Compositions and methods for inhibiting expression of complement component 3

Abstract

To provide compositions and methods for inhibiting expression of a complement component 3.SOLUTION: Described herein is an oligonucleotide (e.g., an RNAi oligonucleotide) comprising sense and antisense strands for targeting a complement component 3 (C3) mRNA. The RNAi oligonucleotide may be used to inhibit C3 expression, level, and / or activity in a cell. A method for using an oligonucleotide (e.g., RNAi oligonucleotide) to prevent or treating diseases, disorders or conditions mediated by the complement pathway activation or dysregulation is also described.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

[0002] The complement system plays a central role in the clearance of immune complexes and in the immune response 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 prepare the body's defense against microbial infections and to maintain tissue hemostasis. Complement is a tightly regulated enzyme cascade that can be activated by one of the following three pathways: the classical pathway in which antibody complexes trigger activation, the alternative pathway that 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 initiated by mannose residues present on certain microorganisms such as specific bacteria, fungi, viruses, etc. Uncontrolled activation or insufficient regulation of the complement pathway can lead to systemic inflammation, cell injury, and tissue damage. Thus, the complement pathway is involved in the etiology of many diverse diseases. Inhibition or regulation of complement pathway activity is recognized as a promising therapeutic strategy. The number of treatment options available for these diseases is limited. Therefore, developing innovative strategies for treating diseases associated with complement pathway activation or dysregulation is an important unmet need.

[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, such as complement activation, opsonization and removal of pathogens, immune complexes, and damaged cells, as well as regulation of humoral and T cell adaptive immune responses.

[0004] C3 is an essential protein for the complement system that helps initiate the complement pathway cascade. Activation of C3 via the classical pathway, alternative pathway, or lectin pathway causes cleavage of C3, resulting in the degradation 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 and leads to further activation of downstream terminal complement. Cleavage of C5 results in the formation of C5a, which is also a potent chemotactic driving factor and anaphylatoxin, and C5b, which rapidly assembles with complement proteins C6, 7, 8, and 9 to construct the pore-forming complex C5b-9 on the pathogen or tissue surface. As a result, C3 can be an ideal target for inhibition or silencing to selectively inhibit the complement pathway as a method for treating diseases associated with complement pathway activation or dysregulation.

Summary of the Invention

Means for Solving the Problems

[0005] This specification describes oligonucleotides (e.g., RNAi oligonucleotides such as sense and antisense strand oligonucleotides) that target complement component (C3), which has been found to play a role in complement pathway activation. An RNAi oligonucleotide or a pharmaceutically acceptable salt thereof (e.g., its sodium salt) can be used to treat a patient having a disease 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, which includes a sense strand and an antisense strand, or a pharmaceutically acceptable salt thereof, wherein the sense strand and the antisense strand form a double-stranded region. The antisense strand includes a region complementary to the C3 mRNA target sequence of SEQ ID NO: 13 or 14, and the complementary region is at least 15 consecutive nucleotides in length. In some embodiments, the sense strand is 15 to 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 to 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 to 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-stranded region that is 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-stranded region that is at least 19 nucleotides in length, optionally at least 20 nucleotides in length. In some embodiments, the complementary region is at least 19 consecutive 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 3 to 5 nucleotides in length between S1 and S2. In some embodiments, L is a triloop or a tetraloop. In some embodiments, L is a tetraloop. In some embodiments, the tetraloop comprises the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, S1 and S2 are 1 to 10 nucleotides in length, and 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 (e.g., at least 95%, 96%, 97%, 98%, 99%, and 100% identity to the sequence number) 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 at most 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 longer. 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 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 and antisense strands are selected from the group consisting of (a) SEQ ID NOs: 1 and 3, respectively, and (b) SEQ ID NOs: 4 and 6, respectively, nu It contains a nucleotide sequence. In some embodiments, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 1, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 3. In some embodiments, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 4, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, as shown in Compound A, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 37, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 38. In some embodiments, as shown in Compound B, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 39, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 40. In some embodiments, as shown in Compound C, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 41, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 42. In some embodiments, as shown in Compound D, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 43, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 44. In some embodiments, as shown in Compound E, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 45, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 46. In some embodiments, as shown in Compound F, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 47, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 48. In some embodiments, as shown in Compound G, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 49, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 50. In some embodiments, as shown in Compound H, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 51, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 52. In some embodiments, as shown in Compound I, the sense strand contains the nucleotide sequence set forth in SEQ ID NO: 53, and the antisense strand contains the nucleotide sequence set forth in SEQ ID NO: 54.

[0010] This specification provides an RNAi oligonucleotide or a pharmaceutically acceptable salt thereof that includes a sense strand and an antisense strand, where 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 pharmaceutically acceptable salt thereof, includes SEQ ID NO: 4 and SEQ ID NO: 6. In other embodiments, the RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof, includes 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 includes 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. In another embodiment, the double helix region has a length of 20 to 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 pharmaceutically acceptable salt thereof, comprises uridine at the 1-position 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-methylphosphonate. In some embodiments, the uridine comprising a phosphate analog has the following structure:

Chemical formula

[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 to 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 to 40 (e.g., 25 to 40, 30 to 40, 35 to 40, 30 to 35, 25 to 35, 20 to 25, 21 to 30, and 31 to 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 comprise a 2'-modification. In some embodiments, the 2'-modification is 2'-fluoro or 2'-O-methyl, where optionally, the 2'-fluoro modification is 2'-fluorodeoxyribonucleoside, and / or the 2'-O-methyl modification is 2'-O-methyl ribonucleoside.

[0016] In one embodiment, the RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof, comprises 40 to 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 pharmaceutically acceptable salt thereof, comprises 40 to 50 (e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50) 2'-O-methyl ribonucleosides. In one embodiment, nucleotides 1 to 7, 11 to 27, and 31 to 36 of the sense strand at least one of (e.g., at least two, at least five, at least ten, at least twenty, and at least thirty), 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, such as 2'-O-methyl ribonucleoside. 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, such as 2'-O-methyl ribonucleoside. 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, such as 2'-O-methyl ribonucleoside. 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, such as 2'-O-methyl ribonucleoside.

[0017] In another embodiment, the RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof, comprises 5 to 15 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15) 2'-fluoro modifications, such as 2'-fluorodeoxyribonucleosides. 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'-fluorodeoxyribonucleoside. In another embodiment, 2 to 4 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'-fluorodeoxyribonucleoside. 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, such as 2'-fluorodeoxyribonucleoside.

[0018] In some embodiments, the RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof, comprises at least one (e.g., at least 2, at least 5, at least 10, at least 20, and at least 30) modified internucleotide linkages. In some embodiments, at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof, has phosphorothioate linkages 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 strand and the antisense strand.

[0020] In some embodiments, at least one nucleotide of the oligonucleotide is conjugated with 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 pharmaceutically acceptable salt thereof, comprises 1 to 5 2'-O-N-acetylgalactosamine (GalNAc) moieties conjugated to the sense strand. In some embodiments, up to 4 nucleotides of the stem-loop L are conjugated to a monovalent GalNAc moiety. In some embodiments, the RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof, comprises 1 to 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 2, or at least 3) 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 positions 28 to 30 of the sense strand are conjugated to a monovalent GalNAc moiety. In some embodiments, each of the nucleotides at positions 28 to 30 of the sense strand is conjugated to a monovalent GalNAc moiety.

[0021] In some embodiments, the nucleotides at positions 28 to 30 of the sense strand have the following structure:

Chemical formula

Chemical formula

[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 includes a double helix between all or part of the sense strand and all or part of the antisense strand. In some embodiments, the complementary region 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 includes 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 pharmaceutically acceptable salt thereof, is double-stranded ribonucleic acid (dsRNA). In some embodiments, the RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof, is single-stranded ribonucleic acid.

[0025] In some embodiments, the RNA oligonucleotide includes a pharmaceutically acceptable salt. In some embodiments, the pharmaceutically 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 pharmaceutically acceptable salt thereof) and a pharmaceutically 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 pharmaceutically acceptable salt thereof.

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

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

[0030] In another aspect, the present disclosure provides a cell comprising any one of the vectors described herein or the oligonucleotides described herein (e.g., any of the RNAi oligonucleotides, or a pharmaceutically acceptable salt thereof).

[0031] In another aspect, the present disclosure provides a method for treating a disease mediated by complement pathway activation or dysregulation, which comprises contacting a cell of a subject with any one of the oligonucleotides described herein (e.g., RNAi oligonucleotides), the pharmaceutical compositions described herein, the vectors described herein, or the cells described herein. In some embodiments, the cell is contacted for a time sufficient to achieve degradation of the C3 mRNA transcript. In some embodiments, the expression of C3 in the cell is reduced. In some embodiments, the transcription of C3 in the cell is reduced. In some embodiments, the level and / or activity of C3 in the cell is decreased. In some embodiments, the level and / or activity of C3 is decreased by 10% to 100% (e.g., 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% decrease) compared to the level and / or activity of C3 in the cells of a subject not administered any one of the RNAi oligonucleotides described herein, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition, the vector, or the cell. In some embodiments, the level and / or activity of C3 is decreased 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 the cells of a subject not administered any one of the RNAi oligonucleotides described herein, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition, the vector, or the cell. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0032] In another aspect, the present disclosure provides a method for reducing C3 expression in a cell, a population of cells, or a subject, the method comprising: i) contacting the cell or population of cells with any one of the RNAi oligonucleotides described herein, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, or a vector; or ii) administering to the subject any one of the RNAi oligonucleotides described herein, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, or a vector. 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., 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% reduction) compared to the level of C3 mRNA, the level of C3 protein, or both in cells of a subject not administered any one of the RNAi oligonucleotides described herein, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, a vector, or a cell.

[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 not administered any one of the RNAi oligonucleotides described herein, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, a vector, or a cell.

[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, classical complement pathway, and / or lectin pathway). In some embodiments, diseases mediated by or associated with complement pathway activation or dysregulation include the following: 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, hypocomplementemic urticarial vasculitis, immune complex small vessel vasculitis, cutaneous small vessel vasculitis, autoimmune necrotizing myopathy, rejection of transplanted organs such as kidney, liver, heart or lung transplantation rejection, e.g., antibody-mediated rejection (AMR) such as chronic AMR (cAMR), antiphospholipid (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, dysfunction of transplanted organs after organ transplantation in acute kidney injury (DGF-AKI), cardiopulmonary bypass-related acute kidney injury, hypoxic-ischemic encephalopathy, dialysis-induced thrombosis, Takayasu arteritis, relapsing polychondritis, acute / preventive graft-versus-host disease, chronic graft-versus-host disease, beta-thalassemia, stem cell transplantation-related 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, lung disease state in cystic fibrosis, hidradenitis suppurativa, non-alcoholic fatty liver disease (NASH), ankylosing spondylitis, hematopoietic stem cell transplantation-related thrombotic microangiopathy (HSCT-TMA) (prevention), coronary artery disease,Atherosclerotic arteriosclerosis, 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, atherosclerotic arteriosclerosis, eosinophilic esophagitis, eosinophilic granulomatosis, eosinophilia syndrome, wound healing, and thrombotic thrombocytopenic purpura (TTP). In some embodiments, the subject is identified as having an antibody-related rejection reaction (AMR) such as chronic AMR.

[0035] In some embodiments, the present disclosure provides a method of treating an antibody-related rejection reaction (AMR) such as chronic AMR (cAMR), which comprises contacting a cell of a subject with any one of the RNAi oligonucleotides described herein, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, a vector, or a cell. In some embodiments, any one of the RNAi oligonucleotides described herein, or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, a vector, or a cell is for use in the prevention or treatment of an antibody-related rejection reaction (AMR) such as chronic AMR (cAMR) in a subject in need thereof.

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

[0037] In one embodiment, the oligonucleotide (e.g., the RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof), or a composition thereof, is formulated for daily, weekly, monthly, or annual 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 dosage 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 dosage 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 other therapeutic agents.

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

[0040] In another aspect, the present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof) described herein, a pharmaceutical composition described herein, a vector described herein, or a cell described herein for use in preventing or treating 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 present disclosure provides an oligonucleotide (e.g., an RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof), a pharmaceutical composition, a composition, a vector, or a cell as described herein, wherein the RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition, the composition, the vector, or the cell is administered either, or formulated for subcutaneous administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0042]

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Figure 37

[0043] Definitions 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 of skill in the art. Suitable methods for administering oligonucleotides include, for example, oral, injection (e.g., intravenous, intraperitoneal, intramuscular, intravitreal, and subcutaneous), infusion formulations, and the like. The method of administering the oligonucleotide 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 is known in the art. The formulation 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" mean, for example, reducing the level, reducing the expression, or reducing the transcription of C3 mRNA and / or C3 protein in a cell or subject 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 C3 protein in a cell or subject). The decrease can be about 5% or more (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100%) of the level, expression, or transcription of C3 mRNA and / or C3 protein compared to before treatment, or compared to the level of C3 mRNA or C3 protein in an untreated subject (e.g., a subject having a disease or disorder related to 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 related to complement activation or dysregulation (e.g., activation or dysregulation of C3))). 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, C3 can be wild-type C3, mutant C3, or transgenic C3 in relation to a genetically engineered cell, cell population, or organism. "Reducing the activity of C3" also means reducing the level of activity associated with C3 (e.g., by reducing the activation of the complement pathway associated with a disease mediated by complement pathway activation or dysregulation).The activity of C3 can 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 can be measured using any method known in the art. The reduction can be 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) in the level, expression, or transcription of C3 mRNA and / or C3 protein compared to cells or subjects not treated with the RNAi oligonucleotides disclosed herein. The above reduction in the level, expression, or transcription of C3 mRNA and / or C3 protein can occur over a period of at least about 1 day or more (e.g., at least 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days, 120 days, or more). The reduction can be a decrease in the amount of C3 protein in the blood of the treatment 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). can be less.

[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 include nucleosides in which the nucleobase moiety is modified by changing a purine or pyrimidine to an alternative purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolo-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 also include nucleosides in which the sugar moiety is modified; for example, 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytosine, 2'-O-methyluridine, 2-fluoro-deoxyadenosine, 2-fluoro-deoxyguanosine, 2-fluoro-deoxycytidine, 2-fluoro-deoxyuridine.

[0047] Exemplary nucleobases having alternative uracils are listed below: 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 nucleobase 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 a substituted cytosine are listed below: 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 (k2C), 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 2cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0049] Exemplary nucleobases having a substituted adenine are listed below: 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-adenosine (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 2A), 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-trimethyl adenosine (m 6 2Am), 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 having alternative guanine are listed below: inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxwybutosine (o2yW), hydroxywybutosine (OhyW), under-modified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (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 2,7G), 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 2Gm), 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] The nucleobase moiety may also be represented by the letter code of each corresponding nucleobase, e.g., A, T, G, C, or U, and in such cases, each letter may 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 part of a gene, primary transcript, or processed mRNA (e.g., the sequence of C3 (e.g., SEQ ID NO: 12)) so as to interfere with the expression of an endogenous gene (e.g., C3).

[0053] The terms "antisense strand" and "guide strand" refer to the strand of an RNAi oligonucleotide (e.g., dsRNA) that contains a region that is substantially complementary to a target sequence, e.g., C3 mRNA (e.g., 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 can be logically encompassed, as apparent 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 the 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 of numbers or a range, "at least" can modify each of the numbers within the series or range. It is understood that each can be modified.

[0055] As used herein, the term "attenuate" means to reduce or substantially stop. By way of non-limiting example, one or more of the treatments provided herein can suppress 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 can be exemplified, for example, by a reduction in one or more aspects of a disease associated with complement pathway activation or dysregulation (e.g., symptoms, tissue characteristics, and cellular, inflammatory or immunological activities, etc.), e.g., 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., having uridine substituted by thymidine). Generally, those skilled in the art understand that the cDNA sequence is the same as the mRNA sequence except that uridine is read as thymidine, such that the terms cDNA and mRNA can be used interchangeably with respect to a particular gene (e.g., the C3 gene).

[0057] As used herein, the terms "C3" and "complement component 3" refer to the protein or gene encoding complement component 3. The term "C3" refers to a natural variant of the wild-type C3 protein, for example, 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 number NP_000055.2 or SEQ ID NO: 11, such as a natural variant of the wild-type C3 protein. The term "C3" also refers to a natural variant of the wild-type C3 polynucleotide, for example, a polynucleotide 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 as set forth in NCBI reference number 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 defines the dosage 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, two or more agents are not co-formulated and are administered sequentially as part of the prescribed regimen. In some embodiments, the administration of two or more combined agents or treatments is such that the reduction of symptoms associated with the disorder or other parameters is greater than would be observed with one agent or treatment alone or in the absence of the other. The effects of the two treatments can be partially additive, fully additive, or greater than additive (e.g., synergistic). The sequential or substantially concurrent administration of each therapeutic agent can be affected by any suitable route, such as, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissues. The therapeutic agents can be administered by the same route or by different routes. For example, the first therapeutic agent of the combination may be administered by intravenous injection and the second therapeutic agent of the combination may be administered orally.

[0059] As used herein, the term "complement pathway activation or dysregulation" refers to any abnormality in the ability of the complement pathways, including the classical pathway, alternative pathway, and lectin pathway, to confer host defense against pathogens, eliminate immune complexes and damaged cells, and their ability to regulate immunity. Activation or dysregulation of the complement pathway can occur in the fluid phase and on cell surfaces, which can lead to excessive complement activation or insufficient regulation, both 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, as understood by one of ordinary skill in the art, refers to the ability of an oligonucleotide containing the first nucleotide or nucleoside sequence to hybridize with an oligonucleotide containing the second nucleotide sequence under certain conditions and form a double-stranded structure. Such conditions may be, for example, stringent conditions, which may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours, followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual," Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered within an organism, can also be applied. One of ordinary skill in the art will be able to determine the set of conditions most suitable for testing the complementarity of two sequences, depending on the ultimate application of the hybridized nucleotides or nucleosides. Also, as used herein, "complementary" sequences can include, or be entirely formed from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and alternative nucleotides or nucleosides, as long as the above requirements regarding the ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen-type base pairs. As described herein, complementary sequences within an oligonucleotide (e.g., an RNAi oligonucleotide) or between an oligonucleotide and a target sequence include base pairs between an oligonucleotide containing the first nucleotide or nucleoside sequence and an oligonucleotide containing the second nucleotide or nucleoside sequence over the entire length of one or both of the nucleotide or nucleoside sequences. Such sequences can be referred to herein as being "fully complementary" to each other.When a first array is said to be "substantially complementary" to a second array, the two arrays are either completely complementary or they can form one or more, but generally five, four, three, or two or fewer mismatched base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application (e.g., reduction of expression via the RISC pathway) during hybridization for a double helix of up to 30 base pairs. "Substantially complementary" may also refer to an oligonucleotide that is substantially complementary to a continuous portion of the mRNA of interest (e.g., the mRNA encoding C3). For example, an oligonucleotide is complementary to at least a portion of C3 mRNA when the sequence is substantially complementary to an uninterrupted portion of the mRNA encoding C3. However, if two oligonucleotides are designed to form one or more single-stranded overhangs during hybridization, such overhangs shall not be considered mismatches with respect to the determination of complementarity. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) comprising one oligonucleotide 22 linked nucleosides in length and another oligonucleotide 20 nucleosides in length can be referred to as "completely complementary" for the purposes described herein even if they have different lengths.

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

[0062] As used herein, the phrase "contacting a cell with an oligonucleotide" includes contacting a cell with an oligonucleotide, such as a single-stranded oligonucleotide or a double-stranded oligonucleotide (e.g., single-stranded RNA or double-stranded RNA forming a double helix), by a method 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. The contact may be carried out either directly or indirectly. Thus, for example, the oligonucleotide may be brought into physical contact with the cell by an individual performing the method, or alternatively, the RNAi oligonucleotide may be placed in a situation that will allow or cause it to contact the cell subsequently. Contacting a cell in vitro can be accomplished, for example, by incubating the cell with the oligonucleotide. Contacting a cell in vivo can be accomplished, for example, by injecting the oligonucleotide into the tissue where the cell is located or in the vicinity thereof, or by injecting the RNAi oligonucleotide into another region, such as the bloodstream or subcutaneous space, such that the drug will reach the tissue where the cell to be contacted is located. For example, the oligonucleotide may include a ligand that directs the oligonucleotide to the site of interest and / or be linked to a ligand, or may be incorporated into a vector (e.g., a viral vector) that delivers the oligonucleotide to the site of interest. Combinations of in vitro and in vivo contact methods are also possible. For example, after contacting a cell with an oligonucleotide in vitro, the cell can also be transplanted into a subject.

[0063] The term "continuous nucleic acid base 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 terms "continuous nucleotide sequence" or "continuous nucleotide base sequence". In some embodiments, all of the nucleotides of the oligonucleotide are present in a continuous nucleotide or nucleoside region. In some embodiments, the oligonucleotide comprises a continuous nucleotide region and optionally may further comprise one or more additional nucleotides or one or more additional nucleosides. The nucleotide linker region may or may not be complementary to the target nucleic acid. The internucleoside linkage present between the nucleotides of the continuous nucleotide region may include phosphorothioate internucleoside linkages. Additionally, the continuous nucleotide region may comprise one or more sugar-modified nucleosides.

[0064] As used herein, the term "deoxyribonucleotide" refers to a nucleotide having a hydrogen in place of a hydroxyl at the 2'-position of its pentose sugar as compared to a ribonucleotide. A modified deoxyribonucleotide is a deoxyribonucleotide having one or more modifications or substitutions of atoms other than the 2'-position and includes modifications or substitutions of the sugar, phosphate group, or base.

[0065] As used herein, the term "disease" refers to an interruption, cessation, or disorder of a bodily function, system, or organ. The diseases or disorders of interest include those that would benefit from treatment with an oligonucleotide as described herein (e.g., a single-stranded or double-stranded RNA construct that forms a duplex as described herein) that targets C3, such as by the treatment 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, the following: 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, 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, hypocomplementemic urticarial vasculitis, immune complex small vessel vasculitis, cutaneous small vessel vasculitis, autoimmune necrotizing myopathy, rejection of transplanted organs such as kidney, liver, heart or lung transplant rejection, for example, antibody-related rejection (AMR) such as chronic AMR (cAMR), antiphospholipid (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, dysfunction of transplanted organs after organ transplantation in acute kidney injury (DGF-AKI), cardiopulmonary bypass-related acute kidney injury, hypoxic-ischemic encephalopathy, dialysis-induced thrombosis, Takayasu arteritis, relapsing polychondritis, acute / preventive graft-versus-host disease, chronic graft-versus-host disease, beta-thalassemia, stem cell transplantation-related thrombotic microangiopathy, biliary atresia, inflammatory liver disease, Behçet's disease, ischemic stroke, intracerebral hemorrhage, scleroderma, scleroderma renal crisis, scleroderma-related 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 conditions in cystic fibrosis, hidradenitis suppurativa, non-alcoholic fatty liver disease (NASH), ankylosing spondylitis, hematopoietic stem cell transplantation-related 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, eosinophilia syndrome, wound healing, and thrombotic thrombocytopenic purpura (TTP).

[0066] As used herein, the term "double helix" refers to the structure formed by complementary base pairing of an antiparallel arrangement of two nucleotides with respect to a nucleic acid (e.g., an oligonucleotide).

[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 clinical outcomes, when administered to a subject such as a human, and thus, the "effective amount" or its synonyms will 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 an amount of an agent that reduces the level and / or activity of C3 sufficient to achieve a therapeutic response as compared to the response obtained without administration of an 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 corresponding to such an amount can vary depending on various factors such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject (e.g., age, sex, and / or weight) or host being treated, but can nevertheless be routinely determined by one of ordinary skill in the art. Also, as used herein, the "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 as compared to a control. As defined herein, the 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 conventional methods known in the art. The dosing regimen can be adjusted to provide an optimal therapeutic response.

[0068] As used herein, the term "excipient" refers to a non-therapeutic agent that can be included in a composition, for example, to impart or contribute to a desired viscosity 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 also contain alternative sugar moieties in addition to ribose and deoxyribose. It is also understood that the term "nucleotide" may refer to alternative nucleotides or surrogate substitution moieties as further detailed below. Those skilled in the art are well aware that guanine, cytosine, adenine, and uracil can be substituted by other moieties without substantially altering the base pairing properties of oligonucleotides containing nucleotides bearing such substitution moieties. For example, without limitation, a nucleotide containing inosine as its base can form a base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be substituted by a nucleotide containing inosine, for example, in the nucleotide sequence of an oligonucleotide particularly taken up in the present disclosure. In another example, any adenine and cytosine in an oligonucleotide can be substituted with guanine and uracil, respectively, to form G-U wobble base pairs that pair with a target mRNA. Sequences containing such substitution moieties are suitable for the compositions and methods particularly taken up in the present 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). The term "reduce" as used herein is used interchangeably with "silencing", "downregulation", "suppression", and other similar terms and includes any level of decrease 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] As used herein, the term "level" means the level or activity of a protein, or 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 means a decrease or increase in the protein level, respectively, compared to the reference (e.g., a decrease or increase of 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 about 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or more than about 200% compared to the reference; 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 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). The level of a protein or mRNA can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, ng / mL) or percentage relative to the total protein or mRNA in a sample.

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

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

[0074] As used herein, the term "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to the corresponding reference nucleotide selected from: adenosine ribonucleotide, guanosine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenosine deoxyribonucleotide, guanosine 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, a modified nucleotide imparts one or more desirable properties to a nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduction of immunogenicity, and the like.

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

[0076] The terms "nucleobase" and "base" include purines (e.g., adenine and guanine) and pyrimidines (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 includes 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 monomer unit of a nucleobase and a sugar moiety or an oligonucleotide having a nucleobase and a sugar moiety. Nucleosides can include naturally occurring ones 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, the term "nucleotide" refers to the monomeric units of oligonucleotides that include nucleosides and internucleoside linkages. The internucleoside linkages may or may not include a phosphate bond. Similarly, "linked nucleosides" may or may not be linked by a phosphate bond. Many "alternative internucleoside linkages" are known in the art and include, but are not limited to, phosphate, phosphorothioate, and boranophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNA) (e.g., locked nucleosides (LNA) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNA), phosphotriesters, phosphorothionates, phosphoramidates, and other variants of the phosphate backbone of natural nucleosides including those described herein.

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

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

[0081] As used herein, the terms "patient in need thereof" or "subject in need thereof" refer to the identification of a subject based on the need for treatment of a disease or disorder, such as a disease mediated by complement dysregulation (e.g., dysregulation related to C3, dysregulation of one or more of the complement pathways (e.g., alternative, classical, and / or lectin pathways)). A subject can be identified, for example, as having a need for treatment of a disease or disorder (e.g., a disease or disorder related to complement pathway activation or dysregulation disclosed herein), based on, for example, early diagnosis by one of ordinary skill in the art (e.g., a physician).

[0082] The "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 the nucleic acids or amino acids in the reference oligonucleotide or polypeptide sequence, after aligning the relevant sequences and introducing gaps as needed to achieve the maximum percent sequence identity. Alignments for the purpose of determining nucleic acid or amino acid sequence identity percentages can be achieved in a variety of ways within the capabilities of one of ordinary skill in the art, using, for example, generally available computer software such as BLAST, BLAST-2, or Megalign software. One of ordinary skill in the art can determine appropriate parameters for aligning the sequences, including any algorithms necessary to achieve the maximum alignment over the full 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 against a given nucleic acid or amino acid sequence, B (or, alternatively expressed, a given nucleic acid or amino acid sequence, A having a particular percent identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows: (Fraction X / Y) × 100 (In the formula, X is the number of nucleotides or amino acids scored as identical matches in the alignment of A and B by an array alignment program (such as BLAST), and Y is the total number of nucleic acids of B. It will be understood that when the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, 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, "pharmaceutically acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving the active compound), and has the property of being substantially non-toxic and non-inflammatory in a patient. Excipients include, for example, the following: antiadhesion agents, antioxidants, binders, coating agents, compression aids, disintegrants, coloring agents (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or coating agents, flavoring agents, fragrances, fluidizing agents (flow promoters), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and water of hydration. Exemplary excipients include, but are not limited to, the following: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethyl cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methyl cellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, 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" means any pharmaceutically acceptable salt of any of the compounds described herein. For example, pharmaceutically acceptable salts of any of the compounds described herein are within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, 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 during the final isolation and purification of the compounds described herein, or individually, in situ, by reacting the free base moiety with a suitable organic acid. The compounds described herein may have ionizable groups such that they can be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts containing an inorganic or organic acid, or the salts may be prepared from inorganic or organic bases in the case of the acidic forms of the compounds described herein. Often, the compounds are prepared or used as pharmaceutically acceptable salts which are 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 can 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, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, 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, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, and further include non-toxic ammonium, quaternary ammonium, and amine cations, for example, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0085] As used herein, the term "pharmaceutical composition" refers to a composition containing the compounds described herein (e.g., RNAi oligonucleotides), which is formulated together with pharmaceutically acceptable excipients and is optionally manufactured or sold upon obtaining approval from a government regulatory agency as part of a therapeutic regimen for the treatment of diseases in mammals. Pharmaceutical compositions can be, for example, for subcutaneous administration, for intravenous administration (e.g., as a sterile solution without particulate embolisms and in a solvent system suitable for intravenous use); for intrathecal injection; for intracerebroventricular injection; for parenchymal injection; for oral administration in unit dosage forms (e.g., tablets, capsules, caplets, gelcaps, or syrups); for topical administration (e.g., as creams, gels, lotions, or ointments; or can be formulated as any other pharmaceutically 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 the oligonucleotide, instead of the 5'-phosphate that 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'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). In some embodiments, the oligonucleotide has a phosphate analog (referred to as a "4'-phosphate analog") at the 4'-carbon position of the sugar at the 5'-terminal nucleotide. An example of a 4'-phosphate analog is oxyethylmethylphosphonate, or an analog thereof, in which the oxygen atom of the oxyethyl group is bonded to the sugar moiety (e.g., at its 4'-carbon). See, e.g., U.S. Patent Application Publication No. 2019 / 0177729 (each content regarding phosphate analogs is incorporated herein by reference). With respect to the 5'-end of the oligonucleotide, other modifications have been developed (see, e.g., International Publication No. 2011 / 133871 pamphlet; U.S. Patent No. 8,927,513; and Prakash et al. (2015), Nucleic Acids Res., 43(6):2993 - 3011; each content regarding phosphate analogs is incorporated herein by reference).

[0087] As used herein, the term "probe" refers to any molecule that can selectively bind to a specific sequence, such as a nucleic acid molecule such as mRNA. Probes are known in the art and can be synthesized using conventional methods 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 "gene expression reduction" refers to a decrease in the amount of RNA transcript or protein encoded by a gene, and / or a decrease in the amount of gene activity in a cell or subject, as compared to an appropriate 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 the C3 mRNA sequence) can result in a decrease in the amount of RNA transcript, protein, and / or activity (e.g., encoded by the C3 gene) as compared to a cell not treated with the RNAi oligonucleotide. Similarly, "reducing expression" as used herein refers to an act that results in a decrease in the expression of a gene (e.g., C3). The reduction in expression can be evaluated by a decrease in the serum concentration of C3, as described herein (e.g., relative to a cell not in contact with the oligonucleotides described herein). Alternatively, the decrease in expression can be evaluated, for example, by a decrease in the level of transcription and / or translation of C3 mRNA (e.g., a decrease of at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, or 60% or more, e.g., a decrease in the range of 1% to 60% or more) as compared to a cell not in contact with the oligonucleotides described herein.

[0089] "Reference" means any useful reference used to compare the level or activity of a protein or mRNA. The reference can be any sample, standard, standard curve, or level used for the purpose of comparison. The reference can be a normal reference sample or reference standard or level. A "reference sample" can be, for example, a control, such as a predetermined 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 normal cells or normal tissue; a sample from a subject without a disease (e.g., cells or tissue); a sample from a subject diagnosed with a disease but not yet treated with the compounds described herein; a sample from a subject treated with the compounds described herein; or a sample of a purified oligonucleotide or protein at a known normal concentration (e.g., any of those 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 indicating a non-disease state, e.g., a value predicted in a healthy control subject. Typically, a normal control value is expressed as a range ("between X and Y"), a high threshold ("less than or equal to X"), or a low threshold ("greater than or equal to X"). A subject having 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 normal subject without a disease or disorder (e.g., a disease or disorder associated with activation or dysregulation of the complement pathway); a value or numerical value obtained from a subject treated with the compounds 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, gender, disease stage, and general health status. The level of a purified oligonucleotide or protein, e.g., a standard curve within any normal reference range described herein, can also be used as a reference.

[0090] As used herein, the term "complementary region" 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 mRNA (e.g., C3) that has been processed to interfere with the expression of an endogenous gene. When the complementary region is not completely complementary to the target sequence, the mismatch may be present in the internal or terminal region of the molecule. Generally, the most tolerated mismatches are within 5, 4, 3, or 2 nucleotides of the terminal region, e.g., the 5' end and / or 3' end 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 atoms other than the 2'-position, including modifications or substitutions of 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 the Argonaute 2 (Ago2) endonuclease upon cleavage of the 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 the Ago2 endonuclease upon cleavage of the target mRNA. In some embodiments, the RNAi oligonucleotide includes a loop region such as a stem-loop, which contains nucleosides as defined by the term herein. RNAi oligonucleotides include, for example, dsRNA, siRNA, and shRNA, which mediate target 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 the expression of C3 in cells, such as cells within a mammalian subject. Generally, most of the nucleosides of an RNAi oligonucleotide are ribonucleosides, but as detailed herein, each strand or both strands may also include one or more non-ribonucleosides, such as deoxyribonucleosides and / or alternative nucleosides. RNAi oligonucleotides are substantially in double-stranded form. In some embodiments, complementary base pairing in the double-stranded region of the RNAi oligonucleotide is formed covalently between the antiparallel sequences of the nucleotides of the individual nucleic acid strands. In some embodiments, complementary base pairing in the double-stranded region of the RNAi oligonucleotide is formed between the antiparallel sequences of the nucleotides of nucleic acid strands covalently linked together. In some embodiments, the complementary base pairs in the double-stranded region of the RNAi oligonucleotide are formed from a folded single nucleic acid strand (e.g., via a hairpin), providing a complementary antiparallel sequence of nucleotides that base pair together. In some embodiments, the RNAi oligonucleotide includes two covalently distinct nucleic acid strands that are fully double-stranded with each other.However, in some embodiments, the RNAi oligonucleotide is partially double-stranded and includes, for example, two covalently distinct nucleic acid strands having overhangs at one or both ends. In some embodiments, the RNAi oligonucleotide includes an antiparallel sequence of nucleotides that are partially complementary 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 the 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 the 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 the target gene (e.g., the 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, for example, over at least 10-36 nucleotides, for example, 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 RNA agents that are about 10-50 nucleotides in length, optionally RNAi agents, and these strands have overhanging ends that include, for example, 1, 2, or 3 protruding linked nucleosides, which can direct or mediate RNA interference. Naturally occurring siRNAs are produced from longer dsRNA molecules (e.g., >25 linked nucleoside length) by the cell's RNAi machinery (e.g., Dicer or its homolog).

[0095] As used herein, the term “strand” refers to a single Refers to a continuous array. 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, e.g., experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal (e.g., a mammal, e.g., a mouse, rat, rabbit, non-human primate, and human). A subject can be a human or an animal seeking treatment, in need of treatment, requiring treatment, receiving treatment, scheduled to receive treatment in the future, or 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 "alternative sugars" defined as structures that can replace the furanose ring of a nucleoside. In certain embodiments, the alternative sugar is a non-furanose (or 4'-substituted furanose) ring or ring system or an open system. Such structures can include simple variations on the natural furanose ring such as a six-membered ring, or can be more complex, as in the case of the acyclic systems used in peptide nucleic acids. Alternative sugars can also include sugar substitutes where the furanose ring is replaced by another ring system, e.g., a morpholino or hexitol ring system. Sugar moieties useful in the preparation of oligonucleotides having motifs 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 alternative sugars (e.g., bicyclic sugars derived from 2'-O-CH2-4' or 2'-O-(CH2)2-4' bridged ribose) and sugar substitutes (e.g., where the ribose ring is replaced by a morpholino or hexitol ring system). The type of heterocyclic base and internucleoside linkage used at each position can vary and is not a factor in determining the motif. In most nucleosides having an alternative sugar moiety, the heterocyclic nucleic acid base 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 and the nucleotides between the two regions form a loop of unpaired nucleotides when read in the 5' to 3' direction on one side and in the 3' to 5' direction on the other side. The 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 that includes a chain of linked nucleosides. "A strand containing a nucleobase sequence" refers to an oligonucleotide that includes 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-phase nucleic acid synthesizer)) or otherwise not 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 that can specifically bind to the C3 gene or C3 mRNA encoding the C3 gene product. For example, an oligonucleotide that can inhibit the gene or the mRNA (e.g., by reducing the level of the protein encoded by the gene or mRNA) by a method 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., receptor) of a target tissue or cell and is capable of being conjugated to another substance for the purpose of targeting the other substance to the target tissue or cell. For example, in some embodiments, the targeting ligand can be conjugated to an oligonucleotide or a vector (e.g., viral vector) containing the oligonucleotide for the purpose of targeting the oligonucleotide to a specific 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 an oligonucleotide or vector facilitates delivery of the oligonucleotide into a specific cell by selective binding to a receptor expressed on the surface of the cell and endosomal internalization by the cell of the complex comprising the oligonucleotide, targeting ligand and receptor. In some embodiments, 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 within the cell.

[0103] As used herein, the term "tetraloop" refers to a loop that enhances the stability of adjacent double helices 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 double helix that is higher than the average predicted Tm of a pair of loops of equal length consisting of randomly selected nucleotide sequences. For example, a tetraloop can confer a melting temperature of at least 50°C, at least 55°C, at least 56°C, at least 58°C, at least 60°C, at least 65°C, or at least 75°C in 10 mM NaHPO4 to a hairpin containing a double helix of at least 2 base pairs. In some embodiments, the tetraloop can stabilize base pairs in adjacent stem double helices by stacking interactions. Further, interactions between nucleotides in the tetraloop include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, 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 to 6 nucleotides, typically 4 to 5 nucleotides. In certain embodiments, the tetraloop comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., may or may not be conjugated to a targeting moiety). In one embodiment, the tetraloop consists of 4 nucleotides. Any nucleotide may be used for 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 (e.g., UUCG), the GNRA family of tetraloops (e.g., 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), the d(GNRA)) family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(fffTNCG) family of tetraloops (e.g., d(TTCG)).For example, see the following: Nakano et al. Biochemistry, 41(48), 14281-14292, 2002. SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL.78th; NO.2; pg.731(2000) (incorporated herein by reference for their relevant disclosures). In some embodiments, the tetraloop is included within a nick tetraloop structure.

[0104] "Therapeutically effective amount" or "preventively effective amount" refers to an amount of an oligonucleotide composition (e.g., an RNAi oligonucleotide such as dsRNA) that produces a desired local or systemic effect (e.g., treatment of one or more symptoms of a disease resulting from complement pathway activation or dysregulation), administered either as a single dose or in multiple doses. The oligonucleotides (e.g., RNAi oligonucleotides) used in the methods of the present disclosure can be administered in an amount sufficient to produce an appropriate 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 the purpose of improving the health and / or well-being of the subject with respect to an existing condition (e.g., a disease, disorder), or for the purpose of preventing or reducing the likelihood of the occurrence of the condition, e.g., by administration of a therapeutic agent (e.g., an oligonucleotide described herein) to the subject. In some embodiments, treatment includes reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., a disease, disorder) experienced by the subject. In some embodiments, a nucleic acid or oligonucleotide described herein (e.g., an RNAi oligonucleotide) is used to control the cellular and clinical manifestations of a complement pathway disorder, e.g., one or more of the diseases associated with complement pathway activation or dysregulation disclosed herein.

[0106] Detailed Description This specification describes oligonucleotides that target a complement component (C3) known to play a specific role in complement pathway activation, such as RNAi oligonucleotides comprising sense and antisense strand oligonucleotides, and pharmaceutically acceptable salts thereof. Administration of the oligonucleotide can reduce the level and / or activity of C3 in cells (e.g., by hepatocytes). For example, the oligonucleotide can be administered in vivo and can be internalized by cells (e.g., hepatocytes; e.g., by binding to the asialoglycoprotein receptor (ASGPR)). After internalization by the cell, the oligonucleotide can bind to the RNA-induced silencing complex (RISC), target C3 mRNA, thereby initiating degradation of C3 mRNA and preventing its translation.

[0107] Diseases mediated by complement dysregulation are often the result of overactivity of the complement. This specification describes 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, the following: skin disorders, neurological disorders, kidney disorders, acute care, rheumatic disorders, lung disorders, dermatological disorders, hematological disorders, and ophthalmic disorders, 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 glomerulosclerosis (FSGS), bullous pemphigoid, epidermolysis bullosa acquisita (EBA), mucous membrane pemphigoid, ANCA vasculitis, hypocomplementemic urticarial vasculitis, immune complex small vessel vasculitis, cutaneous small vessel vasculitis, autoimmune necrotizing myopathy, kidney, liver, heart or lung transplant rejection, etc. Rejection of transplanted organs, such as antibody-related rejection (AMR) such as chronic AMR (cAMR), antiphospholipid (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, post-transplant dysfunction in acute kidney injury (DGF-AKI), cardiopulmonary bypass-related acute kidney injury, hypoxic-ischemic encephalopathy, dialysis-induced thrombosis, Takayasu arteritis, relapsing polychondritis, acute / preventive graft-versus-host disease, chronic graft-versus-host disease, beta-thalassemia, stem cell transplantation-related thrombotic microangiopathy, biliary atresia, inflammatory liver disease, Behçet's disease, ischemic stroke, intracerebral hemorrhage, scleroderma, scleroderma renal crisis, scleroderma-related 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, lung disease state in cystic fibrosis, hidradenitis suppurativa, non-alcoholic fatty liver disease (NASH), ankylosing spondylitis, hematopoietic stem cell transplantation-related 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, eosinophilia syndrome, wound healing, and thrombotic thrombocytopenic purpura (TTP).

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

[0109] Complement pathway activity can be regulated by using an oligonucleotide (e.g., an RNAi oligonucleotide) to reduce the level and / or activity of C3 in a cell (e.g., a hepatocyte), such as a cell of a subject (e.g., a human) in need thereof. The overall design targets C3 of the complement pathway and leaves the activation (protection) of other pathways of the alternative pathway, classical pathway, and lectin pathway intact. Accordingly, 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 C3 activation or dysregulation.

[0110] Complement component 3 target sequence Oligonucleotide-based inhibitors of C3 expression that can be used to achieve a therapeutic benefit are provided herein. From examination of C3 mRNA (see, e.g., Example 3), as well as in vitro and in vivo studies, the sequence of C3 mRNA has been found to be susceptible to oligonucleotide-based inhibition and thus useful as a targeting sequence. For example, the C3 target sequence can include or consist of the sequences set forth in SEQ ID NO: 13 or 14 corresponding to nucleotides 4121-4141 and 780-798 of Homo sapiens complement C3 having the reference sequence NM_0.000064.4 (SEQ ID NO: 12), respectively. These C3 sequences can be the target sequences of Compound A and Compound B, respectively, and their variants described herein having up to 85% sequence identity thereto. Compound A and B (and their variants described herein) can also effectively target Rhesus macaque and Cynomolgus macaques complement C3 having the reference sequences XM_015122636.2 and XM_005587719.2, respectively. Furthermore, the C3 target sequence can be the target of 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), of Mus It may contain or be composed of the sequences described in any of SEQ ID NO: 31 corresponding to nucleotides 2903 to 2922 of complement C3. Compound J may also target Rattus norvegicus complement C3 having the reference sequence NM_016994.2. These regions of C3 mRNA can be targeted using RNAi oligonucleotides such as the dsRNA agents described herein for the purpose of inhibiting C3 mRNA expression and subsequent C3 protein expression.

[0111] In some embodiments, the antisense strand of the oligonucleotide agent provided herein (e.g., an RNAi oligonucleotide) can be designed to have a region complementary to C3 mRNA (e.g., within the target sequence of C3 mRNA) for the purpose of targeting intracellular mRNA and inhibiting its expression. The complementary region generally has a length and base content appropriate to promote annealing of the oligonucleotide (e.g., an RNAi oligonucleotide) or its strand to C3 mRNA for the purpose of inhibiting its transcription. The complementary region 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 oligonucleotide provided herein can have a region complementary to C3 mRNA in the range of 12 - 30 (e.g., 12 - 30, 12 - 22, 15 - 25, 17 - 21, 18 - 27, 19 - 27, or 15 - 30) nucleotides in length. Thus, the oligonucleotide provided herein can have a region complementary 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 oligonucleotide provided herein can have a region complementary to C3 mRNA that is 19 nucleotides in length. In certain embodiments, the complementary region of the oligonucleotide (e.g., the antisense strand of an RNAi oligonucleotide) may be complementary to a contiguous sequence of the nucleotides of the sequence set forth in SEQ ID NO: 12 that is 20 nucleotides in length.

[0112] In certain examples, the RNAi oligonucleotides of the present disclosure can include a region of complementarity that is at least partially complementary to the sequence set forth in SEQ ID NO: 12 (e.g., on the antisense strand of the RNAi oligonucleotide). For example, the oligonucleotides disclosed herein can include a region of complementarity that is completely complementary to the sequence set forth in SEQ ID NO: 12 (e.g., on the antisense strand of the RNAi oligonucleotide). The region of complementarity of the oligonucleotide (e.g., on the antisense strand of the RNAi oligonucleotide) can be complementary to a contiguous sequence of nucleotides of the sequence set forth in SEQ ID NO: 12, and the above sequence ranges from 12 to 20 nucleotides (e.g., 12 - 20, 12 - 18, 12 - 16, 12 - 14, 14 - 20, 14 - 18, 14 - 16, 16 - 20, 16 - 18, or 18 - 20) in length. In some embodiments, the region of complementarity of the oligonucleotide (e.g., on the antisense strand of the RNAi oligonucleotide) can be complementary to a contiguous sequence of 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 the oligonucleotide (e.g., the antisense strand of the RNAi oligonucleotide) can be complementary to a contiguous sequence of 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 contiguous nucleotides of the sequence set forth in SEQ ID NO: 12 can extend over a portion of the full length of the antisense strand. For example, the region of complementarity of the oligonucleotide that is complementary to the contiguous nucleotides of the sequence set forth in SEQ ID NO: 12 can extend over at least 85% (e.g., at least 86%, at least 90%, at least 95%, and at least 99%) of the full length of the antisense strand. In certain embodiments, the region of complementarity of the oligonucleotide that is complementary to the contiguous nucleotides set forth in SEQ ID NO: 12 can extend over the full length of the antisense strand.

[0114] The region complementary to C3 mRNA may have one or more mismatches compared to the corresponding sequence of C3 mRNA. For example, a region of complementarity on an 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 as long as it maintains the ability to form base pairs complementary to C3 mRNA under appropriate hybridization conditions. Alternatively, the region of complementarity on an oligonucleotide may have 1 or less, 2 or less, 3 or less, 4 or less, or 5 or less mismatches as long as it maintains the ability to form base pairs complementary to C3 mRNA under appropriate hybridization conditions. When there are multiple mismatches in the complementary region, the mismatches may be arranged consecutively (e.g., 2, 3, 4, or 5 consecutively) or may be scattered throughout the region of complementarity as long as the oligonucleotide maintains the ability to form base pairs complementary to C3 mRNA under appropriate hybridization conditions. For example, an RNAi oligonucleotide may 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 respect to the corresponding C3 sequence of SEQ ID NO: 12, or the corresponding antisense sequence of SEQ ID NO: 6 and its variants having 1, 2, 3, 4, or 5 mismatches with respect to the sequence of SEQ ID NO: 4.

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

[0116] The compositions described herein that are oligonucleotides (e.g., RNAi oligonucleotides) encode inhibitory constructs (e.g., nucleic acid vectors encoding the same) that target C3 mRNA (e.g., SEQ ID NO: 12). Oligonucleotides for reducing the expression of C3 expression can participate in the RNA interference (RNAi) pathway either upstream or downstream of Dicer involvement. For example, oligonucleotides (e.g., RNAi oligonucleotides) have been developed that include at least one of a sense or antisense strand that is 19-25 nucleotides in length and has a 3' overhang of 1-5 nucleotides (see, e.g., U.S. Patent No. 8,372,968, which is 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. Patent No. 8,883,996, which is incorporated herein by reference). Additionally, extended oligonucleotides (e.g., RNAi oligonucleotides) have been generated, where either or both of the 5' and / or 3' ends of either or both the antisense and sense strands are extended beyond the duplex targeting region such that either the sense or antisense strand includes a thermodynamically stabilized tetraloop structure (see, e.g., U.S. Patent Nos. 8,513,207 and 8,927,705, and International Publication No. WO 2010 / 033225, which are incorporated herein by reference for their disclosures of such oligonucleotides). Such structures can include single-stranded extensions at one or both of the 5' and 3' ends of the molecule, as well as RNAi extensions.

[0117] In addition to or instead of this, the oligonucleotides provided herein can be designed to participate in the RNA interference pathway downstream of Dicer involvement, i.e., after cleavage by Dicer. Such oligonucleotides can have an overhang that includes 1, 2, or 3 nucleotides at the 3' end of the sense strand. Such oligonucleotides, e.g., siRNA It may 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 20-bp double helix and a 2-nucleotide overhang at one or both 3' ends. Longer oligonucleotide designs are also available that include oligonucleotides having a 23-nucleotide guide strand and a 21-nucleotide passenger strand, in which case there are blunt ends at 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 at the 5' end of the passenger strand and the 3' end of the guide strand. Such molecules have a 21-base pair double helix region (see U.S. Patent Nos. 9,012,138, 9,012,621, and 9,193,753, which are incorporated herein by reference for their disclosures regarding longer oligonucleotides).

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

[0119] Other oligonucleotide designs for use with the compositions and methods disclosed herein include the following: for example, 16-mer siRNA (see, e.g., Nucleic Acids in Chemistry and Biology. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNA (e.g., having a stem of 19 bp or less; see, e.g., Moore et al. Methods Mol. Biol. 2010; 629: 141-158), blunt siRNA (e.g., 19 bp in length; see, e.g., Kraynack and Baker, RNA Vol. 12, p163-176 (2006)), asymmetric siRNA (aiRNA; see, e.g., Sun et al., Nat. Biotechnol. 26, 1379-1382 (2008)), asymmetric short double-stranded siRNA (see, e.g., Chang et al., Mol Ther. 2009 Apr; 17(4): 725-32), fork 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 (see, e.g., Abe et al. J Am Chem Soc 129: 15108-15109 (2007)), and 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 disclosure therein. Further non-limiting examples of oligonucleotide structures that may be used in some embodiments to reduce or inhibit the expression of C3 are microRNA (miRNA), short hairpin RNA (shRNA), and small siRNA (Hamilton et al., Embo J., 20 (See also reference 02,21(17):4671-4679; also refer to the specification of US Patent Application Publication No. 2009 / 0099115).

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

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

[0122] In certain embodiments, the sense strand can include 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 include the oligonucleotide sequence of SEQ ID NO: 4, as in the case of Compound B. In other embodiments, the sense strand can include 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 include the oligonucleotide sequence of SEQ ID NO: 1, as in the case of 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 has 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] Furthermore, 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 at least 99%) sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5, and the antisense strand has 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., an RNAi oligonucleotide) may comprise a sense strand comprising the oligonucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5 and an antisense strand comprising the 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 has 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. Further, the oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising the oligonucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and an antisense strand comprising the oligonucleotide sequence of SEQ ID NO: 3, as shown in FIGS. 1D and 1E for Compound A. The oligonucleotides 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] Furthermore, 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 to: 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 4'-O-monomethylphosphonate-2'-O-methyluridine, "-" represents a phosphodiester bond, and "-S-" represents a phosphorothioate bond as shown in FIG. 1E. In some embodiments, the antisense strand can be a pharmaceutically acceptable salt of SEQ ID NO: 38 (e.g., sodium salt). In some embodiments, the sense strand can be a pharmaceutically acceptable salt of SEQ ID NO: 37 (e.g., sodium salt).

[0127] Furthermore, 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, the oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising the oligonucleotide sequence of SEQ ID NO: 1 and an antisense strand comprising the oligonucleotide sequence of SEQ ID NO: 3, as shown for compound A. The sense strand has at least 85% (e.g., at least It may also contain oligonucleotide sequences having a sequence identity of at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%). The antisense strand may contain an oligonucleotide sequence having a sequence identity of at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) with respect to SEQ ID NO: 6. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may contain a sense strand containing the oligonucleotide sequence of SEQ ID NO: 4 and an antisense strand containing the oligonucleotide sequence of SEQ ID NO: 6, as shown for Compound B. The sense strand may contain an oligonucleotide sequence having a sequence identity of at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) with respect to SEQ ID NO: 17, and the antisense strand may contain an oligonucleotide sequence having a sequence identity of at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) with respect to SEQ ID NO: 18. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may contain a sense strand containing the oligonucleotide sequence of SEQ ID NO: 17 and an antisense strand containing the oligonucleotide sequence of SEQ ID NO: 18, as shown for Compound C. The sense strand may contain an oligonucleotide sequence having a sequence identity of at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) with respect to SEQ ID NO: 19, and the antisense strand may contain an oligonucleotide sequence having a sequence identity of at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) with respect to SEQ ID NO: 20. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may contain a sense strand containing the oligonucleotide sequence of SEQ ID NO: 19 and an antisense strand containing 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 the oligonucleotide sequence of SEQ ID NO: 25 and an antisense strand comprising the oligonucleotide sequence of SEQ ID NO: 26, as shown for Compound G.The sense strand has at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97%, and at least 99%) identity to SEQ ID NO: 27. It may include an oligonucleotide sequence having sequence identity with (), and the antisense strand may include 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 include a sense strand including the oligonucleotide sequence of SEQ ID NO: 27 and an antisense strand including the oligonucleotide sequence of SEQ ID NO: 28, as shown for compound H. The sense strand may include 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 include 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 include a sense strand including the oligonucleotide sequence of SEQ ID NO: 29 and an antisense strand including the oligonucleotide sequence of SEQ ID NO: 30, as shown for compound I. The sense strand may include 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 include 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 include a sense strand including the oligonucleotide sequence of SEQ ID NO: 15 and an antisense strand including the oligonucleotide sequence of SEQ ID NO: 16, as shown for compound J. See Table 1 for examples of sense strand and antisense strand pairs.

[0128] [Table 1]

[0129] An oligonucleotide (e.g., an RNAi oligonucleotide) contains a double-stranded region between a sense strand and an antisense strand. The double-stranded structure formed between the sense strand and the antisense strand can be 10 to 30 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, and 30 nucleotides in length). Thus, the double-stranded structure formed between the sense strand and the antisense strand can be 15 to 25 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotides in length). In some embodiments, the double-stranded region can be 20 nucleotides in length.

[0130] The region on the sense strand that forms a double-stranded structure 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 either SEQ ID NO: 2 or SEQ ID NO: 5. For example, the region on the sense strand that forms a double-stranded structure with the antisense strand can have the oligonucleotide sequence of either SEQ ID NO: 2 or SEQ ID NO: 5.

[0131] Furthermore, the double-stranded structure formed between the sense strand and the antisense strand may not extend over the entire length of the sense strand and / or the antisense strand.

[0132] An oligonucleotide (e.g., an 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 in length), such as a 36-nucleotide sense strand, and an antisense strand 18 - 36 nucleotides in length, such as a 22-nucleotide antisense strand. An oligonucleotide (e.g., an RNAi oligonucleotide) has a length such that when acted upon by the Dicer enzyme, an antisense strand incorporated into the mature RISC is obtained as a result.

[0133] The oligonucleotides provided herein can have one 5' end that is thermodynamically less stable compared to 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 in length (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides in length). For example, the 3' overhang on the antisense strand can be 2 nucleotides in length. 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 can be 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 length. In some examples, the oligonucleotide can have an overhang at the 5' end. The overhang can be a 5' overhang that includes 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 in length.

[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] One or more (e.g., 1, 2, 3, 4, 5) mismatches in complementarity may exist between the sense strand and the antisense strand. If 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 at the 3' end of the sense strand of the oligonucleotide. Base mismatches or destabilization of the segment at the 3' end of the sense strand of the oligonucleotide may potentially improve the efficacy of the synthetic double-stranded RNA during RNAi, perhaps by facilitating processing by Dicer. In some embodiments, it should be understood that the sequences presented in the sequence listing may be referred to in representing 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., the RNA counterpart of a DNA nucleotide or the DNA counterpart of an RNA nucleotide) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modifications while retaining complementary properties that are essentially the same as or similar to the specified sequence as compared to the specified sequence.

[0136] In some embodiments, it should be understood that the sequences presented in the sequence listing may be referred to in representing 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., the RNA counterpart of a DNA nucleotide or the DNA counterpart of an RNA nucleotide) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modifications while retaining complementary properties that are essentially the same as or similar to the specified sequence as compared to the specified sequence.

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

[0138] In certain embodiments, the antisense strand is shorter in nucleotide length than the sense strand. In some examples, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) can have an antisense strand that is 10 to 40 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, and 40 nucleotides). Thus, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) can have an antisense strand that is 15 to 30 nucleotides in length (e.g., 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides). For example, the antisense strand can be 20 to 25 nucleotides in length (e.g., 20, 21, 22, 23, 24, and 25 nucleotides). In certain embodiments, the antisense strand can be 22 nucleotides in length.

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

[0140] The oligonucleotides disclosed herein may include an antisense strand having the sequence of either SEQ ID NO: 3 or 6. In some embodiments, the oligonucleotides disclosed herein may include an antisense strand having the amino acid sequence of SEQ ID NO: 6, such as Compound B shown in Figure 2B. In some embodiments, the antisense strand may be a pharmaceutically acceptable salt of SEQ ID NO: 6 (e.g., sodium salt). SEQ ID NO: 6 may have a chemical structure as shown in Figure 1B. Alternatively, the antisense strand may have the sequence of SEQ ID NO: 3, such as Compound A shown in Figures 1D and 1E. In some embodiments, the antisense strand may be a pharmaceutically acceptable salt of SEQ ID NO: 3 (e.g., sodium salt). In addition, the first position at the 5' end of the antisense strand may be uridine. Uridine may include a phosphate analog; for example, uridine may be 4'-O-methylphosphonate-2'-O-methyluridine.

[0141]

[0142] Sense strand The sense strand of an oligonucleotide may also be referred to as the passenger strand. In certain embodiments, the passenger strand is longer in nucleotide number than the guide strand. In some examples, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) may have a sense strand that is 10 to 45 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, and 45 nucleotides). Thus, the oligonucleotide (e.g., RNAi oligonucleotide) may have a sense strand that is 20 to 50 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 nucleotides). 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 that is 7 to 36 nucleotides in length (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) relative to the sequence of SEQ ID NO:12. Thus, the sense strand may include a contiguous sequence that is 10 to 30 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, and 30 nucleotides) of SEQ ID NO:12. In some embodiments, the oligonucleotides disclosed herein may include a sense strand that includes a sequence of contiguous nucleotides relative to the sequence of SEQ ID NO:12, which is 19 nucleotides in length.

[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 to 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). Accordingly, the sense strand including the stem-loop may 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 may be 36 nucleotides in length.

[0145] Furthermore, the stem-loop region on the sense strand may form a double-stranded region with itself. The double-stranded region included 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-stranded region included in the stem-loop may be 6 nucleotides in length. The stem-loop can confer protection against degradation (such as enzymatic degradation) to the RNAi oligonucleotide and can promote targeting properties for delivery to target cells. For example, the loop can be modified without substantially affecting the gene expression inhibitory activity of the oligonucleotide. It can provide additional nucleotides. In certain embodiments, oligonucleotides are provided herein wherein the sense strand comprises a stem-loop described as S1-L-S2 (e.g., at its 3' end), where S1 is complementary to S2 and L forms 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) between S1 and S2. Thus, the loop between S1 and S2 can be 4 nucleotides in length and form a tetraloop, as described herein. In some embodiments, the S1 region is 6 nucleotides in length, the S2 region is 6 nucleotides in length, and the L region is a 4-nucleotide tetraloop.

[0146] The sense strand of the oligonucleotide (e.g., an RNAi oligonucleotide) can include a region that forms a double helix with the stem-loop region and the antisense strand. The stem-loop region can include 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 can be a tetraloop (e.g., within a nick tetraloop structure). The loop of the stem-loop can have the base sequence of SEQ ID NO: 8. The tetraloop can include 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 can include 3-6 nucleotides. For example, the loop of the stem-loop can include 3, 4, 5, or 6 nucleotides. The loop of the stem-loop can include 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 a chemical structure as shown in FIG. 1A. In some embodiments, the sense strand may be a pharmaceutically acceptable salt of SEQ ID NO: 4 (e.g., sodium salt). Alternatively, the sense strand may have the base sequence of SEQ ID NO: 1, such as Compound A shown in FIGS. 1D and 1E. In some embodiments, the sense strand may be a pharmaceutically acceptable salt of SEQ ID NO: 1 (e.g., sodium salt).

[0149] Oligonucleotide Modifications Oligonucleotides can be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance to nuclease degradation, immunogenicity, base pairing properties, RNA distribution and cellular uptake, and other characteristics relevant to therapeutic or research 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 may include one or more suitable modifications. Modified nucleotides may have modifications to their base or nucleobase, sugar (e.g., ribose, deoxyribose), or phosphate group.

[0150] The number of modifications on an oligonucleotide and the positions of those nucleotide modifications can affect the properties of the oligonucleotide. For example, oligonucleotides can be delivered in vivo by conjugating them to lipid nanoparticles (LNPs) or similar carriers or by including them within such carriers. However, when an oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Thus, in any particular embodiment 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, in the case of 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 the desired properties (e.g., protection from enzymatic degradation, the 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 a modified deoxyribose or ribose moiety, in which case 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 present 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 with sugars can include 2'-modifications. The 2'-modifications can be 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid. Typically, the modification is 2'-fluoro, 2'-O-methyl, or 2'-O-methoxyethyl. In some embodiments, the modification is 2'-fluoro and / or 2'-O-methyl. In some embodiments, the 2'-fluoro modification is 2'-fluorodeoxyribonucleoside, and / or the 2'-O-methyl modification is 2'-O-methylribonucleoside. The modification in the sugar may include modification of the sugar ring and may have modification of one or more carbons of the sugar ring. For example, the modification 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, the modified nucleotide may have an acyclic sugar without a 2'-carbon-3'-carbon bond. In some embodiments, the modified nucleotide may have a thiol group, for example, at the 4'-position of the sugar.

[0153] The oligonucleotides described herein (e.g., RNAi oligonucleotides) can include at least one modified nucleotide (e.g., at least one, at least five, at least ten, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least thirty-five, at least forty, at least forty-five, at least fifty, at least fifty-five, at least sixty, or more). For example, the sense strand of the oligonucleotide can include at least one modified nucleotide (e.g., at least one, at least five, at least ten, at least fifteen, at least twenty, at least twenty-five, at least thirty, at least thirty-five, or more). Also, for example, the antisense strand of the oligonucleotide can include at least one modified nucleotide (e.g., at least one, at least five, at least ten, at least fifteen, at least twenty, or more).

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

[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 an oligonucleotide (e.g., an RNAi oligonucleotide) that includes both a sense strand and an antisense strand are modified. The modified nucleotides may be 2'-modifications (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 2'-fluorodeoxyribonucleoside and / or the 2'-O-methyl modification is 2'-O-methyl ribonucleoside.

[0156] The present disclosure provides oligonucleotides having different modification patterns. The oligonucleotides comprising a sense strand and an antisense strand may include 2'-O-methyl modifications of 40 to 50 (e.g., 41, 42, 43, 44, 45, 46, 47, 48, and 49). The modified oligonucleotides may include 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 the sense strand of SEQ ID NO: 4 and the antisense strand of SEQ ID NO: 6, or the RNAi oligonucleotide may have the sense strand of SEQ ID NO: 1 and the antisense strand of SEQ ID NO: 3). In some embodiments, for these oligonucleotides, 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 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 are modified with a 2'-O-methyl modified nucleoside, such as 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 a 2'-O-methyl modified nucleoside, such as 2'-O-methyl ribonucleoside. 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 a 2'-O-methyl modified nucleoside, such as 2'-O-methyl ribonucleoside.In certain embodiments, all of the 1st, 2nd, 4th, 5th, 6th, 7th, 11th, 14th, 15th, 16th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 31st, 32nd, 33rd, 34th, 35th, and 36th positions of the sense strand, and / or all of the 1st, 6th, 9th, 11th, 13th, 15th, 17th, 18th, 20th, 21st, and 22nd positions of the antisense strand are modified with 2'-O-methyl modified nucleosides, such as 2'-O-methyl ribonucleosides.

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

[0159] For example, 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, 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 2'-O-methyl modified nucleosides; 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 2'-fluoro, and 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 FIGS. 1C-1 and 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. Further, in the case of an oligonucleotide having 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 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, in the case of 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 and may be modified; the chemical structures of the sense and antisense strands are shown in FIGS. 2A-1 and 2A-2.

[0161] In some embodiments, the 3' terminal group (e.g., 3'-hydroxyl) may be modified with a phosphate group 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) can enhance its interaction with Argonaute 2. In certain embodiments, the oligonucleotide (e.g., an RNAi oligonucleotide) contains uridine at the first position at the 5' end of the antisense strand. However, oligonucleotides with 5'-phosphate groups are susceptible to degradation by phosphatases or other enzymes, which may limit their bioavailability in vivo. In some embodiments, the oligonucleotide contains an analog of 5' phosphate that is resistant to such degradation. Thus, the uridine at the 5' end of the antisense strand may contain a phosphate analog. The phosphate analog may be an oxymethylphosphonate, vinylphosphonate, or malonylphosphonate. Further, the 5' end of the oligonucleotide strand can be linked to a chemical moiety (a "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 content thereof regarding phosphate analogs is incorporated herein by reference). Many phosphate mimics that can be attached to the 5' end have been developed (see U.S. Patent No. 8,927,513; the content thereof regarding phosphate analogs is incorporated herein by reference). Other modifications have been developed for the 5' end of oligonucleotides (see International Publication No. 2011 / 133871; the content thereof regarding phosphate analogs is incorporated herein by reference). In certain embodiments, a hydroxyl group may be attached to the 5' end of the oligonucleotide.

[0163] The oligonucleotide may have a phosphate analog at the 4'-carbon position of the sugar, which is referred to as a "4'-phosphate analog". For example, refer to International Publication No. WO 2018 / 045317 pamphlet (the content thereof regarding phosphate analogs is incorporated herein by reference). The oligonucleotides provided herein may include a 4'-phosphate analog at the 5'-terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate in which the oxygen atom of the oxymethyl group is bonded to the sugar moiety (e.g., at its 4'-carbon), or an analog thereof. In other embodiments, the 4'-phosphate analog is thiomethylphosphonate or aminomethylphosphonate, and the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bonded to the 4'-carbon of the sugar moiety or an analog thereof. In certain embodiments, the 4'-phosphate analog is oxymethylphosphonate. In some embodiments, oxymethylphosphonate is represented by the formula -O-CH2-PO(OH)2 or -O-CH2-PO(OR)2, where R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group. In certain embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3, or CH2CH3. In some embodiments, R is CH3. In some embodiments, the 4'-phosphate analog is 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 methoxyphosph onate (MOP). The phosphate analog attached to the oligonucleotide may be 5'-monomethyl-protected MOP. In some embodiments, for example, at the first position of the antisense strand, the following uridine nucleotide containing a phosphate analog:

Chemical formula

[0165] Modified nucleoside internucleotide linkage Phosphate modification or substitution in the oligonucleotide can result in an oligonucleotide containing at least one (e.g., at least one, at least two, at least three, at least five, or at least six) modified nucleoside internucleotide linkages. Any one of the oligonucleotides disclosed herein may contain 1 to 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3, or 1 to 2) modified nucleoside internucleotide linkages. For example, any one of the oligonucleotides disclosed herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified nucleoside internucleotide linkages. In some embodiments, the oligonucleotide (e.g., an RNAi oligonucleotide) may contain five modified nucleoside internucleotide linkages. For example, the sense strand of the oligonucleotide may contain one modified nucleoside internucleotide linkage, and the antisense strand may contain four modified nucleoside internucleotide linkages.

[0166] The modified nucleoside internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonic acid linkage, or a boranophosphate linkage. The modified nucleoside internucleotide linkage of any one of the oligonucleotides disclosed herein may be a phosphorothioate linkage. In certain embodiments, all of the modified nucleoside internucleotide linkages of the oligonucleotide may be phosphorothioate linkages.

[0167] The oligonucleotides described in this specification may have phosphorothioate linkages 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 linkages between position 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, the sense strand having the sequence of SEQ ID NO: 1 or 4 may have a phosphorothioate linkage between position 1 and 2, and the antisense strand having the sequence of SEQ ID NO: 3 or 6 may have phosphorothioate linkages between positions 1 and 2, 2 and 3, 20 and 21, and 21 and 22 and may also have a phosphorothioate linkage between positions 20 and 21, and positions 21 and 22.

[0168] Base modification The oligonucleotides provided herein may have one or more modified nucleic acid bases. The modified nucleic acid bases, also referred to herein as base analogs, may be linked to the 1'-position of the nucleotide sugar moiety. The modified nucleic acid bases may be nitrogenous bases. In certain embodiments, the modified nucleic acid bases may contain a nitrogen atom. See, for example, U.S. Patent Application Publication No. 2008 / 0274462 (the content thereof regarding modified nucleic acid bases is incorporated herein by reference). Modified nucleotides may also include universal bases. However, in certain embodiments, the modified nucleotides may not contain a nucleic acid base (e.g., non-basic).

[0169] In some embodiments, the universal base is a heterocyclic moiety located at the 1'-position of the nucleotide sugar moiety in a modified nucleotide or at an equivalent position in a nucleotide sugar moiety substituent, which, when present in a double helix, can face two or more bases without substantially altering 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 fully complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base has a lower T for a double helix formed with a complementary nucleic acid. m forms a double helix with a target nucleic acid having. However, in some embodiments, compared to a reference single-stranded nucleic acid in which a universal base is substituted by one base to generate a single mismatch, a single-stranded nucleic acid containing a universal base has a higher T for a double helix formed with a nucleic acid containing a mismatched base. m forms a double helix with a target nucleic acid having. 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 the above references is incorporated herein by reference for its disclosure regarding base modification).

[0170] Reversible modification Prior to reaching the target cell, certain modifications can be made to protect the oligonucleotide from the in vivo environment, but such modifications may reduce the efficacy or activity of the oligonucleotide once it reaches the cytosol of the target cell. Reversible modifications can be made so that the molecule retains desirable properties outside the cell and are removed upon entry into the cytosolic environment of the cell. Reversible modifications can be removed, for example, by the action of intracellular enzymes or by chemical conditions within the cell (e.g., via reduction by intracellular glutathione).

[0171] The reversibly modified nucleotide can include a glutathione-sensitive moiety. Typically, the nucleic acid molecule is chemically modified with a cyclic disulfide moiety to mask the negative charge generated by the internucleotide pyrophosphate bond and to improve cellular uptake and nuclease resistance. See, e.g., U.S. Patent Application Publication No. 2011 / 0294869, initially assigned to Traversa Therapeutics, Inc. (“Traversa”); PCT Publication No.: WO 2015 / 188197 Pamphlet, assigned to Solstice Biologics, Ltd. (“Solstice”); Meade et al., Nature Biotechnology, 2014, 32:1256-1263 (“Meade”); PCT Publication No.: WO 2014 / 088920 Pamphlet, assigned to Merck Sharp & Dohme Corp. (each of which is incorporated by reference for disclosure of modifications as described above). Reversible modification of the internucleotide pyrophosphate bond is designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g., glutathione). Previous examples include neutral phosphotriester modifications reported to be cleavable intracellularly (see Dellinger et al. J. Am. Chem. Soc. 2003, 125:940-950).

[0172] Such reversible modifications enable protection of the oligonucleotide during in vivo administration (e.g., passage through the bloodstream and / or the lysosomal / endosomal compartments of cells) where the oligonucleotide is exposed to nucleases and other harsh environmental conditions (e.g., pH). When the level of glutathione is released into the cytosol of cells, which has a higher level compared to the extracellular space, the modification is reversed, resulting in the obtained cleaved oligonucleotide. Using a reversible glutathione-sensitive moiety allows for the introduction of a sterically larger chemical group 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 are thus thought not to interfere with the biological activity of the oligonucleotide within the cytosol of the cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. 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 binds to the sugar of the nucleotide. In some embodiments, the glutathione-sensitive moiety binds 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 on the 3'-carbon side 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, U.S. Patent Application Publication No. 2019 / 0177355, the contents of which are incorporated herein by reference in their relevant disclosure.

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

[0175] The targeting ligand can include a carbohydrate, an amino sugar, cholesterol, a peptide, a polypeptide, a protein, or a portion of a protein (e.g., an antibody or antibody fragment) or a lipid. In some embodiments, the targeting ligand is an aptamer. For example, the targeting ligand can be an RGD peptide used to target tumor vasculature or glioma cells, a CREKA peptide used to target tumor vasculature or stroma, transferrin, lactoferrin, or an aptamer that targets the transferrin receptor expressed on the CNS vasculature, or an anti-EGFR antibody that targets EGF R 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 can each be conjugated to an individual targeting ligand. In some cases, 2 to 4 nucleotides of the oligonucleotide are each conjugated to an individual targeting ligand. The targeting ligand may be conjugated to 2 to 4 nucleotides at either the 5' or 3' end of either the sense or antisense strand such that the targeting ligand is similar to the bristles of a toothbrush and the oligonucleotide is similar to a toothbrush (e.g., the ligand is conjugated to a 2 to 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 loop of the stem may each be conjugated individually to the targeting ligand. In some embodiments, the oligonucleotide includes a stem-loop on the 3'-end side of the sense strand, and 3 nucleotides of the loop of the stem are conjugated individually to the targeting ligand.

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

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

[0179] For example, the oligonucleotides of the present disclosure can be conjugated directly or indirectly to monovalent GalNAc. The oligonucleotide may be conjugated directly or indirectly to two or more (e.g., 2, 3, 4, or more) monovalent GalNAcs and is typically conjugated to 3 or 4 monovalent GalNAc moieties. The GalNAc moiety may be present within the loop region of the oligonucleotides described herein. The GalNAc moiety can be used to target the disclosed oligonucleotides to ASGPR on hepatocytes; at that point, the GalNAc-binding oligonucleotide can be internalized and incorporated into an intracellular RNAi mechanism called the RNA-induced silencing complex (RISC). The RISC argonaute-2 (Argo-2) protein within this complex targets the antisense strand of the oligonucleotide duplex to its complementary C3 mRNA, initiating its degradation and thus blocking translation of the target.

[0180] In some embodiments, 2 to 4 nucleotides of the loop (L) of the stem-loop are each conjugated to an individual GalNAc moiety. In some embodiments, 3 nucleotides of the loop of the stem of the oligonucleotide can be conjugated directly or indirectly to 3 individual monovalent GalNAc moieties. In some embodiments, the oligonucleotide is conjugated to one or more divalent GalNAc, trivalent GalNAc, or tetravalent GalNAc moieties.

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

Chemical formula

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

Chemical formula

[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 loops can be present, for example, at nucleotide positions 27 - 30 of the molecule shown in FIG. 1A. In the chemical formula,

Chemical formula

Chemical formula

[0184] A 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. Additionally, an acetal-based linker can be used to conjugate a 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 / 100401 A1 pamphlet published on June 23, 2016, and the content regarding such linkers is incorporated herein by reference. The linker may be a labile linker. However, in other embodiments, the linker is stable (non-labile).

[0185] For a tetraloop containing the nucleotide GAAA (SEQ ID NO: 8) from 5' to 3', an example is shown below, where four (4) GalNAc moieties are attached to the nucleotides of the loop using an acetal linker. 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). In the chemical formula,

Chem.

Chem.

[0186] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) include a sense strand having a tetraloop, where three (3) GalNAc moieties are conjugated to the nucleotides containing the tetraloop, and each GalNAc moiety is conjugated to one (1) nucleotide. In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) include a sense strand having a tetraloop containing GalNAc-conjugated nucleotides, where the tetraloop has the following structure:

Chem.

[0187] In another embodiment, the oligonucleotides described herein (e.g., RNAi oligonucleotides) include a sense strand having a tetraloop containing three (3) GalNAc moieties attached to nucleotides, wherein the tetraloop has the following structure:

Chemical formula

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

[0189] Formulations 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 impart other beneficial properties to the oligonucleotides in the formulation. In some embodiments, a composition comprising an oligonucleotide (e.g., single-stranded or double-stranded oligonucleotide) for reducing the expression of C3 is provided herein. Such a composition, when administered to a subject, can be suitably formulated such that a sufficient portion of the oligonucleotide enters the cell to reduce C3 expression, either in the adjacent environment of the target cell or systemically. As disclosed herein, any of the various suitable oligonucleotide formulations can be used to deliver an oligonucleotide aimed at reducing C3. In some embodiments, the oligonucleotide, pharmaceutical composition, vector, or cell is formulated in a buffered solution such as phosphate buffered saline, liposomes, micellar structures, vectors, and capsids.

[0190] ​The formulations disclosed herein may include excipients. Excipients can impart to the composition improved stability of the active ingredient, improved absorbability, improved solubility, and / or enhanced therapeutic effect. Excipients can be buffering agents (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 oligonucleotide is lyophilized to extend its shelf life and can then be solubilized prior to use (e.g., for administration to a subject). Thus, excipients in a composition containing any one of the oligonucleotides described herein are cryoprotectants (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone), or disintegration temperature modifiers (e.g., dextran, ficoll, or gelatin).

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

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

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

[0194] In some embodiments, a pharmaceutical composition comprising an 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 needed 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 made 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 subcutaneously to a patient as its route of administration.

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

[0196] While many embodiments are directed to liver target delivery of any of the oligonucleotides disclosed herein, targeting of other tissues is also contemplated.

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

[0198] The oligonucleotides disclosed herein can be introduced into the 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 oligonucleotide can be delivered to the cell by injection of a solution containing the oligonucleotide, bombardment with particles coated with the oligonucleotide, exposure of the cell or organism to a solution containing the oligonucleotide, or electroporation of the cell membrane in the presence of the oligonucleotide.

[0199] Formulation of the oligonucleotide using cationic lipids can be used to facilitate transfection of the oligonucleotide into the cell. 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 also be used according to the manufacturer's instructions.

[0200] Thus, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the excipient comprises or can be formulated for administration to the cells, tissues, organs, or body of a subject in need thereof, including liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition, Pharmaceutical Press, 2013).

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

[0202] Other methods for delivering oligonucleotides to cells can also be used, such as lipid-mediated carrier transport, chemical-mediated transport, cationic liposome transfection such as calcium phosphate, and vectors containing oligonucleotides. Vectors used for delivering 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) intracellularly. The transgene can be delivered using a vector as described above, e.g., a viral vector (e.g., an adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus), or a non-viral vector (e.g., a plasmid or synthetic mRNA). In some embodiments, the transgene can be injected directly into a subject, e.g., at or near the source of action (e.g., within 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 C3 gene expression can be demonstrated by a decrease in the amount of mRNA expressed by a first cell or cell population that is substantially identical to, but similarly untreated, a second cell or cell population (control cells not treated with an oligonucleotide (e.g., an RNAi oligonucleotide) or an oligonucleotide targeted to a gene of interest (e.g., an RNAi oligonucleotide)), compared to the first cell or cell population that has been treated such that the C3 gene is transcribed and C3 gene expression is inhibited (e.g., by contacting the cells with an oligonucleotide of the present disclosure (e.g., an RNAi oligonucleotide) or by administering an oligonucleotide of the present disclosure (e.g., an RNAi oligonucleotide) to a subject in which such cells are present or were present). The level of the 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 represented by the following formula:

Number

[0205] Changes in the expression level of the C3 gene can be evaluated in terms of parameters that are functionally related to C3 gene expression, such as C3 protein expression, C3 protein activity, or reduction of the C3 signaling pathway. C3 gene silencing can be determined in any cell expressing either endogenous or heterologous C3 from an expression construct and by any assay known in the art.

[0206] The results of the inhibition of C3 mRNA can be confirmed by an appropriate assay for evaluating one or more properties of the cell or subject, or by biochemical techniques for evaluating molecules (e.g., RNA, protein) that indicate C3 expression. The oligonucleotide provided herein reduces the expression level of C3 to an extent that is evaluated by comparison with an appropriate control of the expression level (e.g., the level of C3 mRNA expression in cells or cell populations to which the oligonucleotide has not been delivered or to which a negative control has been delivered). The appropriate control level of C3 mRNA expression may be a predetermined level or value so that it is not necessary to measure the target level each time. The predetermined level or value can take various forms, including a single cutoff value such as a median or an average value. For example, the predetermined level or value may be at or about the 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 the 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 the sample can also be measured using real-time PCR (RT-PCR). For example, after homogenizing tissue samples with QIAzol Lysis reagent using TissueLyser II (Qiagen), RNA can be extracted by purification using MAGMAX® Technology (ThermoFisher Scientific) according to the manufacturer's instructions. Next, cDNA can be prepared using a high-capacity cDNA reverse transcription kit (ThermoFisher Scientific). Specific primers and probes for C3 and housekeeping controls are used for PCR in a CFX384 real-time PCR detection system (Bio-Rad Laboratories), and the Ct value is estimated using BioRad CFX Maestro software; the expression level is calculated in EXCEL® and plotted in Prism (GraphPad). The primers used for RT-PCR are listed in Table 2.

[0208]

Table 2

[0209] Typical assay formats that use ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, Northern blotting, in situ hybridization, and microarray analysis. Circulating mRNA can be detected using the method described in PCT Publication: WO 2012 / 177906 Pamphlet, the entire content of which is incorporated herein by reference. The expression level of the gene of interest can also be determined using nucleic acid probes.

[0210] The isolated mRNA can be used in hybridization or amplification assays, such 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 a nucleic acid molecule (probe) capable of hybridizing to the mRNA of the gene of interest with the isolated mRNA. For example, the isolated mRNA can be immobilized on a solid surface by electrophoresing the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose and contacting it with the probe. The probe may be immobilized on the solid surface, for example, contacting the mRNA with the probe in an AFFYMETRIX® GENECHIP® array. mRNA detection methods known in the art can be adapted for use in determining the level of mRNA of the gene of interest.

[0211] Alternative methods for determining the expression level of a target gene in a sample include, for example, nucleic acid amplification of, for example, mRNA in a sample by RT-PCR (Mullis, 1987, U.S. Patent 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 system (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., U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques known in the art. These detection schemes are particularly useful for detecting nucleic acid molecules when such molecules are present in very small numbers. In some aspects of the present disclosure, the expression level of a target gene (e.g., C3) is determined by quantitative fluorescent RT-PCR (i.e., the TAQMAN (trademark) System) or the DUAL-GLO (registered trademark) luciferase assay.

[0212] The expression level of the mRNA of the target gene can be monitored using a membrane blot (such as those used in hybridization analysis such as Northern, Southern, dot, etc.), or a microwell, sample tube, gel, bead, or fiber (or any solid support containing the bound nucleic acid). See U.S. Patent No. 5,770,722; 5,874,219; 5,744,305; 5,677,195; 5,445,934, which are incorporated herein by reference. Determination of gene expression levels can also include the use of nucleic acid probes in solution.

[0213] Using the foregoing assays, it is possible to determine the efficacy of treatment with the oligonucleotides described herein based on a decrease in C3 mRNA levels. A decrease in the level of C3 mRNA can be a decrease 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 an appropriate control level of C3 mRNA or the level of C3 in the subject prior to treatment. An appropriate control level can be the level of C3 mRNA expression in cells or cell populations not contacted with the oligonucleotides described herein. In some embodiments, the effect of delivery of the oligonucleotides to cells by the methods disclosed herein is evaluated after a finite period of time. For example, the level of C3 mRNA can be analyzed intracellularly at least 8 hours, 12 hours, 18 hours, 24 hours; or at least 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, or 80 days after introduction of the oligonucleotides 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 the cell or cell population (e.g., the level of protein expressed in a sample derived from a subject). As described above for the evaluation of mRNA inhibition, inhibition of protein expression levels in treated cells or cell populations can likewise be expressed as a percentage of the protein levels in control cells or cell populations.

[0215] The results of the inhibition of C3 protein expression can be confirmed by an appropriate assay for evaluating one or more characteristics of a cell or subject, or by a biochemical technique for evaluating a molecule that exhibits C3 protein expression. The degree to which the oligonucleotides provided herein reduce the expression level of the C3 protein is evaluated by comparing the expression level to an appropriate control (e.g., the level of C3 protein expression in cells or cell populations that have not received the oligonucleotide or have received a negative control). An appropriate control level of C3 protein expression may be a predetermined level or value, such as an amount of C3 protein determined to be within a normal range, e.g., 75-175 mg / dL in serum, so that it is not necessary to measure the control level each time. The predetermined level or value can take various forms, including a single cutoff value such as a median or an average 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 the measurement of 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 for the detection of proteins that indicate the presence or replication of a protein produced by a gene of interest. Further, the above assays can be used to report changes in the mRNA sequence of interest that result in the restoration or alteration of protein function, thereby providing a therapeutic effect and benefit to a subject, treating a disorder of the subject, and / or alleviating the symptoms of a disorder of the subject.

[0217] Using the foregoing assays, a determination can be made regarding the efficacy of treatment with the oligonucleotides described herein based on a decrease in the amount of C3 protein. A decrease in the level of C3 protein can be a decrease 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 an appropriate control level of C3 (e.g., about 75-175 mg / gL). An appropriate control level can be the level of C3 expression in cells or cell populations not contacted with the oligonucleotides described herein. In some embodiments, the effect of delivery of the oligonucleotide to cells by the methods disclosed herein is evaluated after a finite period of time. For example, the level of C3 can be analyzed in cells 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 cells. The level of C3 can be determined to evaluate whether retreatment of the subject is necessary. For example, if the level of C3 rises to the pre-treatment level (or a level that is at least about 20% or more of the pre-treatment level (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more)), the subject is considered to require retreatment.

[0218] Furthermore, inhibition of the C3 gene using the methods described herein can result in a reduction in the 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 such that the cells substantially maintain their native phenotypic characteristics , It may have undergone a limited number of passages. In some embodiments, the cells to which the oligonucleotide is delivered are ex vivo or in vitro (i.e., cells in culture or can be delivered to an organism in which the cells are present). In a specific embodiment, a method is provided for delivering an effective amount of the oligonucleotide disclosed herein to cells for the purpose of reducing only the expression of C3 in hepatocytes.

[0219] The effective amount of the oligonucleotide disclosed herein can be determined as the amount of the oligonucleotide that results in a reduction of a disease or disorder mediated by complement pathway activation or dysregulation, e.g., a symptom of one of the diseases or disorders described herein. The reduction of a symptom of a disease or disorder mediated by complement pathway activation or dysregulation can be, for example, 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% reduction as determined using clinical evaluations known to those of skill in the art. The amount of reduction of a symptom of a disease or disorder mediated by complement pathway activation or dysregulation can be used to determine whether a subject needs to be treated again with the oligonucleotide, pharmaceutical composition, vector, or cell described herein. Examples of assays for determining the reduction of a disease mediated by complement pathway activation or dysregulation include, but are not limited to, measurement and / or quantification of circulating C3 protein, functional assays (e.g., WEISLAB® assay and hemolysis assay). Quantification of C3 (or C3 cleavage products) deposition can be performed via IHC or immunofluorescence through specific disease biomarkers.

[0220] Furthermore, the oligonucleotides described herein that contain both a sense strand and an antisense strand as double-stranded polypeptides can be introduced into the 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 single-stranded oligonucleotides. Vectors used for delivery of 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] Therapeutic method Also disclosed herein are methods for treating in a subject, by administration of a composition described herein (e.g., an oligonucleotide, a vector encoding an oligonucleotide, a cell comprising the vector, a pharmaceutical composition), a complement pathway activation or dysregulation-mediated disease comprising one or more of the diseases associated with complement pathway activation or dysregulation disclosed herein. The method can include treating in a subject a complement pathway activation or dysregulation-mediated disease by administration of a pharmaceutically acceptable salt (e.g., sodium salt) of an RNAi oligonucleotide described herein. The methods described herein typically involve an effective amount of an oligonucleotide, or a pharmaceutically acceptable salt thereof, i.e., an amount capable of producing a desired therapeutic result (e.g., knockdown of C3 expression). A therapeutically acceptable amount can be an amount capable of treating 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 can depend on specific factors including the size of the subject, body surface area, age, the specific composition being administered, the active ingredient in the composition, the time and route of administration, general health status, and other drugs being administered concurrently. Such therapeutic agents can be used to alleviate, arrest, or prevent any type of disease or disorder mediated by complement pathway activation or dysregulation, and these can be administered prophylactically or therapeutically. Administration of a prophylactic agent can be carried out prior to the detection or manifestation of symptoms characteristic of a disease or disorder mediated by complement pathway activation or dysregulation so as to prevent the disease or disorder or slow its progression. Subjects at risk of 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, via a gastrostomy tube, via a duodenal feeding tube, via a gastrostomy, or rectally), parenterally (e.g., by subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intraventricular injection, intrathecal), topically (e.g., transdermally, by inhalation, by eye drops, or via the mucosa), or by direct injection into a target organ (e.g., the liver of the subject). Typically, the oligonucleotides disclosed herein are administered intravenously or subcutaneously. The most suitable route of administration in any given case will depend on the particular composition being 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., the 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 a subject 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), bi-monthly (e.g., once every 2 months), monthly, or weekly. In other cases, the oligonucleotide can be administered every 1, 2, or 3 weeks. In certain embodiments, the oligonucleotide can be administered daily.

[0224] Subjects to be treated for diseases mediated by complement pathway activation or dysregulation can be human or non-human primates or other mammalian subjects (e.g., humans). Other exemplary subjects that can be treated with the oligonucleotides described herein include domesticated animals such as dogs and cats; livestock 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 pharmaceutically acceptable salt thereof) can vary depending on many factors, such as the pharmacodynamic properties of the compound, the mode of administration, the age, health status, and weight of the recipient, the nature and extent of the symptoms, the frequency of treatment and / or the type of co-treatment (if any), and the clearance rate of the compound being treated in the subject. One of ordinary skill in the art can determine an appropriate dosage based on the above factors.

[0226] The oligonucleotide of the present disclosure, or a pharmaceutically acceptable salt thereof, can be administered in an amount and for a time effective to effect one or more (e.g., two or more, three or more, four or more) of the following: (a) reduction of C3 protein expression in a subject cell, (b) reduction of C3 transcription in a subject cell, (c) decrease in the level of C3 protein in a subject cell, (d) decrease in the activity of C3 protein in a subject cell; and / or (e) reduction of one or more symptoms of a disease or disorder mediated by complement pathway activation or dysregulation.

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

[0228] The diseases 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] The treatment of diseases mediated by complement pathway activation or dysregulation can be achieved by administration of oligonucleotides (e.g., RNAi oligonucleotides) that inhibit the expression and / or translation of C3 mRNA (e.g., expression of C3 protein) as described herein.

[0230] The disclosed compositions can be administered in an amount determined by one of ordinary skill in the art to be appropriate. In some embodiments, the oligonucleotides described herein can be initially administered at an appropriate dosage that can be adjusted, if necessary, in response to a clinical response.

[0231] In some cases, the oligonucleotide, or a pharmaceutically acceptable salt thereof, is administered at a dosage of 0.01 to 100 mg / kg of the subject's body weight (for example, 0.01 to 1 mg / kg, 1 to 5 mg / kg, 5 to 20 mg / kg, 20 to 50 mg / kg, 50 to 100 mg / kg). In certain examples, the oligonucleotide is administered at a concentration of 0.01 mg to 50 mg per kg of the subject's body weight (for example, 0.01 to 1 mg / kg, 1 to 5 mg / kg, 5 to 10 mg / kg, 10 to 20 mg / kg, 20 to 30 mg / kg, 30 to 40 mg / kg, 40 to 50 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.01 mg to 20 mg per kg of the subject's body weight (for example, 0.01 to 1 mg / kg, 1 to 5 mg / kg, 5 to 10 mg / kg, 10 to 15 mg / kg, 15 to 20 mg / kg). 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 (for example, 0.01 to 1 mg / kg, 1 to 2 mg / kg, 2 to 5 mg / kg, 5 to 8 mg / kg, 8 to 10 mg / kg, 10 to 12 mg / kg, 12 to 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 (for example, 0.01 to 1 mg / kg, 1 to 2 mg / kg, 2 to 5 mg / kg, 5 to 8 mg / kg, 8 to 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 (for example, 0.01 to 1 mg / kg, 1 to 2 mg / kg, 2 to 3 mg / kg, 3 to 4 mg / kg, 4 to 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 (0.1 to 1 mg / kg, 1 to 5 mg / kg, 5 to 10 mg / kg, 10 to 15 mg / kg, and 15 to 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 (for example, 0.1 to 1 mg / kg, 1 to 2 mg / kg, 2 to 5 mg / kg, 5 to 7 mg / kg, and 7 to 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 (for example, 0.1 to 1 mg / kg, 2 to 3 mg / kg, 3 to 4 mg / kg, and 4 to 5 mg / kg).In other cases, the oligonucleotide is administered at a concentration of 1 mg to 50 mg per kg of the subject's body weight (e.g., 1 to 10 mg / kg, 10 to 20 mg / kg, 20 to 30 mg / kg, 30 to 40 mg / kg, and 40 to 50 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 1 mg to 20 mg per kg of the subject's body weight (e.g., 1 to 5 mg / kg, 5 to 10 mg / kg, 10 to 15 mg / kg and 15 to 20 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 1 mg to 10 mg per kg of the subject's body weight (e.g., 1 to 2 mg / kg, 2 to 5 mg / kg, 5 to 7 mg / kg, and 7 to 10 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 1 mg to 5 mg per kg of the subject's body weight (e.g., 1 to 2 mg / kg, 2 to 3 mg / kg, 3 to 4 mg / kg, and 4 to 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 dosage of less than 10 mg per 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 dosage of about 10 mg / kg or less. In another embodiment, the oligonucleotide is administered at a dosage 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 dosage 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 dosage 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., an RNAi oligonucleotide) is administered at a dosage 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 dosage 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 dosage 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 dosage of about 3 mg / kg or less (e.g., 2.9 mg / kg, 2.5 mg / kg, 2 mg / kg, 1 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dosage 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 dosage 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 dosage of about 0.1 to 10 mg / kg, about 0.2 to 10 mg / kg, about 0.3 to 10 mg / kg, about 0.4 to 10 mg / kg, about 0.5 to 10 mg / kg, about 1 to 10 mg / kg, about 2 to 10 mg / kg, about 3 to 10 mg / kg, about 4 to 10 mg / kg, about 5 to 10 mg / kg, about 6 to 10 mg / kg, about 7 to 10 mg / kg, about 8 to 10 mg / kg, or about 9 mg / kg of the subject's body weight.

[0234] In other cases, the dosage of the composition (e.g., a composition comprising an RNAi oligonucleotide described herein) is an amount effective for prevention or treatment. In some cases, a viral vector (e.g., an rAAV vector) is 10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 、10 12 、10 13 、10 14 、or 10 15 genome copies (GC) per dosage. In some embodiments, rAAV is 10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 、10 12 、10 13 、or 10 14 GC / kg (total weight of the subject) per dosage.

[0235] Optionally, the disclosed oligonucleotide can be administered as part of a pharmaceutically acceptable composition suitable for delivery to a subject, as described herein. The disclosed agent is included in the above composition in an amount sufficient to provide the desired dosage and / or elicit a beneficial effect on treatment, as can be readily determined by one of ordinary skill in the art.

[0236] The disclosed compositions can be administered in an amount (e.g., an effective amount) sufficient to treat a subject or to effect one of the aforementioned outcomes (e.g., reduction of one or more symptoms of a disease in the subject) and for a sufficient period of time. 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, once every two days, 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., by injection) or continuous (e.g., via an implant or infusion pump). The subject can be evaluated for treatment efficacy one week, two weeks, one month, two months, three months, four months, five months, six months, or more after administration of the disclosed composition, depending on the composition and route of administration used for treatment. The subject may be treated for a discrete period (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 can continue over the long term (e.g., over the lifetime of the subject). For example, if an initial round or subsequent round of treatment does not elicit a treatment benefit that includes reduction of any one of the symptoms associated with PNH, such as fatigue, weakness, shortness of breath, bruising or bleeding tendency, recurrent infections, severe headache, thrombosis, and difficulty controlling bleeding, or a decrease in the level of C3 mRNA or C3 protein in the cells or serum of the subject, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional therapeutic agents may be administered to a subject diagnosed with PNH and treated with the compositions disclosed herein.

[0237] Kit The present disclosure also features a kit comprising (a) an oligonucleotide (e.g., an RNAi oligonucleotide) agent that reduces the level and / or activity of C3 in a cell or subject described herein, or a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable carrier, excipient, or diluent. The kit may contain a vector encoding an oligonucleotide (e.g., an RNAi oligonucleotide) described herein, or a cell containing a vector encoding an oligonucleotide (e.g., an RNAi oligonucleotide) described herein. The kit may also include an accompanying document containing instructions for performing any of the methods described herein. In some embodiments, the kit comprises (a) a pharmaceutical composition comprising an oligonucleotide (e.g., an RNAi oligonucleotide) agent that reduces the level and / or activity of C3 in a cell or subject described herein, (b) an additional therapeutic agent, and (c) an accompanying document containing instructions for performing any of the methods described herein.

Example

[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 Synthesis and Purification of Oligonucleotides The RNAi oligonucleotides described in this example and the previous examples were chemically synthesized using the methods described herein. Generally, RNAi oligonucleotides are synthesized by known phosphoramidite synthesis (e.g., see the following: Hughes and Ellington (2017) COLD SPRING HARB PERSPECT BIOL. 9(1) :a023812; Beaucage S.L., Caruthers M.H., Study on Nucleotide Chemistry V: Deoxy nucleoside Phosphoramidites - A New Class of Key Intermediates for Deoxypolynucleotide In addition to Synthesis, TETRAHEDRON LETT. 1981; 22: 1859-1862. doi: 10.1016 / S0040-4039(01)90461-7), it was synthesized using a solid-phase oligonucleotide synthesis method as described for 19-23mer siRNA (for example, see the following: Scaringe et al. (1990) Nucleic Acids Res. 18: 5433-5441 and Usman et 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 having a 19mer core sequence were formatted into constructs having a 25mer sense strand and a 27mer antisense strand to enable processing by the RNAi machinery. The 19mer core sequence was complementary to a region of 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-HPLC) on an Amersham Source 15Q column (1.0 cm × 25 cm; Amersham Pharmacia Biotech) using a 15-minute 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, peaks corresponding to full-length oligonucleotide species were recovered, pooled, desalted on a NAP-5 column, and then lyophilized.

[0242] The purity of each oligomer was determined by capillary electrophoresis using a Beckman PACE 5000 (Beckman Coulter, Inc.; Fullerton, CA). The CE capillary had an inner diameter of 100 μm and contained ssDNA 100RGel (Beckman-Coulter). Typically, approximately 0.6 nmol of oligonucleotide was injected into the capillary, electrophoresed at an electric field of 444 V / cm, and detected by UV absorbance at 260 nm. The denaturing Tris-borate-7M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides that were at least 90% pure as evaluated 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 using a VOYAGER-DE™ BIOSPECTROMETRY™ Work Station (Applied Biosystems; Foster City, CA) according to the manufacturer's recommended protocol. The relative molecular weights of all oligomers were obtained and were, in most cases, within 0.2% of the expected molecular weight .

[0243] Preparation of double-stranded helix Single-stranded RNA oligomers were resuspended in a double-stranded helix buffer consisting of 100 mM potassium acetate and 30 mM HEPES, pH 7.5 (e.g., at a concentration of 100 μM). Complementary sense and antisense strands were mixed in equimolar amounts to obtain a final solution of, for example, 50 μM double-stranded helix. The sample was heated to 100° C. and cooled to room temperature prior to use in 5′ RNA buffer (IDT). The RNAi oligonucleotides were stored at -20° C. The single-stranded RNA oligomers were stored at -80° C. as lyophilized or in nuclease-free water

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

[0245] To generate RNAi oligonucleotide inhibitors of C3 expression, a computer-based algorithm was used to computationally identify C3 mRNA target sequences suitable for assaying inhibition of C3 expression by the RNAi pathway. Over 300 RNAi oligonucleotide guide (antisense) strand sequences (see Table 3) each having a region complementary to an appropriate C3 target sequence of human C3 mRNA were prepared and assayed in vitro for C3 expression inhibition. Nine subsets (see Table 4) were selected from these RNAi oligonucleotides for further study. The subset of nine guide sequences identified by the algorithm was also complementary to the corresponding C3 target sequences of rhesus C3 mRNA (SEQ ID NO: 67; Table 3). C3 RNAi oligonucleotides containing regions complementary to homologous C3 mRNA target sequences having nucleotide sequence similarity are predicted to have the ability to target 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 the RNAi oligonucleotide (Figure 3A) or a subset of the RNAi oligonucleotides screened in Figure 3A at two different concentrations (0.1 and 1 nM) as shown in separate wells of a multiwell cell culture plate (Figure 3B). Cells maintained for 24 hours after transfection were used to measure the level of C3 mRNA derived from the transfected cells using a TAQMAN®-based qPCR assay. Two RT-qPCR assays, a 3' assay and a 5' assay, were used to measure the mRNA levels measured by HEX and FAM probes, respectively. A subset of RNAi oligonucleotide candidates was selected for further in vivo analysis based on the inhibition of C3 mRNA levels determined by RT-qPCR. The levels of C3 mRNA from the transfected cells were measured. Two RT-qPCR assays, a 3' assay and a 5' assay, were used to measure the mRNA levels measured by HEX and FAM probes, respectively. A subset of RNAi oligonucleotide candidates was selected for further in vivo analysis based on the inhibition of C3 mRNA levels 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 the selected compounds (Compounds A-I) at a single subcutaneous dose of 0.5 or 1 mg / kg. Four days later, the 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 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 FIGS. 4B and 4C, and their corresponding sense and antisense strands are summarized in Table 4 and FIG. 4A. The data are presented 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 pre-selected during mouse screening as described in Example 4, Table 4, and FIG. 4A, and tested in cynomolgus macaques (NHPs) for the duration of C3 mRNA silencing after administration of Compounds A-I at a single subcutaneous dose of 4 mg / kg. Liver biopsies were collected from all animals tested (n = 5 / compound) before dosing and on Days 28 and 56 after injection. As shown in FIG. 5, liver C3 mRNA levels decreased by at least 50% with most compounds tested compared to the normalized baseline levels determined by RT-qPCR and the concurrent PBS control Two lead compounds (Compound A and B) were selected based on the knockdown levels of C3 mRNA in the liver of cynomolgus macaques after single administration for testing in multiple-dose studies.

[0251] Compounds A and B were selected from single-dose studies for further evaluation in multiple-dose NHP studies. Cynomolgus macaques were administered a total of 4 doses subcutaneously at 1 mg / kg or 2 mg / kg on days 0, 28, 56, and 84. Liver biopsies were collected before dosing and on days 28, 56, and 112 after the first treatment for evaluation of liver C3 mRNA levels by RT-qPCR (Figure 6A). Serum samples were collected before dosing and on days 1, 14, 28, 42, 56, 70, 84, 98, and 112 after the first dose for evaluation of C3 protein levels by C3 ELISA kit (Figure 6B), complement activity by WIESLAB® AP assay (Figure 8), and hemolysis of rabbit erythrocytes (Figure 9). PBS-treated animals were used as controls for C3 liver mRNA, C3 serum protein, and functional assays. Multiple treatments of cynomolgus macaques with Compound A or B resulted in sustained maintenance of C3 mRNA silencing in the liver, a significant decrease in circulating C3 in serum, a >95% decrease in alternative pathway complement activity, and complete inhibition of lysis of rabbit erythrocytes in the hemolysis assay after multiple administrations 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 28-day results for both single- and multiple-dose NHP studies. The approximate ED of Compound A (0.65 mg / kg) and Compound B (0.55 mg / kg) 50 was calculated from the dose-response curves generated for both compounds (Figure 7).

[0253] Example 6: Pharmacokinetic and Pharmacodynamic Studies of Compound J on C3 Expression in CD-1 Mice CD-1 mice were treated with compound J (a mouse surrogate for compound A), and the percent of liver C3 mRNA knockdown and serum C3 protein levels in the mice as a result of compound J administration were evaluated. The percent of knockdown of liver C3 mRNA 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. The mice received a single subcutaneous dose of compound J at 0.5 mg / kg, 1 mg / kg, or 6 mg / kg. Single administration of compound J showed a dose-dependent percentage of liver C3 mRNA knockdown of liver C3 mRNA, with a reduction of more than 90% of C3 mRNA in the liver from animals receiving the 6 mg / kg dose (n = 5 mice / time point). The nadir of mRNA knockdown was from 3 to 14 days after the 6 mg / Kg dose, as shown in Figure 10A. Measurement of the percentage of C3 protein in the serum of CD-1 mice over the test period also showed a corresponding suppression (Figure 10B).

[0254] The amount of compound J in the 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 over 672 hours after receiving the above dose (Figure 11). Pharmacokinetic analysis showed that the exposure of compound J was highest in the liver, followed by the spleen, kidney, and plasma (Figure 11).

[0255] In a multiple-dose study conducted over 70 days, the percent of C3 mRNA was measured using RT-qPCR, and the amount of C3 protein in serum was measured by mouse C3 ELISA assay. In this case, the 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 knockdowns of approximately 75% and >95% 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. Figure 13 As shown in A and 13B respectively, the 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). PBS-treated CD-1 mice were used as controls for both C3 liver mRNA and C3 serum protein levels.

[0256] In this multiple-dose study, compound J (a mouse surrogate for compound A) was shown to cause a dose-dependent knockdown of liver C3 mRNA, which was demonstrated to be sustained over a 70-day period. The decrease in circulating C3 protein levels corresponded to the decrease in C3 mRNA observed in the liver. Furthermore, plasma and liver concentrations of compound J from dosed animals did not show accumulation of compound J when dosed every other week (1 mg / kg) (Figure 13A and 13B respectively).

[0257] Example 7: Effect of compound J on C3 expression in an NZB / W F1 mouse, lupus nephritis model The NZB / W F1 lupus mouse model was used to test proof of mechanism in a disease model using compound J. NZB / W F1 animals were dosed subcutaneously with 0.5 mg / kg, 3 mg / kg, or 6 mg / kg doses of compound J 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-diseased controls. At 37 weeks of age, C3 and properdin glomerular deposits were evaluated by immunofluorescence imaging of kidneys from compound J-treated and PBS control animals (Figure 14). At 29 weeks of age, the percentage of liver 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-dose treatment with compound J, there was a dose-dependent decrease in C3 and properdin glomerular deposits observed in animals treated with compound J. Multiple-dose treatment of NZB / W F1 mice with compound J resulted in liver C3 It showed a dose-dependent knockdown of mRNA, which was sustained over 16 weeks. The decrease in circulating C3 protein levels (Figure 15A) corresponded to the decrease in C3 mRNA observed in the liver (Figure 15B).

[0258] Serum samples were collected at 29 and 37 weeks of age, and circulating IgG immune complexes (CICs) were measured by ELISA assay 8 and 16 weeks after treatment with compound J, respectively (Figures 16A and 16B). After multiple-dose treatment with compound J, there was no increase in the level of circulating immune complexes due to liver knockdown of C3 expression compared to the CIC levels observed in 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. The mice received multiple doses of 6 mg / kg of compound J. Figure 17 shows a decrease in C3 glomerular deposition from the kidneys of MRL / lpr mice treated with multiple doses of 6 mg / kg of compound J. A decrease in properdin deposition was also observed from kidney samples of animals treated with a subcutaneous dose of 6 mg / kg of compound J every two weeks from 8 to 16 weeks of age, as shown in Figure 17.

[0260] Example 9: cfh - / - Effect of compound J on C3 expression in mice, a complement dysregulation model Complement factor H-deficient mice (Cfh - / - ) were administered 0.5 mg / kg, 3 mg / kg, or 6 mg / kg of compound J at a dose of four times a month from 4 to 8 months of age. Kidneys were harvested from all treatment groups 4 weeks after the last administration 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 C3 glomeruli from the kidneys of CFH- / - mice treated with multiple escalating doses of compound J It shows a dose-dependent decrease in deposition. The decrease in properdin deposition was also observed from kidney samples of animals treated subcutaneously with the compound J every 4 weeks from 16 to 32 weeks of age, as shown in Figure 18. The percentage of liver 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 the serum C5 levels in the above mice (C5 consumption is a characteristic of complement dysregulation in this model).

[0261] Example 10: Effect of compound J on C3 expression in a CAIA-induced arthritis mouse model The effect of compound J in the treatment of symptoms associated with arthritis was verified using a collagen antibody-induced arthritis (CAIA)-induced arthritis mouse model, a simple model of rheumatoid arthritis. The CAIA-induced arthritis mouse model was created by administering collagen antibody 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 the preventive study (Figure 20A), or after the onset of the disease on day 5 for the therapeutic study (Figure 20B). Hind limb 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 limb swelling, a characteristic feature of this model (Figure 21A). Therapeutic treatment with compound J completely reversed the clinical disease manifestation after a single dose compared to PBS-treated control animals (Figure 21B).

[0262] Hematoxylin and eosin (H&E) staining was performed on biopsies of the hindlimbs and knees, showing a decrease in local mononuclear cell infiltration in mice treated prophylactically with 3 doses (Figure 22A) or therapeutically with a single dose (Figures 22B and 24A, respectively) of compound J at a single dose of 6 mg / kg. Furthermore, to demonstrate the reduction of local inflammation as a result of therapeutic treatment with 6 mg / kg of compound J, 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 the cartilage of the knees in the CAIA-induced arthritis mouse model. Animals treated with 6 mg / kg of compound J showed a significant reduction in cartilage erosion compared to PBS-treated mice when treated prophylactically (Figure 23) or therapeutically (Figure 24B). To evaluate complement expression at the local inflammatory sites of CAIA-induced arthritis mice treated or not treated with 6 mg / kg of compound J, experiments using in situ hybridization to C3 and CD45 mRNA were performed on biopsy samples (which is 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 the control group.

[0263] Example 11: Effect of compound J on C3 expression in a multiple sclerosis mouse model. The myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mouse model, a model widely used to investigate the immune-mediated mechanisms of neuroinflammation and demyelination, was prophylactically treated with compound J at a dose of 6 mg / kg (n = 2 experiments). The liver C3 mRNA levels and C3 protein in serum after treatment with compound J were evaluated using RT-qPCR and mouse C3 ELISA assay, respectively, as shown in FIGS. 31A and 31B. Similarly, the percentage of C3 mRNA remaining after treatment with compound J and the amount of C3 in the serum of MOG-induced EAE mice treated with a dose of 6 mg / kg of compound J were evaluated in comparison with C3-deficient mice and MOG-induced EAE C3-deficient mouse strains treated with PBS. The liver knockdown of C3 by compound J reduced the severity of the disease in both experiments performed (FIG. 29). The reduction in severity observed with liver knockdown by compound J treatment was similar to the 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. As shown in FIG. 30, Luxol fast blue staining was performed on spinal cord samples together with H&E staining to visualize myelination and mononuclear cell infiltration. As shown in FIG. 30, Luxol fast blue spinal cord samples were compared among MOG-induced animals treated with 6 mg / kg of compound J, diseased animals treated with PBS, and C3-deficient mice. MOG-induced animals treated with compound J showed a decrease in demyelination and prevention of immune cell infiltration similar to the levels observed in MOG-induced EAE C3-deficient mice.

[0265] Similar results were also observed in the proteolipid protein (PLP)-induced EAE model. Treatment with C3 siRNA reduced the severity of the disease but was not sufficient to prevent the recurrence characteristic of this animal model.

[0266] Example 12: Absorption, Distribution, Metabolism, and Excretion (ADME) Tests in Mice Pharmacokinetic and biodistribution studies were performed in male CD-1 mice by administering PBS (n = 18), or by single subcutaneous (SC) injection of compound A at 3, 10, or 100 mg / kg (n = 39 / cohort), or by single intravenous (IV) injection of 3 mg / kg (n = 36). Bioavailability was approximately 18% based on the comparison of AUC last after IV and SC doses at 3 mg / kg. 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 generally in a dose-proportional manner in the 10 mg / kg group and increased beyond a dose-proportional pattern in the 100 mg / kg group. Liver exposure based on C max and AUC last increased generally in a dose-proportional manner at 10 mg / kg and increased below a dose-proportional pattern at 100 mg / kg compared to the 3 mg / kg dose group, which showed saturation of the distribution process to the liver. The elimination half-life in the liver was 2.1 - 4 days.

[0267] Example 13: Platelet Activation of Compound A Evaluation of platelet activation in human whole blood stimulated with compound A did not induce platelet activation. Whole blood samples (5 male and 5 female donors) were stimulated with PBS or compound A at 10, 100, 200, or 300 μg / mL.

[0268] Example 14: Tolerance of Compound A in Cynomolgus Monkey The tolerability of compound A administration was evaluated in a study involving naive male and female cynomolgus monkeys. Animals were administered phosphate buffered saline (PBS, n = 12) or a subcutaneous dose of compound A at a dose level 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 liver C3 knockdown. Based on this analysis, the dosing levels on days 28, 56, and 84 were adjusted to 3 mg / kg (for the low dose) or 6 mg / kg (for the high dose) 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 a total of 28 pathogen virus, bacteria, and parasite tests were performed before the test, on day 56, and at necropsy to monitor for latent viruses and / or potential reactivation of infections by serology and blood or fecal PCR. A final necropsy was performed for evaluation by a clinical pathologist to determine evidence of potential 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 the low or high dose of compound A, approximately 75 or 80% liver C3 mRNA knockdown was achieved, respectively, and circulating C3 protein levels decreased by approximately 80%. These decreases in liver C3 mRNA and C3 protein levels persisted at all time points evaluated up to day 112 when the study ended. There were no gross or microscopic findings at necropsy, and there were no unexpected deaths. Body weight, liver function tests, blood cell counts, blood chemistry, and lipid metabolism parameters were not affected by chronic compound A treatment, and there was no evidence of increased pathogenic parasite, bacterial, or viral infection in the treated monkeys compared to the cohort administered PBS.

[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 final dose, and blindly evaluated by a pathologist for evidence of viral or bacterial infection and histological changes. No treatment-related histopathological changes or increased infection were observed. Similarly, NZB / W F1 mice were administered 1 or 6 mg / kg of Compound J every 4 weeks from 28 to 40 weeks of age and sacrificed at 44 weeks of age, or administered from 24 to 36 weeks of age and sacrificed at 40 weeks of age. No evidence of increased infection was detected by serological and PCR tests for viral, bacterial pathogen, and parasite populations, and no treatment-related changes were found in the final necropsy blindly evaluated by a veterinary pathologist for the treatment groups.

[0270] Example 16: Safety Pharmacology Test of Compound A in Cynomolgus Monkey Compound A was evaluated in a subcutaneous safety pharmacology test in cynomolgus monkeys. Four animals were administered PBS or a single increasing dose of Compound A (at dose levels of 30, 100, 300 mg / kg) once every 7 days, using the same four animals for each administration. During this test, safety pharmacology including evaluation of cardiovascular (e.g., ECG, blood pressure, heart rate, etc.), respiratory (respiratory rate), and neurological (functional observational battery) endpoints, as well as clinical evaluation, was performed. 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, mild to moderate tremors (limb or whole body) were observed in 3 animals at 4 and 24 hours after administration. The 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 induction of micronuclei and mutagenic activity, respectively. ​

[0271] Example 17: PK / PD Study of Compound A in Cynomolgus Monkey A single-dose PK / PD study was conducted in cynomolgus monkeys. The animals received a single SC or IV dose of PBS (control) at a SC dose or 3 mg / kg of Compound A on Day 0 (n = 5 per group). The concentrations of Compound A in plasma, urine, and tissues were evaluated. Liver biopsies were performed on Days 2, 35, 70, 112, 158, and 252 to evaluate liver C3 mRNA levels (major pharmacodynamic marker). Circulating C3 protein levels and complement functional activity were additional PD markers evaluated throughout the study.

[0272] The concentration of Compound A in plasma, liver, and urine was measured using an anion-exchange high-performance liquid chromatography with fluorescence detection (AEX-HPLC-FD) method based on identification hybridization. The decrease in 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 functional activity (classical pathway, mannose-binding lectin pathway (MBL), and alternative pathway) was measured using the WIESLAB® assay.

[0273] Using the plasma concentration of Compound A over time, a non-compartmental P K profile was created for each animal (group mean is plotted in Figure 32A). The plasma T max range after SC administration was 1 - 6 hours, and the T maxIt was 0.25 hours (collected at the initial time point) for all animals. The plasma concentration of Compound A decreased biphasically, and the distribution phase of the SC route was slower compared to the IV route. The biphasic decrease indicates mainly an initial rapid distribution phase to the liver, followed by a slower excretion phase. Plasma half-lives were reported for 1 out of 5 (2.51 hours) in the SC group and 2 out of 5 (average = 1.21 hours) in the IV group. The half-lives of the remaining animals were not reported because the acceptance criteria for the terminal phase rate constant were not met. The bioavailability of Compound A was approximately 28.5% for the SC dose compared to the IV dose based on AUC last Non-compartmental PK profiles for individual animals were generated using the liver concentration of Compound A over time (group means are plotted in Figure 32B). Liver half-lives were calculated for all 5 animals in both the SC and IV groups and were in the ranges of 13 - 20 days and 20 - 33 days, respectively.

[0274]

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

[0276] After a single administration of 3 mg / kg of Compound A by SC or IV, a reduction in C3 mRNA expression in the liver of monkeys was observed on Day 2 (SC only), and the maximum reduction (about 70% for both administration routes) was reached on Day 35 after administration (Figure 33). A progressive recovery of C3 mRNA expression over time was observed and it returned to near baseline levels by Day 114. This recovery was maintained up to Day 252 for both administration routes.

[0277] ​After a single 3 mg / kg SC administration of Compound A, a decrease in serum C3 protein was observed from day 7 to day 70 (Figure 34). The maximum mean C3 protein level in serum decreased by 54.7% on day 28 after administration and recovered to the pre - administration level by day 168. In contrast to the control, there was no decrease in serum C3 protein level in animals administered a single 3 mg / kg IV dose of Compound A.

[0278] In contrast to the control, there was no effect on classical complement in either the SC group or the IV group (Figure 35). The functionality of the lectin and alternative pathways was decreased in the SC group compared to the control on days 14, 28, and 35. The functionality of the lectin pathway decreased minimally (about 13%) over the three days (Figure 36), while the functionality of the alternative pathway decreased by 87%, 85%, and 73% on days 14, 28, and 35, respectively (Figure 37). The pathway functionality returned to baseline levels by day 70 for the lectin pathway and by day 168 for the alternative pathway.

[0279] After a single 3 mg / kg SC administration of Compound A, the maximum mRNA decrease was 70% on day 35, the maximum decrease in C3 protein was 54.7% on day 28, and the maximum decrease in alternative pathway activity was ≥87% on day 14. The mRNA expression recovered to the baseline level by day 168, the C3 protein expression returned to baseline by day 28, and the alternative pathway function recovered by day 168.

[0280] Example 18: Toxicity Tests in CD - 1 Mice and Cynomolgus Monkeys Six - month toxicity tests in mice and nine - month toxicity tests in cynomolgus monkeys were conducted. The range of dose levels in these tests was selected to achieve at least 10 - fold exposure multiples relative to the expected exposure at the intended maximum clinical dose. selected.

[0281] In the mouse study, the potential toxicity of repeated doses of PBS or Compound A (30, 100, or 300 mg / kg) in CD-1 mice (weekly for 4 weeks; 7 doses), and the potential reversibility of findings after an 8-week recovery period were evaluated. Ten male and ten female mice per dosing cohort were evaluated during the dosing period of the study, and six male and six female mice were maintained during the recovery period. In addition, the toxicokinetic properties of Compound A were determined in a sub-study (n = 111). Compound A administered at levels of 30, 100, and 300 mg / kg was well tolerated, and there were no Compound A-related deaths or adverse findings. Clinical pathology findings included a slight increase in alanine aminotransferase and a slight decrease in triglycerides on Day 171, with complete or partial reversibility evident at the end of the recovery period. Compound A-related non-adverse microscopic findings included minimal or mild mixed cell inflammation of the liver, hepatocyte karyomegaly, increased mitosis, and oval cell hyperplasia at terminal euthanasia, with minimal or mild mitosis and hepatocyte karyomegaly still present at recovery euthanasia. From these results, the NOAEL was considered to be 300 mg / kg, which on Day 169, in male and female, respectively, the mean AUC last values were 760,000 and 543,000 hr*ng / mL, and the mean Cmax values were 160,000 and 96,400 ng / mL, respectively.

[0282] In the monkey study, the potential toxicity of repeated SC administration of Compound A (0, 30, 100, 300 mg / kg every 4 weeks, a total of 10 doses) and the reversibility, persistence, or delayed onset of any effects after an 8-week recovery period were evaluated. Four male and four female monkeys were included in each dose cohort of the main administration study, and two males and two females were included in the 2-month recovery period. In addition, the pharmacokinetics and PD characteristics of Compound A were measured. Nine-month repeated SC administration of Compound A to cynomolgus monkeys 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 weight, qualitative food consumption, hematology, clinical chemistry, urine tests, 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 early deaths during this study, and all animals survived until the scheduled necropsy. A non-adverse increase related to Compound A in fibrinogen occurred at ≥100 mg / kg / dose. At ≥30 mg / kg / dose, there were non-adverse microscopic findings (vacuolization / granular macrophages) in the liver and multiple other tissues. Based on the observations made, the NOAEL was determined to be 300 mg / kg / dose, and the associated AUC last was 1,330,000 hr*ng / mL, and C max was 70,900 ng / mL (male-female combination, day 253).

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

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

[0285] Example 19: Effect of Compound B on C3 expression in cynomolgus monkey, an AMR model A pharmacological test is conducted on 4 sensitized cynomolgus monkeys that have received kidney allografts. In this test, the animals receive an SC dose of Compound B once every 4 weeks for a total of 4 months.

[0286] Example 20: Dose-setting test in humans For a first-in-human (FIH) clinical trial, a single ascending dose (SAD) test is conducted on healthy volunteers in combination with a multiple ascending dose (MAD) cohort in patients with complement-driven diseases. Healthy volunteers and patients receive prophylactic vaccination against Neisseria meningitides serogroups A, C, W, Y, B, Streptococcus pneumoniae, and Haemophilus influenzae type B before receiving Compound A.

[0287] Example 21: Treatment of multiple sclerosis with Compound A in humans Subjects suffering from multiple sclerosis are treated with a pharmaceutical composition containing Compound A (e.g., at a dosage of about 0.01 mg / kg to 50 mg / kg body weight of the subject). The subjects are administered the composition at a frequency of about once a week, e.g., by subcutaneous injection, for a period of about 12 months or more (e.g., until the symptoms resolve or stabilize). Once a month, a clinician evaluates the subject's symptoms and serum C3 levels to assess the efficacy of Compound A. The subject's serum C3 is 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 the serum is decreased, i.e., decreased by at least 10%, compared to the amount of C3 protein in the serum before treatment with Compound A. In addition, a clinician evaluates symptoms of the subject related to multiple sclerosis, e.g., blurred vision, slurred speech, dizziness, percussion pain, lack of coordination, and unsteady gait, etc., and assesses 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 Subjects diagnosed with arthritis are treated with a pharmaceutical compound containing Compound A (e.g., at a dose of about 1.5 mg / kg). The subjects are administered the composition at a frequency of about once a month, e.g., by subcutaneous injection, over a period of about 6 months or more (e.g., until the symptoms resolve or stabilize). To evaluate the efficacy of Compound A every one or two months, the subjects are evaluated by a clinician (e.g., by assessing the subject's symptoms and / or serum C3 levels). 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 administration of Compound A or a control amount of C3 protein, and / or to the amount of C3 protein present in a serum sample from a normal subject (e.g., a disease-free subject), and / or to 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 the serum is decreased, i.e., decreased by at least 10%, compared to the amount of C3 protein in the serum before treatment with Compound A. In addition, by a clinician evaluating the symptoms of the subject associated with arthritis, e.g., pain, stiffness, swelling, redness, and decreased range of motion, etc., it can be evaluated whether there is a reduction in any or all of the symptoms experienced by the subject compared to the symptoms the subject experienced before administration of Compound A.

[0289] Example 23. C3 Evaluation Assay Various assays, including the evaluation of Compound A in plasma or tissue, the WIESLAB® Complement Function Activity Assay, the evaluation of circulating C3, C3 mRNA expression levels, and the pharmacokinetic assay, can be used as described herein to characterize the effect of Compound A on C3 levels.

[0290] Pharmacokinetic Assay of Compound A The concentrations of Compound A or Compound J in the plasma of mice and monkeys were measured. After enzymatically treating plasma samples (blank, unknown, standard, and QC samples), hybridization with a peptide nucleic acid (PNA) probe having sequence complementarity to the antisense strand of Compound A or Compound J was performed. The samples were injected into a high performance liquid chromatography (HPLC) equipped with a fluorescence detector. Chromatographic separation was performed using a gradient system on a Shimadzu Prominence system using a DNAPAC (trademark) PA200 analytical column. The fluorescence detector monitored signals from 436 nm (Ex) to 484 nm (Em). Reference samples of individuals and mixtures were prepared and injected to confirm the retention times of the 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 was performed using linear regression, respectively. This assay was used, for example, in Examples 6, 12, and 17 as described above.

[0291] WIESLAB (registered trademark) Complement Function Activity Assay (CCP, CAP, CLP) To detect the human terminal complement complex (C5b-9) complex resulting from complement activation, the WIESLAB® complement system screening assay was used with a labeled antibody specific for the neoantigen to evaluate complement classical pathway (CCP), CAP, and complement lectin pathway activities. 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. The monkey serum samples were diluted in a diluent containing blockers to ensure that only the respective pathway was activated. The wells were washed and C5b-9 was detected using a specific alkaline phosphatase-labeled antibody against the expressed neoantigen. The amount of complement activation correlated with the chromogenic intensity measured by the 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 a percentage (%) of complement activity and 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 The evaluation of cynomolgus monkey circulating C3 protein levels was performed using a human complement C3 enzyme-linked immunosorbent assay (ELISA) kit (cat# Ab108823, Abcam, Cambridge, UK) designed for the quantitative measurement of complement C3 concentration in human serum. Due to its cross-reactivity with monkey C3, this kit was also used to measure the circulating C3 protein in cynomolgus monkey serum samples. Complement C3-specific antibodies were pre-coated and blocked on a 96-well plate. After adding the standard or test samples to the wells, a complement C3-specific biotinylated detection antibody was added, followed by washing with a wash buffer. Streptavidin-peroxidase conjugate was added, and unbound conjugate was removed with the wash buffer. Tetramethylbenzidine (TMB) was used to visualize the streptavidin-peroxidase enzyme reaction. TMB was oxidized by streptavidin-peroxidase to produce a blue product that changed to yellow after adding an acidic stop solution. The density of the yellow coloration was directly proportional to the amount of complement C3 captured on the plate. The back-calculated concentration of the sample was determined by a curve fitting regression program generated by the calibration standard. This assay was used, for example, in Examples 14 and 17 above.

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

[0294] Pharmacokinetic assay 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 having sequence complementarity to the antisense strand of Compound A. The samples are injected into an HPLC equipped with a fluorescence detector. Chromatographic separation is performed using a gradient system on Shimadzu Prominence systems with a DNAPAC™ PA200 analytical column. The fluorescence detector monitors signals from 436 nm (Ex) to 484 nm (Em). Based on the retention times of potential metabolites of Compound A, the LC gradient conditions are adjusted and determined. Reference samples of individuals and mixtures are prepared and injected to evaluate the retention times of metabolites. The peaks of Compound A and metabolites 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 is planned to be performed using an electrochemiluminescence (ECL) bridging assay. The positive control (PC) is prepared 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 the modified Compound A sequence. The PC, negative control (NC), and test samples are subjected to an acid dissociation step at ambient room temperature and then added to plates containing TRIS, biotin-Compound A, and ruthenium-labeled Compound A to enable the formation of a cross-linked complex between the labeled Compound A present in the sample and the Compound A antibody. After incubation, the NC, PC, and test samples are transferred to streptavidin-coated plates and incubated in the dark for 1 hour, during which the drug binds to the plate and captures the ADA cross-linked complex. Next, the plates are 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 the 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 are described herein, those skilled in the art will recognize that additional modifications and embodiments, including departures from the principles described herein that are within the scope of the art and such departures from the present disclosure that include variations, uses, or adaptations generally following the principles described herein as well as implementations or conventional implementations well known in the art, and that can be applied to the essential features described above and beyond as well as to those described in the following claims. The present invention provides the following items. (Item 1) An RNAi oligonucleotide for reducing complement component 3 (C3) expression, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a double-stranded region, and the antisense strand comprises a region complementary to the C3 mRNA target sequence of SEQ ID NO: 13 or 14, and the complementary region is at least 15 consecutive nucleotides in length, an RNAi oligonucleotide, or a pharmaceutically acceptable salt thereof. (Item 2) The RNAi oligonucleotide according to item 1, or a pharmaceutically acceptable salt thereof, wherein the sense strand is 15 to 50 nucleotides in length. (Item 3) The RNAi oligonucleotide according to item 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the sense strand is 18 to 36 nucleotides in length. (Item 4) The RNAi oligonucleotide according to any one of items 1 to 3, or a pharmaceutically acceptable salt thereof, wherein the antisense strand is 15 to 30 nucleotides in length. (Item 5) The RNAi oligonucleotide according to any one of items 1 to 4, or a pharmaceutically acceptable salt thereof, wherein the antisense strand is 22 nucleotides in length, and the antisense strand and the sense strand form a double-stranded region that is at least 19 nucleotides in length, optionally at least 20 nucleotides in length. (Item 6) The RNAi oligonucleotide according to any one of items 1 to 5, or a pharmaceutically acceptable salt thereof, wherein the sense strand is 36 nucleotides in length, and the antisense strand and the sense strand form a double-stranded region that is at least 19 nucleotides in length, optionally at least 20 nucleotides in length. (Item 7) The RNAi oligonucleotide according to any one of items 1 to 6, or a pharmaceutically acceptable salt thereof, wherein the complementary region is at least 19 consecutive nucleotides in length, optionally at least 20 nucleotides in length. (Item 8) The 3'-end of the sense strand contains a stem-loop described as S1-L-S2, where S1 is complementary to S2 and L forms a loop 3 to 5 nucleotides in length between S1 and S2, the RNAi oligonucleotide according to any one of items 1 to 7, or a pharmaceutically acceptable salt thereof. (Item 9) The RNAi oligonucleotide according to item 8, or a pharmaceutically acceptable salt thereof, wherein L is a triloop or a tetraloop. (Item 10) The RNAi oligonucleotide according to item 9, or a pharmaceutically acceptable salt thereof, wherein L is a tetraloop. (Item 11) The RNAi oligonucleotide according to item 10, or a pharmaceutically acceptable salt thereof, wherein the tetraloop contains the nucleic acid sequence of SEQ ID NO: 8. (Item 12) The RNAi oligonucleotide according to any one of items 8 to 11, or a pharmaceutically acceptable salt thereof, wherein S1 and S2 are 1 to 10 nucleotides in length, and optionally, S1 and S2 have the same length. (Item 13) The RNAi oligonucleotide according to item 12, or a pharmaceutically acceptable salt thereof, wherein 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. (Item 14) The RNAi oligonucleotide according to item 13, or a pharmaceutically acceptable salt thereof, wherein S1 and S2 are 6 nucleotides in length. (Item 15) The RNAi oligonucleotide according to any one of items 8 to 14, or a pharmaceutically acceptable salt thereof, wherein the stem-loop region contains a nucleic acid sequence having at least 85% identity to SEQ ID NO: 7. (Item 16) The RNAi oligonucleotide according to item 15, or a pharmaceutically acceptable salt thereof, wherein the stem-loop region comprises a nucleic acid sequence having at least 95% identity to SEQ ID NO: 7. (Item 17) The RNAi oligonucleotide according to item 16, or a pharmaceutically acceptable salt thereof, wherein the stem-loop region comprises SEQ ID NO: 7. (Item 18) The RNAi oligonucleotide according to any one of items 8 to 16, or a pharmaceutically acceptable salt thereof, wherein the stem-loop comprises a nucleic acid having a maximum of 1, 2, or 3 substitutions, insertions, or deletions relative to SEQ ID NO: 7. (Item 19) The RNAi oligonucleotide according to any one of items 1 to 17, or a pharmaceutically acceptable salt thereof, wherein the antisense strand comprises a 3' overhang sequence that is 1 nucleotide in length or longer. (Item 20) The RNAi oligonucleotide according to item 19, or a pharmaceutically acceptable salt thereof, wherein the antisense strand comprises a linked 3' overhang of at least 2 nucleotides. (Item 21) The RNAi oligonucleotide according to item 20, or a pharmaceutically acceptable salt thereof, wherein the 3' overhang sequence is 2 nucleotides in length, and optionally, the 3' overhang sequence is GG. (Item 22) The RNAi oligonucleotide according to any one of items 1 to 21, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises at least one modified nucleotide. (Item 23) The RNAi oligonucleotide according to item 22, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises 20 to 50 modified nucleotides. (Item 24) The RNAi oligonucleotide according to item 22 or 23, or a pharmaceutically acceptable salt thereof, wherein all of the oligonucleotide is modified. (Item 25) The modified nucleotide is the RNAi oligonucleotide according to any one of Items 22 to 24, which contains a 2'-modification, or a pharmaceutically acceptable salt thereof. (Item 26) The 2'-modification is selected from 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinonucleic acid The RNAi oligonucleotide according to Item 25, or a pharmaceutically acceptable salt thereof, which is a modification selected therefrom. (Item 27) The 2'-modification is 2'-fluoro or 2'-O-methyl. In this case, optionally, the 2'-fluoro modification is 2'-fluorodeoxyribonucleoside, and / or the 2'-O-methyl modification is 2'-O-methylribonucleoside. The RNAi oligonucleotide according to Item 26, or a pharmaceutically acceptable salt thereof. (Item 28) The RNAi oligonucleotide according to Item 27, or a pharmaceutically acceptable salt thereof, which contains 40 to 50 2'-O-methyl modifications, and optionally, the RNAi oligonucleotide contains 40 to 50 2'-O-methylribonucleosides. (Item 29) At least one of nucleotides 1 to 7, 11 to 27, and 31 to 36 of the sense strand, and one or more, or all, of nucleotides 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand are modified with 2'-O-methyl, for example, 2'-O-methylribonucleoside. The RNAi oligonucleotide according to Item 28, or a pharmaceutically acceptable salt thereof. (Item 30) 10 to 30 of nucleotides 1 to 7, 11 to 27, and 31 to 36 of the sense strand, and one or more, or all, of nucleotides 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand are modified with 2'-O-methyl, for example, 2'-O-methylribonucleoside. The RNAi oligonucleotide according to Item 29, or a pharmaceutically acceptable salt thereof. (Item 31) One or more, or all, of nucleotides 1 to 7, 12 to 27, and 31 to 36 of the sense strand, and one or more, or all, of nucleotides 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand are modified with 2'-O-methyl, for example, 2'-O-methyl ribonucleoside, the RNAi oligonucleotide according to item 29, or a pharmaceutically acceptable salt thereof. (Item 32) One or more, or all, of nucleotides 1, 2, 4 to 7, 11, 14 to 16, 18 to 27, and 31 to 36 of the sense strand, and one or more, or all, of nucleotides 1, 6, 9, 11, 13, 15, 17, 18, and 20 to 22 of the antisense strand are modified with 2'-O-methyl, for example, 2'-O-methyl ribonucleoside, the RNAi oligonucleotide according to item 29, or a pharmaceutically acceptable salt thereof. (Item 33) The oligonucleotide according to any one of items 28 to 32, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises a 2'-fluoro modification at 5 to 15, for example, a modification with 2'-fluoro deoxyribonucleoside. (Item 34) One or more, or all, of 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, for example, 2'-fluoro deoxyribonucleoside, the RNAi oligonucleotide according to item 28, or a pharmaceutically acceptable salt thereof. (Item 35) 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, for example, 2'- Fluoro deoxyribonucleoside, the RNAi oligonucleotide according to item 34, or a pharmaceutically acceptable salt thereof. (Item 36) 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, for example, 2'-fluorodeoxyribonucleoside, the RNAi oligonucleotide according to item 34, or a pharmaceutically acceptable salt thereof. (Item 37) All of nucleotides 3, 8 to 10, 12, 13, and 17 of the sense strand, and one or more, or all, of nucleotides 2 to 5, 7, 8, 10, 12, 14, 16, and 19 of the antisense strand are modified with 2'-fluoro, for example, 2'-fluorodeoxyribonucleoside, the RNAi oligonucleotide according to item 34, or a pharmaceutically acceptable salt thereof. (Item 38) The RNAi oligonucleotide according to any one of items 1 to 37, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide contains at least one modified internucleotide linkage. (Item 39) The RNAi oligonucleotide according to item 38, or a pharmaceutically acceptable salt thereof, wherein at least one modified internucleotide linkage is a phosphorothioate linkage. (Item 40) The RNAi oligonucleotide according to item 39, or a pharmaceutically acceptable salt thereof, wherein the RNAi oligonucleotide has phosphorothioate linkages 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. (Item 41) The RNAi oligonucleotide according to any one of items 1 to 40, or a pharmaceutically acceptable salt thereof, wherein there is no internucleotide linkage between the sense strand and the antisense strand. (Item 42) The RNAi oligonucleotide according to any one of items 1 to 41, or a pharmaceutically acceptable salt thereof, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand contains a phosphate analog. (Item 43) The RNAi oligonucleotide according to any one of items 1 to 41, or a pharmaceutically acceptable salt thereof, wherein the RNAi oligonucleotide contains uridine at the 5'-end of the antisense strand. (Item 44) The RNAi oligonucleotide according to item 43, or a pharmaceutically acceptable salt thereof, wherein the uridine contains a phosphate analog. (Item 45) The RNAi oligonucleotide according to any one of items 42 to 44, or a pharmaceutically acceptable salt thereof, wherein the phosphate analog is 4'-O-methylphosphonate. (Item 46) The uridine containing the phosphate analog has the following structure:

Chemical formula

Claims

【Claim 1】 The invention described in the specification.

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