleobase nomenclature.

[0100] 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 is not otherwise derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

[0101] As used herein, the term "target" or "targeting" refers to an oligonucleotide capable of specifically binding to the C3 gene or C3 mRNA encoding the C3 gene product, e.g., an oligonucleotide capable of inhibiting said gene or said mRNA (e.g., by reducing the level of the protein encoded by said gene or mRNA) by methods known to those skilled in the art (e.g., in the fields of antisense and RNA interference).

[0102] As used herein, the term "targeting ligand" refers to a molecule (e.g., 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 can be conjugated to another substance for the purpose of targeting the other substance to the tissue or cell of interest. For example, in some embodiments, the targeting ligand can be conjugated to an oligonucleotide or a vector (e.g., a viral vector) that contains the oligonucleotide for the purpose of targeting the oligonucleotide to a particular tissue or cell of interest. In some embodiments, the targeting ligand selectively binds to a cell surface receptor. Thus, in some embodiments, conjugating the targeting ligand to the oligonucleotide or vector facilitates delivery of the oligonucleotide into a particular cell by selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of a complex that includes the oligonucleotide, the targeting ligand, and the receptor. In some embodiments, the targeting ligand is conjugated to the oligonucleotide via a linker that is cleaved after or during cellular internalization, such that the oligonucleotide is released from the targeting ligand inside the cell.

[0103] As used herein, the term "tetraloop" refers to a loop that enhances the stability of an adjacent duplex formed by hybridization of flanking sequences of nucleotides. The increased stability is detectable as an increase in the melting temperature (Tm) of the adjacent stem duplex that is higher than the Tm of the adjacent stem duplex that would be expected, on average, from a set of loops of comparable length composed of randomly selected nucleotide sequences. For example, a tetraloop can be synthesized by cleaving the tetraloop in 10 mM NaHPO 4can impart 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 to a hairpin comprising a duplex at least 2 base pairs in length. In some embodiments, the tetraloop can stabilize base pairs in the adjacent stem duplex by stacking interactions. Additionally, interactions between nucleotides in the tetraloop include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonds, and contact interactions (Cheong et al., Nature 1990 Aug.16;346(6285):680-2; Heus and Pardi, Science 1991 Jul.12;253(5016):191-4). In some embodiments, the tetraloop comprises or consists of 3-6 nucleotides, typically 4-5 nucleotides. In certain embodiments, the tetraloop comprises or consists of three, four, five, or six nucleotides, which may or may not be modified (e.g., conjugated to a targeting moiety). In one embodiment, the tetraloop consists of four nucleotides. Any nucleotide may be used in the tetraloop, and the standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden (1985) Nucl. Acids Res. 13:3021-3030. For example, the letter "N" may be used to mean that any base may be at that position, the letter "R" may be used to indicate that A (adenine) or G (guanine) may be at that position, and "B" may be used to indicate that C (cytosine), G (guanine), or T (thymine) may be at that position. Examples of tetraloops include the UNCG family of tetraloops (eg, UUCG), the GNRA family of tetraloops (eg, GAAA), and the CUUG tetraloop.(Woese et al., Proc Natl Acad Sci USA. 1990 November;87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov.11;19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family of tetraloops (e.g., d(GTTA), 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, 41(48), 14281-14292, 2002. SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL.78th; NO.2; pg.731(2000), which are incorporated herein by reference for their relevant disclosures. In some embodiments, the tetraloop is comprised within a nicked tetraloop structure.

[0104] A "therapeutically effective amount" or a "prophylactically effective amount" refers to an amount (administered in either single or multiple doses) of a disclosed oligonucleotide composition (e.g., an RNAi oligonucleotide, such as a dsRNA) that produces the desired local or systemic effect (e.g., treatment of one or more symptoms of a disease resulting from complement pathway activation or dysregulation). The oligonucleotides (e.g., RNAi oligonucleotides) used in the methods of the present disclosure may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0105] As used herein, the term "treating" refers to the act of providing medical care to a subject in need thereof, for example, by administering a therapeutic agent (e.g., an oligonucleotide described herein) to the subject for the purpose of improving the health and / or well-being of the subject with respect to an existing condition (e.g., disease, disorder) or for the purpose of preventing or reducing the likelihood of occurrence of a condition. In some embodiments, treating includes reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., disease, disorder) experienced by the subject. In some embodiments, the nucleic acids or oligonucleotides (e.g., RNAi oligonucleotides) described herein are used to control the cellular and clinical symptoms of a disorder of the complement pathway, for example, one or more of the diseases associated with complement pathway activation or dysregulation disclosed herein.

[0106] Detailed Description Described herein are oligonucleotides that target complement component (C3), known to play a specific role in complement pathway activation, such as RNAi oligonucleotides, including sense and antisense oligonucleotides, and pharma- ceutically acceptable salts thereof. Oligonucleotides can be administered to reduce the level and / or activity of C3 in cells (e.g., by hepatocytes). For example, oligonucleotides can be administered in vivo and internalized by cells (e.g., hepatocytes; e.g., by binding to sialoglycoprotein receptor (ASGPR)). After internalization of cells, oligonucleotides can be bound to RNA-induced silencing complex (RISC) to target C3 mRNA, thereby initiating the degradation of C3 mRNA and blocking its translation.

[0107] Diseases mediated by complement dysregulation are often the result of complement hyperactivity. Described herein are methods for treating diseases mediated by or associated with complement pathway activation or dysregulation by administration of the oligonucleotides described herein that reduce the expression level of C3. Examples of disorders mediated by or associated with complement pathway activation or dysregulation that can be treated by the oligonucleotides and compositions described herein include, for example, skin disorders, neurological disorders, renal disorders, acute care, rheumatic disorders, pulmonary disorders, dermatological disorders, hematological disorders, and ophthalmological disorders, such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), IgA nephropathy, lupus nephritis, C3 glomerulopathy (C3 G), dermatomyositis / autoimmune myositis, systemic sclerosis, demyelinating polyneuropathy, pemphigus, membranous nephropathy, focal segmental glomerular sclerosis (FSGS), bullous pemphigoid, epidermolysis bullosa acquisita (EBA), mucous membrane pemphigoid, ANCA vasculitis, hypocomplementemic urticarial vasculitis, immune complex small vasculitis, cutaneous small vasculitis, autoimmune necrotizing myopathy, rejection of transplanted organs such as kidney, liver, heart or lung transplant rejection, antibody-mediated rejection (AMR) such as chronic AMR (cAMR), antiphospholipids, anti-inflammatory drugs ... Lipid (aPL) antibody syndrome, glomerulonephritis, asthma, dense deposit disease (DDD), age-related macular degeneration (AMD), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), severe refractory RA, Felty syndrome, multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, ischemia-reperfusion injury, preeclampsia, impaired function after organ transplant in acute kidney injury (DGF-AKI), cardiopulmonary bypass-associated acute kidney injury, hypoxic-ischemic encephalopathy, dialysis-induced thrombosis, Takayasu's arteritis, relapsing polychondritis, acute / prophylactic Graft-versus-host disease, chronic graft-versus-host disease, beta-thalassemia, stem cell transplant-associated thrombotic microangiopathy, biliary atresia, inflammatory liver disease, Behçet's disease, ischemic stroke, intracerebral hemorrhage, scleroderma, scleroderma renal crisis, scleroderma-associated interstitial lung disease (SSc-ILD), sickle cell disease, autosomal dominant polycystic kidney disease (ADPKD), chemotherapy-induced peripheral neuropathy (CIPN), diabetic neuropathy, amyotrophic lateral sclerosis (ALS), diabetic nephropathy, diabetic retinopathy, geographic atrophy, pulmonary arterial hypertension,Refractory severe asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF), chronic lung allograft dysfunction, pulmonary disease states in cystic fibrosis, hidradenitis suppurativa, nonalcoholic fatty liver disease (NASH), ankylosing spondylitis, hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) (prevention), coronary artery disease, atherosclerosis, osteoporosis (prevention), osteoarthritis, high-risk drusen, inflammatory bowel disease, ulcerative colitis, interstitial cystitis, dialysis-induced complement activation, pyoderma gangrenosum, chronic heart failure, autoimmune myocarditis, nasal polyposis, acute and chronic pancreatitis, atherosclerosis, eosinophilic esophagitis, eosinophilic granulomatosis, hypereosinophilic syndrome, wound healing, and thrombotic thrombocytopenic purpura (TTP).

[0108] The compositions and methods described herein feature oligonucleotides (eg, RNAi oligonucleotides) comprising a sense strand and an antisense strand, which have substantial sequence identity to a region of the C3 gene.

[0109] Oligonucleotides (e.g., RNAi oligonucleotides) can be used to modulate complement pathway activity, for example, by reducing the level and / or activity of C3 in cells (e.g., hepatocytes), such as cells of a subject (e.g., a human) in need thereof. The overall design targets C3 in the complement pathway, leaving other pathways intact (protection), including the alternative, classical, and lectin pathways. Thus, the present disclosure features compositions and methods for treating diseases or disorders mediated by complement pathway activation or dysregulation, such as diseases or disorders mediated by activation or dysregulation of C3.

[0110] Complement component 3 target sequence Provided herein are oligonucleotide-based inhibitors of C3 expression that can be used to achieve therapeutic benefit. Examination of C3 mRNA (see, e.g., Example 3) and in vitro and in vivo testing have found that the sequence of C3 mRNA is amenable to oligonucleotide-based inhibition and therefore useful as a targeting sequence. For example, the C3 target sequence may comprise or consist of the sequence set forth in either SEQ ID NO: 13 or SEQ ID NO: 14, which correspond to nucleotides 4121-4141 and 780-798, respectively, of Homo sapiens complement C3 with reference sequence NM_0.000064.4 (SEQ ID NO: 12). These C3 sequences may be the target sequences of Compound A and Compound B, respectively, and their variants described herein having up to 85% sequence identity thereto. Compounds A and B (and variants thereof described herein) may also effectively target Rhesus macaque and Cynomolgus macaque complement C3, which have reference sequences XM_015122636.2 and XM_005587719.2, respectively. Additionally, the C3 target sequence may comprise or consist of any of the sequences set forth in SEQ ID NO:31, which corresponds to nucleotides 2903-2922 of mus musculus complement C3, which has reference sequence NM_009778.3 (SEQ ID NO:32), which may be targeted by compound J (e.g., an RNAi oligonucleotide having the sense sequence of SEQ ID NO:15 and the antisense sequence of SEQ ID NO:16). Compound J may also target Rattus norvegicus complement C3, which has reference sequence NM_016994.2. These regions of the C3 mRNA can be targeted with RNAi oligonucleotides, such as the dsRNA agents described herein, in order to inhibit C3 mRNA expression and subsequently C3 protein expression.

[0111] In some embodiments, the antisense strand of an oligonucleotide (e.g., RNAi oligonucleotide) agent provided herein can be designed to have a region of complementarity to C3 mRNA (e.g., within the target sequence of C3 mRNA) for the purpose of targeting the mRNA in a cell and inhibiting its expression. The region of complementarity is generally of a length and base content suitable to facilitate annealing of the oligonucleotide (e.g., RNAi oligonucleotide) or strand thereof to C3 mRNA for the purpose of inhibiting its transcription. The region of complementarity can be at least 11, e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides in length. For example, the oligonucleotides provided herein can have a region of complementarity to C3 mRNA ranging from 12-30 (e.g., 12-30, 12-22, 15-25, 17-21, 18-27, 19-27, or 15-30) nucleotides in length. Thus, the oligonucleotides provided herein may have a region of complementarity to C3 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. In some examples, the oligonucleotides provided herein may have a region of complementarity to C3 mRNA that is 19 nucleotides in length. In certain embodiments, the region of complementarity of the oligonucleotide (e.g., the antisense strand of an RNAi oligonucleotide) may be complementary to a contiguous sequence of nucleotides of the sequence set forth in SEQ ID NO: 12 that is 20 nucleotides in length.

[0112] In certain instances, the RNAi oligonucleotides of the present disclosure may comprise a region of complementarity (e.g., on the antisense strand of the RNAi oligonucleotide) that is at least partially complementary to the sequence set forth in SEQ ID NO: 12. For example, the oligonucleotides disclosed herein may comprise a region of complementarity (e.g., on the antisense strand of the RNAi oligonucleotide) that is fully complementary to the sequence set forth in SEQ ID NO: 12. The region of complementarity of the oligonucleotide (e.g., on the antisense strand of the RNAi oligonucleotide) may be complementary to a contiguous sequence of nucleotides of the sequence set forth in SEQ ID NO: 12, said sequence ranging from 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, the region of complementarity of an oligonucleotide (e.g., on the antisense strand of an RNAi oligonucleotide) may be complementary to a sequence of contiguous nucleotides of the sequence set forth in SEQ ID NO: 12 that is 19 nucleotides in length. In certain embodiments, the region of complementarity of an oligonucleotide (e.g., the antisense strand of an RNAi oligonucleotide) may be complementary to a sequence of contiguous nucleotides of the sequence set forth in SEQ ID NO: 12 that is 20 nucleotides in length.

[0113] The region of complementarity of the oligonucleotide that is complementary to the consecutive nucleotides of the sequence described in SEQ ID NO: 12 can span a part of the entire length of the antisense strand.For example, the region of complementarity of the oligonucleotide that is complementary to the consecutive nucleotides of the sequence described in SEQ ID NO: 12 can span at least 85% (for example, at least 86%, at least 90%, at least 95%, and at least 99%) of the entire length of the antisense strand.In certain embodiments, the region of complementarity of the oligonucleotide that is complementary to the consecutive nucleotides of the sequence described in SEQ ID NO: 12 can span the entire length of the antisense strand.

[0114] The region of complementarity to C3 mRNA may have one or more mismatches compared to the corresponding sequence of C3 mRNA. For example, the region of complementarity on the oligonucleotide (e.g., an oligonucleotide 20-50 nucleotides in length, e.g., an oligonucleotide 20-25 nucleotides in length (e.g., 22 nucleotides in length) may have up to 1, up to 2, up to 3, up to 4, or up to 5 mismatches, so long as the oligonucleotide maintains the ability to form complementary base pairs with C3 mRNA under appropriate hybridization conditions. Alternatively, the region of complementarity on the 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, so long as the oligonucleotide maintains the ability to form complementary base pairs with C3 mRNA under appropriate hybridization conditions. If there are multiple mismatches in the region of complementarity, it is preferred that the oligonucleotide is capable of forming complementary base pairs with C3 mRNA under appropriate hybridization conditions. As long as the ability to form complementary base pairs with the mRNA is maintained, the mismatches can be consecutive (e.g., 2, 3, 4, or 5 consecutive) or can be scattered throughout the region of complementarity. For example, the RNAi oligonucleotide can include a sense oligonucleotide having the sequence of SEQ ID NO: 4 and its variants having up to 1, 2, 3, 4, or 5 mismatches with the corresponding C3 sequence of SEQ ID NO: 12, or a corresponding antisense sequence of SEQ ID NO: 6 and its variants having 1, 2, 3, 4, or 5 mismatches with the sequence of SEQ ID NO: 4.

[0115] Types of oligonucleotides There are a variety of oligonucleotide structures useful for targeting C3 in the methods of the present disclosure, including RNAi, antisense miRNA, shRNA, etc. Any of the structures described herein or elsewhere can be used as a framework to incorporate or target the sequences described herein (e.g., hotspot sequences of C3, such as those in SEQ ID NO: 13 or 14).

[0116] The compositions described herein, which are oligonucleotides (e.g., RNAi oligonucleotides), encode inhibitory constructs (e.g., nucleic acid vectors encoding same) that target C3 mRNA (e.g., SEQ ID NO: 12). Oligonucleotides for reducing expression of C3 expression can participate in the RNA interference (RNAi) pathway upstream or downstream of Dicer involvement. For example, oligonucleotides (e.g., RNAi oligonucleotides) have been developed that are 19-25 nucleotides in length and include at least one sense or antisense strand with a 1-5 nucleotide 3' overhang (see, e.g., U.S. Pat. No. 8,372,968, incorporated herein by reference). Longer oligonucleotides have also been developed that are processed by Dicer to produce active RNAi products (see, e.g., U.S. Pat. No. 8,883,996, incorporated herein by reference). Additionally, extended oligonucleotides (e.g., RNAi oligonucleotides) have also been generated in which either or both of the 5' or 3' ends of either or both of the antisense and sense strands are extended beyond the double helix targeting region such that either the sense or antisense strand contains a thermodynamically stabilized tetraloop structure (see, e.g., U.S. Pat. Nos. 8,513,207 and 8,927,705, and WO 2010033225, which are incorporated herein by reference for their disclosure of these oligonucleotides). Such structures may include single-stranded extensions at either or both of the 5' and 3' ends of the molecule, as well as RNAi extensions.

[0117] Additionally or alternatively, the oligonucleotides provided herein can be designed to participate in the RNA interference pathway downstream of Dicer participation, i.e., after cleavage by Dicer. Such oligonucleotides can have an overhang of 1, 2, or 3 nucleotides at the 3' end of the sense strand. Such oligonucleotides, for example siRNAs, can include a 22-nucleotide guide strand (e.g., SEQ ID NOs: 13 and 14) that is antisense to the target RNA and a complementary passenger strand, where both strands anneal to form a 20bp double helix and a 2-nucleotide overhang at one or both 3' ends. Longer oligonucleotide designs are also available, including oligonucleotides with a 23-nucleotide guide strand and a 21-nucleotide passenger strand, where the 3' end of the passenger strand and the 5' end of the guide strand are blunt-ended, and the 5' end of the passenger strand and the 3' end of the guide strand have a 2-nucleotide 3' guide strand overhang to the left of the molecule. In such molecules, there is a 21 base pair double helical region (see U.S. Pat. Nos. 9,012,138, 9,012,621, and 9,193,753, which are incorporated by reference herein for their disclosures regarding longer oligonucleotides).

[0118] The oligonucleotides disclosed herein can include sense and antisense strands, both of which are 17-26 (e.g., 17-26, 20-25, or 21-23) nucleotides in length. For example, the oligonucleotides disclosed herein can include sense and antisense strands, both of which are 19-22 nucleotides in length. The sense and antisense strands can also be the same length. Alternatively, the oligonucleotides can include sense and antisense strands, such that there is a 3'-overhang on either the sense or antisense strand, or on both the sense and antisense strands. For example, the 3' overhangs on the sense strand, antisense strand, or both the sense and antisense strands can be 1 or 2 nucleotides in length. In some embodiments, the oligonucleotides have a 22 nucleotide antisense strand and a 20 nucleotide sense strand, with a blunt end on the "right side" of the molecule (i.e., the 3' end of the passenger strand and the 5' end of the guide strand) and a 2 nucleotide 3'-guide strand overhang on the "left side" of the molecule (i.e., the 5' end of the passenger strand and the 3' end of the guide strand). In such a molecule, there may be, for example, a 20 base pair double helical region.

[0119] Other oligonucleotide designs for use with the compositions and methods disclosed herein include, for example, 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., with stems of 19 bp or less; see, e.g., Moore et al. Methods Mol. Biol. 2010; 629:141-158), blunt siRNAs (e.g., 19 bp in length; see, e.g., Kraynack and Baker, RNA Vol. 12, p163-176 (2006)), asymmetric siRNAs (aiRNAs; see, e.g., Sun et al., Nat. Biotechnol. 26, 1379-1382 (2008)), asymmetric short duplex siRNAs (see, e.g., Chang et al., Mol Ther. 2009), and the like. Apr;17(4):725-32), forked siRNA (e.g., Hohjoh, FEBS Letters, Vol 557, issue 1-3; Jan 2004, p 193-198), single-stranded siRNA (Elsner; Nature Biotechnology 30,1063(2012)), dumbbell-shaped circular siRNA (e.g., Abe et al. J Am Chem Soc 129:15108-15109(2007)), as well as internally segmented small interfering RNA (siRNA; see, e.g., Bramsen et al., Nucleic Acids Res. 2007 Sep;35(17):5886-5897). Each of the foregoing references is incorporated by reference in its entirety for the relevant disclosures therein. Further non-limiting examples of oligonucleotide structures that may be used in some embodiments to reduce or inhibit expression of C3 are microRNAs (miRNAs), short hairpin RNAs (shRNAs), and small siRNAs (see Hamilton et al., Embo J., 2002, 21(17):4671-4679; see also U.S. Patent Application Publication No. 2009 / 0099115).

[0120] Oligonucleotides Oligonucleotides (e.g., RNAi oligonucleotides) for targeting C3 expression via the RNAi pathway generally have sense and antisense strands that form a double helix with each other. Oligonucleotides (e.g., RNAi oligonucleotides) may be single-stranded or double-stranded ribonucleic acid (dsRNA). In addition, the sense and antisense strands may not be covalently linked; for example, the oligonucleotide may have a nick between the sense and antisense strands. Oligonucleotides (e.g., RNAi oligonucleotides) may be in the form of a pharma- ceutically acceptable salt. For example, oligonucleotides (e.g., RNAi oligonucleotides) may be in the form of a sodium salt.

[0121] Although the aforementioned oligonucleotide (e.g., RNAi oligonucleotide) sequences are represented as RNA sequences that can be synthesized in a cell; these sequences can also be represented as corresponding DNA (e.g., cDNA) that can be incorporated into the vectors of the present disclosure. Those skilled in the art will understand that cDNA sequences are equivalent to mRNA sequences, except for the replacement of uridine with thymidine, and can be used herein for the same purpose, i.e., to generate antisense oligonucleotides to inhibit the expression of C3 mRNA. In the case of DNA, the polynucleotide comprising the antisense nucleic acid is a DNA sequence. The DNA sequence may correspond to the antisense strand of Compound A or Compound B, and may have the polynucleotide sequence of SEQ ID NO: 34 or SEQ ID NO: 35, respectively, or have at least 85% or more sequence identity thereto. The DNA sequence may correspond to the sense strand of Compound A or Compound B, and may have the polynucleotide sequence of SEQ ID NO: 33 or SEQ ID NO: 35, respectively, or have at least 85% or more sequence identity thereto. In the case of RNA vectors, the transgene cassette incorporates the RNA equivalent of the antisense DNA sequence described herein.

[0122] In certain embodiments, the sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:4 or SEQ ID NO:5. For example, the sense strand may comprise an oligonucleotide sequence of SEQ ID NO:4, as in Compound B. In other embodiments, the sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2. For example, the sense strand may comprise an oligonucleotide sequence of SEQ ID NO:1, as in Compound A.

[0123] In some embodiments, the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 6. In other embodiments, the antisense strand may comprise at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 3. For example, the antisense strand may comprise the oligonucleotide sequence of SEQ ID NO: 6, as in the case of Compound B, and / or the antisense strand may comprise the oligonucleotide sequence of SEQ ID NO: 3, as in the case of Compound A.

[0124] Further, the sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:4 or SEQ ID NO:5, and the antisense strand may comprise at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:6. The oligonucleotide (e.g., RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO:4 or SEQ ID NO:5, and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO:6, as shown in FIG. 2B for Compound B.

[0125] In addition, the sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and the antisense strand may comprise at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:3. Furthermore, the oligonucleotide (e.g., RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2, and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO:3, as shown in Figures 1D and 1E for Compound A. The oligonucleotide provided herein may comprise a sense strand having a sequence set forth in any of SEQ ID NOs:1, 2, 4, and 5, and an antisense strand comprising a complementary sequence selected from SEQ ID NOs:3 and 6.

[0126] Additionally, the sense strand may comprise the oligonucleotide sequence of SEQ ID NO:37 and the antisense strand may comprise the oligonucleotide sequence of SEQ ID NO:38 as shown below. Sense strand (SEQ ID NO:37): 5'mA-S-mU-mC-mA-mA-mC-mU-fC-fA-fC-fC-mU-mG-mU-mA-mA-mU-mA-mA-mA-mG-mC-mA- mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' Hybridized with: Antisense strand (SEQ ID NO:38): 5'[MePhosphonate-4O-mU]-S-fU-S-fU-fA-fU-mU-fA-mC-mA-fG-mG-mU-mG-fA-mG-mU-mU-mG-mA-mU-S-mG-S-mG 3' (mX is a 2'-O-methyl ribonucleotide, fX is a 2'-fluoro-deoxyribonucleotide, [ademA-GalNAc] is a 2'-O-GalNAc modified adenosine, [Mephosphonate-4O-mU] is a 4'-O-monomethylphosphonate-2'-O-methyluridine, "-" indicates a phosphodiester bond, and "-S-" indicates a phosphorothioate bond, as shown in FIG. 1E. In some embodiments, the antisense strand can be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 38. In some embodiments, the sense strand can be a pharma-ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 37.

[0127] Further, the sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:1, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:3. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO:1 and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO:3, as shown for Compound A. The sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:4, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:6. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO: 4 and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO: 6, as shown for compound B. The sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 17, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 18. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO: 17 and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO: 18, as shown for compound C.The sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 19, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 20. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising the oligonucleotide sequence of SEQ ID NO: 19 and an antisense strand comprising the oligonucleotide sequence of SEQ ID NO: 20, as shown for Compound D. The sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 21, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 22. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising the oligonucleotide sequence of SEQ ID NO: 21 and an antisense strand comprising the oligonucleotide sequence of SEQ ID NO: 22, as shown for Compound E. The sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 23, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 24. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising the oligonucleotide sequence of SEQ ID NO: 23 and an antisense strand comprising the oligonucleotide sequence of SEQ ID NO: 24, as shown for Compound F.The sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:25, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:26. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO:25 and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO:26, as shown for compound G. The sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:27, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO:28. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO: 27 and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO: 28, as shown for compound H. The sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 29, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 30. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO: 29 and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO: 30, as shown for compound I.The sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 15, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) sequence identity to SEQ ID NO: 16. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO: 15 and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO: 16, as shown for compound J. See Table 1 for examples of sense and antisense strand pairs.

[0128] [Table 1]

[0129] The oligonucleotide (e.g., RNAi oligonucleotide) comprises a double helix region between the sense strand and the antisense strand. The double helix formed between the sense strand and the antisense strand can be 10-30 nucleotides long (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides long). Thus, the double helix formed between the sense strand and the antisense strand can be 15-25 nucleotides long (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotides long). In some embodiments, the double helix region can be 20 nucleotides long.

[0130] The region on the sense strand that forms a duplex with the antisense strand can have a nucleotide sequence that is at least 85% identical (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the oligonucleotide sequence of any of SEQ ID NOs: 2 and 5. For example, the region on the sense strand that forms a duplex with the antisense strand can have the oligonucleotide sequence of any of SEQ ID NOs: 2 and 5.

[0131] Furthermore, the double helix formed between the sense and antisense strands does not have to span the entire length of the sense and / or antisense strand.

[0132] The oligonucleotide (e.g., RNAi oligonucleotide) can include a sense strand longer than 22 nucleotides (e.g., 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long), e.g., a 36 nucleotide sense strand, and an antisense strand 18-36 nucleotides long, e.g., a 22 nucleotide antisense strand. The oligonucleotide (e.g., RNAi oligonucleotide) has a length such that, when acted upon by the Dicer enzyme, it results in the antisense strand being incorporated into mature RISC.

[0133] The oligonucleotides provided herein can have one 5' end that is less thermodynamically stable than the other 5' end. The oligonucleotides provided herein can be asymmetric oligonucleotides that include a blunt end at the 3' end of the sense strand and an overhang at the 3' end of the antisense strand. The 3' overhang on the antisense strand can be 1-8 nucleotides long (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides long). For example, the 3' overhang on the antisense strand can be 2 nucleotides long. Typically, oligonucleotides for RNAi have a 2 nucleotide overhang at the 3' end of the antisense, guide, strand; however, other overhangs are possible. In other embodiments, the 3' overhang may have a length of 1-6 nucleotides, optionally 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 3-5, 5-6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides. In some examples, the oligonucleotide may have an overhang at the 5' end. The overhang may be a 5' overhang comprising a length of 1-6 nucleotides, optionally 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-6, 4-5, 3-4, 4-6, 4-5, 5-6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides.

[0134] The two terminal nucleotides at the 3' end of the antisense strand may be modified. In certain embodiments, the two terminal nucleotides at the 3' end of the antisense strand may be complementary to the target C3 mRNA. Alternatively, the two terminal nucleotides at the 3' end of the antisense strand may not be complementary to the target C3 mRNA. In some embodiments, the two terminal nucleotides at the 3' end of the antisense strand may be GG. Typically, one or both of the two terminal GG nucleotides at each 3' end of the oligonucleotide are not complementary to the target.

[0135] There may be one or more (e.g., 1, 2, 3, 4, 5) mismatches in complementarity between the sense strand and the antisense strand. When there are two or more mismatches between the sense strand and the antisense strand, they may be located consecutively (e.g., two, three or more consecutively) or may be scattered throughout the region of complementarity. For example, the 3' end of the sense strand may contain one or more mismatches. Thus, two mismatches may be incorporated into the 3' end of the sense strand. Base mismatches or destabilization of the segment at the 3' end of the sense strand of the oligonucleotide may improve the efficacy of synthetic duplexes in RNAi, possibly by promoting processing by Dicer.

[0136] It should be understood that in some embodiments, the sequences presented in the sequence listing may be referenced to represent 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., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modifications compared to the designated sequence while retaining essentially the same or similar complementary properties as the designated sequence.

[0137] Antisense strand The antisense strand of oligonucleotide may also be called guide strand.For example, if antisense strand can participate in RNA-induced silencing complex (RISC) and bind to Argonaute protein, or participate in or bind to one or more similar factors, and can direct the direct silencing of target gene, it can be called guide strand.

[0138] In certain embodiments, the antisense strand is less in nucleotide length than the sense strand. In some examples, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) can have an antisense strand that comprises 10-40 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 nucleotides) in length. Thus, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) can have an antisense strand that comprises 15-30 nucleotides (e.g., 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides) in length. For example, the antisense strand can comprise 20-25 nucleotides (e.g., 20, 21, 22, 23, 24, and 25 nucleotides) in length. In certain embodiments, the antisense strand can be 22 nucleotides in length.

[0139] The oligonucleotides disclosed herein may comprise an antisense strand that comprises a contiguous sequence between 12-22 nucleotides (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 nucleotides) in length that is complementary to the sequence of SEQ ID NO: 12. For example, the oligonucleotide may comprise an antisense strand that comprises a contiguous sequence between 15-21 nucleotides (e.g., 15, 16, 17, 18, 19, 20, and 21 nucleotides) in length that is complementary to the sequence of SEQ ID NO: 12. In some embodiments, the oligonucleotide may comprise an antisense strand having a contiguous sequence 19 nucleotides in length that is complementary to the sequence of SEQ ID NO: 12.

[0140] The oligonucleotides disclosed herein may comprise an antisense strand having the sequence of either SEQ ID NO: 3 or 6. In some embodiments, the oligonucleotides disclosed herein may comprise an antisense strand having the amino acid sequence of SEQ ID NO: 6, such as compound B shown in FIG. 2B. In some embodiments, the antisense strand may be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 6. SEQ ID NO: 6 may have a chemical structure as shown in FIG. 1B. Alternatively, the antisense strand may have the sequence of SEQ ID NO: 3, such as compound A shown in FIG. 1D and FIG. 1E. In some embodiments, the antisense strand may be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 3.

[0141] In addition, the first position of the 5' end of the antisense strand can be a uridine. The uridine can include a phosphate analog; for example, the uridine can be 4'-O-monomethylphosphonate-2'-O-methyluridine.

[0142] Sense strand The sense strand of an oligonucleotide may also be referred to as a passenger strand. In certain embodiments, the passenger strand is longer in length and has more nucleotides than the guide strand. In some examples, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) may have a sense strand that includes 10 to 45 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45 nucleotides) in length. Thus, an oligonucleotide (e.g., an RNAi oligonucleotide) can have a sense strand that comprises 20-50 nucleotides (e.g., 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, and 50 nucleotides) in length. In certain embodiments, the sense strand can be 20 nucleotides in length. In other embodiments, the sense strand can be 36 nucleotides in length.

[0143] The oligonucleotide may have a sense strand that includes a contiguous sequence of 7 to 36 nucleotides (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36 nucleotides) long relative to the sequence of SEQ ID NO: 12. Thus, the sense strand may include a contiguous sequence of 10 to 30 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides) long relative to the sequence of SEQ ID NO: 12. In some embodiments, the oligonucleotide disclosed herein may include a sense strand that includes a contiguous sequence of nucleotides relative to the sequence of SEQ ID NO: 12, which is 19 nucleotides long.

[0144] The sense strand may include a stem loop at its 3' end. In some embodiments, the sense strand includes a stem loop at its 5' end. The sense strand including the stem loop may be 10-50 nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 nucleotides in length). Thus, the sense strand including the stem loop can be 20 to 40 nucleotides in length (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 nucleotides in length). For example, the sense strand including the stem loop can be 36 nucleotides in length.

[0145] Furthermore, the stem loop region on the sense strand may form a double helical region with itself. The double helical region contained in the stem loop may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides in length. For example, the double helical region contained in the stem loop may be 6 nucleotides in length. The stem loop may provide protection against degradation (such as enzymatic degradation) to the RNAi oligonucleotide and may promote targeting properties for delivery to target cells. For example, the loop may provide additional nucleotides that may be modified without substantially affecting the gene expression inhibitory activity of the oligonucleotide. In certain embodiments, the sense strand may be (e.g., at its 3' end) S 1 -LS 2 Provided herein is an oligonucleotide comprising a stem loop described as: 1 is S 2 and L is complementary to S 1 and S. 2 A loop of up to 10 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length) is formed between S 1 and S. 2The loop between S and S may be 4 nucleotides long and form a tetraloop, as described herein. 1 The region is 6 nucleotides long and 2 The L region is 6 nucleotides long and the L region is a 4 nucleotide tetraloop.

[0146] The sense strand of the oligonucleotide (e.g., RNAi oligonucleotide) may comprise a stem-loop region and a region that forms a double helix with the antisense strand. The stem-loop region may comprise a nucleotide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the oligonucleotide sequence of SEQ ID NO:7. In some embodiments, the stem-loop region has the oligonucleotide sequence of SEQ ID NO:7.

[0147] The loop (L) of the stem-loop may be a tetraloop (e.g., in a nick tetraloop structure). The loop of the stem-loop may have the base sequence of SEQ ID NO: 8. The tetraloop may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, the loop of the stem-loop has 4-5 nucleotides. However, in some embodiments, the loop of the stem-loop may comprise 3-6 nucleotides. For example, the loop of the stem-loop may comprise 3, 4, 5, or 6 nucleotides. The loop of the stem-loop may comprise a combination of guanosine and adenosine nucleic acid residues.

[0148] The oligonucleotides disclosed herein may include a sense strand sequence having any one of the polynucleotide sequences of SEQ ID NO: 1, 2, 4, and 5. The sense strand may have the sequence of SEQ ID NO: 4, such as compound B shown in FIG. 2B. SEQ ID NO: 4 may have the chemical structure shown in FIG. 1A. In some embodiments, the sense strand may be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 4. Alternatively, the sense strand may have the base sequence of SEQ ID NO: 1, such as compound A shown in FIG. 1D and FIG. 1E. In some embodiments, the sense strand may be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 1.

[0149] Oligonucleotide Modification Oligonucleotides can be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, base pairing properties, RNA distribution and cellular uptake, and other characteristics relevant for therapeutic or research applications (Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881; Bramsen et al., Frontiers in Genetics, 3(2012):1-22). Thus, in some embodiments, the oligonucleotides of the present disclosure can include one or more suitable modifications. Modified nucleotides can have modifications in their base or nucleobase, sugar (e.g., ribose, deoxyribose), or phosphate groups.

[0150] The number of modifications on an oligonucleotide and the location of those nucleotide modifications can affect the properties of the oligonucleotide. For example, oligonucleotides can be delivered in vivo by conjugating them to or incorporating them into lipid nanoparticles (LNPs) or similar carriers. However, when an oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least a portion of the nucleotides to be modified. Thus, in certain embodiments of any of the oligonucleotides provided herein, all or substantially all of the nucleotides of the oligonucleotide are modified. In certain embodiments, more than half of the nucleotides are modified. In other embodiments, less than half of the nucleotides are modified. Typically, for naked delivery, all sugars are modified at the 2' position. These modifications can be reversible or irreversible. The oligonucleotides disclosed herein can have a sufficient number and type of modified nucleotides to provide desired properties (e.g., protection from enzymatic degradation, ability to target desired cells after in vivo administration, and / or thermodynamic stability).

[0151] sugar modification Modified sugars, also referred to herein as sugar analogs, include modified deoxyribose or ribose moieties where one or more modifications are present at the 2', 3', 4', and / or 5' carbon positions of the sugar. Modified sugars can also include non-natural alternative carbon structures such as those found in locked nucleic acids ("LNAs") (see Koshkin et al. (1998), Tetrahedron 54, 3607-3630), unlocked nucleic acids ("UNAs") (see Snead et al. (2013), Molecular Therapy-Nucleic Acid, 2, e103), and bridged nucleic acids ("BNAs") (see Imanishi and Obika (2002), The Royal Society of Chemistry, Chem. Commun., 1653-1659). Koshkin et al., Snead et al., and Imanishi and Obika are incorporated herein by reference for their disclosures regarding sugar modifications.

[0152] Nucleotide modifications at the sugar may include 2'-modifications. The 2'-modifications may be 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid. Typically, the modifications are 2'-fluoro, 2'-O-methyl, or 2'-O-methoxyethyl. In some embodiments, the modifications are 2'-fluoro and / or 2'-O-methyl. In some embodiments, the 2'-fluoro modification is a 2'-fluoro deoxyribonucleoside and / or the 2'-O-methyl modification is a 2'-O-methylribonucleoside. Modifications at the sugar may include modifications of the sugar ring or may have modifications of one or more carbons of the sugar ring. For example, modifications of the sugar of a nucleotide may include the 2'-oxygen of the sugar linked to the 1'-carbon or 4'-carbon of the sugar, or the 2'-oxygen linked to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In certain embodiments, modified nucleotides may have acyclic sugars that lack a 2'-carbon-3'-carbon bond. In some embodiments, modified nucleotides may have a thiol group, for example, at the 4' position of the sugar.

[0153] The oligonucleotides (e.g., RNAi oligonucleotides) described herein can contain 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). For example, the sense strand of the oligonucleotide can contain 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). Also, for example, the antisense strand of the oligonucleotide can contain at least one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).

[0154] In certain embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) may contain 20-50 (e.g., 20-30, 24-30, 28-30, 30-40, 34-40, 38-44, 44-50, and 48-50) modified nucleotides.

[0155] All nucleotides of the sense strand of the oligonucleotide may be modified. Additionally, all nucleotides of the antisense strand of the oligonucleotide may be modified. In some embodiments, all nucleotides of the oligonucleotide (e.g., RNAi oligonucleotide) including both the sense strand and the antisense strand are modified. The modified nucleotide may be 2'-modified (e.g., 2'-fluoro or 2'-O-methyl). The 2'-modification to the nucleotide may be 2'-fluoro and / or 2'-O-methyl, and optionally, the 2'-fluoro modification is a 2'-fluoro deoxyribonucleoside and / or the 2'-O-methyl modification is a 2'-O-methyl ribonucleoside.

[0156] The present disclosure provides oligonucleotides with different modification patterns. An oligonucleotide comprising a sense strand and an antisense strand may comprise 40-50 (e.g., 41, 2, 43, 44, 45, 46, 47, 48, and 49) 2'-O-methyl modifications. The modified oligonucleotide may comprise a sense strand having a nucleotide sequence of either SEQ ID NO: 1 or 4 and an antisense strand having a nucleotide sequence of either SEQ ID NO: 3 or 6 (e.g., an RNAi oligonucleotide may have a sense strand of SEQ ID NO: 4 and an antisense strand of SEQ ID NO: 6, or an RNAi oligonucleotide may have a sense strand of SEQ ID NO: 1 and an antisense strand of SEQ ID NO: 3). In some embodiments, these oligonucleotides are modified at one or more of positions 1, 2, 3, 4, 5, 6, 7, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, and 36 of the sense strand and / or at one or more of positions 1, 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, and 22 of the antisense strand with a 2'-O-methyl modified nucleoside, e.g., a 2'-O-methyl ribonucleoside. In some embodiments, all of positions 1, 2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, and 36 of the sense strand and all of positions 1, 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, and 22 of the antisense strand are modified with 2'-O-methyl modified nucleosides, e.g., 2'-O-methyl ribonucleosides. In other embodiments, one or more of positions 1, 2, 4, 5, 6, 7, 11, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, and 36 of the sense strand and / or one or more of positions 1, 6, 9, 11, 13, 15, 17, 18, 20, 21, and 22 of the antisense strand are modified with 2'-O-methyl modified nucleosides, e.g., 2'-O-methyl ribonucleosides.In certain embodiments, all of positions 1, 2, 4, 5, 6, 7, 11, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, and 36 of the sense strand and / or all of positions 1, 6, 9, 11, 13, 15, 17, 18, 20, 21, and 22 of the antisense strand are modified with 2'-O-methyl modified nucleosides, e.g., 2'-O-methyl ribonucleosides.

[0157] Oligonucleotides, including the sense strand and the antisense strand, may have 5-15 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, and 14) 2'-fluoro modifications. For these oligonucleotides, one or more of positions 8, 9, 10, 11, 12, 13, and 17 of the sense strand and / or one or more of positions 2, 3, 4, 5, 7, 10, 14, 16, and 19 of the antisense strand may be modified with a 2'-fluoro modified nucleoside. For example, all of positions 8, 9, 10, and 11 of the sense strand and / or all of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand may be modified with a 2'-fluoro modified nucleoside. In other 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, 3, 4, 5, 7, 8, 10, 12, 14, 16, and 19 of the antisense strand may be modified. In another example, all of positions 3, 8, 9, 10, 12, 13, and 17 of the sense strand and / or all of positions 2, 3, 4, 5, 7, 8, 10, 12, 14, 16, and 19 of the antisense strand may be modified with 2'-fluoro modified nucleosides.

[0158] In the case of an oligonucleotide comprising a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 3, one or more of positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand and / or one or more of positions 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand may be modified with a 2'-O-methyl modified nucleoside. Furthermore, all of positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand and / or one or more of positions 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand may be modified with a 2'-O-methyl modified nucleoside. In the case of an oligonucleotide having a sense strand having the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 3, one or more of positions 8 to 11 of the sense strand and one or more of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand may be modified with a 2'-fluoro modified nucleoside. Thus, all of positions 8 to 11 of the sense strand and all of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand may be modified with a 2'-fluoro modified nucleoside.

[0159] For example, in the case of an oligonucleotide having a sense strand with the sequence of SEQ ID NO:1 and an antisense strand with the sequence of SEQ ID NO:3, all of positions 1-7, 12-27, and 31-36 of the sense strand and / or one or more of positions 1, 6, 8, 9, 11-13, and 15-22 of the antisense strand may be modified with a 2'-O-methyl modified nucleoside; all of positions 8-11 of the sense strand and all of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand may be modified with a 2'-fluoro, the chemical structure of the sense strand is shown in FIG. 1A, the antisense strand is shown in FIG. 1B, and the RNAi oligonucleotide is shown in FIG. 1C-1 and FIG. 1C-2.

[0160] In the case of an oligonucleotide comprising a sense strand having the sequence of SEQ ID NO:4 and an antisense strand having the sequence of SEQ ID NO:6, one or more of positions 1, 2, 4, 5, 6, 7, 11, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, and 36 of the sense strand and / or one or more of positions 1, 6, 9, 11, 13, 15, 17, 18, 20, 21, and 22 of the antisense strand may be modified with 2'-O-methyl. In some embodiments, all of positions 1, 2, 4, 5, 6, 7, 11, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, and 36 of the sense strand and all of positions 1, 6, 9, 11, 13, 15, 17, 18, 20, 21, and 22 of the antisense strand may be modified with 2'-O-methyl. Additionally, for an oligonucleotide having a sense strand with the sequence of SEQ ID NO: 4 and an antisense strand with the sequence of SEQ ID NO: 6, one or more of positions 3, 8, 9, 10, 12, 13, 17 of the sense strand and / or one or more of positions 2, 3, 4, 5, 7, 8, 10, 12, 14, 16, and 19 of the antisense strand may be modified with 2'-fluoro. In some embodiments, all of positions 3, 8, 9, 10, 12, 13, and 17 of the sense strand and all of positions 2, 3, 4, 5, 7, 8, 10, 12, 14, 16, and 19 of the antisense strand are modified with 2'-fluoro. For example, for an oligonucleotide having a sense strand comprising the sequence of SEQ ID NO:4 and an antisense strand having the sequence of SEQ ID NO:6, all of positions 1, 2, 4, 5, 6, 7, 11, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, and 36 of the sense strand and all of positions 1, 6, 9, 11, 13, 15, 17, 18, 20, 21, and 22 of the antisense strand may be modified with 2'-O-methyl; all of positions 3, 8, 9, 10, 12, 13, and 17 of the sense strand and all of positions 2, 3, 4, 5, 7, 8, 10, 12, 14, 16, and 19 of the antisense strand may be modified with 2'-fluoro; the chemical structures of the sense and antisense strands are shown in Figures 2A-1 and 2A-2.

[0161] In some embodiments, the terminal 3' end group (e.g., 3'-hydroxyl) may be modified with a phosphate or other group, which can be used, for example, to attach a linker, adapter, or label, or to directly ligate the oligonucleotide to another nucleic acid.

[0162] 5'-Terminal Phosphate The 5'-terminal phosphate group of an oligonucleotide (e.g., an RNAi oligonucleotide) may enhance the interaction with Argonaute 2. In certain embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide) includes a uridine at the first position of the 5'-end of the antisense strand. However, oligonucleotides with a 5'-phosphate group may be susceptible to degradation by phosphatases or other enzymes, limiting their bioavailability in vivo. In some embodiments, an oligonucleotide includes a 5'-phosphate analog that is resistant to such degradation. Thus, the uridine at the 5'-end of the antisense strand may include a phosphate analog. The phosphate analog may be an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate. Additionally, the 5' end of the oligonucleotide chain may be attached to a chemical moiety ("phosphate mimic") that mimics the electrostatic and steric properties of the natural 5'-phosphate group (see Prakash et al., Nucleic Acids Res. 2015 Mar 31; 43(6):2993-3011, the contents of which are incorporated herein by reference with respect to phosphate analogues). Many phosphate mimetics have been developed that can be attached to the 5' end (see U.S. Pat. No. 8,927,513, the contents of which are incorporated herein by reference with respect to phosphate analogues). Other modifications have been developed for the 5' end of oligonucleotides (see WO 2011 / 133871, the contents of which are incorporated herein by reference with respect to phosphate analogues). In certain embodiments, a hydroxyl group may be attached to the 5' end of the oligonucleotide.

[0163] Oligonucleotides may have a phosphate analog at the 4'-carbon position of the sugar, referred to as a "4'-phosphate analog." See, for example, WO 2018 / 045317, the contents of which regarding phosphate analogs are incorporated herein by reference. Oligonucleotides provided herein may include a 4'-phosphate analog at the 5'-terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate, or an analog thereof, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., at its 4'-carbon). In other embodiments, the 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 attached to the 4'-carbon of the sugar moiety or analog thereof. In certain embodiments, the 4'-phosphate analog is an oxymethylphosphonate. In some embodiments, the oxymethylphosphonate has the formula -O-CH 2 -PO(OH) 2 or -O-CH2-PO(OR) 2 where R is H, CH 3 , alkyl group, CH 2 CH 2 CN, CH 2 OCOC(CH 3 ) 3 , C.H. 2 OCH 2 CH 2 Si(CH 3 ) 3 or a protecting group. In certain embodiments, the alkyl group is independently selected from 2 CH 3 More typically, R is H, CH 3 , or C.H. 2 CH 3 In some embodiments, R is CH3. In some embodiments, the 4'-phosphate analog is 5'-methoxyphosphanate-4'-oxy. In some embodiments, the 4'-phosphate analog is 4'-(methylmethoxyphosphonate). In some embodiments, the phosphate analog is a 4'-O-monomethylphosphonate analog.

[0164] In some embodiments, the phosphate analog attached to the oligonucleotide is a methoxyphosphonate (MOP). The phosphate analog attached to the oligonucleotide may be a 5' monomethyl protected MOP. In some embodiments, for example, at position 1 of the antisense strand, the following uridine nucleotide containing a phosphate analog: [ka] A modified nucleotide may be used, referred to as [MePhosphonate-4O-mU] or 5'-methoxy, phosphonate-4'oxy-2'-O-methyluridine. 5'-Methoxy, phosphonate-4'oxy-2'-O-methyluridine may be the first nucleotide at the 5' end of the antisense strand. For example, the first nucleotide at the 5' end of either SEQ ID NO: 3 or 6 may be 5'-methoxy, phosphonate-4'oxy-2'-O-methyluridine.

[0165] Modified Internucleoside Linkages Phosphate modifications or substitutions in an oligonucleotide can result in an oligonucleotide that includes at least one (e.g., at least one, at least two, at least three, at least five, or at least six) modified internucleotide linkages. Any one of the oligonucleotides disclosed herein can include 1-10 (e.g., 1-10, 2-8, 4-6, 3-10, 5-10, 1-5, 1-3, or 1-2) modified internucleotide linkages. For example, any one of the oligonucleotides disclosed herein can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages. In some embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide) can include five modified internucleotide linkages. For example, the sense strand of the oligonucleotide can include one modified internucleotide linkage and the antisense strand can include four modified internucleotide linkages.

[0166] The modified internucleotide bond can be phosphorodithioate bond, phosphorothioate bond, phosphotriester bond, thionoalkylphosphonate bond, thionealkylphosphotriester bond, phosphoramidite bond, phosphonate bond or boranophosphate bond.The modified internucleotide bond of any one of the oligonucleotides disclosed herein can be phosphorothioate bond.In certain embodiments, all modified internucleotide bonds of the oligonucleotide can be phosphorothioate bond.

[0167] The oligonucleotides described herein may have phosphorothioate bonds 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 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. For example, the sense strand of the oligonucleotide may have phosphorothioate bonds between 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. Thus, a sense strand having a sequence of SEQ ID NO: 1 or 4 may have a phosphorothioate bond between positions 1 and 2, positions 2 and 3, positions 20 and 21, and positions 21 and 22.

[0168] Base Modification The oligonucleotides provided herein may have one or more modified nucleobases. The modified nucleobases, also referred to herein as base analogs, may be linked to the 1' position of the nucleotide sugar moiety. The modified nucleobases may be nitrogenous bases. In certain embodiments, the modified nucleobases may contain nitrogen atoms. See, for example, US Published Patent Application No. 2008 / 0274462, the contents of which regarding modified nucleobases are incorporated herein by reference. The modified nucleotides may also include universal bases. However, in certain embodiments, the modified nucleotides may not contain nucleobases (e.g., non-basic).

[0169] 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 at an equivalent position in a nucleotide sugar moiety substitute, which, when present in a double helix, can be located opposite more than one type of base without substantially changing the structure of the double helix. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide or polynucleotide) that is perfectly complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base has a lower T than a double helix formed with a complementary nucleic acid. m However, in some embodiments, compared to a reference single-stranded nucleic acid in which the universal base is replaced with one base to create a single mismatch, the single-stranded nucleic acid containing the universal base has a higher T than the double helix formed with the nucleic acid containing the mismatched base. m Non-limiting examples of universal binding nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and / or 1-β-D-ribofuranosyl-3-nitropyrrole (see U.S. Patent Application Publication No. 2007 / 0254362; Van Aerschot et al., Nucleic Acids Res. 1995 Nov 11; 23(21):4363-70; Loakes et al., Nucleic Acids Res. 1995 Jul 11; 23(13):2361-6; Loakes et al., Nucleic Acids Res. 1994 Oct 11; 22(20):4039-43, each of which is incorporated herein by reference for its disclosure regarding base modifications).

[0170] reversible modification Before reaching target cells, oligonucleotides can be modified to protect them from the in vivo environment, but such modifications may reduce the efficacy or activity of oligonucleotides when they reach the cytosol of target cells.Reversible modifications can be performed so that molecules retain desirable properties outside cells, and are subsequently removed when they enter the cytosolic environment of cells.Reversible modifications can be removed, for example, by the action of intracellular enzymes or by chemical conditions inside cells (e.g., via reduction by intracellular glutathione).

[0171] Reversibly modified nucleotides may include glutathione-sensitive moieties. Typically, nucleic acid molecules are chemically modified with cyclic disulfide moieties to mask the negative charge generated by internucleotide diphosphate bonds and to improve cellular uptake and nuclease resistance. See U.S. Patent Application Publication No. 2011 / 0294869, originally assigned to Traversa Therapeutics, Inc. ("Traversa"), PCT Publication No. WO 2015 / 188197, assigned to Solstice Biologics, Ltd. ("Solstice"), Meade et al., Nature Biotechnology, 2014, 32:1256-1263 ("Meade"), PCT Publication No. WO 2014 / 088920, assigned to Merck Sharp & Dohme Corp, each of which is incorporated by reference for disclosure of such modifications. Reversible modifications of the internucleotide diphosphate bond are designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g., glutathione). Previous examples include neutralizing phosphotriester modifications that have been reported to be cleavable intracellularly (see Dellinger et al. J. Am. Chem. Soc. 2003, 125:940-950).

[0172] Such reversible modifications allow protection during in vivo administration (e.g., passage through blood and / or lysosomal / endosomal compartments of cells) where the oligonucleotide is exposed to nucleases and other harsh environmental conditions (e.g., pH). Upon release into the cytosol of cells, where glutathione levels are higher compared to the extracellular space, the modification is reversed, resulting in a cleaved oligonucleotide. The use of reversible glutathione-sensitive moieties allows the introduction of sterically larger chemical groups into the oligonucleotide of interest compared to the options available using irreversible chemical modifications. This is because these larger chemical groups are removed in the cytosol and therefore are not likely to interfere with the biological activity of the oligonucleotide in the cytosol of the cell. As a result, these larger chemical groups can be engineered to confer various advantages on the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, reduced immunogenicity, etc. The structure of the glutathione-sensitive moiety can be engineered to modify the kinetics of its release.

[0173] In some embodiments, the glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, the glutathione-sensitive moiety is attached to the 2' carbon of the sugar of the modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5'-carbon of the sugar, for example, 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 the sugar, for example, when the modified nucleotide is the 3'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, for example, US Patent Publication No. 2019 / 0177355, the contents of which are incorporated herein by reference for their relevant disclosures.

[0174] Targeting Ligands It may be desirable to target the oligonucleotides of the present disclosure to one or more cells or one or more organs (e.g., cells of the liver). Such a strategy may help to avoid undesirable effects in other organs, or may avoid excessive loss of oligonucleotide to cells, tissues, or organs that do not benefit from the oligonucleotide. Thus, in some embodiments, the oligonucleotides disclosed herein can be modified to facilitate targeting of a particular tissue, cell, or organ, for example, to facilitate delivery of the oligonucleotide to the liver. In certain embodiments, the oligonucleotides disclosed herein can be modified to facilitate delivery of the oligonucleotide to hepatocytes of the liver. The oligonucleotides may include nucleotides conjugated to one or more targeting ligands.

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

[0176] One or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide may each be conjugated to a separate targeting ligand. In some cases, 2-4 nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. The targeting ligand may be conjugated to 2-4 nucleotides at either the sense or antisense end such that the targeting ligand resembles the bristles of a toothbrush and the oligonucleotide resembles a toothbrush (e.g., the ligand is conjugated to a 2-4 nucleotide overhang or extension at the 5' or 3' end of the sense or antisense strand). For example, the oligonucleotide may include a stem loop at either the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the stem loop may be individually conjugated to a targeting ligand. In some embodiments, the oligonucleotide includes a stem loop at the 3' end of the sense strand, and 3 nucleotides of the stem loop are individually conjugated to a targeting ligand.

[0177] In some embodiments, it is desirable to target the oligonucleotide that reduces expression of C3 to hepatocytes in the liver of a subject. Any suitable hepatocyte targeting moiety can be used for this purpose.

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

[0179] For example, the oligonucleotide of the present disclosure can be directly or indirectly conjugated with a monovalent GalNAc. The oligonucleotide can be directly or indirectly conjugated with two or more (e.g., 2, 3, 4, or more) monovalent GalNAc, typically conjugated with 3 or 4 monovalent GalNAc moieties. The GalNAc moiety can be present in the loop region of the oligonucleotide described herein. The GalNAc moiety can be used to target the disclosed oligonucleotide to ASGPR on liver cells; at that point, the GalNAc-linked oligonucleotide can be internalized and incorporated into the intracellular RNAi machinery called RNA-induced silencing complex (RISC). The RISC Argonaute-2 (Argo-2) protein in this complex targets the antisense strand of the oligonucleotide duplex to its complementary C3 mRNA and initiates its degradation, thus blocking translation of the target.

[0180] In some embodiments, 2-4 nucleotides of the loop (L) of the stem loop are each conjugated to a separate GalNAc moiety. In some embodiments, three nucleotides of the loop of the stem of the oligonucleotide may be directly or indirectly conjugated to three separate monovalent GalNAc moieties. In some embodiments, the oligonucleotide is conjugated to one or more divalent, trivalent, or tetravalent GalNAc moieties.

[0181] The oligonucleotides described herein may include a monovalent GalNAc linked to a guanine nucleobase, as depicted below, which is referred to as [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanine-GalNAc: [ka]

[0182] Additionally or alternatively, the oligonucleotides described herein may comprise a monovalent GalNAc linked to an adenine nucleobase, as depicted below, which is referred to as [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc. [ka]

[0183] An example of such a linkage is shown below for a loop (L=linker, X=heteroatom) containing the nucleotide sequence GAAA (SEQ ID NO:8) from 5' to 3', with the stem attachment points indicated. Such a loop may be present, for example, at nucleotide positions 27-30 of the molecule shown in FIG. 1A. In the formula: [ka] is the point of attachment to the oligonucleotide chain. [ka]

[0184] The targeting ligand can be linked to the nucleotide using a suitable method or chemistry (e.g., click chemistry). The targeting ligand can be conjugated to the nucleotide using a click linker. In addition, an acetal-based linker can be used to conjugate the targeting ligand to any one of the nucleotides of the oligonucleotides described herein. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO 2016 / 100401A1, published on June 23, 2016, the contents of which regarding such linkers are incorporated herein by reference. The linker can be a labile linker. However, in other embodiments, the linker is stable (non-labile).

[0185] An example is shown below for a tetraloop containing the nucleotides GAAA (SEQ ID NO: 8) 5' to 3', in which four (4) GalNAc moieties are attached to the nucleotides of the loop using acetal linkers. Such loops can be present in the oligonucleotides disclosed herein (see, for example, positions 27-30 of the oligonucleotides having the sequences of SEQ ID NOs: 1 and 4). [ka] is the point of attachment to the oligonucleotide chain. [ka]

[0186] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise a sense strand having a tetraloop, where three (3) GalNAc moieties are conjugated to a nucleotide comprising the tetraloop, and each GalNAc moiety is conjugated to one (1) nucleotide. In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise a sense strand having a tetraloop comprising a GalNAc-conjugated nucleotide, where the tetraloop has the following structure: [ka] Including, During the ceremony: Z represents a bond, click chemistry handle, or linker of 1 to 20 contiguous covalently bonded atoms in length (inclusive) 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; X is O, S, or N.

[0187] In another embodiment, an oligonucleotide (e.g., an RNAi oligonucleotide) described herein comprises a sense strand having a tetraloop comprising three (3) GalNAc moieties attached to a nucleotide, wherein the tetraloop has the following structure: [ka] Includes.

[0188] In some embodiments, a double helix extension (e.g., up to 3, 4, 5, or 6 base pairs in length) is provided between the targeting ligand (e.g., the GalNAc moiety) and the oligonucleotide (e.g., the RNAi oligonucleotide). In some embodiments, the double helix extension between the targeting ligand (e.g., the GalNAc moiety) and the oligonucleotide (e.g., the RNAi oligonucleotide) is 6 base pairs in length.

[0189] formulation Various formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides can be delivered to a subject or cellular environment using formulations that minimize degradation, facilitate delivery and / or uptake, or confer other beneficial properties to the oligonucleotide in the formulation. In some embodiments, compositions are provided herein that include oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) for reducing the expression of C3. Such compositions can be suitably formulated such that, upon administration to a subject, a sufficient portion of the oligonucleotide enters the cell to reduce C3 expression, either in the immediate environment of the target cell or throughout the body. As disclosed herein, any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for the purpose of reducing C3. In some embodiments, oligonucleotides, pharmaceutical compositions, vectors, or cells are formulated in a buffer solution, such as phosphate buffered saline, liposomes, micellar structures, vectors, and capsids.

[0190] The formulations disclosed herein may include excipients. The excipients may provide the composition with improved stability, improved absorption, improved solubility, and / or enhanced therapeutic effect of the active ingredient. The excipients may be buffers (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide) or vehicles (e.g., buffer solutions, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotides can be lyophilized to extend their shelf life and then dissolved prior to use (e.g., administration to a subject). Thus, the excipients in the compositions comprising any one of the oligonucleotides described herein are cryoprotectants (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone), or disintegration temperature regulators (e.g., dextran, ficoll, or gelatin).

[0191] Pharmaceutical compositions containing oligonucleotides can be formulated to be compatible with their intended route of administration, including parenteral, e.g., subcutaneous, intravenous, intradermal, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal (e.g., subcutaneous) administration.

[0192] Pharmaceutical compositions suitable for injection use include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid propylene glycol, etc.), and suitable mixtures thereof. In many cases, it is optional to include isotonicity agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of oligonucleotide in a selected solvent together with one or a combination of the above-listed ingredients, as needed, followed by filter sterilization.

[0193] In some embodiments, the pharmaceutical composition comprising the oligonucleotide comprises sterile water (or water for injection (WFI)). In some embodiments, the pharmaceutical composition comprising the oligonucleotide comprises PBS.

[0194] In some embodiments, the pharmaceutical composition comprising the oligonucleotide comprises a preservative-free, sterile solution in WFI. In some embodiments, the pH of the pharmaceutical composition is about 7.2 (e.g., pH 7.2). In some embodiments, 0.1N NaOH or 0.1N HCl may be titrated as necessary to adjust the pH of the solution to a target of 7.2. In some embodiments, the concentration of the free acid form of the RNAi oligonucleotide in the pharmaceutical composition is about 160 mg / mL (e.g., 160 mg / mL). Using WFI, in some embodiments, the total concentration as the free acid form can be about 160 mg / mL. In some embodiments, the target fill volume is about 1.3 mL in a 2 mL glass vial. In some embodiments, the solution is expected to be administered to a patient via subcutaneous administration.

[0195] In some embodiments, the composition may contain at least about 0.1% of a therapeutic agent (e.g., an oligonucleotide for reducing C3 expression) or more, although the percentage of active ingredient may be from about 1% to about 80% or more by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0196] Many embodiments are directed to liver-targeted delivery of any of the oligonucleotides disclosed herein, although targeting of other tissues is also contemplated.

[0197] Pharmaceutical Use Disclosed herein are methods for delivering an effective amount of any one of the oligonucleotides (e.g., RNAi oligonucleotides) disclosed herein to a cell or subject for the purpose of reducing expression of C3 in the cell or subject.

[0198] The oligonucleotides disclosed herein can be introduced into cells of a subject having a disease or disorder mediated by complement pathway activation or dysregulation (e.g., activation or dysregulation of C3) using any suitable nucleic acid delivery method. For example, the oligonucleotides can be delivered to cells by injection of a solution containing the oligonucleotide, bombardment with particles coated with the oligonucleotide, exposure of cells or organisms to a solution containing the oligonucleotide, or electroporation of the cell membrane in the presence of the oligonucleotide.

[0199] Formulation of oligonucleotides with cationic lipids can be used to facilitate the transfection of oligonucleotides into cells.For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used.Suitable lipids include oligofectamine, lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche), all of which can be used according to the manufacturer's instructions.

[0200] Thus, in some embodiments, the formulation comprises a lipid nanoparticle. In some embodiments, the excipient comprises a liposome, lipid, lipid complex, microsphere, microparticle, nanosphere, or nanoparticle, or may otherwise be formulated for administration to a cell, tissue, organ, or body of a subject in need thereof (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition, Pharmaceutical Press, 2013).

[0201] Effective intracellular concentrations of the oligonucleotides disclosed herein can also be achieved through stable expression of a polynucleotide encoding the oligonucleotide (e.g., by integration into the nuclear or mitochondrial genome of a mammalian cell) or by transient expression in cells contacted with a polynucleotide (e.g., a plasmid or other vector (e.g., a viral vector) encoding the oligonucleotide). Examples of expression vectors are disclosed, for example, in WO 1994 / 011026, which is incorporated herein by reference. Expression vectors for use in the compositions and methods described herein contain oligonucleotide sequences that reduce C3 expression and additional sequence elements that are used, for example, for the expression of these agents and / or for the integration of these polynucleotide sequences into the genome of a mammalian cell. The expression vector can be a viral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0202] Other methods for delivering oligonucleotides to cells, such as lipid-mediated carrier transport, chemical-mediated transport, cationic liposome transfection such as calcium phosphate, and vectors containing oligonucleotides can also be used. The vectors used to deliver the oligonucleotides described herein can be viral vectors, such as retroviral vectors (e.g., lentiviral vectors), adenoviral vectors (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), and adeno-associated viral vectors (AAV) (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10).

[0203] In some examples, the oligonucleotides described herein can be delivered in the form of a transgene engineered to express the oligonucleotide (e.g., its sense and antisense strands) in a cell. The transgene can be delivered using a vector, such as a viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus), or a non-viral vector (e.g., a plasmid or synthetic mRNA), as described above. In some embodiments, the transgene can be injected directly into the subject, for example, at or near the source of action (e.g., in or near the liver) or into the bloodstream.

[0204] C3 inhibition Upon administration, the oligonucleotides of the present disclosure can bind to C3 mRNA and inhibit its expression. Inhibition of expression of the C3 gene can be evidenced by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells can be present, for example, in a sample derived from (e.g., obtained from) a subject) in which the C3 gene is transcribed and which has been treated to inhibit expression of the C3 gene (e.g., by contacting the cells with an oligonucleotide (e.g., an RNAi oligonucleotide) of the present disclosure or administering an oligonucleotide (e.g., an RNAi oligonucleotide) of the present disclosure to a subject in which the cells are or were present), compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been similarly treated (control cells that have not been treated with an oligonucleotide (e.g., an RNAi oligonucleotide) or an oligonucleotide (e.g., an RNAi oligonucleotide) targeted to a gene of interest). The level of target mRNA can be measured using techniques well known to those of skill in the art, such as RT-qPCR. The degree of inhibition can be expressed by the following formula:

number

[0205] The change in the expression level of the C3 gene can be evaluated with respect to a parameter functionally related to C3 gene expression, such as a reduction in C3 protein expression, C3 protein activity, or C3 signaling pathway. C3 gene silencing can be determined in any cell that expresses either endogenous or heterologous C3 from an expression construct, and by any assay known in the art.

[0206] The results of inhibition of C3 mRNA can be confirmed by a suitable assay to evaluate one or more characteristics of the cell or subject, or by biochemical techniques to evaluate molecules indicative of C3 expression (e.g., RNA, protein). The extent to which the oligonucleotides provided herein reduce the expression level of C3 is assessed by comparing the expression level to an appropriate control (e.g., the level of C3 mRNA expression in a cell or cell population to which no oligonucleotide has been delivered or to which a negative control has been delivered). A suitable control level of C3 mRNA expression may be a predetermined level or value, so that subject levels do not need to be measured every time. The predetermined level or value can take a variety of forms, including a single cutoff value, such as a median or mean value. For example, the predetermined level or value may be at or about a level of 75-175 mg / dL of C3 protein, which corresponds to the level of C3 protein typically found in the serum of healthy subjects.

[0207] The expression level of C3 mRNA in a sample can be determined, for example, by detecting a transcribed polynucleotide or a part thereof, such as mRNA. RNA can be extracted from cells using RNA extraction techniques, including, for example, acid phenol / guanidine isothiocyanate extraction (RNAZOL™ B; Biogenesis), RNEASY™ RNA preparation kit (Qiagen) or PAXGENE™ (PreAnalytix, Switzerland). C3 mRNA in a sample can also be measured using real-time PCR (RT-PCR). For example, RNA can be extracted by homogenizing tissue samples with QIAzo Lysis reagent using a TissueLyser II (Qiagen) followed by purification using MAGMAX® Technology (ThermoFisher Scientific) according to the manufacturer's instructions. cDNA can then be prepared using a High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Specific primers and probes for C3 and housekeeping controls were used for PCR on a CFX384 Real-Time PCR Detection System (Bio-Rad Laboratories), and Ct values ​​were estimated using BioRad CFX Maestro software; expression levels were calculated in EXCEL® and plotted in Prism (GraphPad). Primers used for RT-PCR are listed in Table 2.

[0208] [Table 2]

[0209] Typical assay formats using ribonucleic acid hybridization include nuclear run-on assay, RT-PCR, RNase protection assay, Northern blotting, in situ hybridization, and microarray analysis. Circulating mRNA can be detected using the methods described in PCT Publication WO 2012 / 177906, the entire contents of which are incorporated herein by reference. Expression levels of genes of interest can also be determined using nucleic acid probes.

[0210] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Northern or Southern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA of a gene of interest. For example, the isolated mRNA can be immobilized on a solid surface and contacted with a probe by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. The probe can also be immobilized on a solid surface, for example, contacting the mRNA with the probe in an AFFYMETRIX® GENECHIP® array. Methods for detecting mRNA known in the art can be adapted for use in determining the level of mRNA of a gene of interest.

[0211] Alternative methods for determining the expression level of a gene of interest in a sample include, for example, RT-PCR (Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Qβ replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al. (1989) Bio / Technology 6:1197), and the like. The process includes nucleic acid amplification of, for example, mRNA in a sample by PCR (U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method and / or reverse transcriptase (to prepare cDNA), followed by detection of the amplified molecules using techniques known in the art. These detection schemes are particularly useful for detection of nucleic acid molecules when such molecules are present in very low numbers. In some embodiments of the present disclosure, the expression level of a gene of interest (e.g., C3) is determined by quantitative fluorescent RT-PCR (i.e., TAQMAN™ System) or DUAL-GLO® luciferase assay.

[0212] The expression level of the mRNA of the gene of interest can be monitored using membrane blots (such as those used in hybridization analysis such as Northern, Southern, dot, etc.), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acid). See U.S. Patent Nos. 5,770,722; 5,874,219; 5,744,305; 5,677,195; and 5,445,934, which are incorporated herein by reference. Determining gene expression levels can also include using nucleic acid probes in solution.

[0213] The above assays can be used to determine the effectiveness of treatment with the oligonucleotides described herein based on the reduction in C3 mRNA levels. The reduction in the level of C3 mRNA can be a reduction of 1% or less, 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 70% or less, 80% or less, or 90% or less compared to a suitable control level of C3 mRNA or the level of C3 in the subject before treatment. A suitable control level can be the level of C3 mRNA expression in a cell or cell population that has not been contacted with the oligonucleotides described herein. In some embodiments, the effect of delivery of oligonucleotides to cells by the methods disclosed herein is evaluated after a finite period of time. For example, levels of C3 mRNA can be analyzed in cells for at least 8 hours, 12 hours, 18 hours, 24 hours; or for at least 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, or 80 days following introduction of the oligonucleotide into the cells.

[0214] Furthermore, inhibition of the C3 gene can result in inhibition of C3 protein expression, which can be manifested by a decrease in the level of C3 protein expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject).As explained above, for assessment of mRNA suppression, inhibition of protein expression levels in a treated cell or group of cells can also be expressed as a percentage of protein levels in a control cell or group of cells.

[0215] The results of inhibiting C3 protein expression can be confirmed by a suitable assay to evaluate one or more characteristics of the cell or subject, or by a biochemical technique to evaluate molecules indicative of C3 protein expression. The extent to which the oligonucleotides provided herein reduce the expression level of C3 protein is assessed by comparing the expression level to a suitable control (e.g., the level of C3 protein expression in a cell or cell population to which no oligonucleotide has been delivered or to which a negative control has been delivered). A suitable control level of C3 protein expression may be a predetermined level or value, e.g., an amount of C3 protein determined to be in the normal range, e.g., 75-175 mg / dL in serum, so that it is not necessary to measure the control level every time. The predetermined level or value can take a variety of forms, including a single cutoff value, such as a median or mean value.

[0216] The level of C3 protein produced by the expression of the C3 gene can be determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC / MS / MS), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, and the like. Such assays can also be used to detect proteins that indicate the presence or replication of a protein produced by a gene of interest. In addition, the above assays can also be used to report changes in the mRNA sequence of interest that result in restoration or alteration of protein function, thereby providing a therapeutic effect and benefit to the subject, treating a disorder in the subject, and / or alleviating symptoms of a disorder in the subject.

[0217] The aforementioned assays can be used to make a determination about the efficacy of treatment with the oligonucleotides described herein based on a reduction in the amount of C3 protein. The reduction in the level of C3 protein can be a reduction of 1% or less, 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 70% or less, 80% or less, or up to 90% or less compared to an appropriate control level of C3 (e.g., about 75-175 mg / gL). An appropriate control level can be the level of C33 expression in a cell or cell population that has not been contacted with an oligonucleotide described herein. In some embodiments, the effect of delivery of an oligonucleotide to a cell by the methods disclosed herein is evaluated after a finite period of time. For example, the level of C3 can be analyzed in the cell for at least 8 hours, 12 hours, 18 hours, 24 hours; or for at least 1, 2, 3, 4, 5, 6, 7, or 14 days after introduction of the oligonucleotide into the cell. The level of C3 can be determined to assess whether the subject requires retreatment. For example, the subject is considered to require retreatment if the level of C3 rises to pretreatment levels (or to levels that are at least about 20% or more (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) of the pretreatment levels).

[0218] Furthermore, inhibition of the C3 gene using the methods described herein may result in reduced transcription of C3 mRNA in cells of a subject identified as having a disease mediated by complement pathway activation or dysregulation. The methods provided herein are useful in any suitable cell type (e.g., cells that express C3, such as hepatocytes). In some embodiments, the cells are primary cells obtained from a subject, which may have undergone a limited number of passages, such that the cells substantially maintain their native phenotypic characteristics. In some embodiments, the cells to which the oligonucleotides are delivered are ex vivo or in vitro (i.e., cells in culture or may be delivered to an organism in which the cells reside). In specific embodiments, methods are provided for delivering an effective amount of the oligonucleotides disclosed herein to cells for the purpose of reducing the expression of C3 only in hepatocytes.

[0219] The effective amount of the oligonucleotide disclosed herein can be determined as the amount of oligonucleotide that results in the alleviation of symptoms of a disease or disorder mediated by complement pathway activation or dysregulation, such as one of the diseases or disorders described herein. The alleviation of symptoms of a disease or disorder mediated by complement pathway activation or dysregulation can be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% alleviation, for example, as determined using clinical evaluations known to those skilled in the art. The amount of alleviation of symptoms of a disease or disorder mediated by complement pathway activation or dysregulation can be used to determine whether the subject needs to be treated again with the oligonucleotide, pharmaceutical composition, vector, or cell described herein. Examples of assays for determining the alleviation of diseases mediated by complement pathway activation or dysregulation include, but are not limited to, the measurement and / or quantification of circulating C3 protein, functional assays (e.g., WEISLAB® assay and hemolytic assay). Quantitation of C3 (or C3 cleavage products) deposition can be performed via specific disease biomarkers via IHC or immunofluorescence.

[0220] Furthermore, the oligonucleotides described herein, which include both the sense and antisense strands as double-stranded polypeptides, can be introduced into cells of a subject using any suitable nucleic acid delivery. The double-stranded oligonucleotides can be delivered to cells by injection of a solution containing the oligonucleotide, bombardment with particles coated with the oligonucleotide, exposure of cells or organisms to a solution containing the oligonucleotide, or electroporation of cell membranes in the presence of the oligonucleotide. The double-stranded oligonucleotides can also be delivered to cells using lipid-mediated carrier transport, chemical-mediated transport, cationic liposome transfection such as calcium phosphate, and vectors encoding the nucleic acid of the single-stranded oligonucleotide. The vectors used to deliver the double-stranded oligonucleotides can be viral vectors, such as retroviral vectors (e.g., lentiviral vectors), adenoviral vectors (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), and adeno-associated viral vectors (AAV) (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).

[0221] Treatment method Also disclosed herein are methods for treating a disease mediated by complement pathway activation or dysregulation, including, for example, one or more of the diseases associated with complement pathway activation or dysregulation disclosed herein, in a subject by administering a composition described herein (e.g., an oligonucleotide, a vector encoding the oligonucleotide, a cell comprising the vector, a pharmaceutical composition). The method may include treating a disease mediated by complement pathway activation or dysregulation in a subject by administering a pharma- ceutically acceptable salt (e.g., a sodium salt) of an RNAi oligonucleotide described herein. The methods described herein typically involve administering an effective amount of the oligonucleotide, or a pharma- ceutically acceptable salt thereof, i.e., an amount that can produce a desired therapeutic result (e.g., knockdown of C3 expression). A therapeutically acceptable amount may be an amount that can treat a disease or disorder mediated by complement pathway activation or dysregulation (e.g., activation or dysregulation of C3). The appropriate dosage for any one subject may depend on certain factors, including the subject's size, body surface area, age, the specific composition administered, the active ingredients in the composition, the time and route of administration, general health, and other drugs administered concomitantly. Such therapeutic agents can be used, for example, to slow, stop, or prevent any type of disease or disorder mediated by complement pathway activation or dysregulation, and can be administered prophylactically or therapeutically. Administration of a prophylactic agent may occur prior to detection or manifestation of symptoms characteristic of a disease or disorder mediated by complement pathway activation or dysregulation, such that the disease or disorder is prevented or its progression is delayed. Subjects at risk for a disease mediated by complement pathway activation or dysregulation can be identified, for example, by one or a combination of diagnostic or prognostic assays known in the art.

[0222] The compositions disclosed herein can be administered to a subject using any standard method. For example, any one of the compositions disclosed herein can be administered 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, intraarterial injection or infusion, intraosseous injection, intramuscular injection, intracerebral injection, intraventricular injection, intrathecal injection), locally (e.g., transdermally, by inhalation, eye drops, or via mucosa), or by direct injection into a target organ (e.g., the liver of a subject). Typically, the oligonucleotides disclosed herein are administered intravenously or subcutaneously. The most suitable route for administration in any given case may depend on the particular composition administered, the subject, the particular disease or disorder mediated by complement pathway activation or dysregulation to be treated, the pharmaceutical formulation method, the method of administration (e.g., time and route of administration), the age, weight, sex of the subject, the severity of the disease to be treated, the diet of the subject, and the excretion rate of the subject.

[0223] The oligonucleotides described herein can be administered to subjects suffering from a disease or disorder mediated by complement pathway activation or dysregulation, for example, annually (e.g., once every 12 months), semi-annually (e.g., once every 6 months), quarterly (e.g., once every 3 months), bimonthly (e.g., once every 2 months), monthly, or weekly. In other cases, the oligonucleotides can be administered every 1, 2, or 3 weeks. In certain embodiments, the oligonucleotides can be administered daily.

[0224] The subject to be treated for a disease mediated by complement pathway activation or dysregulation may be a human or a non-human primate or another mammalian subject (e.g., a human). Other exemplary subjects that may be treated with the oligonucleotides described herein include domestic animals such as dogs and cats; livestock animals such as horses, cows, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters.

[0225] Dosage The dosage of the compositions of the present disclosure (e.g., compositions comprising an RNAi oligonucleotide as described herein, or a pharma- ceutically acceptable salt thereof) may vary depending on many factors, such as the pharmacodynamic properties of the compound, the mode of administration, the age, health, and weight of the recipient, the nature and extent of symptoms, the frequency of treatment and / or type of concurrent treatment (if any), and the clearance rate of the compound being treated. One of skill in the art can determine the appropriate dosage based on the above factors.

[0226] The oligonucleotides of the present disclosure, or pharma- ceutically acceptable salts thereof, may be administered in an amount and for a time effective to result in one or more (e.g., two or more, three or more, four or more) of the following: (a) reducing expression of C3 protein in cells of the subject; (b) reducing transcription of C3 in cells of the subject; (c) reducing the level of C3 protein in cells of the subject; (d) reducing the activity of C3 protein in cells of the subject; and / or (e) alleviating one or more symptoms of a disease or disorder mediated by activation or dysregulation of the complement pathway.

[0227] Thus, the present disclosure relates to a method for treating a disease mediated by complement pathway activation or dysregulation in a subject in need thereof, comprising administering an effective amount of a described oligonucleotide that specifically binds to C3 mRNA and inhibits expression of C3 protein in the subject. For example, the present disclosure provides a method for treating a disease mediated by complement pathway activation or dysregulation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an oligonucleotide, pharmaceutical composition, vector, or cell disclosed herein.

[0228] The disease mediated by complement pathway activation or dysregulation to be treated using the disclosed methods and compositions can be, for example, one or more of the diseases associated with complement pathway activation or dysregulation disclosed herein.

[0229] Treatment of diseases mediated by complement pathway activation or dysregulation can be achieved by administration of oligonucleotides (e.g., RNAi oligonucleotides) that inhibit expression and / or translation of C3 mRNA (e.g., expression of C3 protein) as described herein.

[0230] The disclosed compositions can be administered in amounts determined to be appropriate by one of skill in the art. In some embodiments, the oligonucleotides described herein can be initially administered at appropriate dosages that can be adjusted, if necessary, depending on the clinical response.

[0231] In some cases, the oligonucleotide, or a pharma- ceutically acceptable salt thereof, is administered at a dose of 0.01-100 mg / kg (e.g., 0.01-1 mg / kg, 1-5 mg / kg, 5-20 mg / kg, 20-50 mg / kg, 50-100 mg / kg) of the subject's body weight. In certain examples, the oligonucleotide is administered at a concentration of 0.01 mg-50 mg / kg (e.g., 0.01-1 mg / kg, 1-5 mg / kg, 5-10 mg / kg, 10-20 mg / kg, 20-30 mg / kg, 30-40 mg / kg, 40-50 mg / kg) of the subject's body weight. In other examples, the oligonucleotide is administered at a concentration of 0.01 mg-20 mg / kg (e.g., 0.01-1 mg / kg, 1-5 mg / kg, 5-10 mg / kg, 10-15 mg / kg, 15-20 mg / kg) of the subject's body weight. In other examples, the oligonucleotide is administered at a concentration of 0.01 mg to 15 mg per kg of the subject's body weight (e.g., 0.01-1 mg / kg, 1-2 mg / kg, 2-5 mg / kg, 5-8 mg / kg, 8-10 mg / kg, 10-12 mg / kg, 12-15 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.01 mg to 10 mg per kg of the subject's body weight (e.g., 0.01-1 mg / kg, 1-2 mg / kg, 2-5 mg / kg, 5-8 mg / kg, 8-10 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.01 mg to 5 mg per kg of the subject's body weight (e.g., 0.01-1 mg / kg, 1-2 mg / kg, 2-3 mg / kg, 3-4 mg / kg, 4-5 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.1 mg to 20 mg per kg of the subject's body weight (e.g., 0.1-1 mg / kg, 1-5 mg / kg, 5-10 mg / kg, 10-15 mg / kg, and 15-20 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.1 mg to 10 mg per kg of the subject's body weight (e.g., 0.1-1 mg / kg, 1-2 mg / kg, 2-5 mg / kg, 5-7 mg / kg, and 7-10 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.1 mg to 5 mg per kg of the subject's body weight (e.g., 0.1-1 mg / kg, 2-3 mg / kg, 3-4 mg / kg, and 4-5 mg / kg).In other instances, the oligonucleotide is administered at a concentration of 1 mg to 50 mg per kg of the subject's body weight (e.g., 1-10 mg / kg, 10-20 mg / kg, 20-30 mg / kg, 30-40 mg / kg, and 40-50 mg / kg). In other instances, the oligonucleotide is administered at a concentration of 1 mg to 20 mg per kg of the subject's body weight (e.g., 1-5 mg / kg, 5-10 mg / kg, 10-15 mg / kg, and 15-20 mg / kg). In other instances, the oligonucleotide is administered at a concentration of 1 mg to 10 mg per kg of the subject's body weight (e.g., 1-2 mg / kg, 2-5 mg / kg, 5-7 mg / kg, and 7-10 mg / kg). In other instances, the oligonucleotide is administered at a concentration of 1 mg to 5 mg per kg of the subject's body weight (e.g., 1-2 mg / kg, 2-3 mg / kg, 3-4 mg / kg, and 4-5 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 30 mg / kg to 300 mg / kg (e.g., 30 to 200 mg / kg, 30 to 100 mg / kg, 30 to 50 mg / kg, 50 to 300 mg / kg, 100 to 300 mg / kg, 200 to 300 mg / kg, and 250 to 300 mg / kg).

[0232] In certain embodiments, the oligonucleotide is administered at a dose of less than 10 mg / kg of the subject's body weight (e.g., 9 mg / kg or less, 8 mg / kg or less, 7 mg / kg or less, 6 mg / kg or less, 5 mg / kg or less, 4 mg / kg or less, 3 mg / kg or less, 2 mg / kg or less, 1 mg / kg or less). In other embodiments, the oligonucleotide is administered at a dose of about 10 mg / kg or less. In another embodiment, the oligonucleotide is administered at a dose of about 9 mg / kg or less (e.g., 8.9 mg / kg, 8 mg / kg, 7 mg / kg, 5 mg / kg, 3 mg / kg, and 1 mg / kg or less). In other embodiments, the oligonucleotide is administered at a dose of about 8 mg / kg or less (e.g., 7.9 mg / kg, 7 mg / kg, 5 mg / kg, 3 mg / kg, and 1 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dose of about 7 mg / kg or less (e.g., 6.9 mg / kg, 6 mg / kg, 4 mg / kg, 2 mg / kg, and 1 mg / kg or less). In another embodiment, the oligonucleotide (e.g., RNAi oligonucleotide) is administered at a dose of about 6 mg / kg or less (e.g., 5.9 mg / kg, 5 mg / kg, 3 mg / kg, and 1 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dose of about 5 mg / kg or less (e.g., 4.9 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, and 1 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dose of about 4 mg / kg or less (e.g., 3.9 mg / kg, 3 mg / kg, 2 mg / kg, and 1 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dose of about 3 mg / kg or less (e.g., 2.9 mg / kg, 2.5 mg / kg, 2 mg / kg, and 1 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dose of about 2 mg / kg or less (e.g., 1.9 mg / kg, 1.5 mg / kg, 1 mg / kg, and 0.5 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dose of about 1 mg / kg or less (e.g., 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, and 0.1 mg / kg or less).

[0233] In another embodiment, the oligonucleotide is administered at a dose of about 0.1-10 mg / kg, about 0.2-10 mg / kg, about 0.3-10 mg / kg, about 0.4-10 mg / kg, about 0.5-10 mg / kg, about 1-10 mg / kg, about 2-10 mg / kg, about 3-10 mg / kg, about 4-10 mg / kg, about 5-10 mg / kg, about 6-10 mg / kg, about 7-10 mg / kg, about 8-10 mg / kg, or about 9 mg / kg of the subject's body weight.

[0234] In other cases, the composition (e.g., a composition comprising an RNAi oligonucleotide described herein) is administered in an amount that is effective for prophylactic or therapeutic purposes. In some cases, the viral vector (e.g., an rAAV vector) is administered in an amount of 10 or more per subject. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 In some embodiments, the rAAV is administered at a dose of 10 genome copies (GC). 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 The dose is given in GC / kg (total weight of subject).

[0235] Optionally, the disclosed oligonucleotides can be administered as part of a pharma- ceutically acceptable composition suitable for delivery to a subject, as described herein. The disclosed agents are included in the composition in an amount sufficient to provide the desired dosage and / or elicit a therapeutically beneficial effect, as can be readily determined by one of skill in the art.

[0236] The disclosed compositions described herein can be administered in an amount (e.g., an effective amount) sufficient and for a sufficient time to treat a subject or to result in one of the aforementioned outcomes (e.g., reduction of one or more symptoms of a disease in a subject). The disclosed compositions can be administered one or more times. The disclosed compositions can be administered once a day, twice a day, three times a day, every other day, once a week, twice a week, three times a week, once every two weeks, once a month, once every two months, twice a year, or once a year. Treatment can be discrete (e.g., injections) or continuous (e.g., treatment via an implant or infusion pump). Subjects can be evaluated for therapeutic efficacy one week, two weeks, one month, two months, three months, four months, five months, six months, or more after administration of the disclosed compositions, depending on the composition and route of administration used for treatment. A subject may be treated for a discrete period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months) or until the disease or condition is alleviated, or, depending on the severity and nature of the disease or condition being treated, treatment may continue long term (e.g., for the life of the subject). For example, a subject diagnosed with PNH and treated with a composition disclosed herein may be administered one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional therapeutic agents if an initial round or subsequent rounds of treatment do not elicit a therapeutic benefit, including reduction in any one of the symptoms associated with PNH, such as fatigue, weakness, shortness of breath, easy bruising or bleeding, recurrent infections, severe headaches, blood clots, and difficulty controlling bleeding, or a reduction in the level of C3 mRNA or C3 protein levels in the subject's cells or serum.

[0237] kit The present disclosure also features a kit comprising (a) a pharmaceutical composition comprising an oligonucleotide (e.g., RNAi oligonucleotide) agent that reduces the level and / or activity of C3 in a cell or subject as described herein, or a pharma- ceutically acceptable salt thereof, and, optionally, a pharma- ceutically acceptable carrier, excipient, or diluent. The kit may contain a vector encoding an oligonucleotide (e.g., RNAi oligonucleotide) as described herein, or a cell comprising a vector encoding an oligonucleotide (e.g., RNAi oligonucleotide) as described herein. The kit may also include a package insert containing instructions for carrying out any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition comprising an oligonucleotide (e.g., RNAi oligonucleotide) agent that reduces the level and / or activity of C3 in a cell or subject as described herein, (b) an additional therapeutic agent, and (c) a package insert containing instructions for carrying out any of the methods described herein. EXAMPLES

[0238] The following examples are for illustrative purposes only and are not intended to limit the present disclosure in any way.

[0239] Example 1: Preparation of RNAi oligonucleotides Oligonucleotide synthesis and purification The RNAi oligonucleotides described in this and the previous examples were chemically synthesized using methods described herein. Generally, RNAi oligonucleotides can be prepared using a variety of methods, including phosphoramidite synthesis (see, e.g., Hughes and Ellington (2017) COLD SPRING HARB PERSPECT BIOL. 9(1):a023812; Beaucage SL, Caruthers MH, Study on Nucleotide Chemistry V: Deoxy nucleoside phosphoramidites-A New Class of Key Intermediates for Deoxypolynucleotide Synthesis, TETRAHEDRON LETT. 1981;22:1859-1862. doi:10.1016 / S0040-4039(01)90461-7), as well as solid-phase oligonucleotide synthesis methods as described for 19-23mer siRNAs (see, e.g., Scaringe et al. (1990) Nucleic Acids Res. 18:5433-5441 and Usman et al. (1999) Nucleic Acids Res. 18:5433-5441). al. (1987) J. Am. Chem. Soc. 109:7845-7845; U.S. Patent Nos. 5,804,683; 5,831,071; 5,998,203; 6,008,400; 6,111,086; 6,117,657; 6,353,098; 6,362,323; 6,437,117 and 6,469,158).

[0240] RNAi oligonucleotides with a 19-mer core sequence were formatted into constructs with a 25-mer sense strand and a 27-mer antisense strand to allow processing by the RNAi machinery. The 19-mer core sequence was complementary to a region of the C3 mRNA.

[0241] Individual RNA strands were synthesized and HPLC purified according to standard methods (Integrated DNA Technologies; Coralville, IA). For example, RNA oligonucleotides were synthesized using solid-phase phosphoramidite chemistry, deprotected, and then desalted on a NAP-5 column (Amersham Pharmacia Biotech; Piscataway, NJ) using standard techniques (Damha & Olgivie (1993) METHODS MOL. BIOL. 20:81-114; Wincott et al. (1995) NUCLEIC ACID RES. 23:2677-2684). Oligomers were purified using ion-exchange high-performance liquid chromatography (IE-HPC) on an Amersham Source 15Q column (1.0 cm x 25 cm; Amersham Pharmacia Biotech) using a 15 min step linear gradient. The gradient varied from 90:10 buffer A:B to 52:48 buffer A:B, where buffer A was 100 mM Tris pH 8.5 and buffer B was 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm and peaks corresponding to full-length oligonucleotide species were collected, pooled, desalted on a NAP-5 column and then lyophilized.

[0242] The purity of each oligomer was determined by capillary electrophoresis on a Beckman PACE 5000 (Beckman Coulter, Inc.; Fullerton, CA). The CE capillary had an inner diameter of 100 μm and contained ssDNA 100 R gel (Beckman-Coulter). Typically, approximately 0.6 nmoles of oligonucleotide were injected into the capillary, run at an electric field of 444 V / cm, and detected by UV absorbance at 260 nm. Denaturing Tris-borate-7M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides that were at least 90% pure, as assessed by CE, were obtained for use in the experiments described below. The identity of the compounds was verified by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry on a VOYAGER-DE™ BIOSPECTROMETRY™ Work Station (Applied Biosystems; Foster City, CA) following the manufacturer's recommended protocol. Relative molecular weights of all oligomers were obtained and were often within 0.2% of the predicted molecular weights.

[0243] Preparation of the double helix Single-stranded RNA oligomers were resuspended (e.g., at a concentration of 100 μM) in duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense and antisense strands were mixed in equimolar amounts to give a final solution of, e.g., 50 μM duplex. Samples were heated to 100°C in 5'RNA buffer (IDT) and allowed to cool to room temperature before use. RNAi oligonucleotides were stored at -20°C. Single-stranded RNA oligomers were lyophilized or stored in nuclease-free water at -80°C.

[0244] Example 2: Generation of C3-targeting RNAi oligonucleotides Identification of C3 mRNA target sequence Complement is a tightly regulated enzyme cascade that can be activated by several different pathways, including the classical complement pathway (CCP), in which antibody complexes trigger activation. Regardless of which pathway initiates the process, complement activation converges on C3 in the cascade. Once activated, C3 is cleaved to form the effector molecules C3a and C3b, which cause inflammation, deposition of C3b in tissues, and activation of terminal complement and further tissue damage.

[0245] To generate RNAi oligonucleotide inhibitors of C3 expression, a computer-based algorithm was used to computationally identify suitable C3 mRNA target sequences for assaying inhibition of C3 expression by the RNAi pathway. Over 300 RNAi oligonucleotide guide (antisense) strand sequences (see Table 3), each with a region of complementarity to the appropriate C3 target sequence of human C3 mRNA, were prepared and assayed in vitro for C3 expression inhibition. From these RNAi oligonucleotides, nine subsets (see Table 4) were selected for further study. A subset of the nine guide sequences identified by the algorithm was also complementary to the corresponding C3 target sequence of monkey C3 mRNA (SEQ ID NO: 67; Table 3). C3 RNAi oligonucleotides that contain a region of complementarity to a homologous C3 mRNA target sequence with nucleotide sequence similarity are predicted to have the ability to target the homologous C3 mRNA.

[0246] [Table 3]

[0247] Example 3: Identification of RNAi oligonucleotides that inhibit C3 expression in vitro The activity of RNAi oligonucleotides (formatted as dsiRNA oligonucleotides) generated as described in Examples 1 and 2 to reduce C3 mRNA was measured using an in vitro cell-based assay. Briefly, HepG2 human hepatocytes expressing endogenous C3 were transfected with 1 nM of RNAi oligonucleotide (FIG. 3A) or a subset of the RNAi oligonucleotides screened in FIG. 3A at two different concentrations (0.1 and 1 nM) as shown in separate wells of a multi-well cell culture plate (FIG. 3B). Cells were maintained for 24 hours after transfection and the levels of C3 mRNA from transfected cells were measured using a TAQMAN®-based qPCR assay. Two RT-qPCR assays, the 3′ assay and the 5′ assay, were used to measure mRNA levels measured by HEX and FAM probes, respectively. A subset of RNAi oligonucleotide candidates was selected for further in vivo analysis based on inhibition of C3 mRNA levels as determined by RT-qPCR.

[0248] Example 4: Screening of RNAi oligonucleotides in mice expressing human C3 cDNA (HDI mice) A subset of RNAi oligonucleotide candidates (or "compounds") from Example 3 was screened in mice expressing human C3 cDNA. CD-1 mice transfected with a vector expressing human C3 cDNA were administered a single subcutaneous dose of 0.5 or 1 mg / kg of selected compounds (compound AI). Four days later, animals were sacrificed for evaluation of human C3 mRNA levels from liver homogenates determined by RT-qPCR using a specific probe. Compounds that showed at least 50% knockdown efficacy in transfected mice were selected for testing in cynomolgus macaques. The results of the in vivo screening of a subset of nine compounds (i.e., compounds A, B, C, D, E, F, G, H, and I) are shown in Figures 4B and 4C, and their corresponding sense and antisense strands are summarized in Table 4 and Figure 4A. Data are expressed as the percentage of C3 mRNA remaining in the liver compared to PBS-treated mice.

[0249] [Table 4]

[0250] Example 5: Screening of RNAi oligonucleotides in Cynomolgus Macaques All compounds A-I were preselected during mouse screening and tested in cynomolgus macaques (NHPs) for duration of C3 mRNA silencing after administration of a single subcutaneous dose of 4 mg / kg of compounds A-I, as described in Example 4, Table 4, and Figure 4A. Liver biopsies (n=5 / compound) of all animals tested were collected pre-dose and at days 28 and 56 post-injection. As shown in Figure 5, most compounds tested reduced hepatic C3 mRNA levels by at least 50% compared to normalized baseline levels and time-matched PBS controls as determined by RT-qPCR. Two lead compounds (compounds A and B) were selected for testing in a multiple dose study based on knockdown levels of C3 mRNA in cynomolgus macaque livers after a single dose.

[0251] Compounds A and B were selected from the single dose study for further evaluation in a multiple dose NHP study. Cynomolgus macaques were administered a total of four doses at 1 mg / kg or 2 mg / kg subcutaneously on days 0, 28, 56, and 84. Liver biopsies were collected pre-dose and 28, 56, and 112 days after the first treatment for evaluation of liver C3 mRNA levels by RT-qPCR (Figure 6A). Serum samples were collected pre-dose and 1, 14, 28, 42, 56, 70, 84, 98, and 112 days after the first treatment for evaluation of C3 protein levels by C3 ELISA kit (Figure 6B), complement activity by WIESLAB® AP assay (Figure 8), and rabbit red blood cell hemolysis (Figure 9). PBS-treated animals were used as controls from the C3 liver mRNA, C3 serum protein, and function assays. Multiple treatment of cynomolgus macaques with Compounds A or B resulted in sustained C3 mRNA silencing in the liver, a significant reduction in circulating C3 in serum, a >95% reduction in alternative pathway complement activity, and complete inhibition of rabbit red blood cell lysis in a hemolysis assay after multiple doses of Compounds A and B, as shown in Figures 6A, 6B, 8, and 9, respectively.

[0252] The potencies of Compounds A and B were calculated by combining the results at Day 28 for both single and multiple dose NHP studies. Approximate EDs for Compound A (0.65 mg / kg) and Compound B (0.55 mg / kg) were calculated as 50 was calculated from dose-response curves generated for both compounds (Figure 7).

[0253] Example 6: Pharmacokinetic and pharmacodynamic study of compound J on C3 expression in CD-1 mice CD-1 mice were treated with Compound J (a mouse surrogate for Compound A) to assess percent liver C3 mRNA knockdown and serum C3 protein levels in mice as a result of Compound J administration. Percent liver C3 mRNA knockdown was measured using RT-qPCR as a result of Compound J administration. The amount of C3 in serum was measured using a mouse C3 ELISA assay. Mice received a single subcutaneous dose of Compound J at 0.5 mg / kg, 1 mg / kg, or 6 mg / kg. A single dose of Compound J demonstrated a dose-dependent liver C3 mRNA knockdown percentage, with livers from animals receiving the 6 mg / kg dose (n=5 mice / time point) showing a greater than 90% reduction in C3 mRNA. The nadir of mRNA knockdown was 3-14 days from the 6 mg / Kg dose, as shown in Figure 10A. The percentage of C3 protein in the serum of CD-1 mice was measured over the study period and was correspondingly suppressed (Figure 10B).

[0254] The amount of Compound J in plasma, liver, kidney, and spleen tissues of CD-1 mice administered a single subcutaneous dose of 6 mg / kg of Compound J was measured using stem-loop qPCR for 672 hours after receiving the dose (Figure 11). Pharmacokinetic analysis showed that the highest exposure of Compound J was in the liver, followed by the spleen, kidney, and plasma (Figure 11).

[0255] In a multiple dose study conducted over 70 days, the percent C3 mRNA was measured using RT-qPCR and the amount of C3 protein in serum was measured by mouse C3 ELISA assay, where CD-1 mice received 4 doses of Compound J at 1 mg / kg or 6 mg / kg on days 0, 14, 28, and 42, as shown in Figures 12A and 12B, respectively. This regimen resulted in approximately 75% and >95% knockdown of liver C3 mRNA and serum protein levels, respectively. Liver biopsies and serum collections were performed on days 3, 14, 17, 28, 31, 42, 45, 56, and 70 after the first dose. Liver and plasma concentrations of Compound J after 4 doses of 1 mg / kg were analyzed from liver biopsies and plasma samples using stem-loop qPCR (SL-qPCR), as shown in Figures 13A and 13B, respectively. PBS-treated CD-1 mice were used as a control for both C3 liver mRNA and C3 serum protein levels.

[0256] This multiple dose study revealed that Compound J (a mouse surrogate for Compound A) showed a dose-dependent knockdown of hepatic C3 mRNA that was sustained over a 70 day period. The reduction in circulating C3 protein levels corresponded to the observed reduction in C3 mRNA in the liver. Furthermore, plasma and liver concentrations of Compound J from treated animals showed no accumulation of Compound J when administered every other week (1 mg / kg) (Figures 13A and 13B, respectively).

[0257] Example 7: Effect of Compound J on C3 expression in NZB / W F1 mice, a lupus nephritis model Proof of mechanism was tested in disease models with Compound J using the NZB / W F1 lupus mouse model. NZB / W F1 animals were administered Compound J at doses of 0.5 mg / kg, 3 mg / kg, or 6 mg / kg subcutaneously every 4 weeks starting at 21 weeks of age (n=10 / group). PBS-treated animals were used as negative controls, and kidneys from CD1 mice were used as non-disease controls. At 37 weeks of age, C3 and properdin glomerular deposition was assessed by immunofluorescence imaging of kidneys from Compound J-treated and PBS control animals (Figure 14). At 29 weeks of age, percent hepatic C3 mRNA was measured using RT-qPCR (Figure 15A), and the amount of C3 protein in serum was quantified using a mouse C3 ELISA assay for each dose level of Compound J (Figure 15B). After multiple doses of treatment with Compound J, there was a dose-dependent decrease in C3 and properdin glomerular deposition observed from Compound J-treated animals. Multiple dose treatment of NZB / W F1 mice with Compound J showed a dose-dependent knockdown of hepatic C3 mRNA that was sustained over 16 weeks. The reduction in circulating C3 protein levels (FIG. 15A) paralleled the observed reduction in C3 mRNA in the liver (FIG. 15B).

[0258] Serum samples were collected at 29 and 37 weeks of age to measure circulating IgG immune complexes (CICs) by ELISA assay after 8 and 16 weeks of treatment with Compound J, respectively (Figures 16A and 16B). After multiple doses of treatment with Compound J, there was no increase in the levels of circulating immune complexes due to hepatic knockdown of C3 expression compared to the CIC levels observed from the PBS-treated control group.

[0259] Example 8: Effect of Compound J on C3 expression in MRL / lpr mice, a lupus nephritis model The MRL / Ipr lupus mouse model was used and treated with Compound J. Mice received multiple doses of 6 mg / kg Compound J. Figure 17 shows a reduction in C3 glomerular deposition from kidneys of MRL / lpr mice treated with multiple doses of 6 mg / kg Compound J. A reduction in properdin deposition is also observed from kidney samples of animals treated with a subcutaneous dose of 6 mg / kg Compound J every 2 weeks from 8-16 weeks of age, as shown in Figure 17.

[0260] Example 9: cfh - / - Effect of Compound J on C3 expression in a mouse model of complement dysregulation Complement factor H-deficient mice (Cfh - / - ) were administered 4 monthly doses of 0.5 mg / kg, 3 mg / kg, or 6 mg / kg Compound J from 4 to 8 months of age. Kidneys were harvested from all treatment groups 4 weeks after the last dose of Compound J, and immunofluorescence analysis was performed to visualize both C3 and properdin deposition in the glomeruli of CFH- / - treated animals. Figure 18 shows a dose-dependent reduction in C3 glomerular deposition from kidneys of CFH- / - mice treated with multiple ascending doses of Compound J. Reduction in properdin deposition was also observed in kidney samples from animals treated with subcutaneous doses of Compound J every 4 weeks from 16-32 weeks of age, as shown in Figure 18. Percent hepatic C3 mRNA was measured using RT-qPCR (Figure 19). Treatment reduced C3 and properdin deposition in the kidney. In addition, treatment with Compound J normalized serum C5 levels in these mice (C5 consumption is a hallmark of complement dysregulation in this model).

[0261] Example 10: Effect of Compound J on C3 expression in CAIA-induced arthritis mouse model The effect of compound J in treating symptoms related to arthritis was examined using a collagen antibody-induced arthritis (CAIA)-induced arthritis mouse model, a simple model of rheumatoid arthritis. The CAIA-induced arthritis mouse model was generated by administering collagen antibodies to mice on day 0, followed by an LPS booster on day 3. Compound J was tested in both preventive and therapeutic studies. Animals were administered 3 or 6 mg / kg of compound J on day -7 for preventive studies (Figure 20A) or administered after disease onset on day 5 for therapeutic studies (Figure 20B). Hind paw inflammation was visually analyzed on day 10, and the results of both preventive and therapeutic studies are shown in Figures 21A and 21B, respectively. Preventive treatment with compound J prevented hind paw swelling, a unique feature of this model (Figure 21A). Therapeutic treatment with compound J completely reversed clinical disease expression after a single dose when compared to PBS-treated control animals (Figure 21B).

[0262] Hematoxylin and eosin (H&E) staining was performed on hind paw and knee biopsies, showing a reduction in local mononuclear cell infiltration in mice treated with Compound J at a single dose of 6 mg / kg, either prophylactically at three doses (Figure 22A) or therapeutically at a single dose (Figures 22B and 24A, respectively). To further demonstrate the reduction in local inflammation as a result of therapeutic treatment with Compound J at 6 mg / kg, lymphocyte (CD45 positive cells), leukocyte (CD11b positive cells) and macrophage (F4 / 80 positive cells) marker staining was performed on biopsy samples as shown in Figures 25, 26, and 27, respectively. Biopsy samples were also stained with Safranin O to visualize knee cartilage in the CAIA-induced arthritis mouse model. Animals treated with Compound J at 6 mg / kg showed a significant reduction in cartilage erosion compared to PBS-treated mice when treated prophylactically (Figure 23) or therapeutically (Figure 24B). To assess complement expression at the local inflammatory site in CAIA-induced arthritic mice with or without treatment with 6 mg / kg Compound J, experiments using in situ hybridization to C3 and CD45 mRNA were performed on biopsy samples, which are shown in Figure 28. Hepatic knockdown of C3 by Compound J reduced lymphocyte (CD45 positive cells) infiltration and local C3 mRNA expression in the case of therapeutic treatment with Compound J compared to PBS-treated animals as a control group.

[0263] Example 11: Effect of Compound J on C3 expression in a mouse model of multiple sclerosis. Myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mouse model, a widely used model to investigate immune-mediated mechanisms of neuroinflammation and demyelination, was prophylactically treated with a 6 mg / kg dose of Compound J (n=2 experiments). Liver C3 mRNA levels as well as C3 protein in serum after treatment with Compound J were assessed using RT-qPCR and mouse C3 ELISA assays, respectively, as shown in Figures 31A and 31B. Similarly, the percentage of C3 mRNA remaining after treatment with Compound J, as well as the amount of C3 in serum of MOG-induced EAE mice after treatment with a 6 mg / kg dose of Compound J, were assessed in comparison to PBS-treated C3-deficient mice and MOG-induced EAE C3-deficient mouse strains. Liver knockdown of C3 with Compound J reduced disease severity in both experiments performed (Figure 29). The reduction in severity observed with liver knockdown with Compound J treatment was similar to clinical observations in C3-deficient animals (global knockout).

[0264] Lumbar spinal cord samples were also obtained from MOG-induced EAE mice treated with Compound J. Luxol fast blue staining was performed on spinal cord samples along with H&E staining to visualize myelination as well as mononuclear cell infiltration, as shown in Figure 30. Luxol fast blue spinal cord samples were compared between diseased animals treated with 6 mg / kg Compound J, PBS, and C3-deficient mice, as shown in Figure 30. MOG-induced animals treated with Compound J showed reduced demyelination and prevention of immune cell infiltration similar to the levels observed in MOD-induced EAE C3-deficient mice.

[0265] Similar results were observed in a proteolipid protein (PLP)-induced EAE model: C3 siRNA treatment reduced disease severity but was not sufficient to prevent relapse, a hallmark of this animal model.

[0266] Example 12: Absorption, distribution, metabolism, and excretion (ADME) study in mice Pharmacokinetic and biodistribution studies were performed in male CD-1 mice administered PBS (n=18) or Compound A at 3, 10, or 100 mg / kg by single subcutaneous (SC) injection (n=39 / cohort) or 3 mg / kg by single intravenous (IV) injection (n=36). Bioavailability was measured using the AUC last The liver exposure was approximately 18% based on a comparison of the 3 mg / kg IV and SC cohorts. However, liver exposure was similar between the 3 mg / kg IV and SC cohorts. Plasma exposure compared to the 3 mg / kg dose group increased in an approximately dose-proportional manner in the 10 mg / kg group and in a greater than dose-proportional manner in the 100 mg / kg group. max and AUC last Liver exposure based on 10 mg / kg increased approximately dose-proportionally at 10 mg / kg and in a less than dose-proportional manner at 100 mg / kg compared with the 3 mg / kg dose group indicating saturation of the hepatic distribution process. The hepatic elimination half-life ranged from 2.1 to 4 days.

[0267] Example 13: Platelet activation of Compound A Assessment of platelet activation in human whole blood stimulated with Compound A did not induce platelet activation. Whole blood samples (five male and five female donors) were stimulated with PBS or Compound A at 10, 100, 200, or 300 μg / mL.

[0268] Example 14: Tolerability of Compound A in Cynomolgus Monkeys The tolerability of Compound A administration was evaluated in a study in naive male and female cynomolgus monkeys. Animals were dosed with phosphate buffered saline (PBS, n=12) or a SC dose of Compound A at dose levels of 1.5 mg / kg (low) or 3 mg / kg (high) on day 0 (n=6 / cohort), and liver biopsies were performed on day 21 to determine the level of hepatic C3 knockdown. Based on this analysis, dose levels were adjusted to 3 mg / kg (for the low dose) or 6 mg / kg (for the high dose) on days 28, 56, and 84 to attempt approximately 75% and 90% C3 mRNA knockdown, respectively. Blood samples were collected on days -21, -7, -3, 0, 28, 56, and 112, and viral, bacterial, and parasitic testing for a total of 28 pathogens was performed pre-study, on day 56, and at necropsy to monitor potential reactivation of latent virus and / or infection by serology and blood or fecal PCR. A terminal necropsy for evaluation by a clinical pathologist was performed to determine potential evidence of infection. Interim measurements of circulating C3 protein levels in serum were also performed along with CBC, coagulation, clinical chemistry, and urinalysis. By day 49 after administration of either low or high doses of Compound A, approximately 75 or 80% hepatic C3 mRNA knockdown was achieved, with approximately 80% reduction in circulating C3 protein levels, respectively. These reductions in hepatic C3 mRNA and C3 protein levels persisted at all time points evaluated until day 112, when the study was terminated. There were no gross or microscopic findings at necropsy, and no unscheduled deaths. Body weight, liver function tests, blood counts, blood chemistry, and lipid metabolism parameters were not affected by chronic Compound A treatment, and there was no evidence of increased pathogenic parasitic, bacterial, or viral infections in treated monkeys compared to PBS-treated cohorts.

[0269] Example 15: Tolerability of Compound J in CD-1 and NZB / W F1 mice The tolerability of chronic administration of Compound J was evaluated in CD-1 and NZB / W F1 mice. CD-1 mice were administered PBS or Compound J (1 or 100 mg / kg) for a total of 4 months, sacrificed 1 month after the last dose, and evaluated by a blinded pathologist for evidence of viral or bacterial infection and histological changes. No treatment-related histopathological changes or increased infections were observed. Similarly, NZB / W F1 mice were administered Compound J at 1 or 6 mg / kg every 4 weeks from 28 to 40 weeks of age and terminally sacrificed at 44 weeks of age, or from 24 to 36 weeks of age and terminally sacrificed at 40 weeks of age. No evidence of increased infections was detected by serological and PCR testing for viral, bacterial pathogen, and parasite populations, and no treatment-related changes were found at terminal necropsy, evaluated by a veterinary pathologist blinded to treatment groups.

[0270] Example 16: Safety Pharmacology Study of Compound A in Cynomolgus Monkeys Compound A was evaluated in a subcutaneous safety pharmacology study in cynomolgus monkeys. Four animals received either PBS or single ascending doses of Compound A (at dose levels of 30, 100, and 300 mg / kg) once every 7 days, with the same four animals at each dose. During the study, safety pharmacology was performed, including assessment of cardiovascular (e.g., ECG, blood pressure, heart rate, etc.), respiratory (respiratory rate), and neurological (functional observational battery) endpoints, as well as clinical evaluations. No cardiovascular or respiratory effects were observed at any dose level. No neurological effects were observed with Compound A at 30 or 100 mg / kg. At 300 mg / kg, clinical findings of minimal to mild tremors (limbs or whole body) were noted in three animals at 4 and 24 hours post-dose. Neurological findings observed at 300 mg / kg were considered adverse. Therefore, the no observed adverse effect level (NOAEL) was determined to be 100 mg / kg. Furthermore, genotoxicity evaluations including in vitro micronucleus assay and in vitro bacterial reverse mutation assay were negative for micronucleus induction and mutagenic activity, respectively.

[0271] Example 17: PK / PD Study of Compound A in Cynomolgus Monkeys A single-dose PK / PD study was conducted in cynomolgus monkeys. Animals received a SC dose of PBS (control) or a single SC or IV dose of 3 mg / kg Compound A on day 0 (n=5 per group). Compound A concentrations were assessed in plasma, urine, and tissues. Liver biopsies were performed on days 2, 35, 70, 112, 158, and 252 to assess hepatic C3 mRNA levels (the primary pharmacodynamic marker). Circulating C3 protein levels and complement function activity were additional PD markers evaluated throughout the study.

[0272] Compound A concentrations were measured in plasma, liver, and urine using a certified hybridization-based anion exchange high performance liquid chromatography (AEX-HPLC-FD) method with fluorescence detection. Reduction of complement component 3 (C3) mRNA expression in monkey liver was measured using real-time quantitative polymerase chain reaction (RT-qPCR). C3 protein in monkey serum was measured using ELISA, and complement function activity (classical pathway, mannose-binding lectin pathway (MBL), and alternative pathway) was measured using the WIESLAB® assay.

[0273] Plasma concentrations of Compound A over time were used to generate non-compartmental PK profiles for individual animals (group means are plotted in Figure 32A). Plasma T max The range is 1 to 6 hours, and the T maxwas 0.25 hours (collected at the first time point) for all animals. Plasma concentrations of Compound A declined biphasically with a slower distribution phase for the SC route compared to the IV route. The biphasic decline indicates an initial rapid distribution phase, mainly to the liver, followed by a slower elimination phase. Plasma half-lives were reported for 1 of 5 animals in the SC group (2.51 hours) and for 2 of 5 animals in the IV group (mean = 1.21 hours). Half-lives for the remaining animals were not reported because the acceptance criteria for the terminal phase rate constant were not met. Bioavailability of Compound A was measured by AUC last The mean mean mean CI for the SC dose was approximately 28.5% compared with the IV dose.

[0274] Liver concentrations of Compound A over time were used to generate non-compartmental PK profiles for individual animals (group means are plotted in Figure 32B). Liver half-lives were calculated for all five animals in both the SC and IV groups and ranged from 13 to 20 days and 20 to 33 days, respectively.

[0275] The total amount of Compound A excreted in urine by each animal at each collection time was used to calculate total drug excreted and % drug excreted. The mean urinary excretion of Compound A was 4.3% and 4.8% in the SC and IV groups, respectively.

[0276] After a single dose of 3 mg / kg Compound A administered SC or IV, a reduction in C3 mRNA expression in monkey liver was observed on day 2 (SC only), reaching a maximum reduction (approximately 70% for both routes of administration) on day 35 post-administration (FIG. 33). A gradual recovery of C3 mRNA expression over time was observed, returning to near baseline levels by day 114. This recovery was maintained through day 252 for both routes of administration.

[0277] Following a single 3 mg / kg SC dose of Compound A, a decrease in serum C3 protein was observed from days 7 to 70 (Figure 34). Maximum mean C3 protein levels in serum were decreased by 54.7% at day 28 post-dosing and recovered to pre-dosing levels by day 168. There was no decrease in serum C3 protein levels in animals receiving a single 3 mg / kg IV dose of Compound A compared to controls.

[0278] There was no effect on classical complement in either the SC or IV groups compared to controls (Figure 35). Lectin and alternative pathway functionality was decreased in the SC group on days 14, 28, and 35 compared to controls. Lectin pathway functionality was minimally decreased (approximately 13%) on all three days (Figure 36), whereas alternative pathway functionality was decreased by 87%, 85%, and 73% on days 14, 28, and 35, respectively (Figure 37). Pathway functionality returned to baseline levels by day 70 for the lectin pathway and by day 168 for the alternative pathway.

[0279] Following a single 3 mg / kg SC dose of Compound A, maximal mRNA reduction was 70% on day 35, maximal C3 protein reduction was 54.7% on day 28, and maximal reduction in alternative pathway activity was ≧87% on day 14. mRNA expression returned to baseline levels by day 168, C3 protein expression returned to baseline by day 28, and alternative pathway function was restored by day 168.

[0280] Example 18: Toxicity studies in CD-1 mice and Cynomolgus monkeys A 6-month toxicity study in mice and a 9-month toxicity study in cynomolgus monkeys were conducted. The range of dose levels in these studies was selected to achieve at least a 10-fold exposure multiple over the expected exposure of the maximum intended clinical dose.

[0281] A mouse study evaluated the potential toxicity of repeated doses (every 4 weeks; 7 doses) of PBS or Compound A (30, 100, or 300 mg / kg) in CD-1 mice, and possible reversibility of findings after an 8-week recovery period. Ten male and 10 female mice per dose cohort were evaluated during the dosing period of the study, and six males and six females were maintained during the recovery period. In addition, the toxicokinetic properties of Compound A were determined in a substudy (n=111). Compound A administered at levels of 30, 100, and 300 mg / kg was well tolerated, with no Compound A-related deaths or adverse findings. Clinical pathology findings included a slight increase in alanine aminotransferase along with a slight decrease in triglycerides at day 171, revealing full or partial reversibility at the end of the recovery period. Compound A-related non-adverse microscopic findings included minimal or mild mixed cell inflammation, hepatocellular karyocytic hypertrophy, increased mitosis, and oval cell hyperplasia in the liver at terminal euthanasia, with minimal or mild mitosis and hepatocellular karyocytic hypertrophy still present at recovery euthanasia. From these results, the NOAEL was considered to be 300 mg / kg, which was approximately 1.0 times the mean AUC last The mean values ​​were 760,000 and 543,000 hr*ng / mL, and the mean Cmax values ​​were 160,000 and 96,400 ng / mL.

[0282] The monkey study assessed the potential toxicity of repeated SC administration of Compound A (0, 30, 100, and 300 mg / kg every 4 weeks for a total of 10 doses) and the reversibility, persistence, or delayed onset of any effects after an 8-week recovery period. Four male and four female monkeys were included in each dose cohort of the main dosing study, and two males and two females were included in the 2-month recovery period. In addition, the toxicokinetic and PD properties of Compound A were measured. Repeated SC administration of Compound A to cynomolgus monkeys for 9 months was well tolerated. Repeated dose administration of Compound A did not result in test article-related changes in the following parameters: clinical observations, neurological examinations, ophthalmological examinations, body weights, qualitative food consumption, hematology, clinical chemistry, urinalysis, cytokines (i.e., MCP-1, TNF-α, IL-8, IL-1RA, G-CSF, IFN-γ, IL-1β, and IP-1), complement factors Bb and C3a, gross pathology, or organ weights during the dosing period of the study. Furthermore, there were no premature deaths during this study and all animals survived to scheduled necropsy. Compound A-related non-adverse increases in fibrinogen occurred at ≥ 100 mg / kg / dose. At ≥ 30 mg / kg / dose, there were non-adverse microscopic findings (vacuolated / granular macrophages) in the liver as well as multiple other tissues. Based on the findings observed, the NOAEL was determined to be 300 mg / kg / dose with associated AUC last is 1330000hr*ng / mL, C max The mean serum concentration was 70,900 ng / mL (male and female combination, day 253).

[0283] Genotoxicity assessments including in vitro micronucleus assays and in vitro bacterial reverse mutation assays were negative for mutagenic activity and micronucleus induction, respectively.

[0284] Based on the totality of the data, the NOAEL is considered to be 300 mg / kg. Neurological clinical signs (e.g., tremors) observed in the weekly escalation dose study were not observed in the 9-month monthly dosing monkey toxicity study.

[0285] Example 19: Effect of Compound B on C3 Expression in Cynomolgus Monkeys, an AMR Model A pharmacology study in four sensitized cynomolgus monkeys receiving renal allografts is conducted in which the animals receive an SC dose of Compound B once every four weeks for a total period of four months.

[0286] Example 20: Dose-finding study in humans For the First-in-Human (FIH) clinical trial, a single ascending dose (SAD) study will be conducted in healthy volunteers in combination with a multiple ascending dose (MAD) cohort in patients with complement-driven diseases. Healthy volunteers and patients will be prophylactically vaccinated against Neisseria meningitides types A, C, W, Y, and B, Streptococcus pneumoniae, and Haemophilus influenzae type B prior to administration of Compound A.

[0287] Example 21: Treatment of multiple sclerosis with Compound A in humans A subject suffering from multiple sclerosis is treated with a pharmaceutical composition containing Compound A (e.g., at a dose of about 0.01 mg / kg to 50 mg / kg body weight of the subject). The subject is administered the composition, e.g., by subcutaneous injection, about once a week for a period of about 12 months or more (e.g., until symptoms resolve or stabilize). About once a month, the subject's symptoms and serum C3 level are evaluated by a clinician to assess the effectiveness of Compound A. The subject's serum C3 can be quantified using a serum sample and compared to the amount of C3 protein found in the subject's serum before administration of Compound A or a control amount of C3 protein, or the amount of C3 protein present in a serum sample from a normal subject (e.g., a disease-free subject). Treatment with Compound A is determined to be effective if the amount of C3 protein in serum is reduced, i.e., reduced by at least 10%, compared to the amount of C3 protein in serum before treatment with Compound A. In addition, the subject's symptoms associated with multiple sclerosis, such as blurred vision, slurred speech, dizziness, percussion tenderness, lack of coordination, and unsteady gait, can be evaluated by a clinician to assess whether there is a reduction in any or all of the symptoms experienced by the subject compared to the symptoms experienced by the subject before administration of Compound A and / or compared to a placebo control subject.

[0288] Example 22: Treatment of Arthritis with Compound A in Humans A subject diagnosed with arthritis is treated with a pharmaceutical compound containing compound A (e.g., at a dose of about 1.5 mg / kg). The subject is administered the composition, e.g., by subcutaneous injection, about once a month for a period of about 6 months or more (e.g., until symptoms resolve or stabilize). Every one or two months, the subject is evaluated by a clinician (e.g., by evaluating the subject's symptoms and / or serum C3 level) to evaluate the effectiveness of compound A. The subject's serum C3 is quantified using a serum sample from the subject and compared to the amount of C3 protein found in the subject's serum before compound A is administered or a control amount of C3 protein, or the amount of C3 protein present in a serum sample from a normal subject (e.g., disease-free subject), and / or the amount of C3 protein present in a serum sample from a placebo-treated patient. Treatment with compound A is determined to be effective if the amount of C3 protein in serum is reduced, i.e., reduced by at least 10%, compared to the amount of C3 protein in serum before treatment with compound A. In addition, the clinician can assess the subject's symptoms associated with arthritis, such as pain, stiffness, swelling, redness, and reduced range of motion, to assess whether there is a reduction in any or all of the symptoms the subject is experiencing compared to the symptoms the subject was experiencing before Compound A was administered.

[0289] Example 23. C3 evaluation assay A variety of assays can be used to characterize the effect of Compound A on C3 levels, including assessment of Compound A in plasma or tissues, the WIESLAB® complement function activity assay, assessment of circulating C3, C3 mRNA expression levels, and pharmacokinetic assays, as described herein.

[0290] Pharmacokinetic assay of compound A The concentrations of Compound A or Compound J were measured in mouse and monkey plasma. Plasma samples (blank, unknown, standard, and QC samples) were enzymatically treated, followed by hybridization with a peptide nucleic acid (PNA) probe with sequence complementarity to the antisense strand of Compound A or Compound J. Samples were injected into a high performance liquid chromatography (HPLC) equipped with a fluorescence detector. Chromatographic separation was performed using a gradient system on Shimadzu Prominence systems using a DNAPAC™ PA200 analytical column. The fluorescence detector monitored signals from 436 nm (Ex) to 484 nm (Em). Individual and mixture reference samples were prepared and injected to confirm the retention times of metabolites. The peaks of Compound A and its expected metabolites were successfully separated. Quantification of Compound A or Compound J in monkey or mouse plasma, respectively, was performed using linear regression. This assay was used, for example, in Examples 6, 12, and 17, as described above.

[0291] WIESLAB® Complement Function Activity Assays (CCP, CAP, CLP) Classical pathway (CCP), CAP, and lectin pathway activity were assessed using WIESLAB® Complement System Screening Assays using labeled antibodies specific for neoantigens to detect human terminal complement complex (C5b 9) complexes resulting from complement activation. These assays can also detect cynomolgus monkey C5b-9. The amount of neoantigen generated was proportional to the level of functional activity of the individual pathways. The wells of the assay microtiter strips were coated with specific activators of the classical, alternative, or lectin pathways. Monkey serum samples were diluted in diluents containing blockers to ensure that only the respective pathway was activated. The wells were washed and C5b-9 was detected using specific alkaline phosphatase-labeled antibodies against the expressed neoantigens. The amount of complement activation correlated with the intensity of color development measured by absorbance at 405 nm. The value of the positive control provided in the test kit was defined as 100% complement activation. All measurements were expressed as percent (%) complement activity and were determined as follows: [(sample-negative control) / (positive control-negative control)]*100 This assay was used, for example, in Example 17 above.

[0292] Circulating C3 protein levels Assessment of cynomolgus monkey circulating C3 protein levels was assessed using a human complement C3 enzyme-linked immunosorbent assay (ELISA) kit (cat#Ab108823, Abcam, Cambridge, UK) designed for quantitative measurement of complement C3 concentration in human serum. Due to cross-reactivity with monkey C3, this kit was also used to measure circulating C3 protein in cynomolgus monkey serum samples. Complement C3-specific antibodies were precoated onto 96-well plates and blocked. Standards or test samples were added to the wells, followed by washing with wash buffer. Streptavidin-peroxidase conjugate was added and unbound conjugate was removed with wash buffer. Tetramethylbenzidine (TMB) was used to visualize the streptavidin-peroxidase enzymatic reaction. TMB was oxidized with streptavidin-peroxidase to produce a blue product that turned yellow after the addition of an acidic stop solution. The density of the yellow color was directly proportional to the amount of complement C3 captured by the plate. The back-calculated concentrations of the samples were determined by a curve-fitting regression program generated by calibration standards. This assay was used, for example, in Examples 14 and 17 above.

[0293] C3 mRNA expression level Assessment of C3 mRNA expression was determined in cynomolgus monkey liver samples using a multiplex relative quantitative real-time reverse transcriptase PCR assay. mRNA was isolated from frozen liver tissue, followed by mRNA quantification and transcription into complementary DNA (cDNA). cDNA was used as a template for qPCR reactions to measure C3 mRNA levels by normalization to peptidyl-prolyl cis-trans isomerase B (PPIB). The extent of C3 mRNA in the treatment group was calculated as a percentage of expression (normalized to PPIB mRNA levels) relative to the untreated or pre-treatment group, where C3 mRNA expression in the control group was set to 100%.

[0294] Pharmacokinetic assays The concentration of Compound A in human plasma is measured using HPLC-FD analysis. Plasma samples (blank, unknown, standard, and quality control [QC] samples) are enzymatically treated with proteinase K, followed by hybridization with a PNA probe with sequence complementarity to the antisense strand of Compound A. Samples are injected into an HPLC equipped with a fluorescence detector. Chromatographic separation is performed using a gradient system on Shimadzu Prominence systems using a DNAPAC™ PA200 analytical column. The fluorescence detector monitors signals from 436 nm (Ex) to 484 nm (Em). The LC gradient conditions are adjusted and determined based on the retention times of potential metabolites of Compound A. Individual and mixture reference samples are prepared and injected to evaluate the retention times of metabolites. Compound A and metabolite peaks are separated. Quantification of Compound A in human plasma is performed using linear regression, respectively.

[0295] Anti-drug antibody assay An anti-drug antibody (ADA) assay for Compound A in human serum is under development and will be performed using an electrochemiluminescence (ECL) bridging assay. Positive controls (PC) will be made from rabbits immunized against an immunogenic cocktail consisting of keyhole limpet hemocyanin (KLH)-conjugated Compound A and KLH-conjugated oligonucleotides of various lengths corresponding to modified Compound A sequences. PC, negative controls (NC), and test samples will be subjected to an acid dissociation step at ambient room temperature and then added to a plate containing TRIS, biotin-Compound A, and ruthenium-labeled Compound A to allow the formation of a crosslinked complex between the labeled Compound A present in the sample and the Compound A antibody. After incubation, the NC, PC, and test samples will be transferred to a streptavidin-coated plate and incubated in the dark for 1 hour, during which the drug will bind to the plate and capture the ADA crosslinked complex. The plate is then washed and Meso Scale Discovery® (MSD®) Read Buffer is added to generate an ECL signal that is directly proportional to the amount of ADA present in the sample. The ADA assay is validated prior to evaluation of clinical samples.

[0296] Other embodiments All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although specific embodiments have been described herein, those skilled in the art will recognize that further modifications and embodiments are encompassed and may be applied to the essential features described above, including variations, uses, or modifications that generally follow the principles described herein, and including such departures from the present disclosure that are within the scope of known or customary practice in the art, and are set forth in the following claims.

Claims

1. 1. A composition for reducing complement component 3 (C3) expression, comprising an RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, said oligonucleotide comprising a sense strand and an antisense strand, wherein said sense strand and said antisense strand form a double helical region, said antisense strand comprising a region of complementarity to a C3 mRNA target sequence of SEQ ID NO: 13 or 14, said region of complementarity being at least 15 contiguous nucleotides in length. (a) the sense strand is 15 to 50 nucleotides in length; and / or (b) the antisense strand is 15 to 30 nucleotides in length; The composition of claim 1.

3. 2. The composition of claim 1, wherein the region of complementarity is at least 19 contiguous nucleotides in length, optionally at least 20 nucleotides in length.

4. 2. The composition of claim 1, wherein the 3' end of the sense strand comprises a stem loop described as S1-L-S2, where S1 is complementary to S2 and L forms a loop 3 to 5 nucleotides long between S1 and S2. (a) L is a tetraloop, the tetraloop comprising the nucleic acid sequence of SEQ ID NO: 8; and / or (b) the stem loop comprises a nucleic acid sequence having at least 85% identity to SEQ ID NO:7; and / or (c) the stem loop comprises a nucleic acid having up to one, two, or three substitutions, insertions, or deletions relative to SEQ ID NO:7; The composition of claim 4.

6. The composition of claim 1 , wherein the oligonucleotide comprises at least one modified nucleotide.

7. 7. The composition of claim 6, wherein the modified nucleotide comprises a 2'-modification, the 2'-modification being selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinoic acid. (a) at least one of nucleotides 1 to 7, 11 to 27, and 31 to 36 of the sense strand and one or more of nucleotides 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand, or all of the nucleotides are modified with 2'-O-methyl, e.g., with a 2'-O-methyl ribonucleoside; or (b) at least one of nucleotides 3, 8, 9, 10, 11, 12, 13, and 17 of the sense strand and one or more or all of nucleotides 2, 3, 4, 5, 7, 8, 10, 12, 14, 16, and 19 of the antisense strand are modified with 2'-fluoro, e.g., a 2'-fluoro deoxyribonucleoside; The composition of claim 7. (a) the oligonucleotide comprises at least one modified internucleotide bond, and / or (b) there is no internucleotide bond between the sense strand and the antisense strand; and / or (c) the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog; and / or (d) the RNAi oligonucleotide comprises a uridine at position 1 of the 5' end of the antisense strand; The composition of claim 1.

10. The composition of claim 1, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, and optionally each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

11. The composition of claim 10, wherein the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

12. The sense strand and the antisense strand are (a) SEQ ID NOs: 1 and 3, respectively; and (b) SEQ ID NOs: 4 and 6, respectively. The composition of claim 1, comprising a nucleotide sequence selected from the group consisting of: (a) the sense strand comprises a nucleotide sequence set forth in SEQ ID NO:37 and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO:38; or (b) the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 39, and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 40; The composition of claim 1.

14. A pharmaceutical composition comprising the composition of claim 1 and a pharma- ceutically acceptable carrier, excipient, or diluent.

15. The composition of claim 1 for use in a method for treating a disease mediated by complement pathway activation or dysregulation, the method comprising contacting a subject's cells with the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof.

16. wherein the cells are contacted for a period of time sufficient to effect degradation of C3 mRNA transcripts; (a) expression of C3 in the cell is reduced; and / or (b) translation of C3 mRNA in said cell is reduced; and / or (c) the level and / or activity of C3 in the cell is reduced; The composition of claim 15.

17. The composition of claim 15, wherein the subject has been identified as having a disease, disorder, or condition mediated by complement pathway activation or dysregulation.

18. The disease mediated by complement pathway activation or dysregulation includes, but is not limited to, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), IgA nephropathy, lupus nephritis, C3 glomerulopathy (C3G), dermatomyositis / autoimmune myositis, systemic sclerosis, demyelinating polyneuropathy, pemphigus, membranous nephropathy, focal segmental glomerular sclerosis (FSGS), bullous pemphigoid, epidermolysis bullosa acquisita (EBA), mucous membrane pemphigoid, ANCA vasculitis, hypocomplementemic urticarial vasculitis, immune complex small vasculitis, cutaneous small vasculitis, autoimmune necrotizing myopathy, renal, hepatic, cardiac or rejection of transplanted organs such as lung transplant rejection, antibody-mediated rejection (AMR) such as chronic AMR (cAMR), antiphospholipid (aPL) syndrome, glomerulonephritis, asthma, dense deposit disease (DDD), age-related macular degeneration (AMD), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), severe refractory RA, Felty's syndrome, multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, ischemia-reperfusion injury, preeclampsia, post-transplant dysfunction in acute kidney injury (DGF-AKI), cardiopulmonary bypass-associated acute kidney injury, hypoxic-ischemic encephalopathy, dialysis-induced thrombosis , Takayasu's arteritis, relapsing polychondritis, acute / prophylactic graft-versus-host disease, chronic graft-versus-host disease, beta-thalassemia, stem cell transplant-associated thrombotic microangiopathy, biliary atresia, inflammatory liver disease, Behçet's disease, ischemic stroke, intracerebral hemorrhage, scleroderma, scleroderma renal crisis, scleroderma-associated interstitial lung disease (SSc-ILD), sickle cell disease, autosomal dominant polycystic kidney disease (ADPKD), chemotherapy-induced peripheral neuropathy (CIPN), diabetic neuropathy, amyotrophic lateral sclerosis (ALS), diabetic nephropathy, diabetic retinopathy, geographic atrophy, pulmonary arterial hypertension, refractory severe asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF), chronic lung allograft dysfunction, pulmonary disease in cystic fibrosis, hidradenitis suppurativa, nonalcoholic fatty liver disease (NASH), ankylosing spondylitis, hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) (prevention), coronary artery disease, atherosclerosis, osteoporosis (prevention), osteoarthritis, high-risk drusen, inflammatory bowel disease, ulcerative colitis, interstitial cystitis, dialysis-induced complement activation, pyoderma gangrenosum, chronic heart failure, autoimmune myocarditis, nasal polyposis, acute and chronic pancreatitis, atherosclerosis, eosinophilic esophagitis,16. The composition of claim 15, which is for treating eosinophilic granulomatosis, hypereosinophilic syndrome, wound healing, and thrombotic thrombocytopenic purpura (TTP).

19. 13. The composition of claim 1 for use in a method for reducing C3 expression in a cell, a population of cells, or a subject, the method comprising: i) contacting said cell or said population of cells with said RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof; or ii) administering said composition to said subject. and optionally, reducing C3 expression comprises decreasing the amount or level of C3 mRNA, the level of C3 protein, or both, by about 10% to 100% compared to the level of C3 mRNA, the level of C3 protein, or both, in cells of a subject not administered the composition. composition.

20. A kit comprising the composition of claim 1.

21. 2. The treatment of diseases such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), IgA nephropathy, lupus nephritis, C3 glomerulopathy (C3G), dermatomyositis / autoimmune myositis, systemic sclerosis, demyelinating polyneuropathy, pemphigus, membranous nephropathy, focal segmental glomerular sclerosis (FSGS), bullous pemphigoid, epidermolysis bullosa reticularis (EBA), mucous membrane pemphigoid, ANCA vasculitis, hypocomplementemic urticarial vasculitis, immune complex small vasculitis, cutaneous small vasculitis, autoimmune necrotizing myopathy, kidney, liver, heart or lung transplant rejection, in a subject in need thereof. Rejection of transplanted organs, e.g., antibody-mediated rejection (AMR), antiphospholipid (aPL) syndrome, glomerulonephritis, asthma, dense deposit disease (DDD), age-related macular degeneration (AMD), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), severe refractory RA, Felty's syndrome, multiple sclerosis (MS), traumatic brain injury (TBI), spinal cord injury, ischemia-reperfusion injury, preeclampsia, impaired function after transplantation in acute kidney injury (DGF-AKI), cardiopulmonary bypass-associated acute kidney injury, hypoxic-ischemic encephalopathy, dialysis-induced thrombosis, Takayasu's arteritis, relapsing polychondritis, acute / prophylactic transplant Graft-versus-host disease, chronic graft-versus-host disease, beta-thalassemia, stem cell transplant-associated thrombotic microangiopathy, biliary atresia, inflammatory liver disease, Behçet's disease, ischemic stroke, intracerebral hemorrhage, scleroderma, scleroderma renal crisis, scleroderma-associated interstitial lung disease (SSc-ILD), sickle cell disease, autosomal dominant polycystic kidney disease (ADPKD), chemotherapy-induced peripheral neuropathy (CIPN), diabetic neuropathy, amyotrophic lateral sclerosis (ALS), diabetic nephropathy, diabetic retinopathy, geographic atrophy, pulmonary arterial hypertension, refractory severe asthma, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis (IPF), chronic pulmonary Allograft dysfunction, Pulmonary conditions in cystic fibrosis, Hidradenitis suppurativa, Nonalcoholic fatty liver disease (NASH), Ankylosing spondylitis, Hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA) (prevention), Coronary artery disease, Atherosclerosis, Osteoporosis (prevention), Osteoarthritis, High-risk drusen, Inflammatory bowel disease, Ulcerative colitis, Interstitial cystitis, Dialysis-induced complement activation, Pyoderma gangrenosum, Chronic heart failure, Autoimmune myocarditis, Nasal polyposis, Acute and chronic pancreatitis, Atherosclerosis, Eosinophilic esophagitis, Eosinophilic granulomatosis, Hypereosinophilic syndrome, Wound healing,and the pharmaceutical composition according to claim 14 for use in the prevention or treatment of thrombotic thrombocytopenic purpura (TTP).

22. The composition of claim 1 , wherein the RNA oligonucleotide comprises a pharma- ceutically acceptable salt, and the pharma- ceutically acceptable salt is a sodium salt.

23. The composition of claim 15, wherein the disease or dysregulation comprises antibody-mediated rejection, membranous nephropathy, and / or pulmonary arterial hypertension.

24. A pharmaceutical composition as described in claim 14 for use in the prevention or treatment of a disease or disorder in a subject in need thereof, wherein the disease or disorder comprises antibody-mediated rejection, membranous nephropathy, and / or pulmonary arterial hypertension.

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