Compositions and methods for inhibiting complement factor B

Abstract

Described herein is an oligonucleotide (e.g., RNAi oligonucleotide) that comprises sense and antisense strands for targeting complement factor B (CFB) mRNA.RNAi oligonucleotide can be used to inhibit CFB expression, level and / or activity in cells.Also described is a method of using oligonucleotide (e.g., RNAi oligonucleotide) for prevention or treatment of disease, disorder or condition mediated by complement pathway activation or dysregulation.

Description

[Technical field]

[0001] Sequence Listing This application is filed together with an electronic format sequence listing. The sequence listing is provided as a file named 50694-092WO3_Sequence_Listing_1_19_23_ST26.xml, created on January 19, 2023, and is 394.2 kilobytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. [Background technology]

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

[0003] Complement factor B (CFB) is a component of the complement pathway that initiates the alternative complement pathway cascade. CFB is cleaved into Ba and Bb fragments. The Bb fragment associates with C3b, together forming C3 convertase, which is essential for the activation of the alternative complement pathway. Dysregulation and excessive activation of CFB are associated with several diseases, including paroxysmal nocturnal hemoglobinuria (PNH), multiple sclerosis, and rheumatoid arthritis. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need for compositions and methods that can be used to inhibit or silence CFB in subjects with diseases associated with activation or dysregulation of the complement pathway. [Means for solving the problem]

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

[0006] The first aspect of the present disclosure provides an RNAi oligonucleotide or its pharma- ceutically acceptable salt (e.g., its sodium salt) for reducing complement factor B (CFB) expression, the oligonucleotide comprises a sense strand and an antisense strand. The sense strand and the antisense strand of the oligonucleotide form a double helix region. The antisense strand of the oligonucleotide comprises a region of complementarity to the CFB mRNA target sequence, for example, SEQ ID NO: 13 or 14, and the region of complementarity is at least 15 consecutive nucleotides in length (e.g., at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length). In one embodiment, the sense strand is 15-50 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length). In one embodiment, the sense strand is 18-36 nucleotides in length (e.g., 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 nucleotides in length). In one embodiment, the antisense strand is 15-30 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length).

[0007] In one embodiment, the antisense strand of the oligonucleotide is 22 nucleotides long, and the antisense strand and the sense strand form a double helical region that is at least 19 nucleotides long, optionally at least 20 nucleotides long. In some embodiments, the sense strand is 36 nucleotides long, and the antisense strand and the sense strand form a double helical region that is at least 19 nucleotides long, optionally at least 20 nucleotides long. In some embodiments, the region of complementarity is at least 19 consecutive nucleotides long, optionally at least 20 nucleotides long.

[0008] In some embodiments, the 3' end of the sense strand of the oligonucleotide comprises a stem loop described as S1-L-S2, where S1 is complementary to S2, and L forms a loop between S1 and S2 that is 3 to 5 nucleotides (e.g., 3, 4 or 5 nucleotides) long. In some embodiments, L is a triloop or tetraloop. In one embodiment, L is a tetraloop. In one embodiment, the tetraloop comprises a nucleic acid sequence of 5'GAAA3'.

[0009] In some embodiments, S1 and S2 of the stem loop are 1-10 nucleotides in length (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). S1 and S2 can have the same length. In some embodiments, S1 and S2 are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In one embodiment, S1 and S2 are 6 nucleotides in length.

[0010] In some 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%, 99% or 100%) identity to SEQ ID NO:7. In some embodiments, the stem-loop region comprises a nucleic acid sequence having at least 95% (e.g., at least 96%, 97%, 98%, 99% or 100%) identity to SEQ ID NO:7. In some embodiments, the stem-loop region comprises the sequence 5'-GCAGCCGAAAGGCUGC-3' (SEQ ID NO:7). In some embodiments, the stem-loop comprises a nucleic acid having up to 1, 2 or 3 nucleic acid substitutions, insertions or deletions relative to SEQ ID NO:7.

[0011] In some embodiments, the antisense strand of oligonucleotide comprises a 3' overhang sequence that is 1 nucleotide or more in length.In some embodiments, the antisense strand comprises a 3' overhang of at least two linked nucleotides.In one embodiment, the 3' overhang sequence is 2 nucleotides in length, for example, the sequence is GG.In some embodiments, the sense strand comprises a 5' overhang of at least two linked nucleotides.

[0012] In some embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, the oligonucleotide comprises 20-50 modified nucleotides (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 modified nucleotides). In some embodiments, the oligonucleotide comprises 20-40 (e.g., 25-40, 30-40, 35-40, 30-35, 25-35, 20-25, 21-30, and 31-40) modified nucleotides. In one embodiment, all of the nucleotides of the oligonucleotide are modified.

[0013] Modified nucleotides include 2'-modifications. In some embodiments, the 2'-modifications are 2'-aminoethyl, 2'-fluoro 2'-O-methyl 2'-O-methoxymethyl, and 2'-deoxy 2'-fluoro β-arabinonucleic acid. In some embodiments, the 2'-modifications are 2'-fluoro or 2'-O-methyl, and optionally, the 2'-fluoro modification is a 2'-fluoro deoxyribonucleoside and / or the 2'-O-methyl modification is a 2'-O-methylribonucleoside. In some embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises between 40 and 50 (e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) 2'-O-methyl modifications, and optionally, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises between 40 and 50 (e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) 2'-O-methyl ribonucleosides.

[0014] In some embodiments, at least one of nucleotides 1-7, 11-27, and 31-36 of the sense strand and at least one of nucleotides 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with 2'-O-methyl, e.g., a 2'-O-methyl ribonucleoside. In some embodiments, 10 to 29 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29) nucleotides 1-7, 12-27, and 31-36 of the sense strand and 10 to 16 (e.g., 11, 12, 13, 14, 15 or 16) nucleotides 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with 2'-O-methyl, e.g., 2'-O-methyl ribonucleosides. In one embodiment, all of nucleotides 1-7, 12-27, and 31-36 of the sense strand and all of nucleotides 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with 2'-O-methyl, e.g., 2'-O-methyl ribonucleosides. In one embodiment, all of nucleotides 1-7, 12-27, and 31-36 of the sense strand and all of nucleotides 1, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with 2'-O-methyl, e.g., 2'-O-methyl ribonucleosides.

[0015] In some embodiments, the RNAi oligonucleotide or a pharma- ceutically acceptable salt thereof comprises 5 to 15 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) 2'-fluoro modified nucleotides, such as 2'-fluoro deoxyribonucleosides. In some embodiments, at least one of nucleotides 8, 9, 10, and 11 of the sense strand and at least one of nucleotides 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with a 2'-fluoro modified nucleotide, such as a 2'-fluoro deoxyribonucleoside. In some embodiments, 2 to 4 (e.g., 2, 3, and 4) of nucleotides 8, 9, 10, and 11 of the sense strand and 2 to 7 (e.g., 2, 3, 4, 5, 6, and 7) of nucleotides 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with a 2'-fluoro modified nucleotide, such as a 2'-fluoro deoxyribonucleoside. In one embodiment, all of nucleotides 8, 9, 10, and 11 of the sense strand and all of nucleotides 2, 3, 5, 7, 10, and 14 of the antisense strand are modified with 2'-fluoro modified nucleotides, such as 2'-fluoro deoxyribonucleosides. In one embodiment, all of nucleotides 8, 9, 10, and 11 of the sense strand and all of nucleotides 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with 2'-fluoro modified nucleotides, such as 2'-fluoro deoxyribonucleosides.

[0016] In one embodiment, the sense strand has the nucleic acid sequence of SEQ ID NO: 37 and the antisense strand has the nucleic acid sequence of SEQ ID NO: 38. In one embodiment, the sense strand has the nucleic acid sequence of SEQ ID NO: 66 and the antisense strand has the nucleic acid sequence of SEQ ID NO: 67.

[0017] In some embodiments, the RNAi oligonucleotide or its pharmaceutically acceptable salt comprises at least one modified internucleotide bond. In one embodiment, at least one modified internucleotide bond is phosphorothioate bond. In some embodiments, the RNAi oligonucleotide or its pharmaceutically acceptable salt has phosphorothioate bond 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. In some embodiments, there is no internucleotide bond between the sense strand and the antisense strand.

[0018] In some embodiments, the 4'-carbon of the sugar of the 5' nucleotide of the antisense strand comprises a phosphate analog. In some embodiments, the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises a uridine at position 1 of the 5' end of the antisense strand. In one embodiment, the uridine comprises a phosphate analog. In one embodiment, the phosphate analog is 4'-O-monomethylphosphonate. In one embodiment, the uridine comprising a phosphate analog has the following structure: [ka] Includes.

[0019] In some embodiments, at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands. In some embodiments, each targeting ligand comprises a carbohydrate, an amino sugar, cholesterol, a polypeptide, or a lipid. In some embodiments, each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the GalNAc moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety. In some embodiments, the RNAi oligonucleotide comprises 1 to 5 (e.g., 1, 2, 3, 4, and 5) 2'-ON-acetylgalactosamine (GalNAc) moieties conjugated to the sense strand. In one embodiment, up to 4 nucleotides of L of the stem loop are conjugated to monovalent GalNAc moieties. In one embodiment, 3 nucleotides of L of the stem loop are conjugated to monovalent GalNAc moieties. In some embodiments, one or more of the nucleotides at nucleotide positions 28-30 of the sense strand are conjugated to a monovalent GalNAc moiety. In one embodiment, each of the nucleotides at positions 28-30 of any one of SEQ ID NOs: 1, 4, 17, 19, 21, 23, 25, 27, and 29 (and variants thereof having at least 85% sequence identity thereto) is conjugated to a monovalent GalNAc moiety. In one embodiment, the nucleotides at positions 28-30 of any one of SEQ ID NOs: 1, 4, 17, 19, 21, 23, 25, 27, and 29 (and variants thereof having at least 85% sequence identity thereto) have the following structure: [ka] wherein Z represents a bond, a click chemistry handle, or a linker of 1 to 20 contiguous covalently bonded atoms in length (inclusive) selected from the group consisting of substituted and unsubstituted alkylene, substituted and unsubstituted alkenylene, substituted and unsubstituted alkynylene, substituted and unsubstituted heteroalkylene, substituted and unsubstituted heteroalkenylene, substituted and unsubstituted heteroalkynylene, and combinations thereof; and X is O, S, or N. In some embodiments, Z is an acetal linker. In some embodiments, X is O. In one embodiment, the nucleotides at positions 28-30 of any one of SEQ ID NOs: 1, 4, 17, 19, 21, 23, 25, 27, and 29 (and variants thereof having at least 85% sequence identity thereto) have the following structure: [ka] Includes.

[0020] In some embodiments, the RNAi oligonucleotides described herein, or pharma- ceutically acceptable salts thereof, comprise a sense strand having a tetraloop, and three GalNAc moieties are conjugated to a nucleotide comprising the tetraloop, each GalNAc moiety being conjugated to one nucleotide. In some embodiments, the oligonucleotides (e.g., RNAi oligonucleotides) described herein comprise a sense strand having a tetraloop comprising a GalNAc-conjugated nucleotide, and the tetraloop has the following structure: [ka] Includes.

[0021] In some embodiments, the sense strand of the RNAi oligonucleotide, or a pharma- ceutically acceptable salt thereof, comprises a nucleotide 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 SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the antisense strand of the oligonucleotide (e.g., RNAi oligonucleotide) comprises a nucleotide 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 SEQ ID NO:3 or SEQ ID NO:6. In some embodiments, the sense strand comprises a nucleotide sequence having at least 95% (e.g., at least 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the antisense strand comprises a nucleotide sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:3 or SEQ ID NO:6. In some embodiments, the sense strand comprises the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:4. In some embodiments, the antisense strand comprises the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:6. In some embodiments, the sense strand and the antisense strand comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs:1 and 3, respectively, and SEQ ID NOs:4 and 6, respectively.

[0022] In one embodiment, the sense strand comprises a nucleotide 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 SEQ ID NO:1, and the antisense strand comprises a nucleotide 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 SEQ ID NO:3. In another embodiment, the sense strand comprises a nucleotide 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 SEQ ID NO:4, and the antisense strand comprises a nucleotide 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 SEQ ID NO:6. In one embodiment, the sense strand comprises a nucleotide sequence having at least 95% (e.g., at least 95%, at least 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:1, and the antisense strand comprises a nucleotide sequence having at least 95% (e.g., at least 95%, at least 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:3. In another embodiment, the sense strand comprises a nucleotide sequence having at least 95% (e.g., at least 95%, at least 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:4, and the antisense strand comprises a nucleotide sequence having at least 95% (e.g., at least 95%, at least 96%, 97%, 98%, 99% or 100%) sequence identity to SEQ ID NO:6. In another embodiment, the sense strand comprises a nucleotide sequence set forth in SEQ ID NO:1, and the antisense strand comprises a nucleotide sequence set forth in SEQ ID NO:3. In another embodiment, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO:4 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:6.In one embodiment, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 37 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 38. In one embodiment, the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 66 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO: 67.

[0023] In some embodiments, the RNA oligonucleotide comprises a pharma- ceutically acceptable salt. In some embodiments, the pharma- ceutically acceptable salt is a sodium salt.

[0024] In a second aspect, the present disclosure provides a pharmaceutical composition comprising any one of the RNAi oligonucleotides described herein, or any pharma- ceutically acceptable salts thereof, and a pharma- ceutically acceptable carrier, excipient, or diluent.

[0025] In a third aspect, the present disclosure provides vectors encoding one or both of the sense and antisense strands of the RNAi oligonucleotides described herein, or pharma- ceutically acceptable salts thereof.

[0026] In a fourth aspect, the disclosure provides a cell comprising a vector encoding all or a portion of any of the RNAi oligonucleotides described herein, or a pharma- ceutically acceptable salt thereof.

[0027] In a fifth aspect, the present disclosure provides a method of treating a subject having a disease, disorder, or condition associated with complement pathway activation or dysregulation (e.g., activation or dysregulation of CFB), comprising administering to the subject a therapeutically effective amount of any one or more of the RNAi oligonucleotides or pharma- ceutically acceptable salts thereof described herein, or pharmaceutical compositions comprising the same, vectors encoding the same, or cells comprising the oligonucleotides or vectors. In some embodiments, the RNAi oligonucleotides degrade CFB mRNA transcripts in cells of the subject. In some embodiments, expression of CFB in cells of the subject is reduced. In some embodiments, expression of CFB in cells of the subject is reduced by 10% to 100% (e.g., 10% to 90%, 10% to 70%, 10% to 50%, 10% to 30%, 20% to 100%, 40% to 100%, 60% to 100%, and 80% to 100%) compared to the expression level of CFB in cells of a subject not administered the RNAi oligonucleotides, pharmaceutical compositions, vectors, or cells described herein. In some embodiments, the level and / or activity of CFB is reduced in the subject. In some embodiments, the level and / or activity of CFB is reduced by 10% to 100% (e.g., 10% to 90%, 10% to 70%, 10% to 50%, 10% to 30%, 20% to 100%, 40% to 100%, 60% to 100%, and 80% to 100%) compared to the level and / or activity of CFB in a subject not administered an RNAi oligonucleotide, pharmaceutical composition, vector, or cell described herein. In some embodiments, the level and / or activity of CFB is reduced by 50% to 100% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100% and 90% to 100%) compared to the level and / or activity of CFB in a subject not administered an RNAi oligonucleotide, pharmaceutical composition, vector or cell described herein.In some embodiments, administration of the RNAi oligonucleotides, pharmaceutical compositions, vectors, or cells described herein to a subject in need thereof reduces the amount of CFB circulating in the blood of the subject compared to a subject not receiving the RNAi oligonucleotides, pharmaceutical compositions, vectors, or cells described herein (an untreated subject). The amount of CFB in the blood of a treated subject can be reduced to less than or equal to 1,000 μg / mL, 900 μg / mL, 800 μg / mL, 700 μg / mL, 600 μg / mL, 500 μg / mL, 400 μg / mL, 300 μg / mL, 200 μg / mL, 100 μg / mL, or 50 μg / mL. For example, administration of the RNAi oligonucleotides, pharmaceutical compositions, vectors, or cells described herein may reduce the amount of CFB in the blood of a treated subject to within the range of 50-1000 μg / mL (e.g., within the range of 50-900 μg / mL, 50-800 μg / mL, 50-700 μg / mL, 50-600 μg / mL, 50-500 μg / mL, 50-400 μg / mL, 50-300 μg / mL, or 50-200 μg / mL) or to less than 50 μg / mL.

[0028] In one embodiment of the fifth aspect, the subject is a mammal, such as a human.

[0029] In some embodiments, the RNAi oligonucleotide or its pharmaceutically acceptable salt, pharmaceutical composition, vector or cell is formulated for daily, weekly, monthly or yearly administration.In some embodiments, the RNAi oligonucleotide or its pharmaceutically acceptable salt, pharmaceutical composition, vector or cell is formulated for intravenous, subcutaneous, intramuscular, oral, intranasal, sublingual, intrathecal and intradermal administration.In one embodiment, the RNAi oligonucleotide or its pharmaceutically acceptable salt, pharmaceutical composition, vector or cell is formulated for subcutaneous administration. In one embodiment, the RNAi oligonucleotide or a pharma- ceutically acceptable salt thereof, pharmaceutical composition, vector, or cell is formulated for administration at a dose of about 0.1 mg / kg to about 150 mg / kg (e.g., 0.1 mg / kg to 100 mg / kg, 0.1 mg / kg to 50 mg / kg, 0.1 mg / kg to 1 mg / kg, 1 mg / kg to 150 mg / kg, 50 mg / kg to 150 mg / kg, and 100 mg / kg to 150 mg / kg).

[0030] In a sixth aspect, the present disclosure provides a method of reducing CFB expression in a cell, a population of cells, or a subject by contacting the cell, a population of cells, or a subject with an oligonucleotide (e.g., an RNAi oligonucleotide) of the present disclosure, or a pharmaceutical composition comprising the oligonucleotide, a vector encoding the oligonucleotide, or a cell comprising the vector, as described herein. The oligonucleotide (e.g., an RNAi oligonucleotide) of the present disclosure, or a pharmaceutical composition comprising the oligonucleotide, a vector encoding the oligonucleotide, or a cell comprising the vector, as described herein, can be administered to the subject. In some embodiments, reducing CFB expression includes reducing the amount or level of CFB mRNA, the amount or level of CFB protein, or both. In some embodiments, the level of CFB mRNA, the level of CFB protein, or both, is reduced by 10% to 100% (e.g., 10% to 80%, 10% to 60%, 10% to 40%, 10% to 20%, 20% to 100%, 40% to 100%, 60% to 100%, and 80% to 100%) compared to the level of CFB mRNA, the level of CFB protein, or both, in cells of a subject that has not been administered an oligonucleotide (e.g., an RNAi oligonucleotide) or a pharmaceutical composition comprising the oligonucleotide, a vector encoding the oligonucleotide, or a cell comprising the vector described herein. In some embodiments, the level of CFB mRNA, the level of CFB protein, or both is reduced by 50% to 100% (e.g., 50% to 90%, 50% to 80%, 50% to 70%, 50% to 60%, 60% to 100%, 70% to 100%, 80% to 100%, and 90% to 100%) compared to the level of CFB mRNA, the level of CFB protein, or both in cells of a subject that has not been administered an RNAi oligonucleotide, pharmaceutical composition, vector, or cell as described herein. In some embodiments, administration of an RNAi oligonucleotide, pharmaceutical composition, vector, or cell as described herein to a subject in need thereof reduces the amount of CFB circulating in the blood of the subject compared to a subject that has not been administered an RNAi oligonucleotide, pharmaceutical composition, vector, or cell as described herein (an untreated subject).The treated subject's blood CFB level may be reduced to less than or equal to 1,000 μg / mL, 900 μg / mL, 800 μg / mL, 700 μg / mL, 600 μg / mL, 500 μg / mL, 400 μg / mL, 300 μg / mL, 200 μg / mL, 100 μg / mL or 50 μg / mL. For example, administration of the RNAi oligonucleotides, pharmaceutical compositions, vectors, or cells described herein may reduce the amount of CFB in the blood of a treated subject to within the range of 50-1000 μg / mL (e.g., within the range of 50-900 μg / mL, 50-800 μg / mL, 50-700 μg / mL, 50-600 μg / mL, 50-500 μg / mL, 50-400 μg / mL, 50-300 μg / mL, or 50-200 μg / mL) or to less than 50 μg / mL.

[0031] In a seventh aspect, the disclosure provides a kit comprising an oligonucleotide (e.g., an RNAi oligonucleotide), a pharmaceutical composition, a vector, or a cell described herein. In some embodiments, the kit comprises a pharmaceutical composition containing an RNAi oligonucleotide or a pharmaceutically acceptable salt thereof, an agent that reduces the level and / or activity of CFB in a cell or subject described herein, and optionally a pharmaceutically acceptable carrier, excipient, or diluent. In some embodiments, the kit comprises a vector encoding any one of the RNAi oligonucleotides, pharmaceutical compositions, vectors, or cells described herein. In some embodiments, the kit comprises a package insert with instructions for carrying out any of the methods described herein. In some embodiments, the kit comprises a pharmaceutical composition containing an RNAi oligonucleotide agent, pharmaceutical composition, vector, or cell described herein that reduces the level and / or activity of CFB in a cell or subject; an additional therapeutic agent; and a package insert with instructions for carrying out any of the methods described herein.

[0032] In an eighth aspect, the disclosure features the use of an oligonucleotide (e.g., an RNAi oligonucleotide), pharmaceutical composition, vector, or cell described herein for use in preventing or treating a disease, disorder, or condition mediated by complement pathway activation or dysregulation (e.g., CFB activation or dysregulation) in a subject in need thereof.

[0033] In any one of the fifth, sixth or eighth aspects, the subject is identified as having a disease, disorder or condition mediated by complement pathway activation or dysregulation (e.g., dysregulated CFB activation). In some embodiments, the disease is as follows: paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN), including primary MN, Escherichia coli (E.coli)-induced or typical hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration, geographic atrophy, diabetic retinopathy, uveitis, intermediate uveitis, Behçet's uveitis, retinitis pigmentosa, macular edema, multifocal choroiditis, Vogt-Koyanagi-Harada syndrome, scattershot choroidal retinopathy, sympathetic ophthalmia, ocular cicatricial pemphigoid (OCP), ocular pemphigoid, non-arthritic ischemic optic neuropathy, postoperative inflammation, retinal vein occlusion, neuropathy, multiple sclerosis, stroke during surgery, Guillain-Barré syndrome, traumatic brain injury, Parkinson's disease, hemodialysis complications, hyperacute graft rejection, xenograft rejection, interleukin-2 induced toxicity during IL-2 treatment, inflammatory diseases, inflammation in autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, myocarditis, post-ischemia reperfusion state, myocardial infarction, balloon angioplasty, post-pump syndrome in cardiopulmonary or renal bypass, atherosclerosis, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infections or sepsis, immune complex disorders and autoimmune diseases, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, proliferative nephritis, liver fibrosis, hemolytic anemia, myasthenia gravis, tissue regeneration, nerve regeneration, dyspnea, hemoptysis, acute respiratory distress syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolism and pulmonary infarction, pneumonia, fibrous dust disease, pulmonary fibrosis, allergies, bronchoconstriction, hypersensitivity pneumonitis, parasitic diseases, Goodpasture's syndrome, pulmonary vasculitis, microimmune type ( Pauci-immune vasculitis, immune complex-associated inflammation, antiphospholipid syndrome, glomerulonephritis, obesity, arthritis, autoimmune heart disease, inflammatory bowel disease, ischemia-reperfusion injury, Barraquer-Simons syndrome, hemodialysis, antineutrophil cytoplasmic antibody (ANCA) vasculitis, cryoglobulinemia, psoriasis, transplantation, central nervous system diseases such as Alzheimer's disease and other neurodegenerative conditions, dense deposition diseases, bullous skin diseases, membranoproliferative glomerulonephritis type II (MPGN II), chronic graft-versus-host disease, Felty syndrome, pyoderma gangrenosum (PG), hidradenitis suppurativa (HS), pulmonary arterial hypertension, primary Sjogren's syndrome, primary biliary cholangitis, autosomal dominant polycystic kidney disease, and myelin oligodendrocyte glycoprotein antibody disease (MOGAD). In some embodiments, the disease is rheumatoid arthritis.

[0034] In some embodiments, the RNA oligonucleotides described herein include pharmaceutically acceptable salts.In some embodiments, the pharmaceutically acceptable salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfonate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, 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, methylamine, dimethylamine, trimethylamine, triethylamine, or ethylamine, or is an alkali or alkaline earth metal salt. In some embodiments, the alkali or alkaline earth metal salt is selected from the group consisting of sodium, lithium, potassium, calcium, magnesium, and ammonium (e.g., quaternary ammonium and tetramethylammonium). In some embodiments, the pharmaceutically acceptable salt is a sodium salt.

[0035] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon payment of the necessary fee. [Brief description of the drawings]

[0036] [Figure 1A] 1 shows the chemical structure of the sense strand of Compound A (SEQ ID NO: 66). [Figure 1B] 1 shows the chemical structure of the antisense strand of Compound A (SEQ ID NO: 67). [Figure 1C] The nucleic acid sequences of the sense strand (SEQ ID NO: 1) and the antisense strand (SEQ ID NO: 3) of Compound A are shown. [Figure 2A] 1 shows the chemical structure of the sense strand of Compound B (SEQ ID NO: 37). [Figure 2B] 2 shows the nucleic acid sequence of the antisense strand of Compound B (SEQ ID NO: 38). [Figure 2C] The nucleic acid sequences of the sense strand (SEQ ID NO: 4) and the antisense strand (SEQ ID NO: 6) of Compound B are shown. [Figure 2D] 1 shows a schematic diagram of the sense and antisense strands of compound B. [Figure 2E-1-2E-2] 1 shows the chemical structures of Compound B RNAi oligonucleotides (SEQ ID NOs: 37 and 38). [Figure 3A] 1 is a graph showing the percentage of CFB mRNA remaining after in vitro treatment of HuH-7 cells with 1 nM amounts of various oligonucleotides, specifically identifying the effect of compound A and B on promoted CFB knockdown. [Figure 3B] 1 is a graph showing the percentage of CFB mRNA remaining after treatment of HuH-7 cells with various oligonucleotides at 0.03 nM, 0.1 nM, and 1 nM amounts, specifically identifying the effect of Compound B-facilitated CFB knockdown. [Figure 4A] FIG. 1 is a graph showing the amount of CFB mRNA remaining in mice 4 days after co-administration of Compound A or Compound C at a single dose: 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg or 3.0 mg / k. [Figure 4B] FIG. 13 is a graph showing the amount of CFB mRNA remaining in mice 4 days after co-administration of Compound A or Compound D at a single dose: 0.3 mg / k. [Figure 4C] 1 is a graph showing the amount of CFB mRNA remaining in mice 4 days after co-administration of Compounds A, B, E, F, G, H or I at a single dose: 0.5 mg / kg. [Diagram 5]FIG. 1 is a graph showing measurement of the percent of CFB mRNA in the liver of cynomolgus macaques pre-dose and 28 and 56 days after treatment with a single dose of 4 mg / kg of Compounds AI, compared to PBS administered as a control. [Figure 6A] FIG. 1 is a graph showing the measurement of the percent of CFB mRNA in the liver of cynomolgus macaques following treatment with 1 mg / kg or 2 mg / kg of Compound A or Compound B on days 0, 28, 56, and 84, compared to PBS administered as a control. [Figure 6B] FIG. 1 is a graph showing the measurement of the percent of CFB in serum of cynomolgus macaques following treatment with 1 mg / kg or 2 mg / kg of Compound A or Compound B compared to PBS administered as a control. [Figure 7] 1 is a graph showing the estimated ED50 for Compound A and Compound B measured as CFB mRNA in liver of cynomolgus macaques 28 days after administration of a single dose of 2 mg / kg Compound A or Compound B. [Figure 8] Graph showing percent complement activity (AP) in serum of cynomolgus monkeys after treatment with 2 mg / kg Compound A or Compound B on days 0, 28, 56, and 84, as measured by a WIESLAB® ELISA-based functional assay. PBS was administered in the same multiple-dose regimen as the control group. [Figure 9] Graph showing percent lysis from serum of cynomolgus monkeys after treatment with 1 mg / kg or 2 mg / kg of Compound B on days 0, 28, 56 and 84, as measured by rabbit erythrocyte hemolysis. PBS was administered in the same multiple dose regimen as the control group. [Figure 10A]Figure 1 shows RT-qPCR measurement of CFB mRNA (%) in liver of CD-1 mice following administration of a single subcutaneous dose of 0.25 mg / kg, 0.5 mg / kg, and 3 mg / kg of Compound J, compared to PBS administered as a control. The level of liver knockdown was followed for 63 days, with 5 mice sacrificed at each time point for measurement. [Figure 10B] FIG. 1 shows quantitative immunoblot measurement of CFB circulating protein (%) in serum of CD-1 mice over a 42 day period following administration of a single subcutaneous dose of Compound J: 0.25 mg / kg (second column from the left), 0.5 mg / kg (third column from the left) and 3 mg / kg (right-most column) compared to PBS administered as a control (left-most column). [Figure 11] Graph showing stem-loop-qPCR measurement of siRNA exposure in plasma, spleen, liver and kidney tissues of CD-1 mice administered a single subcutaneous dose of 3 mg / kg Compound J. The time course of the measurement was over a period of 672 hours. Five mice were sacrificed at each time point for measurement. [Figure 12A] FIG. 13 shows RT-qPCR measurements of percent CFB mRNA in the liver of CD-1 mice over a 70 day period following administration of four doses of 0.5 mg / kg (day 56 only) or 3 mg / kg Compound J on days 0, 14, 28 and 42 compared to PBS administered as a control. [Figure 12B] Graph showing quantitative measurement by immunoblot of CFB serum protein in CD-1 mice over a 70 day period following administration of four doses of 0.5 mg / kg or 3 mg / kg Compound J on days 0, 14, 28 and 42. CFB levels were calculated as a percentage of CFB serum levels measured from the PBS control group (n=5 / time point). [Figure 13A] FIG. 13 is a graph showing stem-loop-qPCR measurement of Compound J concentrations in liver tissue of CD-1 mice administered four doses of 0.5 mg / kg Compound J on days 0, 14, 28 and 42. [Figure 13B]FIG. 13 is a graph showing stem-loop-qPCR measurement of Compound J concentrations in plasma of CD-1 mice administered four doses of 0.5 mg / kg Compound J on days 0, 14, 28 and 42. [Figure 14A] 1 is a graph showing clinical scores of hind paws from a collagen antibody-induced arthritis model in which arthritis was induced on day 0, an LPS booster on day 3, followed by prophylactic treatment with three doses of Compound J at 0.5 mg / kg or 3 mg / kg doses on days -7, 0, and 7. PBS-treated CAIA animals served as the control group. [Figure 14B] Graph showing clinical scores of hind paws from a collagen antibody-induced arthritis model in which arthritis was induced on day 0, boosted with LPS on day 3, and then therapeutically treated with a single dose of 0.5 mg / kg or 3 mg / kg dose of Compound J on day 5 after disease induction. PBS-treated CAIA animals served as the control group. [Figure 15A] Images of hind paw inflammation on day 11 in a CAIA mouse model in which arthritis was induced by collagen antibody administered on day 0 and an LPS booster on day 3, followed by prophylactic treatment with three doses of Compound J at a dose of 3 mg / kg on days -7, 0, and 7. PBS-treated CAIA animals served as a control group. [Figure 15B] Images of hind paw inflammation on day 13 in a CAIA mouse model in which arthritis was induced with collagen antibody administered on day 0 and an LPS booster on day 3, followed by therapeutic treatment with a single 3 mg / kg dose of Compound J on day 5 after disease induction. PBS-treated CAIA animals served as a control group. [Figure 16] 13 shows images of H&E staining demonstrating a reduction in mononuclear cell infiltration into the hind paws following prophylactic treatment with three doses of 3 mg / kg dose of Compound J on days -7, 0, and 7. Naïve and PBS-treated CAIA animals were used as negative and positive controls for inflammation, respectively. [Figure 17]Images of Safranin O staining demonstrating prevention of cartilage erosion and pannus formation and H&E staining demonstrating reduction of mononuclear cell infiltration in knee joints of a CAIA-induced arthritis model after animals were prophylactically treated with three doses of 3 mg / kg Compound J on days -7, 0, and 7. Naive and PBS-treated CAIA animals were used as negative and positive controls, respectively. [Figure 18] 13 shows images of Safranin O staining demonstrating prevention of cartilage erosion and pannus formation in the knee joints of the CAIA-induced arthritis model after prophylactic treatment of animals with three doses of 3 mg / kg Compound J on days -7, 0, and 7. Naïve and PBS-treated CAIA animals were used as negative and positive controls, respectively. [Figure 19] Images of lymphocyte (CD45+) staining in the hind paws of CAIA-induced arthritic animals demonstrating a reduction in immune cell infiltration following therapeutic treatment with a single dose of 3 mg / kg Compound J on day 5 after disease induction. Naive and PBS-treated CAIA animals were used as negative and positive controls, respectively. [Figure 20] Images of neutrophil and macrophage (CD11b+) staining of hind paws of CAIA-induced arthritic animals demonstrating a reduction in immune cell infiltration following therapeutic treatment with a single dose of 3 mg / kg Compound J on day 5 after disease induction. Naive and PBS-treated CAIA animals were used as negative and positive controls, respectively. [Figure 21] Images of macrophage (F4 / 80+) staining of the hind paws of CAIA-induced arthritic animals demonstrating a reduction in immune cell infiltration following therapeutic treatment with a single dose of 3 mg / kg Compound J on day 5 after disease induction. Naive and PBS-treated CAIA animals were used as negative and positive controls, respectively. [Figure 22] Shown are images of in situ hybridization of fluorescent tags to CFB mRNA (red) to monitor local complement expression and CD45+ cell (green-lymphocyte) infiltration into the hind paws of CAIA-induced animals following therapeutic treatment with a single 3 mg / kg dose of Compound J on day 5 after disease induction. [Diagram 23]Graph showing the mean clinical scores from two experiments with MOG-induced experimental autoimmune encephalomyelitis (EAE) mice in which disease was induced on day 0, received two doses of pertussis toxin on days 0 and 1, and then were therapeutically treated with five weekly doses of Compound J at a dose of 3 mg / kg starting on day 7 after disease induction. PBS-treated EAE animals were used as disease positive controls. [Figure 24] Representative images of luxol fast blue spinal cord staining of MOG-induced EAE mice after receiving 5 weekly doses of 3 mg / kg Compound J compared to naive PBS-treated EAE mice used as disease positive controls are shown. [Figure 25A] FIG. 13 is a graph showing the amount of hepatic CFB mRNA in MOG-induced EAE mice after receiving 5 weekly doses of 3 mg / kg Compound J compared to naive PBS-treated EAE mice (disease positive). [Figure 25B] FIG. 13 is a graph showing the amount of serum CFB in MOG-induced EAE mice after receiving 5 weekly doses of 3 mg / kg Compound J compared to naive PBS-treated EAE mice (disease positive control). [Figure 26] Graph showing proteinuria:creatinine ratios measured from spot urine collections from a passive Heymann nephritis (PHN) rat model in which proteinuria was induced with a single dose of sheep anti-rat Fx1A on day 0 and treated prophylactically with three doses of 12 mg / kg Compound J on days -14, -7 and 0 compared to PBS-treated PHN animals (disease positive control) and healthy animals. [Figure 27] Graph showing percent lysis from serum of a passive Heymann nephritis (PHN) rat model after treatment with 12 mg / kg Compound J on days -14, -7 and 0, as measured by rabbit red blood cell hemolysis assay before disease induction (day -1) or 6 days after disease induction, compared to PBS-treated PHN animals (disease positive control) and healthy animals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] definition As used herein, the terms "about" and "approximately" refer to an amount that is ±10% of the recited value, optionally ±5% of the recited value, or more optionally ±2% of the recited value.

[0038] As used herein, "administer" and "administration" refer to any method of providing a pharmaceutical preparation to a subject. The oligonucleotides described herein can be administered by any method known to those skilled in the art. Suitable methods for administering oligonucleotides can include, for example, oral, injection (e.g., intravenous, intraperitoneal, intramuscular, intravitreal and subcutaneous), infusion formulations, and the like. The method of administering oligonucleotides can include subcutaneous administration. The oligonucleotides prepared as described herein can be administered in a variety of forms, depending on the disorder to be treated and the age, condition and weight of the subject, as known in the art. The preparations can be administered prophylactically; i.e., to reduce the likelihood of developing a disease or condition.

[0039] As used herein, an "agent that reduces the level and / or activity of CFB" refers to an oligonucleotide (e.g., an RNAi oligonucleotide) disclosed herein that can be used (e.g., administered) to reduce the level or expression of CFB in a cell or subject, e.g., in the cells or serum of a subject. "Reducing the level of CFB," "reducing the expression of CFB," and "reducing the transcription of CFB" refer to reducing the level, reducing the expression, or reducing the transcription of CFB mRNA and / or CFB protein in a cell or subject, e.g., by administering an oligonucleotide agent (such as those described herein) to the cell or subject. The level of CFB mRNA and / or CFB protein can be measured using any method known in the art (e.g., by measuring the level of CFB mRNA or the level of CFB protein in a cell or subject). The reduction 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 CFB mRNA or protein levels compared to before treatment, or compared to levels in an untreated subject (e.g., a subject having a disease or disorder associated with complement activation or dysregulation (e.g., activation or dysregulation of CFB)), or compared to a control subject (e.g., a healthy subject (e.g., a subject not having a disease or disorder associated with complement activation or dysregulation (e.g., activation or dysregulation of CFB)). The level, expression, transcription of mRNA and / or CFB protein may be reduced. The CFB may be any CFB (e.g., mouse CFB, rat CFB, monkey CFB, or human CFB), as well as a variant or mutant of CFB. Thus, the CFB may be wild-type CFB, mutant CFB, or transgenic CFB in the context of a genetically engineered cell, cell population, or organism. "Reducing the activity of CFB" also means reducing the level of activity associated with CFB (e.g., by reducing complement pathway activation associated with a disease mediated by complement pathway activation or dysregulation).The activity of CFB may be reduced by about 5% or more (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or about 100%). The activity level of CFB may be measured using any method known in the art. The reduction may be a decrease in the level, expression or transcription of CFB mRNA and / or CFB protein by at least about 5% or more (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or about 100% or more relative to a cell or subject not treated with an oligonucleotide agent disclosed herein). The reduction in CFB mRNA and / or CFB protein level, expression or transcription can be over a period of at least one day or more (e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 days or more). The reduction can be, for example, a decrease of at least 5 μg / mL (e.g., 5-1000 μg / mL) of the amount of CFB protein in the blood of a treated subject (e.g., a human subject) over a period of at least one day or more (e.g., at least 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80 days or more).

[0040] The term "alternative nucleoside" or "alternative nucleotide" refers to a nucleoside having an alternative sugar or alternative nucleobase, such as those described herein. Alternative nucleosides can include nucleosides in which the nucleobase moiety has been modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or pyrimidine, such as an "alternative nucleobase" selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uridine, 5-bromouridine, 5-thiazolo-uridine, 2-thio-uridine, pseudouridine, 1-methylpseudouridine, 5-methoxyuridine, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine. Alternative nucleosides can also include nucleosides where the sugar moiety has been modified; for example, 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O-methyl cytosine, 2'-O-methyl uridine, 2-fluoro-deoxy adenosine, 2-fluoro-deoxy guanosine, 2-fluoro-deoxycytidine, 2-fluoro-deoxyuridine.

[0041] Exemplary nucleobases with substituted uracil include the following: pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, i.e., having the nucleic acid base deoxythymine), 1-methyl-pseudouridine (m 1 ψ), 5-methyl-2-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 S 4 ψ), 4-thio-1-methylpseudouridine, 3-methyl-pseudouridine (m 3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-uridine (ψm), 2-thio-2'-O-methyluridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um) and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine and 5-[3-(1-E-propenylamino)uridine.

[0042] Exemplary nucleobases having an alternative cytosine include the following: 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetylcytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysine (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.

[0043] Exemplary nucleobases with an alternative adenine include the following: 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl-adenine (m 2 A), N6-methyl-adenosine (m 6 A), 2-methylthio-N6-methyl-adenosine (ms 2 m 6 A), N6-isopentenyl-adenosine (i 6 A), 2-methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A), N6-(cis-hydroxyisopentenyl)adenosine (io 6 A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A), N6-glycinylcarbamoyl-adenosine (g 6 A), N6-threonylcarbamoyl-adenosine (t 6 A), N6-methyl-N6-threonylcarbamoyl-adenosine (m 6 t 6 A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms 2 g 6 A), N6,N6-dimethyladenosine (m 6 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-trimethyladenosine (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.

[0044] Exemplary nucleobases with substituted guanine include the following: inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxywyosine (o2yW), hydroxywyosine (OhyW), undermodified hydroxywyosine (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 2,7 G), N2,N2,7-dimethyl-guanosine (m 2,2,7G), 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.

[0045] Nucleobase moieties may also be represented by the letter code of each corresponding nucleobase, e.g., A, T, G, C, or U, where each letter can optionally include alternative nucleobases of equivalent function.

[0046] As used herein, the term "alternative complement pathway" refers to one of three pathways of complement activation, the others being the classical pathway and the lectin pathway.

[0047] As used herein, the term "antisense" refers to an oligonucleotide that is sufficiently complementary to all or a portion of a gene, primary transcript or processed mRNA (e.g., the sequence of CFB (e.g., SEQ ID NO: 12)) so as to disrupt expression of an endogenous gene (e.g., CFB).

[0048] The terms "antisense strand" and "guide strand" refer to the strand of an RNAi oligonucleotide that includes a region that is substantially complementary to a target sequence, such as a CFB mRNA (eg, SEQ ID NO: 12).

[0049] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically encompassed, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 10 nucleotides of a 21-nucleotide nucleic acid molecule" means that a range of 10 to 21 nucleotides, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides, has the indicated property. When "at least" is present before a series or range of numbers, it is understood that "at least" can modify each of the numbers in the series or range.

[0050] As used herein, the term "attenuate" means to reduce or substantially stop. As a non-limiting example, one or more of the treatments provided herein can inhibit or substantially stop the onset or progression of a disease mediated by complement pathway activation or dysregulation (e.g., CFB 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 activity, etc.), such as the diseases described herein.

[0051] The term "cDNA" refers to a nucleic acid sequence that is the DNA equivalent of an mRNA sequence (i.e., has uridines substituted for thymidines). Generally, those of skill in the art will understand that a cDNA sequence is the same as an mRNA sequence, except that uridine is read as thymidine, and therefore the terms cDNA and mRNA may be used interchangeably with respect to a particular gene (e.g., the CFB gene).

[0052] As used herein, "CFB" and "complement factor B" refer to the protein or gene encoding complement factor B, depending on the context in which the term is used. The term "CFB" also encompasses naturally occurring variants of wild-type CFB proteins, such as proteins having at least 85% sequence identity (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% or more sequence identity) to the amino acid sequence of wild-type human CFB set forth in NCBI Reference No. NP_001701.2 (SEQ ID NO: 11). The term "CFB" also refers to naturally occurring variants of the wild-type CFB gene, such as those 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 CFB set forth in NCBI Reference No. NM_001710.5 (SEQ ID NO: 12).

[0053] As used herein, the term "complement pathway activation or dysregulation" refers to abnormalities in the ability of the complement pathways, including the classical, alternative and lectin pathways, to confer host defense against pathogens, to eliminate immune complexes and damaged cells, and to regulate immunity. Activation or dysregulation of the alternative complement pathway can occur in the fluid phase and on cell surfaces and can lead to excessive complement activation or insufficient regulation, either of which can cause tissue damage.

[0054] As used herein, "complementary," when used to describe a first nucleotide or nucleoside sequence in relation to a second nucleotide or nucleoside sequence, refers to the ability of an oligonucleotide containing the first nucleotide or nucleoside sequence to hybridize and form a double helix structure with an oligonucleotide containing the second nucleotide sequence under specified conditions, as will be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, which may include: 400 mM NaCl, 40 mM PIPE pH 6.4, 1 mM EDTA, at 50° C. or 70° C. for 12-16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions may be applied, such as physiologically relevant conditions such as those that may be encountered inside an organism. Those skilled in the art will be able to determine the set of conditions most suitable for testing the complementarity of two sequences according to the ultimate application of the hybridized nucleotides or nucleosides. As used herein, "complementary" sequences may also include or be formed entirely from non-Watson-Crick base pairs and / or base pairs formed from non-natural and alternative nucleotides or nucleosides, so long as the above requirements for ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsten base pairs. As described herein, a complementary sequence within an oligonucleotide (e.g., an RNAi oligonucleotide) or between an oligonucleotide and a target sequence includes base pairing between an oligonucleotide containing a first nucleotide or nucleoside sequence and an oligonucleotide containing a second nucleotide or nucleoside sequence over the entire length of one or both nucleotide or nucleoside sequences. Such sequences may be referred to herein as "fully complementary" to one another.When a first sequence is referred to as "substantially complementary" with respect to a second sequence, the two sequences may be fully complementary or both may form one or more, but generally no more than 5, 4, 3 or 2 mismatched base pairs, while retaining the ability to hybridize under conditions most relevant to their ultimate application (e.g., reducing expression via the RISC pathway) upon hybridization for a duplex of up to 30 base pairs. "Substantially complementary" may also refer to an oligonucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding CFB). For example, an oligonucleotide is complementary to at least a portion of a CFB mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding CFB. However, if two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered as mismatches for purposes of determining complementarity. For example, oligonucleotides (e.g., RNAi oligonucleotides) comprising one oligonucleotide 22 linked nucleosides in length and another oligonucleotide 20 nucleosides in length can be referred to as "fully complementary" for the purposes described herein even though they have different lengths.

[0055] As used herein, a "complementary oligonucleotide" is one that is capable of base pairing according to standard Watson-Crick complementarity rules. Specifically, purines base pair with pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with thymine (A:T) in the case of DNA and adenine paired with uracil (A:U) in the case of RNA. It is understood that two oligonucleotides can hybridize to each other even if they are not completely complementary to each other, so long as each has at least one region that is substantially complementary to the other.

[0056] As used herein, the phrase "contacting a cell with an oligonucleotide" includes contacting a cell with an oligonucleotide, e.g., an RNAi oligonucleotide (e.g., a single-stranded oligonucleotide or a double-stranded oligonucleotide that forms a double helix), by methods known in the art. Contacting a cell with an oligonucleotide includes contacting a cell with an oligonucleotide in vitro or contacting a cell with an oligonucleotide in vivo. Contacting can be performed either directly or indirectly. Thus, for example, the oligonucleotide can be physically contacted with the cell by the individual performing the method, or the oligonucleotide agent can be placed in a situation that allows or will cause it to subsequently contact the cell. Contacting a cell in vitro can be performed, for example, by incubating the cell with the oligonucleotide. Contacting a cell in vivo can be performed, for example, by injecting the oligonucleotide into or near the tissue in which the cell is located, or by injecting the oligonucleotide agent into another area, e.g., the bloodstream or subcutaneous space, so that the agent subsequently reaches the tissue in which the cell to be contacted is located. For example, the oligonucleotide may contain and / or be linked to a ligand that directs the oligonucleotide to the desired site, or may be incorporated into a vector (e.g., a viral vector) that delivers the oligonucleotide to the desired site. A combination of in vitro and in vivo contact methods is also possible. For example, cells may be contacted with oligonucleotides in vitro and then transplanted into a subject.

[0057] The term "contiguous nucleobase region" refers to a region of an oligonucleotide that is complementary to a target nucleic acid (e.g., the antisense strand of an RNAi oligonucleotide). This term may be used interchangeably herein with the term "contiguous nucleotide sequence" or "contiguous nucleotide base sequence". In some embodiments, all of the nucleotides of an oligonucleotide are present in a contiguous nucleotide or nucleoside region. In some embodiments, an oligonucleotide comprises a contiguous nucleotide region and may optionally further comprise one or more nucleotides or one or more nucleosides. The nucleotide linker region may or may not be complementary to the target nucleic acid. The internucleoside linkages present between the nucleotides of the contiguous nucleotide region may include phosphorothioate internucleoside linkages. In addition, the contiguous nucleotide region may include one or more sugar-modified nucleosides.

[0058] As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen instead of a hydroxyl at the 2' position of its pentose sugar compared to a ribonucleotide. Modified deoxyribonucleotides are deoxyribonucleotides that have one or more modifications or substitutions of atoms other than the 2' position, including modifications or substitutions of the sugar, phosphate group, or base.

[0059] As used herein, the term "disease" refers to the interruption, cessation or disorder of a bodily function, system or organ. Diseases or disorders of interest include those that would benefit from treatment with the oligonucleotides described herein (e.g., single-stranded or double-stranded RNA constructs that form a duplex as described herein) that target CFB, such as by the therapeutic methods described herein. Non-limiting examples of diseases or disorders mediated by or associated with complement pathway activation or dysregulation (e.g., dysregulation or activation of CFB) that can be treated using the compositions and methods described herein include, for example, skin disorders, neurological disorders, nephrological disorders, acute care, rheumatic disorders, pulmonary disorders, dermatological disorders, hematological disorders and ophthalmological disorders, such as paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN), including primary MN, Escherichia coli (E.coli)-induced or typical hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration, geographic atrophy, diabetic retinopathy, uveitis, intermediate uveitis, Behçet's uveitis, retinitis pigmentosa, macular edema, multifocal choroiditis, Vogt-Koyanagi-Harada syndrome, scattershot choroidal retinopathy, sympathetic ophthalmia, ocular cicatricial pemphigoid, ocular pemphigoid, nonarthritic ischemic optic neuropathy, postoperative inflammation, retinal vein occlusion, neuropathy, multiple sclerosis , stroke, Guillain-Barre syndrome, traumatic brain injury, Parkinson's disease, hemodialysis complications, hyperacute graft rejection, xenograft rejection, interleukin-2 induced toxicity during IL-2 treatment, inflammatory diseases, inflammation in autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, myocarditis, post-ischemia-reperfusion state, myocardial infarction, balloon angioplasty, post-pump syndrome in cardiopulmonary or renal bypass, atherosclerosis, hemodialysis, renal ischemia, aortic reconstruction mesenteric artery reperfusion after pulmonary artery disease, infection or sepsis, immune complex disorders and autoimmune diseases, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, proliferative nephritis, liver fibrosis, hemolytic anemia, myasthenia gravis, tissue regeneration, nerve regeneration, dyspnea, hemoptysis, acute respiratory distress syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolism and infarction, pneumonia, fibrous dust disease, pulmonary fibrosis, allergies, bronchoconstriction, hypersensitivity pneumonitis, parasitic diseases, Goodpasture's syndrome, pulmonary vasculitis, microimmune vasculitis, immune complex-associated inflammation, antiphospholipid syndrome, glomerulonephritis, obesity, arthritis, autoimmune heart disease, inflammatory bowel disease, ischemia-reperfusion injury, Barraquer-Simons syndrome, hemodialysis, antineutrophil cytoplasmic antibody (ANCA) vasculitis, cryoglobulinemia, psoriasis, transplantation, central nervous system diseases such as Alzheimer's disease and other neurodegenerative conditions, dense deposition diseases, bullous skin diseases, membranoproliferative glomerulonephritis type II (MPGN II), chronic graft-versus-host disease, Felty's syndrome, pyoderma gangrenosum (PG), hidradenitis suppurativa (HS), pulmonary arterial hypertension, primary Sjogren's syndrome, primary biliary cholangitis, autosomal dominant polycystic kidney disease and myelin oligodendrocyte glycoprotein antibody disease (MOGAD).

[0060] As used herein, the term "double helix," with respect to nucleic acids (eg, oligonucleotides), refers to the structure formed by complementary base pairing of antiparallel sequences of two nucleotides.

[0061] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of an agent (e.g., an oligonucleotide described herein) that reduces the level and / or activity of CFB (e.g., in a cell or subject) refer to an amount sufficient to produce a beneficial or desired result, including a clinical result, when administered to a subject, such as a human, and thus "effective amount" or its synonyms vary depending on the context in which it is applied. For example, in the context of treating a disease associated with activation or dysregulation of the complement pathway, it is the amount of an agent that reduces the level and / or activity of CFB sufficient to achieve a therapeutic response compared to the response obtained without administration of the agent that reduces the level and / or activity of CFB. The amount of a given agent that reduces the level and / or activity of CFB described herein that corresponds to such an amount may vary depending on a variety of factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, subject identity (e.g., age, sex, and / or weight), or host being treated, but can still be routinely determined by one of skill in the art. As used herein, a "therapeutically effective amount" of an agent that reduces the level and / or activity of the disclosed CFB is also an amount that produces a beneficial or desired result in a subject compared to a control. As defined herein, a therapeutically effective amount of an agent that reduces the level and / or activity of the disclosed CFB can be readily determined by a person skilled in the art by routine methods known in the art. Dosage regimens can be adjusted to provide an optimal therapeutic response.

[0062] As used herein, the term "excipient" refers to a non-therapeutic agent that may be included in a composition to, for example, impart or contribute a desired consistency or a stabilizing effect.

[0063] Each of "G", "C", "A", "T" and "U" generally represents a nucleotide containing guanine, cytosine, adenine, thymidine and uracil as a base, respectively, but may contain alternative sugar moieties other than ribose and deoxyribose. It is also understood that the term "nucleotide" may refer to alternative nucleotides or surrogate replacement moieties as further detailed below. Those skilled in the art will appreciate that guanine, cytosine, adenine and uracil may be substituted by other moieties without substantially altering the base pairing properties of an oligonucleotide containing a nucleotide bearing such a replacement moiety. For example, but not by way of limitation, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine may be substituted by a nucleotide containing, for example, inosine in the nucleotide sequence of an oligonucleotide specifically featured in this disclosure. In another example, any adenine and cytosine in the oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair that pairs with the target mRNA. Sequences containing such substitutions are suitable for the compositions and methods specifically featured in this disclosure.

[0064] As used herein, the term "inhibitor" refers to any agent that reduces the level and / or activity of a protein (e.g., CFB). Non-limiting examples of inhibitors include oligonucleotides (e.g., dsRNA, siRNA or shRNA). As used herein, the term "reduce" is used interchangeably with "silencing," "downregulation," "suppression" and other similar terms, and includes any level reduction of 5% or more (e.g., 10%, 15%, 25%, 35%, 50%, 75% and 100%). A typical level of CFB protein found in the serum of a healthy human is about 200 μg / mL; thus, a reduced level of CFB protein can be, for example, an amount less than about 200 μg / mL (e.g., 5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL, 150 μg / mL and 190 μg / mL).

[0065] "Level" refers to the level or activity of a protein or an mRNA encoding a protein (e.g., CFB), optionally compared to a reference. The reference can be any useful standard, as defined herein. A "decreased level" or "increased level" of a protein refers to a decrease or increase, respectively, in the protein level compared to the reference (e.g., a decrease or increase of, e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more compared to the reference; e.g., a decrease or increase of, e.g., about 10%, about 15%, about 20%, about 30%, about 40%, about 500% or more compared to the reference). By "decreased or increased" is meant a decrease or increase of more than about 20%, about 50%, about 75%, about 100%, or about 200%; e.g., a decrease or increase of less than about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold or less compared to the reference; e.g., a decrease or increase of more than about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, or about 1000-fold or more compared to the reference. Protein or mRNA levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, ng / mL) or percentage of total protein or mRNA in the sample.

[0066] As used herein, the term "loop" refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) flanked by two antiparallel regions of nucleic acid that are sufficiently complementary to each other such that under appropriate hybridization conditions (e.g., in a phosphate buffer or inside a cell), the two antiparallel regions flanking the unpaired region hybridize to form a double helix (called the "stem").

[0067] As used herein, the term "modified internucleotide bond" refers to an internucleotide bond that has one or more chemical modifications compared to a reference internucleotide bond that includes a phosphodiester bond. In some embodiments, the modified nucleotide is a non-naturally occurring linkage. Typically, the modified internucleotide bond confers one or more desirable properties to the nucleic acid in which the modified internucleotide bond is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0068] As used herein, the term "modified nucleotide" refers to a nucleotide that has one or more chemical modifications compared to a corresponding reference nucleotide selected from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. In some embodiments, the modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, the modified nucleotide has one or more chemical modifications in its sugar, nucleobase, and / or phosphate group. In some embodiments, the modified nucleotide has one or more chemical moieties conjugated to the corresponding reference nucleotide. Typically, the modified nucleotide confers one or more desirable properties to the nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, reduced immunogenicity, etc.

[0069] A "nick tetraloop structure" is a structure of an RNAi oligonucleotide characterized by the presence of separate sense (passenger) and antisense (guide) strands, where the sense strand has a region of complementarity with the antisense strand, and at least one of the strands, typically the sense strand, has a tetraloop configured to stabilize the adjacent stem region formed in at least one strand. The nick tetraloop structure creates a single break in the nucleotides of the sense and antisense strands, thereby preventing the strands from being covalently linked at that site.

[0070] The terms "nucleobase" and "base" include purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds during nucleic acid hybridization. In the context of the present disclosure, the term nucleobase also encompasses alternative nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as alternative nucleobases. Such variants are described, for example, in Hirao et al. (Accounts of Chemical Research, vol. 45: page 2055, 2012) and Bergstrom (Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1, 2009).

[0071] The term "nucleoside" refers to a monomeric unit of a nucleobase and a sugar moiety or an oligonucleotide having a nucleobase and a sugar moiety. Nucleosides can include naturally occurring nucleosides as well as alternative nucleosides such as those described herein. The nucleobase of a nucleoside can be a naturally occurring nucleobase or an alternative nucleobase. Similarly, the sugar moiety of a nucleoside can be a naturally occurring sugar or an alternative sugar.

[0072] As used herein, a "nucleotide" refers to a monomeric unit of an oligonucleotide that includes a nucleoside and an internucleoside linkage. The internucleoside linkage may or may not include a phosphate linkage. Similarly, a "linked nucleoside" may or may not be linked by a phosphate linkage. Many "alternative internucleoside linkages" are known in the art, including, but not limited to, phosphate, phosphorothioate, and boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNAs) (e.g., locked nucleosides (LNAs) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNAs), phosphotriesters, phosphorothioates, phosphoramidates, and other variants of the phosphate backbone of natural nucleosides, including those described herein.

[0073] As used herein, the term "oligonucleotide" refers to a short nucleic acid, for example, a nucleic acid less than 100 nucleotides in length. The oligonucleotide can be single-stranded or RNAi. The oligonucleotide can have or not have a double helix region. As a non-limiting set of examples, the oligonucleotide can be, but is not limited to, a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a dicer substrate interfering RNA (dsiRNA), an antisense oligonucleotide, a short siRNA, or a single-stranded siRNA. In some embodiments, the oligonucleotide is an RNAi oligonucleotide.

[0074] As used herein, the term "overhang" refers to terminal non-base-paired nucleotides resulting from one strand or region that extends beyond the end of the complementary strand that forms a double helix with the strand or region. In some embodiments, the overhang comprises one or more unpaired nucleotides that extend from the double helix region at the 5'-end or 3'-end of the oligonucleotide (e.g., RNAi oligonucleotide). In certain embodiments, the overhang is a 3' or 5' overhang on the antisense strand or sense strand of the oligonucleotide (e.g., RNAi oligonucleotide).

[0075] As used herein, the term "patient in need thereof" or "subject in need thereof" refers to the identification of a subject based on the need for treatment of a disease or disorder, e.g., a disease mediated by alternative complement dysregulation (e.g., dysregulation associated with CFB, dysregulation of one or all of the complement pathways (e.g., alternative, classical and / or lectin pathways), etc.). A subject can be identified, for example, as having a need for treatment of a disease or disorder based on early diagnosis, e.g., by a skilled artisan (e.g., a physician).

[0076] "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 sequences and introducing gaps, if necessary, to achieve the maximum percent of sequence identity. Alignment for the purpose of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways that are within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2 or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with or against a given nucleic acid or amino acid sequence, B (or can be expressed as 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 (where X is the number of nucleotides or amino acids that are scored as identical matches by a sequence alignment program (such as BLAST) in its alignment of A and B, and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0077] As used herein, "pharmaceutical acceptable excipient" refers to any component other than the compounds described herein (e.g., a vehicle that can suspend or dissolve active compounds), and has the property of being substantially non-toxic and non-inflammatory in patients.Excipients include, for example, anti-adhesive agents, antioxidants, binders, coating agents, compression aids, disintegrants, dyes (coloring agents), emollients, emulsifiers, fillers (diluents), film-forming or coating agents, flavors, fragrances, flow agents (glidants), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0078] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt of any of the compounds described herein. For example, pharmaceutically acceptable salts of any of the compounds described herein include those that are within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid. The compounds described herein may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts, including inorganic or organic acids, or salts may be prepared from inorganic or organic bases in the case of the acidic form of the compounds described herein. Often, compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases and methods for preparing suitable salts are well known in the art. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases.Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogensulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, and the like. Representative salts include phosphate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, such as, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0079] The term "pharmaceutical composition" as used herein refers to a composition containing a compound (e.g., an oligonucleotide agent) described herein, which is formulated together with a pharmaceutical acceptable excipient and is optionally manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal. The pharmaceutical composition can be formulated, for example, for subcutaneous administration, for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); for intrathecal injection; for intraventricular injection; for intraparenchymal injection; for oral administration in unit dosage form (e.g., tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment; or any other pharmaceutical acceptable formulation.

[0080] As used herein, the term "phosphate analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate group. In some embodiments, the phosphate analog is located at the 5'-terminal nucleotide of an oligonucleotide in place of the 5'-phosphate, which is often susceptible to enzymatic removal. In some embodiments, the 5' phosphate analog contains a phosphatase-resistant bond. Examples of phosphate analogs include 5' phosphonates, such as 5' methylene phosphonates (5'-MP) and 5'-(E)-vinyl phosphonates (5'-VP). In some embodiments, the oligonucleotide has a phosphate analog at the 4'-carbon position of the sugar at the 5'-terminal nucleotide (referred to as a "4'-phosphate analog"). One example of a 4'-phosphate analog is an oxymethyl phosphonate or analog thereof, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., at its 4'-carbon). See, e.g., U.S. Patent Application Publication No. 2019 / 0177729 (the contents of each of which regarding phosphate analogs are incorporated herein by reference). Other modifications have been developed for the 5' end of oligonucleotides (see, e.g., WO 2011 / 133871; U.S. Pat. No. 8,927,513; and Prakash et al. (2015), Nucleic Acids Res., 43(6):2993-3011; the contents of each concerning phosphate analogs are incorporated herein by reference).

[0081] The term "probe" as used herein refers to any molecule that can selectively bind to a specific sequence, for example, a nucleic acid molecule such as mRNA. Probes can be synthesized using methods known and routine in the art, or derived from appropriate biological preparations. Probes can be specifically 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.

[0082] As used herein, the term "reduced expression" of a gene refers to a reduction in the amount of RNA transcripts or protein encoded by the gene and / or a reduction in the amount of activity of the gene in a cell or subject, as compared to a suitable reference cell or subject. For example, the act of treating a cell with an RNAi oligonucleotide (e.g., one having an antisense strand complementary to a CFB mRNA sequence) can result in a reduction in the amount of RNA transcripts, protein and / or activity (e.g., encoded by the CFB gene) as compared to a cell that has not been treated with the RNAi oligonucleotide. Similarly, "reducing expression" as used herein refers to an act that results in a decrease in expression of a gene (e.g., CFB). Reduction in expression can be assessed by a reduction in serum concentration of CFB, as described herein (e.g., for cells that have not been contacted with an oligonucleotide as described herein). Alternatively, decreased expression can be assessed by a reduction in the level of CFB mRNA transcription and / or translation (e.g., at least a 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 55%, or 60% or more decrease, e.g., a decrease ranging from 1% to 60% or more) compared to a cell not contacted with an oligonucleotide described herein. Reduced expression of CFB can be measured using a WIESLAB® Complement assay, an ELISA assay, a hemolytic assay, or any assay known in the art.

[0083] "Reference" refers to any useful standard used to compare protein or mRNA levels or activity. A reference can be any sample, standard, standard curve, or level used for comparison purposes. A reference can be a normal reference sample or reference standard or level. A "reference sample" can be, for example, a control, e.g., a predefined negative control value such as a "normal control" or a previous sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (e.g., cell or tissue) from a subject without a disease; a sample from a subject who has been diagnosed with a disease but has not yet been treated with a compound described herein; a sample from a subject who has been treated with a compound described herein; or a sample of a known normal concentration of a purified oligonucleotide or protein (e.g., any described herein). "Reference standard or level" refers to a value or numerical value obtained from a reference sample. A "normal control value" is a predetermined value indicative of a non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range ("between X and Y"), a high threshold value ("below X"), or a low threshold value ("above X"). A subject having a measured value within the normal control value for a particular biomarker is typically considered to be "within the normal range" for that biomarker. A normal reference standard or level can be a value or value obtained from a normal subject without a disease or disorder (e.g., a disease or disorder associated with activation or dysregulation of the complement pathway); a subject treated with a compound described herein. In some embodiments, the reference sample, standard or level is matched to the subject sample by at least one of the following criteria: age, weight, sex, disease stage, and general health. A standard curve of purified oligonucleotide or protein levels, such as any described herein, within the normal reference range can also be used as a reference.

[0084] As used herein, the term "region of complementarity" refers to a region on the antisense strand of an oligonucleotide that is substantially complementary to all or a portion of a gene, primary transcript, sequence (e.g., a target sequence, e.g., a CFB nucleotide sequence), or mRNA (e.g., CFB) that is processed to disrupt the expression of an endogenous gene. If the region of complementarity is not completely complementary to the target sequence, mismatches may occur in the internal or terminal regions of the molecule. In general, mismatches are most tolerated in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the oligonucleotide (e.g., an RNAi oligonucleotide).

[0085] As used herein, the term "ribonucleotide" refers to a nucleotide having ribose as its pentose sugar, which contains a hydroxyl group at its 2' position. A modified ribonucleotide is a ribonucleotide having one or more modifications or substitutions of an atom other than the 2' position, including modifications or substitutions of the ribose, phosphate group, or base.

[0086] As used herein, the term "RNAi oligonucleotide" refers to either (a) a double-stranded oligonucleotide having a sense strand (passenger) and an antisense strand (guide), where the antisense strand or a portion of the antisense strand is used by Argonaute 2 (Ago2) endonuclease in cleaving a target mRNA, or (b) a single-stranded oligonucleotide having a single antisense strand, where the antisense strand (or a portion of the antisense strand) is used by Ago2 endonuclease in cleaving a target mRNA. In some embodiments, the RNAi oligonucleotide comprises a loop region, such as a stem-loop, which comprises nucleosides as that term is defined herein. RNAi oligonucleotides include, for example, dsRNA, siRNA, and shRNA, which mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. RNAi oligonucleotides direct sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). RNAi oligonucleotides reduce expression of C3 in cells, for example, cells in a subject, such as a mammalian subject. Generally, the majority of nucleosides in an RNAi oligonucleotide are ribonucleosides, but as described in detail herein, each or both strands may also include one or more non-ribonucleosides, such as deoxyribonucleosides and / or alternative nucleosides. The RNAi oligonucleotide is substantially in a double helical form. In some embodiments, the complementary base pairing of the double helical region of the RNAi oligonucleotide is formed between antiparallel sequences of nucleotides of covalently separate nucleic acid strands. In some embodiments, the complementary base pairing of the double helical region of the RNAi oligonucleotide is formed between antiparallel sequences of nucleotides of covalently linked nucleic acid strands. In some embodiments, the complementary base pairing of the double helical region of the RNAi oligonucleotide is formed from a single nucleic acid strand that is folded back (e.g., via a hairpin) to provide a complementary antiparallel sequence of nucleotides that base pair together. In some embodiments, the RNAi oligonucleotide comprises two covalently separate nucleic acid strands that are completely double helical with each other.However, in some embodiments, the RNAi oligonucleotide is partially double-stranded, e.g., comprises two covalently separate nucleic acid strands with overhangs at one or both ends. In some embodiments, the RNAi oligonucleotide comprises an antiparallel sequence of partially complementary nucleotides, and thus may have one or more mismatches, which may include internal or terminal mismatches.

[0087] As used herein, the terms "sense strand" and "passenger strand" refer to a strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand. The region of the sense strand that is complementary to a region of the antisense strand is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%) identical to a portion of a target gene (e.g., CFB 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, e.g., 10-31 nucleotides, 10-26 nucleotides, 10-20 nucleotides or 10-15 nucleotides.

[0088] The terms "siRNA" and "short interfering RNA," also known as "small interfering RNA," refer to an RNA agent, optionally an RNAi agent, of about 10-50 nucleotides in length, whose strands have overhangs containing, for example, one, two, or three overhanging linked nucleosides, which can direct or mediate RNA interference. Naturally occurring siRNAs are produced from longer dsRNA molecules (e.g., >25 linked nucleosides in length) by the cellular RNAi machinery (e.g., Dicer or its homologs).

[0089] As used herein, the term "strand" refers to a single continuous sequence of nucleotides linked together via internucleotide bonds (e.g., phosphodiester bonds, phosphorothioate bonds). In some embodiments, the strand has two free ends, e.g., a 5' end and a 3' end.

[0090] As used herein, the term "subject" refers to any organism to which a composition according to the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans). A subject may be a human or animal seeking or in need of treatment, in need of treatment, undergoing treatment, scheduled to undergo future treatment, or under the care of a professional trained in a particular disease or condition.

[0091] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring. Sugar also includes "sugar substitutes," defined as structures that can replace the furanose ring of a nucleoside. In certain embodiments, sugar substitutes are non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures can include simple variations on the natural furanose ring, such as six-membered rings, or can be more complex, as in the case of the acyclic systems used in peptide nucleic acids. Sugar substitutes can also include sugar substitutes in which the furanose ring is replaced with another ring system, such as a morpholino or hexitol ring system. Sugar moieties useful for preparing oligonucleotides having a motif include, but are not limited to, β-D-ribose, β-D-2'-deoxyribose, substituted sugars (e.g., 2', 5' and bis-substituted sugars), 4'-S-sugars (e.g., 4'-S-ribose, 4'-S-2'-deoxyribose and 4'-S-2' substituted ribose), bicyclic sugar surrogates (e.g., bicyclic sugars derived from 2'-O-CH2-4' or 2'-O-(CH2)2-4' bridged ribose) and sugar surrogates (e.g., where the ribose ring is replaced with a morpholino or hexitol ring system). The type of heterocyclic base and internucleoside linkage used at each position varies and is not a determining factor of the motif. In most nucleosides having surrogate sugar moieties, the heterocyclic nucleobase is generally maintained to allow hybridization.

[0092] As used herein, the term "stem loop" refers to a region of an oligonucleotide in which two regions have complementary nucleotide sequences when one is read in the 5' to 3' direction and the other is read in the 3' to 5' direction, and the nucleotides between the two regions form an unpaired loop. A stem loop region may also be referred to as a hairpin or hairpin loop.

[0093] As used herein, the term "strand" refers to an oligonucleotide comprising a chain of linked nucleosides. A "strand comprising a nucleobase sequence" refers to an oligonucleotide comprising a chain of linked nucleosides represented by a sequence designated using standard nucleobase nomenclature.

[0094] As used herein, the term "synthetic" refers to a nucleic acid or other molecule that is artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or is not otherwise derived from a natural source (e.g., a cell or organism) that normally produces the molecule.

[0095] As used herein, the term "target" or "targeting" refers to an oligonucleotide capable of specifically binding to a CFB gene or CFB mRNA encoding a CFB gene product, e.g., an oligonucleotide capable of inhibiting said gene or said mRNA (e.g., by reducing the level of the protein encoded by said gene or mRNA) by methods known to those skilled in the art (e.g., in the antisense and RNA interference fields).

[0096] As used herein, the term "targeting ligand" refers to a molecule (e.g., carbohydrate, amino sugar, cholesterol, polypeptide, or lipid) that selectively binds to a cognate molecule (e.g., a receptor) of a tissue or cell of interest and can be conjugated to another substance for the purpose of targeting the other substance to the tissue or cell of interest. For example, in some embodiments, the targeting ligand can be conjugated to an oligonucleotide or a vector (e.g., a viral vector) that contains the oligonucleotide for the purpose of targeting the oligonucleotide to a 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 the 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 a complex that includes the oligonucleotide, the targeting ligand, and the receptor. In some embodiments, the targeting ligand is conjugated to the oligonucleotide via a linker that is cleaved after or during cellular internalization, such that the oligonucleotide is released from the targeting ligand inside the cell.

[0097] As used herein, the term "tetraloop" refers to a loop that enhances the stability of adjacent duplexes formed by hybridization of flanking sequences of nucleotides. The increased stability is detectable as an increase in the melting temperature (Tm) of adjacent stem duplexes that is higher than the Tm of adjacent stem duplexes that would be expected on average from a set of loops of comparable length made 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 that includes a duplex of at least 2 base pairs in length. In some embodiments, the tetraloop can stabilize the base pairs in adjacent stem duplexes by stacking interactions. Additionally, interactions between nucleotides in a tetraloop include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug.16;346(6285):680-2; Heus and Pardi, Science 1991 Jul.12;253(5016):191-4). In some embodiments, the tetraloop comprises or consists of 3-6 nucleotides, typically 4-5 nucleotides. In certain embodiments, the tetraloop comprises or consists of 3, 4, 5, or 6 nucleotides, which may or may not be modified (e.g., conjugated to a targeting moiety). In one embodiment, the tetraloop consists of 4 nucleotides. Any nucleotide may be used in the tetraloop and the standard IUPAC-IUB symbols for such nucleotides may be used as described in Cornish-Bowden (1985) Nucl. Acids Res. 13:3021-3030.For example, the letter "N" can be used to mean that any base can be at that position, the letter "R" can be used to indicate that A (adenine) or G (guanine) can be at that position, and "B" can be used to indicate that C (cytosine), G (guanine) or T (thymine) can 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), d(GNRA) family of tetraloops), the d(GNAB) family of tetraloops, the d(CNNG) family of tetraloops, and the d(TNCG) family of tetraloops (e.g., d(TTCG)). See, for example, Nakano et al. Biochemistry, 41(48), 14281-14292, 2002. SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL.78th; NO.2; pg.731(2000) (incorporated herein by reference for their relevant disclosures). In some embodiments, the tetraloop is comprised within a nick tetraloop structure.

[0098] A "therapeutically effective amount" or a "prophylactically effective amount" refers to an amount (administered in either single or multiple doses) of a disclosed oligonucleotide composition (e.g., an RNAi oligonucleotide, such as a dsRNA) that produces the desired local or systemic effect (e.g., treatment of one or more symptoms of a disease resulting from complement pathway activation or dysregulation). The oligonucleotides (e.g., RNAi oligonucleotides) used in the methods of the present disclosure may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0099] As used herein, the term "treating" refers to the act of providing medical care to a subject in need thereof, for example, by administering a therapeutic agent (e.g., an oligonucleotide described herein) to the subject for the purpose of improving the health and / or well-being of the subject with respect to an existing condition (e.g., disease, disorder) or for the purpose of preventing or reducing the likelihood of the occurrence of a condition. In some embodiments, treating includes reducing the frequency or severity of at least one sign, symptom, or contributing factor of a condition (e.g., disease, disorder) experienced by the subject. In some embodiments, the nucleic acid or RNAi oligonucleotide agents (e.g., dsRNA) described herein are used to control the cellular and clinical symptoms of a complement pathway disorder (e.g., a disorder caused by activation or dysregulation of CFB).

[0100] Detailed Description Described herein are oligonucleotides (e.g., RNAi oligonucleotides), including sense and antisense strand oligonucleotides, targeting complement factor B (CFB), known to play a specific role in alternative complement pathway activation, and pharma- ceutically acceptable salts thereof. The oligonucleotides can be administered to reduce the level and / or activity of CFB in a cell (e.g., a hepatocyte) or a subject (e.g., a human). For example, the oligonucleotides can be administered in vivo and internalized by a cell (e.g., a hepatocyte; e.g., by binding to a sialoglycoprotein receptor (ASGPR). After internalization of the cell, the oligonucleotides can be bound to an RNA-induced silencing complex (RISC) to target CFB mRNA, thereby initiating degradation of CFB mRNA and blocking its translation.

[0101] Diseases mediated by alternative complement dysregulation are often the result of complement hyperactivity. Described herein are methods of treating diseases mediated by or associated with complement pathway activation or dysregulation by administration of oligonucleotides described herein that reduce the expression and / or activity levels of CFB. Examples of disorders mediated by or associated with complement pathway activation that can be treated with the oligonucleotides and compositions described herein include, for example, skin disorders, neurological disorders, nephrological disorders, acute care, rheumatic disorders, pulmonary disorders, dermatological disorders, hematological disorders, and ophthalmological disorders, such as paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN), including primary MN, Escherichia coli (E.coli)-induced or typical hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration, geographic atrophy, diabetic retinopathy, uveitis, intermediate uveitis, Behçet's uveitis, retinitis pigmentosa, macular edema, multifocal choroiditis, Vogt-Koyanagi-Harada syndrome, scattershot choroidal retinopathy, sympathetic ophthalmia, ocular cicatricial pemphigoid, ocular pemphigoid, nonarthritic ischemic optic neuropathy, postoperative inflammation, retinal vein occlusion, neuropathy, multiple sclerosis , stroke, Guillain-Barré syndrome, traumatic brain injury, Parkinson's disease, hemodialysis complications, hyperacute graft rejection, xenograft rejection, interleukin-2 induced toxicity during IL-2 treatment, inflammatory diseases, inflammation in autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, myocarditis, post-ischemia-reperfusion states, myocardial infarction, balloon angioplasty, post-pump syndrome in cardiopulmonary or renal bypass, atherosclerosis, hemodialysis, renal ischemia, post-aortic reconstruction mesenteric artery reperfusion, infection or sepsis, immune complex disorders and autoimmune diseases, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, proliferative nephritis, liver fibrosis, hemolytic anemia, myasthenia gravis, tissue regeneration, nerve regeneration, dyspnea, hemoptysis, acute respiratory distress syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolism and pulmonary infarction, pneumonia, fibrous dust disease, pulmonary fibrosis, allergies, bronchoconstriction, hypersensitivity pneumonitis, parasitic diseases, glaucoma, : Adpasture syndrome, pulmonary vasculitis, microimmune vasculitis, immune complex-associated inflammation, antiphospholipid syndrome, glomerulonephritis, obesity, arthritis, autoimmune heart disease, inflammatory bowel disease, ischemia-reperfusion injury, Barraquer-Simons syndrome, hemodialysis, antineutrophil cytoplasmic antibody (ANCA) vasculitis, cryoglobulinemia, psoriasis, transplantation, central nervous system diseases such as Alzheimer's disease and other neurodegenerative conditions, dense deposition diseases, bullous skin diseases, membranoproliferative glomerulonephritis type II (MPGN II), chronic graft-versus-host disease, Felty's syndrome, pyoderma gangrenosum (PG), hidradenitis suppurativa (HS), pulmonary arterial hypertension, primary Sjogren's syndrome, primary biliary cholangitis, autosomal dominant polycystic kidney disease and myelin oligodendrocyte glycoprotein antibody disease (MOGAD).

[0102] The compositions and methods described herein feature oligonucleotides (e.g., RNAi oligonucleotides) and pharma- ceutically acceptable salts thereof (e.g., sodium salts thereof), which include sense and antisense strands that have substantial sequence identity to a region of a CFB gene (e.g., the human CFB gene).

[0103] Oligonucleotides (e.g., RNAi oligonucleotides) can be used to modulate complement pathway activity by reducing the level and / or activity of CFB in cells (e.g., hepatocytes), such as cells of a subject (e.g., a human) in need thereof. The oligonucleotide agents target the CFB of the complement pathway, leaving other pathways intact for activation (protection), including the alternative, classical, and lectin pathways. Thus, the present disclosure features compositions and methods for treating diseases or disorders mediated by complement pathway activation or dysregulation (e.g., diseases or disorders mediated by activation or dysregulation of CFB).

[0104] Complement factor B target sequence Provided herein are oligonucleotide-based inhibitors of CFB expression that can be used to achieve therapeutic benefit. From examination of CFB mRNA (see, e.g., Example 1) and in vitro and in vivo testing, it has been found that the sequence of CFB mRNA is amenable to oligonucleotide-based inhibition and therefore useful as a targeting sequence. For example, the CFB target sequence may comprise or consist of the sequence set forth in either SEQ ID NO: 13 or SEQ ID NO: 14, which correspond to nucleotides 1827-1845 and 489-507, respectively, of Homo sapiens complement factor B, which has reference sequence NM_001710.6 (SEQ ID NO: 12). These CFB sequences may be the target sequences of Compound A and Compound B, respectively, and their variants described herein having up to 85% sequence identity thereto. Compounds A and B (and their variants described herein) may also effectively target Rhesus macaque CFB, which has reference sequence XM_015122636.2. Additionally, the CFB target sequence may comprise or consist of the sequence set forth in SEQ ID NO:31, which corresponds to nucleotides 770-789 of mus musculus complement factor B having reference sequence NM_008198.2 (SEQ ID NO:32), which is 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). Compound J may also target Rattus norvegicus complement CFB, having reference sequence NM_212466.3. These regions of the CFB mRNA can be targeted with oligonucleotides, such as dsRNA agents described herein, for the purpose of inhibiting CFB mRNA expression and subsequent CFB protein expression.

[0105] In some embodiments, the antisense strand of an oligonucleotide (e.g., RNAi oligonucleotide) agent described herein can be designed to have a region of complementarity to CFB mRNA (e.g., within the target sequence of CFB mRNA) for the purpose of targeting the mRNA in a cell and inhibiting its expression. The region of complementarity is generally of a length and base content appropriate to facilitate annealing of the oligonucleotide (e.g., RNAi oligonucleotide) or strand thereof to CFB mRNA for the purpose of inhibiting its transcription. The region of complementarity can be at least 11, e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 nucleotides in length. For example, the oligonucleotides provided herein can have a region of complementarity to CFB mRNA ranging from 12-30 (e.g., 12-30, 12-22, 15-25, 17-21, 18-27, 19-27, or 15-30) nucleotides in length. Thus, the oligonucleotides provided herein can have a region of complementarity to CFB that is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some examples, the oligonucleotides provided herein can have a region of complementarity to CFB mRNA that is 19 nucleotides in length.

[0106] In certain examples, an oligonucleotide agent of the present disclosure may comprise a region of complementarity (e.g., on the antisense strand of an RNAi oligonucleotide) that is at least partially complementary to a sequence set forth in SEQ ID NO: 12. For example, an oligonucleotide disclosed herein may comprise a region of complementarity (e.g., on the antisense strand of an RNAi oligonucleotide) that is fully complementary to a sequence set forth in SEQ ID NO: 12. The region of complementarity of an oligonucleotide (e.g., on the antisense strand of an RNAi oligonucleotide) may be complementary to a contiguous sequence of nucleotides of the sequence set forth in SEQ ID NO: 12, said sequence ranging from 12 to 20 nucleotides (e.g., 12 to 20, 12 to 18, 12 to 16, 12 to 14, 14 to 20, 14 to 18, 14 to 16, 16 to 20, 16 to 18, or 18 to 20) in length. In some embodiments, the region of complementarity of an oligonucleotide (e.g., on the antisense strand of an RNAi oligonucleotide) may be complementary to a 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 an oligonucleotide (eg, the antisense strand of an RNAi oligonucleotide) may be complementary to a sequence of contiguous nucleotides of the sequence set forth in SEQ ID NO:12 that is 20 nucleotides in length.

[0107] The region of complementarity of the oligonucleotide that is complementary to the consecutive nucleotides of the sequence described in SEQ ID NO: 12 can span a part of the entire length of the antisense strand. For example, the region of complementarity of the oligonucleotide that is complementary to the consecutive nucleotides of the sequence described in SEQ ID NO: 12 can span at least 85% (e.g., at least 86%, at least 90%, at least 95% and at least 99%) of the entire length of the antisense strand. In certain embodiments, the region of complementarity of the oligonucleotide that is complementary to the consecutive nucleotides of the sequence described in SEQ ID NO: 12 can span the entire length of the antisense strand.

[0108] A region of complementarity to CFB mRNA may have one or more mismatches compared to the corresponding sequence of CFB mRNA. For example, a region of complementarity on an oligonucleotide (e.g., an oligonucleotide 20-50 nucleotides in length, such as an oligonucleotide 20-25 nucleotides in length (e.g., 22 nucleotides in length) may have up to 1, up to 2, up to 3, up to 4, or up to 5 mismatches, so long as the oligonucleotide maintains the ability to form complementary base pairs with CFB mRNA under appropriate hybridization conditions. Alternatively, a region of complementarity on an oligonucleotide may have no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches, so long as the oligonucleotide maintains the ability to form complementary base pairs with CFB mRNA under appropriate hybridization conditions. If there are multiple mismatches in the region of complementarity, it is preferred that the oligonucleotide has at least one mismatch in the region of complementarity to the CFB mRNA under appropriate hybridization conditions. The mismatches can be consecutive (e.g., 2, 3, 4, or 5 consecutive) or scattered throughout the region of complementarity, so long as they maintain the ability to form complementary base pairs with the mRNA. For example, an oligonucleotide agent can include a sense oligonucleotide having the sequence of SEQ ID NO:4 and variants thereof having up to 1, 2, 3, 4, or 5 mismatches to the corresponding CFB sequence of SEQ ID NO:12, or the corresponding antisense sequence of SEQ ID NO:6 and variants thereof having 1, 2, 3, 4, or 5 mismatches to the sequence of SEQ ID NO:4.

[0109] Types of oligonucleotides In the methods of the present disclosure, there are a variety of oligonucleotide structures useful for targeting CFB, including RNAi, antisense miRNA, shRNA, etc. Any of the structures described herein or elsewhere can be used as a framework to incorporate or target the sequences described herein (e.g., hotspot sequences of CFB, such as those in SEQ ID NOs: 13 and 14).

[0110] The compositions described herein that are oligonucleotides (e.g., RNAi oligonucleotides) encode inhibitory constructs (e.g., nucleic acid vectors encoding same) that target CFB mRNA (e.g., SEQ ID NO: 12). Oligonucleotides for reducing expression of CFB expression can participate in the RNA interference (RNAi) pathway upstream or downstream of Dicer involvement. For example, oligonucleotides (e.g., RNAi oligonucleotides) that are 19-25 nucleotides in length and include at least one sense or antisense strand with a 1-5 nucleotide 3' overhang have been developed (see, e.g., U.S. Pat. No. 8,372,968, incorporated herein by reference). Longer oligonucleotides have also been developed that are processed by Dicer to produce active RNAi products (see, e.g., U.S. Pat. No. 8,883,996, incorporated herein by reference). In addition, extended oligonucleotides (e.g., RNAi oligonucleotides) have also been generated in which either or both of the 5' or 3' ends of either or both of the antisense and sense strands are extended beyond the double helix targeting region such that either the sense or antisense strand contains a thermodynamically stabilized tetraloop structure (see, for example, U.S. Pat. Nos. 8,513,207 and 8,927,705 and WO2010033225, which are incorporated herein by reference for their disclosure of these oligonucleotides). Such structures can include single-stranded extensions at either or both of the 5' and 3' ends of the molecule as well as RNAi extensions.

[0111] Additionally or alternatively, the oligonucleotides provided herein can be designed to participate in the RNA interference pathway downstream of Dicer participation, i.e., after cleavage by Dicer. Such oligonucleotides can have an overhang of 1, 2 or 3 nucleotides at the 3' end of the sense strand. Such oligonucleotides, for example siRNAs, can include a 22 nucleotide guide strand (e.g., SEQ ID NOs: 13 and 14) that is antisense to the target RNA and a complementary passenger strand, where both strands anneal to form a 20bp double helix and a 2 nucleotide overhang at one or both 3' ends. Longer oligonucleotide designs are also available, including oligonucleotides with a 23 nucleotide guide strand and a 21 nucleotide passenger strand, with blunt ends at the 3' end of the passenger strand and the 5' end of the guide strand, and with a 2 nucleotide 3' guide strand overhang to the left of the molecule at the 5' end of the passenger strand and the 3' end of the guide strand. In such molecules, there is a 21 base pair double helical region (see U.S. Pat. Nos. 9,012,138, 9,012,621 and 9,193,753, which are incorporated by reference herein for their disclosures regarding longer oligonucleotides).

[0112] The oligonucleotides disclosed herein can include sense and antisense strands that are both 17-26 (e.g., 17-26, 20-25, or 21-23) nucleotides in length. For example, the oligonucleotides disclosed herein can include sense and antisense strands that are both 19-22 nucleotides in length. The sense and antisense strands can be the same length. Alternatively, the oligonucleotides can include sense and antisense strands such that there is a 3'-overhang on either the sense or antisense strand or on both the sense and antisense strands. For example, the 3' overhangs on the sense strand, antisense strand, or both the sense and antisense strands can be 1 or 2 nucleotides in length. In some embodiments, the oligonucleotides have a 22 nucleotide antisense strand and a 20 nucleotide sense strand, with a blunt end on the "right side" of the molecule (i.e., the 3' end of the passenger strand and the 5' end of the guide strand) and a 2 nucleotide 3'-guide strand overhang on the "left side" of the molecule (i.e., the 5' end of the passenger strand and the 3' end of the guide strand). In such a molecule, there may be, for example, a 20 base pair double helical region.

[0113] Other oligonucleotide designs for use with the compositions and methods disclosed herein include, for example, 16-mer siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., with stems of 19 bp or less; see, e.g., Moore et al. Methods Mol. Biol. 2010; 629:141-158), blunt siRNAs (e.g., 19 bp in length; see, e.g., Kraynack and Baker, RNA Vol. 12, p163-176 (2006)), asymmetric siRNAs (aiRNAs; see, e.g., Sun et al., Nat. Biotechnol. 26, 1379-1382 (2008)), asymmetric short duplex siRNAs (see, e.g., Chang et al., Mol Ther. 2009), and the like. Apr;17(4):725-32), forked siRNA (e.g., Hohjoh, FEBS Letters, Vol 557, issue 1-3; Jan 2004, p 193-198), single-stranded siRNA (Elsner; Nature Biotechnology 30,1063(2012)), dumbbell-shaped circular siRNA (e.g., Abe et al. J Am Chem Soc 129:15108-15109(2007)), as well as internally segmented small interfering RNA (siRNA; e.g., Bramsen et al., Nucleic Acids Res. 2007 Sep;35(17):5886-5897). Each of the foregoing references is incorporated by reference in its entirety for the relevant disclosures therein.Further non-limiting examples of oligonucleotide structures that may be used in some embodiments to reduce or inhibit expression of CFB are microRNAs (miRNAs), short hairpin RNAs (shRNAs), and small siRNAs (see Hamilton et al., Embo J., 2002, 21(17):4671-4679; see also U.S. Patent Application Publication No. 2009 / 0099115).

[0114] Oligonucleotides Oligonucleotides (e.g., RNAi oligonucleotides) for targeting CFB expression via the RNAi pathway generally have sense and antisense strands that form a double helix with each other. Oligonucleotides (e.g., RNAi oligonucleotides) can be single-stranded or double-stranded ribonucleic acid (dsRNA). In addition, the sense and antisense strands do not have to be covalently linked; for example, the oligonucleotide can have a nick between the sense and antisense strands. Oligonucleotides (e.g., RNAi oligonucleotides) can be in the form of a pharma- ceutically acceptable salt. For example, oligonucleotides (e.g., RNAi oligonucleotides) can be in the form of a sodium salt.

[0115] Although the aforementioned oligonucleotide (e.g., RNAi oligonucleotide) sequences are represented as RNA sequences that can be synthesized in a cell; these sequences can also be represented as corresponding DNA (e.g., cDNA) that can be incorporated into the vectors of the present disclosure. Those skilled in the art will understand that cDNA sequences are equivalent to mRNA sequences, except for the replacement of uridine with thymidine, and can be used herein for the same purpose, i.e., to generate antisense oligonucleotides to inhibit the expression of CFB mRNA. In the case of DNA, the polynucleotide comprising the antisense nucleic acid is a DNA sequence. The DNA sequence can correspond to the antisense strand of Compound A or Compound B, and can have the polynucleotide sequence of SEQ ID NO: 34 or SEQ ID NO: 36, respectively, or have at least 85% or more sequence identity thereto. The DNA sequence can correspond to the sense strand of Compound A or Compound B, and can have the polynucleotide sequence of SEQ ID NO: 33 or SEQ ID NO: 35, respectively, or have at least 85% or more sequence identity thereto. In the case of RNA vectors, the transgene cassette incorporates the RNA equivalent of the antisense DNA sequence described herein.

[0116] In certain embodiments, the sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97% and at least 99%) sequence identity to SEQ ID NO:4 or SEQ ID NO:5. For example, the sense strand may comprise an oligonucleotide sequence of SEQ ID NO:4, as in Compound B. In other embodiments, the sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97% and at least 99%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2. For example, the sense strand may comprise an oligonucleotide sequence of SEQ ID NO:1, as in Compound A.

[0117] In some embodiments, the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97% and at least 99%) sequence identity to SEQ ID NO: 6. In other embodiments, the antisense strand may comprise at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97% and at least 99%) sequence identity to SEQ ID NO: 3. For example, the antisense strand may comprise an oligonucleotide sequence of SEQ ID NO: 6, as in the case of Compound B, and / or the antisense strand may comprise an oligonucleotide sequence of SEQ ID NO: 3, as in the case of Compound A.

[0118] Further, the sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97% and at least 99%) sequence identity to SEQ ID NO:4 or SEQ ID NO:5, and the antisense strand may comprise at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97% and at least 99%) sequence identity to SEQ ID NO:6. The oligonucleotide (e.g., RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO:4 or SEQ ID NO:5, and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO:6, as shown in FIG. 2C for Compound B.

[0119] In addition, the sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97% and at least 99%) sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and the antisense strand may comprise at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97% and at least 99%) sequence identity to SEQ ID NO:3. Furthermore, the oligonucleotide (e.g., RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2, and an antisense strand comprising an oligonucleotide sequence of SEQ ID NO:3, as shown in FIG. 1C for Compound A.

[0120] Additionally, the sense strand may comprise the oligonucleotide sequence of SEQ ID NO:37 and the antisense strand may comprise the oligonucleotide sequence of SEQ ID NO:38 as shown below. Sense strand (SEQ ID NO:37): 5'mA-S-mC-mA-mA-mU-mG-mU-fG-fA-fG-fU-mG-mA-mU-mG-mA-mG-mA-mU-mA-mG-mC-mA- mG-mC-mC-mG-[ademA-GalNAc]-[ademA-GalNAc]-[ademA-GalNAc]-mG-mG-mC-mU-mG-mC 3' Hybridized with: Antisense strand (SEQ ID NO:38): 5'[MePhosphonate-4O-mU]-S-fA-S-fU-fC-fU-mC-fA-mU-mC-fA-mC-mU-mC-fA-mC-mA-mU-mU-mG-mU-S-mG-S-mG 3' (mX is a 2'-O-methyl ribonucleotide, fX is a 2'-fluoro-deoxyribonucleotide, [ademA-GalNAc] is a 2'-O-GalNAc modified adenosine, [Mephosphonate-4O-mU] is a 4'-O-monomethylphosphonate-2'-O-methyluridine, "-" indicates a phosphodiester bond, and "-S-" indicates a phosphorothioate bond, as shown in Figures 2D, 2E-1 and 2E-2. In some embodiments, the antisense strand can be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 38. In some embodiments, the sense strand can be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 37.

[0121] The sense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97% and at least 99%) sequence identity to SEQ ID NO: 15, and the antisense strand may comprise an oligonucleotide sequence having at least 85% (e.g., at least 87%, at least 90%, at least 95%, at least 97% and at least 99%) sequence identity to SEQ ID NO: 16. For example, an oligonucleotide (e.g., an RNAi oligonucleotide) may comprise a sense strand comprising an oligonucleotide sequence of SEQ ID NO: 15 and a sense strand comprising an oligonucleotide sequence of SEQ ID NO: 16, as shown for compound J. See Table 1 for examples of sense and antisense strands.

[0122] [Table 1]

[0123] The oligonucleotide (e.g., RNAi oligonucleotide) comprises a double helix region between the sense strand and the antisense strand. The double helix formed between the sense strand and the antisense strand can be 10-30 nucleotides long (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides long). Thus, the double helix formed between the sense strand and the antisense strand can be 15-25 nucleotides long (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 nucleotides long). In some embodiments, the double helix region can be 20 nucleotides long.

[0124] The region on the sense strand that forms a duplex with the antisense strand may have a nucleotide sequence that is at least 85% identical (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the oligonucleotide sequence of any of SEQ ID NOs: 2 and 5. For example, the region on the sense strand that forms a duplex with the antisense strand may have the oligonucleotide sequence of any of SEQ ID NOs: 2 and 5.

[0125] Furthermore, the double helix formed between the sense and antisense strands does not have to span the entire length of the sense and / or antisense strand.

[0126] The oligonucleotide (e.g., RNAi oligonucleotide) can include a sense strand longer than 22 nucleotides (e.g., 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long), such as a 36 nucleotide sense strand, and an antisense strand 18-36 nucleotides long, such as a 22 nucleotide antisense strand. The oligonucleotide (e.g., RNAi oligonucleotide) has a length such that, when acted upon by the Dicer enzyme, it results in the antisense strand being incorporated into mature RISC.

[0127] The oligonucleotides provided herein can have one 5' end that is less thermodynamically stable than the other 5' end. The oligonucleotides provided herein can be asymmetric oligonucleotides that include a blunt end at the 3' end of the sense strand and an overhang at the 3' end of the antisense strand. The 3' overhang on the antisense strand can be 1-8 nucleotides long (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides long). For example, the 3' overhang on the antisense strand can be 2 nucleotides long. Typically, oligonucleotides for RNAi have a 2 nucleotide overhang at the 3' end of the antisense, guide, strand. However, other overhangs are possible. In other embodiments, the 3' overhang may have a length of 1-6 nucleotides, optionally 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 3-5, 5-6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides. In some examples, the oligonucleotide may have an overhang at the 5' end. The overhang may be a 5' overhang comprising a length of 1-6 nucleotides, optionally 1-5, 1-4, 1-3, 1-2, 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-6, 4-5, 3-4, 4-6, 4-5, 5-6 nucleotides, or 1, 2, 3, 4, 5, or 6 nucleotides.

[0128] 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 CFB mRNA. Alternatively, the two terminal nucleotides at the 3' end of the antisense strand may not be complementary to the target CFB 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.

[0129] There may be one or more (e.g., 1, 2, 3, 4, 5) mismatches in complementarity between the sense strand and the antisense strand. When there are two or more mismatches between the sense strand and the antisense strand, they may be located consecutively (e.g., two, three or more consecutively) or may be scattered throughout the region of complementarity. For example, the 3' end of the sense strand may contain one or more mismatches. Thus, two mismatches may be incorporated into the 3' end of the sense strand. Base mismatches or destabilization of the segment at the 3' end of the sense strand of the oligonucleotide may improve the efficacy of synthetic duplexes in RNAi, possibly by promoting processing by Dicer.

[0130] It should be understood that in some embodiments, the sequences presented in the sequence listing may be referenced to represent the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modifications compared to the designated sequence while retaining essentially the same or similar complementary properties as the designated sequence.

[0131] Antisense strand The antisense strand of oligonucleotide may also be called guide strand.For example, if antisense strand can participate in RNA-induced silencing complex (RISC), bind to Argonaute protein, or participate in or bind to one or more similar factors, and direct the direct silencing of target gene, it can be called guide strand.

[0132] In certain embodiments, the antisense strand is less in nucleotide length than the sense strand. In some examples, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) can have an antisense strand that comprises 10-40 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 nucleotides) in length. Thus, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) can have an antisense strand that comprises 15-30 nucleotides (e.g., 15, 16, 17, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides) in length. For example, the antisense strand can comprise 20-25 nucleotides (e.g., 20, 21, 22, 23, 24, and 25 nucleotides) in length. In certain embodiments, the antisense strand can be 22 nucleotides in length.

[0133] The oligonucleotides disclosed herein may comprise an antisense strand that comprises a contiguous sequence between 12-22 nucleotides (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22 nucleotides) in length that is complementary to the sequence of SEQ ID NO: 12. For example, the oligonucleotides may comprise an antisense strand that comprises a contiguous sequence between 15-21 nucleotides (e.g., 15, 16, 17, 18, 19, 20, and 21 nucleotides) in length that is complementary to the sequence of SEQ ID NO: 12. In some embodiments, the oligonucleotides may comprise an antisense strand that has a contiguous sequence 19 nucleotides in length that is complementary to the sequence of SEQ ID NO: 12.

[0134] The oligonucleotides disclosed herein may comprise an antisense strand having the sequence of either SEQ ID NO: 3 or 6. For example, the oligonucleotides disclosed herein may comprise an antisense strand having the amino acid sequence of SEQ ID NO: 6, such as compound B shown in FIG. 2C. In some embodiments, the antisense strand may be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 6. SEQ ID NO: 6 may have the chemical structure shown in FIG. 2B. Alternatively, the antisense strand may have the sequence of SEQ ID NO: 3, such as compound A shown in FIG. 1C. In some embodiments, the antisense strand may be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 3.

[0135] In addition, the first position of the 5' end of the antisense strand can be a uridine. The uridine can include a phosphate analog; for example, the uridine can be 4'-O-monomethylphosphonate-2'-O-methyluridine.

[0136] Sense strand The sense strand of an oligonucleotide may also be referred to as a passenger strand. In certain embodiments, the passenger strand is longer in length and has more nucleotides than the guide strand. In some examples, the oligonucleotides provided herein (e.g., RNAi oligonucleotides) may have a sense strand that includes 10 to 45 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45 nucleotides) in length. Thus, an oligonucleotide (e.g., an RNAi oligonucleotide) can have a sense strand that comprises 20-50 nucleotides (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 nucleotides) in length. In certain embodiments, the sense strand can be 20 nucleotides in length. In other embodiments, the sense strand can be 36 nucleotides in length.

[0137] The oligonucleotide may have a sense strand that comprises a contiguous sequence of 7 to 36 nucleotides (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, and 36 nucleotides) relative to the sequence of SEQ ID NO: 12. Thus, the sense strand may comprise a contiguous sequence of 10 to 30 nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30 nucleotides) relative to the sequence of SEQ ID NO: 12. In some embodiments, the oligonucleotide disclosed herein may comprise a sense strand that comprises a contiguous sequence of nucleotides relative to the sequence of SEQ ID NO: 12, which is 19 nucleotides in length.

[0138] 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). Thus, the sense strand including the stem loop can be 20 to 40 nucleotides in length (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 nucleotides in length). For example, the sense strand including the stem loop can be 36 nucleotides in length.

[0139] Additionally, the stem loop region on the sense strand may form a double helical region with itself. The double helical region contained in the stem loop may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleotides in length. For example, the double helical region contained in the stem loop may be 6 nucleotides in length. The stem loop may provide protection against degradation (such as enzymatic degradation) to the oligonucleotide agent and may facilitate targeting properties for delivery to a target cell. For example, the loop may provide additional nucleotides that may be modified without substantially affecting the gene expression inhibitory activity of the oligonucleotide. In certain embodiments, oligonucleotides are provided herein in which the sense strand comprises (e.g., at its 3' end) a stem loop described as S1-L-S2, 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 long and form a tetraloop, as described herein, hi some embodiments, the S1 region is 6 nucleotides long, the S2 region is 6 nucleotides long, and the L region is a 4 nucleotide tetraloop.

[0140] The sense strand of the oligonucleotide (e.g., RNAi oligonucleotide) may comprise a stem-loop region and a region that forms a double helix with the antisense strand. The stem-loop region may comprise a nucleotide sequence that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the oligonucleotide sequence of SEQ ID NO:7. In some embodiments, the stem-loop region has the oligonucleotide sequence of SEQ ID NO:7.

[0141] The loop (L) of the stem loop can be a tetraloop (e.g., in a nick tetraloop structure). The loop of the stem loop can have a base sequence of GAAA. 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.

[0142] 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. 2C. SEQ ID NO: 4 may have a chemical structure as shown in FIG. 2A. In some embodiments, the sense strand may be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 4. Alternatively, the sense strand may have the base sequence of SEQ ID NO: 1, such as compound A shown in FIG. 1C. SEQ ID NO: 1 may have a chemical structure as shown in FIG. 1A. In some embodiments, the sense strand may be a pharma- ceutically acceptable salt (e.g., sodium salt) of SEQ ID NO: 1.

[0143] Oligonucleotide Modification Oligonucleotides can be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation, immunogenicity, base pairing properties, RNA distribution and cellular uptake, and other characteristics relevant for therapeutic or research applications (Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881; Bramsen et al., Frontiers in Genetics, 3(2012):1-22). Thus, in some embodiments, the oligonucleotides of the present disclosure can include one or more suitable modifications. Modified nucleotides can have modifications in their base or nucleobase, sugar (e.g., ribose, deoxyribose), or phosphate groups.

[0144] The number of modifications on an oligonucleotide and the position of those nucleotide modifications can affect the properties of the oligonucleotide. For example, oligonucleotides can be delivered in vivo by conjugating them to or incorporating them into lipid nanoparticles (LNPs) or similar carriers. However, when the oligonucleotide is not protected by LNPs or similar carriers, it may be advantageous for at least a portion of the nucleotides to be modified. Thus, in certain embodiments of any of the oligonucleotides provided herein, all or substantially all of the nucleotides of the oligonucleotide are modified. In certain embodiments, more than half of the nucleotides are modified. In other embodiments, less than half of the nucleotides are modified. Typically, for direct delivery, all sugars are modified at the 2' position. These modifications can be reversible or irreversible. The oligonucleotides disclosed herein can have a sufficient number and type of modified nucleotides to provide desired properties (e.g., protection from enzymatic degradation, ability to target desired cells after in vivo administration, and / or thermodynamic stability).

[0145] sugar modification Modified sugars, also referred to herein as sugar analogs, contain modified deoxyribose or ribose moieties with one or more modifications at the 2', 3', 4' and / or 5' carbon positions of the sugar. Modified sugars can also contain non-natural alternative carbon structures such as those found in locked nucleic acids ("LNAs") (see Koshkin et al. (1998), Tetrahedron 54, 3607-3630), unlocked nucleic acids ("UNAs") (see Snead et al. (2013), Molecular Therapy-Nucleic Acid, 2, e103), and bridged nucleic acids ("BNAs") (see Imanishi and Obika (2002), The Royal Society of Chemistry, Chem. Commun., 1653-1659). Koshkin et al., Snead et al., and Imanishi and Obika are incorporated herein by reference for their disclosures regarding sugar modifications.

[0146] Nucleotide modifications at the sugar 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 modifications are 2'-fluoro, 2'-O-methyl, or 2'-O-methoxyethyl. In some embodiments, the modifications are 2'-fluoro and / or 2'-O-methyl. Modifications at the sugar can include modifications of the sugar ring and can have modifications of one or more carbons of the sugar ring. For example, modifications of the sugar of a nucleotide can include the 2'-oxygen of the sugar linked to the 1'-carbon or 4'-carbon of the sugar, or the 2'-oxygen linked to the 1'-carbon or 4'-carbon via an ethylene or methylene bridge. In certain embodiments, modified nucleotides can have acyclic sugars that lack a 2'-carbon-3'-carbon bond. In some embodiments, modified nucleotides can have a thiol group, for example, at the 4' position of the sugar.

[0147] The oligonucleotides (e.g., RNAi oligonucleotides) described herein may contain at least one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 or more). For example, the sense strand of the oligonucleotide may contain at least one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35 or more). For example, the antisense strand of the oligonucleotide may also contain at least one modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20 or more).

[0148] In certain embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) may contain 20-50 (e.g., 20-30, 24-30, 28-30, 30-40, 34-40, 38-44, 44-50, and 48-50) modified nucleotides.

[0149] All nucleotides of the sense strand of the oligonucleotide may be modified. Additionally, all nucleotides of the antisense strand of the oligonucleotide may be modified. In some embodiments, all nucleotides of the oligonucleotide (e.g., RNAi oligonucleotide), including both the sense strand and the antisense strand, are modified. The modified nucleotides may be 2'-modified (e.g., 2'-fluoro or 2'-O-methyl). The 2'-modification to the nucleotide may be 2'-fluoro and / or 2'-O-methyl, and optionally, the 2'-fluoro modification is a 2'-fluoro deoxyribonucleoside and / or the 2'-O-methyl modification is a 2'-O-methyl ribonucleoside.

[0150] The present disclosure provides oligonucleotides having different modification patterns. An oligonucleotide comprising a sense strand and an antisense strand can comprise 40-50 (e.g., 41, 2, 43, 44, 45, 46, 47, 48, and 49) 2'-O-methyl modifications. A modified oligonucleotide can comprise a sense strand having a nucleotide sequence of either SEQ ID NO: 1 or 4 and an antisense strand having a nucleotide sequence of either SEQ ID NO: 3 or 6 (e.g., an oligonucleotide agent can have a sense strand of SEQ ID NO: 4 and an antisense strand of SEQ ID NO: 6, or an oligonucleotide agent can have a sense strand of SEQ ID NO: 1 and an antisense strand of SEQ ID NO: 3). In some embodiments, these oligonucleotides are modified at one or more of positions 1, 2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, and 36 of the sense strand and / or at one or more of positions 1, 4, 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, and 22 of the antisense strand with a 2'-O-methyl modified nucleoside, e.g., a 2'-O-methyl ribonucleoside. In some embodiments, all of positions 1, 2, 3, 4, 5, 6, 7, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 31, 32, 33, 34, 35, and 36 of the sense strand and all of positions 1, 4, 6, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, and 22 of the antisense strand are modified with 2'-O-methyl modified nucleosides, e.g., 2'-O-methyl ribonucleosides. In other embodiments, one or more of positions 1, 2, 4, 5, 6, 7, 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, 13, 15, 16, 17, 18, 19, 20, 21, and 22 of the antisense strand are modified with a 2'-O-methyl modified nucleoside, e.g., a 2'-O-methyl ribonucleoside.In certain embodiments, all of positions 1, 2, 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 / or all of positions 1, 6, 8, 9, 11, 13, 15, 17, 18, 20, 21, and 22 of the antisense strand are modified with 2'-O-methyl modified nucleosides, e.g., 2'-O-methyl ribonucleosides.

[0151] Oligonucleotides, including sense and antisense strands, may have 5-15 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, and 14) 2'-fluoro modifications. For these oligonucleotides, one or more of positions 8, 9, 10, and 11 of the sense strand and / or one or more of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand may be modified with a 2'-fluoro modified nucleoside. For example, all of positions 8, 9, 10, and 11 of the sense strand and / or all of positions 2, 3, 5, 7, 10, and 14 of the antisense strand may be modified with a 2'-fluoro modified nucleoside. In another example, all of positions 8, 9, 10, and 11 of the sense strand and / or all of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand may be modified with a 2'-fluoro modified nucleoside.

[0152] In the case of an oligonucleotide having a sense strand having a sequence of SEQ ID NO: 4 and an antisense strand having a sequence of SEQ ID NO: 6, one or more of positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand and / or one or more of positions 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand may be modified with a 2'-O-methyl modified nucleoside. Furthermore, all of positions 1 to 7, 12 to 27, and 31 to 36 of the sense strand and / or all of positions 1, 6, 8, 9, 11 to 13, and 15 to 22 of the antisense strand may be modified with a 2'-O-methyl modified nucleoside. In the case of an oligonucleotide having a sense strand having a sequence of SEQ ID NO: 4 and an antisense strand having a sequence of SEQ ID NO: 6, one or more of positions 8 to 11 of the sense strand and one or more of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand may be modified with a 2'-fluoro modified nucleoside. Thus, all of positions 8-11 of the sense strand and all of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand can be modified with 2'-fluoro modified nucleosides.

[0153] For example, 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, all of positions 1-7, 12-27, and 31-36 of the sense strand and / or all of positions 1, 6, 8, 9, 11-13, and 15-22 of the antisense strand can be modified with 2'-O-methyl modified nucleosides; all of positions 8-11 of the sense strand and all of positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand can be modified with 2'-fluoro modified nucleosides as shown in Figures 2A and 2B, respectively.

[0154] In the case of an oligonucleotide comprising a sense strand having a sequence of SEQ ID NO: 1 and an antisense strand having a sequence of SEQ ID NO: 3, one or more of positions 1-7, 12-27, and 31-36 of the sense strand and / or one or more of positions 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand may be modified with a 2'-O-methyl modified nucleoside. In some embodiments, all of positions 1-7, 12-27, and 31-36 of the sense strand and all of positions 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand may be modified with a 2'-O-methyl modified nucleoside. Furthermore, in the case of an oligonucleotide comprising a sense strand having a sequence of SEQ ID NO: 1 and an antisense strand having a sequence of SEQ ID NO: 3, one or more of positions 8-11 of the sense strand and / or one or more of positions 2, 3, 5, 7, 10, and 14 of the antisense strand may be modified with a 2'-fluoro modified nucleoside. In some embodiments, all of positions 8-11 of the sense strand and all of positions 2, 3, 5, 7, 10, 12, and 14 of the antisense strand are modified with 2'-fluoro modified nucleosides. For example, in the case of an oligonucleotide having a sense strand comprising the sequence of SEQ ID NO: 1 and an antisense strand having the sequence of SEQ ID NO: 3, all of positions 1-7, 12-27, and 31-36 of the sense strand and all of positions 1, 4, 6, 8, 9, 11-13, and 15-22 of the antisense strand can be modified with 2'-O-methyl modified nucleosides; all of positions 8-11 of the sense strand and all of positions 2, 3, 5, 7, 10, and 14 of the antisense strand can be modified with 2'-fluoro modified nucleosides as shown in Figures 1A and 1B, respectively.

[0155] In some embodiments, the terminal 3' end group (e.g., 3'-hydroxyl) can 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.

[0156] 5'-Terminal Phosphate The 5'-terminal phosphate group of an oligonucleotide (e.g., an RNAi oligonucleotide) may enhance the interaction with Argonaute 2. In certain embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide) includes a uridine at position 1 of the 5'-end of the antisense strand. However, oligonucleotides with a 5'-phosphate group are susceptible to degradation by phosphatases or other enzymes, which may limit their bioavailability in vivo. In some embodiments, an oligonucleotide includes a 5'-phosphate analog that is resistant to such degradation. Thus, the uridine at the 5'-end of the antisense strand may include a phosphate analog. The phosphate analog may be an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate. Additionally, the 5' end of the oligonucleotide chain may be attached to a chemical moiety ("phosphate mimic") that mimics the electrostatic and steric properties of the natural 5'-phosphate group (see Prakash et al., Nucleic Acids Res. 2015 Mar 31; 43(6): 2993-3011; the contents of which are incorporated herein by reference with respect to phosphate analogues). Many phosphate mimetics have been developed that can be attached to the 5' end (see U.S. Pat. No. 8,927,513; the contents of which are incorporated herein by reference with respect to phosphate analogues). Other modifications have been developed for the 5' end of oligonucleotides (see WO 2011 / 133871; the contents of which are incorporated herein by reference with respect to phosphate analogues). In certain embodiments, a hydroxyl group may be attached to the 5' end of the oligonucleotide.

[0157] Oligonucleotides may have a phosphate analog at the 4'-carbon position of the sugar, referred to as a "4'-phosphate analog." See, for example, WO 2018 / 045317, the contents of which regarding phosphate analogs are incorporated herein by reference. Oligonucleotides provided herein may include a 4'-phosphate analog at the 5'-terminal nucleotide. In some embodiments, the phosphate analog is an oxymethylphosphonate or an analog thereof, in which the oxygen atom of the oxymethyl group is attached to the sugar moiety (e.g., at its 4'-carbon). In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is attached to the 4'-carbon of the sugar moiety or an analog thereof. In certain embodiments, the 4'-phosphate analog is an oxymethylphosphonate. In some embodiments, the 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, CHOCH2CH2Si(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.

[0158] In some embodiments, the phosphate analog attached to the oligonucleotide is a methoxyphosphonate (MOP). The phosphate analog attached to the oligonucleotide can be a 5' monomethyl protected MOP. In some embodiments, for example, at position 1 of the antisense strand, the following uridine nucleotide containing a phosphate analog: [ka] may be used, and the modified nucleotide is referred to as 4'-O-monomethylphosphonate-2'-O-methyluridine ([MePhosphonate-4O-mU]) or 5'-methoxy, phosphonate-4'oxy-2'-O-methyluridine (5'-MeMOP). 5'-Methoxy, phosphonate-4'oxy-2'-O-methyluridine (5'-MeMOP) may be the first nucleotide at the 5' end of the antisense strand. In some embodiments, the first nucleotide at the 5' end of either SEQ ID NO: 3 or 6 may be 5'-methoxy, phosphonate-4'oxy-2'-O-methyluridine.

[0159] Modified Internucleoside Linkages Phosphate modifications or substitutions in an oligonucleotide can result in an oligonucleotide that includes at least one (e.g., at least one, at least two, at least three, at least five, or at least six) modified internucleotide linkages. Any one of the oligonucleotides disclosed herein can include 1-10 (e.g., 1-10, 2-8, 4-6, 3-10, 5-10, 1-5, 1-3, or 1-2) modified internucleotide linkages. For example, any one of the oligonucleotides disclosed herein can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified internucleotide linkages. In some embodiments, an oligonucleotide (e.g., an RNAi oligonucleotide) can include five modified internucleotide linkages. For example, the sense strand of the oligonucleotide can include one modified internucleotide linkage and the antisense strand can include four modified internucleotide linkages.

[0160] The modified internucleotide bond can be phosphorodithioate bond, phosphorothioate bond, phosphotriester bond, thionoalkylphosphonate bond, thionealkylphosphotriester bond, phosphoramidite bond, phosphonate bond or boranophosphate bond.The modified internucleotide bond of any one of the oligonucleotides disclosed herein can be phosphorothioate bond.In certain embodiments, all modified internucleotide bonds of the oligonucleotide can be phosphorothioate bond.

[0161] The oligonucleotides described herein may have phosphorothioate bonds between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. For example, the sense strand of the oligonucleotide may have phosphorothioate bonds between positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. Thus, a sense strand having a sequence of SEQ ID NO: 1 or 4 may have phosphorothioate bonds between positions 1 and 2, positions 2 and 3, positions 20 and 21, and positions 21 and 22.

[0162] Base Modification The oligonucleotides provided herein may have one or more modified nucleobases. Modified nucleobases, also referred to herein as base analogs, may be linked to the 1' position of a nucleotide sugar moiety. Modified nucleobases may be nitrogenous bases. In certain embodiments, modified nucleobases may contain nitrogen atoms. See, for example, US Published Patent Application No. 20080274462 (the contents of which regarding modified nucleobases are incorporated herein by reference). Modified nucleotides may also include universal bases. However, in certain embodiments, modified nucleotides may not contain nucleobases (e.g., non-basic).

[0163] In some embodiments, a universal base is a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide or at an equivalent position in a nucleotide sugar moiety substitute, which, when present in a double helix, can be located opposite more than one type of base without substantially changing the structure of the double helix. In some embodiments, compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide or polynucleotide) that is completely complementary to a target nucleic acid, a single-stranded nucleic acid containing a universal base has a lower T than a double helix formed with a complementary nucleic acid. m However, in some embodiments, compared to a reference single-stranded nucleic acid in which the universal base is replaced with one base to create a single mismatch, the single-stranded nucleic acid containing the universal base has a higher T than the double helix formed with the nucleic acid containing the mismatched base. m Non-limiting examples of universal binding nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and / or 1-β-D-ribofuranosyl-3-nitropyrrole (see U.S. Patent Application Publication No. 2007 / 0254362; Van Aerschot et al., Nucleic Acids Res. 1995 Nov 11; 23(21):4363-70; Loakes et al., Nucleic Acids Res. 1995 Jul 11; 23(13):2361-6; Loakes et al., Nucleic Acids Res. 1994 Oct 11; 22(20):4039-43, each of which is incorporated herein by reference for its disclosure regarding base modifications).

[0164] reversible modification Before reaching the target cell, certain modifications can be carried out to protect the oligonucleotide from the in vivo environment, but such modifications may reduce the efficacy or activity of the oligonucleotide when it reaches the cytosol of the target cell.Reversible modifications can be carried out so that the molecule retains the desired properties outside the cell, and are subsequently removed when it enters the cytosol environment of the cell.Reversible modifications can be removed, for example, by the action of intracellular enzymes or chemical conditions inside the cell (e.g., via reduction by intracellular glutathione).

[0165] Reversibly modified nucleotides may include glutathione-sensitive moieties. Typically, nucleic acid molecules are chemically modified with cyclic disulfide moieties to mask the negative charge generated by internucleotide diphosphate bonds and to improve cellular uptake and nuclease resistance. See U.S. Patent Application Publication No. 2011 / 0294869, originally assigned to Traversa Therapeutics, Inc. ("Traversa"), PCT Publication No. WO 2015 / 188197, assigned to Solstice Biologics, Ltd. ("Solstice"), Meade et al., Nature Biotechnology, 2014, 32:1256-1263 ("Meade"), PCT Publication No. WO 2014 / 088920, assigned to Merck Sharp & Dohme Corp, each of which is incorporated by reference for disclosure of such modifications. Reversible modifications of the internucleotide diphosphate bond are designed to be cleaved intracellularly by the reducing environment of the cytosol (e.g., glutathione). Previous examples include neutralizing phosphotriester modifications that have been reported to be cleavable intracellularly (see Dellinger et al. J. Am. Chem. Soc. 2003, 125:940-950).

[0166] Such reversible modifications allow protection during in vivo administration (e.g., passage through blood and / or lysosomal / endosomal compartments of cells) where the oligonucleotide is exposed to nucleases and other harsh environmental conditions (e.g., pH). Upon release into the cytosol of cells, where glutathione levels are higher compared to the extracellular space, the modification is reversed, resulting in a cleaved oligonucleotide. The use of reversible glutathione-sensitive moieties allows the introduction of sterically larger chemical groups into the oligonucleotide of interest compared to the options available using irreversible chemical modifications. This is because these larger chemical groups are removed in the cytosol and therefore are not likely to interfere with the biological activity of the oligonucleotide in the cytosol of the cell. As a result, these larger chemical groups can be engineered to confer various advantages on the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, reduced immunogenicity, etc. The structure of the glutathione-sensitive moiety can be engineered to alter the kinetics of its release.

[0167] In some embodiments, the glutathione-sensitive moiety is attached to the sugar of the nucleotide. In some embodiments, the glutathione-sensitive moiety is attached to the 2' carbon of the sugar of the modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5'-carbon of the sugar, especially when the modified nucleotide is the 5'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3'-carbon of the sugar, especially when the modified nucleotide is the 3'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, for example, US Patent Publication No. 2019 / 0177355, the contents of which are incorporated herein by reference for their relevant disclosures.

[0168] Targeting Ligands It may be desirable to target the oligonucleotides of the present disclosure to one or more cells or one or more organs (e.g., cells of the liver). Such a strategy may help to avoid undesirable effects in other organs, or may avoid excessive loss of oligonucleotide to cells, tissues, or organs that do not benefit from the oligonucleotide. Thus, in some embodiments, the oligonucleotides disclosed herein can be modified to facilitate targeting of a particular tissue, cell, or organ, for example, to facilitate delivery of the oligonucleotide to the liver. In certain embodiments, the oligonucleotides disclosed herein can be modified to facilitate delivery of the oligonucleotide to hepatocytes of the liver. The oligonucleotides may include nucleotides conjugated to one or more targeting ligands.

[0169] The targeting ligand may include carbohydrates, amino sugars, cholesterol, peptides, polypeptides, proteins or protein parts (e.g., antibodies or antibody fragments), or lipids. In some embodiments, the targeting ligand is an aptamer. For example, the targeting ligand may be an RGD peptide used to target tumor vasculature or glioma cells, a CREKA peptide (SEQ ID NO: 78) that targets tumor vasculature or stroma, an aptamer that targets transferrin, lactoferrin, or transferrin receptor expressed in the CNS vasculature, or an anti-EGFR antibody that targets EGFR on glioma cells. In some embodiments, the targeting ligand is one or more N-acetylgalactosamine (GalNAc) moieties.

[0170] One or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide may each be conjugated to a separate targeting ligand. In some cases, 2-4 nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. The targeting ligand may be conjugated to 2-4 nucleotides at either the sense or antisense end such that the targeting ligand resembles the bristles of a toothbrush and the oligonucleotide resembles a toothbrush (e.g., the ligand is conjugated to a 2-4 nucleotide overhang or extension at the 5' or 3' end of the sense or antisense strand). For example, the oligonucleotide may include a stem loop at either the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the stem loop may be individually conjugated to a targeting ligand. In some embodiments, the oligonucleotide includes a stem loop at the 3' end of the sense strand, and 3 nucleotides of the stem loop are individually conjugated to a targeting ligand.

[0171] In some embodiments, it is desirable to target the oligonucleotide that reduces the expression of CFB to hepatocytes in the liver of a subject. Any suitable hepatocyte targeting moiety can be used for this purpose.

[0172] GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGPR), which is expressed primarily on the sinusoidal surface of hepatocyte cells, and plays a major role in the binding, internalization, and subsequent clearance of circulating glycoproteins that contain terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins). Indirect or direct conjugation of GalNAc moieties to the oligonucleotides of the present disclosure can be used to target these oligonucleotides to the ASGPR expressed in the hepatocyte cells.

[0173] For example, the oligonucleotide of the present disclosure can be directly or indirectly conjugated with monovalent GalNAc. The oligonucleotide can be directly or indirectly conjugated with two or more (e.g., 2, 3, 4 or more) monovalent GalNAc, typically conjugated with 3 or 4 monovalent GalNAc moieties. The GalNAc moiety can be present in the loop region of the oligonucleotide described herein. The GalNAc moiety can be used to target the disclosed oligonucleotide to ASGPR on hepatocytes; at that point, the GalNAc-linked oligonucleotide can be internalized and incorporated into the intracellular RNAi machinery called RNA-induced silencing complex (RISC). The RISC Argonaute-2 (Argo-2) protein in this complex targets the antisense strand of the oligonucleotide duplex to its complementary CFB mRNA and initiates its degradation, thus blocking translation of the target.

[0174] In some embodiments, 2-4 nucleotides of the loop (L) of the stem loop are each conjugated to a separate GalNAc moiety. In some embodiments, three nucleotides of the loop of the stem of the oligonucleotide may be directly or indirectly conjugated to three separate monovalent GalNAc moieties. In some embodiments, the oligonucleotide is conjugated to one or more divalent, trivalent or tetravalent GalNAc moieties.

[0175] Oligonucleotides described herein can include a monovalent GalNAc linked to a guanine nucleobase, as depicted below, which is referred to as [ademG-GalNAc] or 2'-aminodiethoxymethanol-guanine-GalNAc. [ka]

[0176] Additionally or alternatively, the oligonucleotides described herein may contain a monovalent GalNAc linked to an adenine nucleobase, as depicted below, which is referred to as 2'-O-GalNAc modified adenosine [ademA-GalNAc] or 2'-aminodiethoxymethanol-adenine-GalNAc. [ka]

[0177] An example of such a linkage is shown below for a loop (L=linker, X=heteroatom) containing the nucleotide sequence GAAA from 5' to 3', with the stem attachment points indicated. Such a loop may be present, for example, at nucleotide positions 27-30 of the molecule shown in Figures 1A and 2A (see also Figures 1C and 2C). In the formula: [ka] is the point of attachment to the oligonucleotide chain. [ka]

[0178] The targeting ligand can be linked to the nucleotide using a suitable method or chemistry (e.g., click chemistry). The targeting ligand can be conjugated to the nucleotide using a click linker. In addition, the targeting ligand can be conjugated to any one of the nucleotides of the oligonucleotides described herein using an acetal-based linker. Acetal-based linkers are disclosed, for example, in International Patent Application Publication No. WO 2016 / 100401A1, published on June 23, 2016, the contents of which regarding such linkers are incorporated herein by reference. The linker can be a labile linker. However, in other embodiments, the linker is stable (non-labile).

[0179] An example is shown below of a loop containing the nucleotides GAAA 5' to 3', where the GalNAc moiety is 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 formula: [ka] is the point of attachment to the oligonucleotide chain. [ka]

[0180] In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise a sense strand having a tetraloop, where three (3) GalNAc moieties are conjugated to a nucleotide comprising the tetraloop, and each GalNAc moiety is conjugated to one (1) nucleotide. In some embodiments, the oligonucleotides described herein (e.g., RNAi oligonucleotides) comprise a sense strand having a tetraloop comprising a GalNAc-conjugated nucleotide, where the tetraloop has the following structure: [ka] Includes.

[0181] In some embodiments, a double helix extension (e.g., up to 3, 4, 5, or 6 base pairs in length) is provided between the targeting ligand (e.g., the GalNAc moiety) and the oligonucleotide (e.g., the RNAi oligonucleotide). In some embodiments, the double helix extension between the targeting ligand (e.g., the GalNAc moiety) and the oligonucleotide (e.g., the RNAi oligonucleotide) is 6 base pairs in length.

[0182] formulation Various formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides can be delivered to a subject or cellular environment using formulations that minimize degradation, facilitate delivery and / or uptake, or confer another beneficial property to the oligonucleotide in the formulation. In some embodiments, compositions are provided herein that include oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) for reducing the expression of CFB. Such compositions can be suitably formulated such that, upon administration to a subject, a sufficient portion of the oligonucleotide enters the cell to reduce CFB expression, either in the immediate environment of the target cell or throughout the body. As disclosed herein, any of a variety of suitable oligonucleotide formulations can be used to deliver the oligonucleotide for the purpose of reducing CFB. In some embodiments, the oligonucleotide, pharmaceutical composition, vector, or cell is formulated in a buffer solution, such as phosphate buffered saline, liposomes, micellar structures, vectors, and capsids.

[0183] The formulations disclosed herein may include excipients. The excipients may provide the composition with improved stability, improved absorption, improved solubility and / or enhanced therapeutic effect of the active ingredient. The excipients may be buffers (e.g., sodium citrate, sodium phosphate, Tris base or sodium hydroxide) or vehicles (e.g., buffer solutions, petrolatum, dimethyl sulfoxide or mineral oil). In some embodiments, the oligonucleotides can be lyophilized to extend their shelf life and then dissolved prior to use (e.g., administration to a subject). Thus, the excipients in the compositions comprising any one of the oligonucleotides described herein are cryoprotectants (e.g., mannitol, lactose, polyethylene glycol or polyvinylpyrrolidone) or disintegration temperature regulators (e.g., dextran, ficoll or gelatin).

[0184] Pharmaceutical compositions containing oligonucleotides can be formulated to be compatible with their intended route of administration, including parenteral, e.g., subcutaneous, intravenous, intradermal, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.

[0185] Pharmaceutical compositions suitable for injection use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid propylene glycol, etc.) and suitable mixtures thereof. In many cases, it is optional to include isotonicity agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Sterile injectable solutions can be prepared by incorporating the required amount of oligonucleotide in a selected solvent together with one or a combination of the above-listed ingredients, as needed, followed by filter sterilization.

[0186] In some embodiments, the pharmaceutical composition comprising the oligonucleotide comprises sterile water (WFI). In some embodiments, the pharmaceutical composition comprising the oligonucleotide comprises PBS.

[0187] In some embodiments, the pharmaceutical composition comprising oligonucleotide is a preservative-free solution. In some embodiments, the pharmaceutical composition comprises a sterile solution in WFI. In some embodiments, 0.1N NaOH or 0.1N HCl can be titrated to adjust the pH of the solution to a target of about 7.2 (e.g., pH 7.2). In some embodiments, the concentration of oligonucleotide can be about 160 mg / mL (e.g., 160 / mg / mL) as free acid form. In some embodiments, WFI can be added to bring the pharmaceutical composition to a desired total concentration of oligonucleotide. In some embodiments, the target fill volume is about 1.3 mL in a 2 mL glass vial. In some embodiments, the solution is administered subcutaneously to a subject.

[0188] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., an oligonucleotide for reducing CFB expression), although the percentage of active ingredient may be from about 1% to about 80% or more by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be taken into account by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0189] Many embodiments are directed to liver-targeted delivery of any of the oligonucleotides disclosed herein, although targeting of other tissues is also contemplated.

[0190] Pharmaceutical Use Disclosed herein is a method of delivering an effective amount of any one of the oligonucleotides disclosed herein to a cell or subject for the purpose of reducing expression of CFB in the cell or subject.

[0191] The oligonucleotides disclosed herein can be introduced into cells of a subject having a disease or disorder mediated by complement pathway activation or dysregulation (e.g., activation or dysregulation of CFB) using any suitable nucleic acid delivery method. For example, 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.

[0192] Formulation of oligonucleotides with cationic lipids can be used to facilitate the transfection of oligonucleotides into cells.For example, cationic lipids such as lipofectin, cationic glycerol derivatives and polycationic molecules (e.g., polylysine) can be used.Suitable lipids include oligofectamine, lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.) or FuGene 6 (Roche), all of which can be used according to the manufacturer's instructions.

[0193] Thus, in some embodiments, the formulation comprises a lipid nanoparticle. In some embodiments, the excipient comprises a liposome, lipid, lipid complex, microsphere, microparticle, nanosphere, or nanoparticle, or may otherwise be formulated for administration to a cell, tissue, organ, or body of a subject in need thereof (see, e.g., Remington: THE SCIENCE AND PRACTICE OF PHARMACY, 22nd edition, Pharmaceutical Press, 2013).

[0194] Effective intracellular concentrations of the oligonucleotides disclosed herein may also be achieved through stable expression of a polynucleotide encoding the oligonucleotide (e.g., by integration into the nuclear or mitochondrial genome of a mammalian cell) or by transient expression in cells contacted with a polynucleotide (e.g., a plasmid or other vector (e.g., a viral vector) encoding the oligonucleotide. Examples of expression vectors are disclosed, for example, in WO 1994 / 011026, incorporated herein by reference. Expression vectors for use in the compositions and methods described herein contain oligonucleotide sequences that reduce CFB expression as well as additional sequence elements used, for example, for expression of these agents and / or integration of these polynucleotide sequences into the genome of a mammalian cell. The expression vector may be a viral vector, a retroviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0195] 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. The vectors used to deliver the oligonucleotides described herein can be viral vectors, such as retroviral vectors (e.g., lentiviral vectors), adenoviral vectors (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), and adeno-associated viral vectors (AAV) (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10).

[0196] In some examples, the oligonucleotides described herein can be delivered in the form of a transgene engineered to express the oligonucleotide (e.g., its sense and antisense strands) in a cell. The transgene can be delivered using a vector, such as a viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or a non-viral vector (e.g., a plasmid or synthetic mRNA), as described above. In some embodiments, the transgene can be injected directly into the subject, for example, at or near the source of action (e.g., in or near the liver) or into the bloodstream.

[0197] CFB Inhibition Upon administration, the oligonucleotides of the present disclosure can bind to CFB mRNA and inhibit its expression. Inhibition of expression of the CFB gene can be evidenced by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells can be present, for example, in a sample derived from (e.g., obtained from) a subject) in which the CFB gene is transcribed and which has been treated to inhibit expression of the CFB gene (e.g., by contacting the cells with an oligonucleotide (e.g., an RNAi oligonucleotide) of the present disclosure or administering an oligonucleotide (e.g., an RNAi oligonucleotide) of the present disclosure to a subject in which the cells are or were present), compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been similarly treated (a control cell that has not been treated with an oligonucleotide (e.g., an RNAi oligonucleotide) or an oligonucleotide (e.g., an RNAi oligonucleotide) targeted to a gene of interest). The level of target mRNA can be measured using techniques well known to those skilled in the art, such as RT-qPCR. The degree of inhibition can be expressed by the following formula:

number

[0198] Changes in the expression level of the CFB gene can be assessed with respect to parameters functionally related to CFB gene expression, such as reduction in CFB protein expression, CFB protein activity, or the CFB signaling pathway. CFB gene silencing can be determined in any cell expressing either endogenous or heterologous CFB from an expression construct and by any assay known in the art.

[0199] The results of inhibition of CFB mRNA can be confirmed by a suitable assay to evaluate one or more characteristics of a cell or subject or a biochemical technique to evaluate molecules (e.g., RNA, protein) indicative of CFB expression. The extent to which the oligonucleotides provided herein reduce the expression level of CFB is assessed by comparing the expression level to a suitable control (e.g., the level of CFB mRNA expression in a cell or cell population to which no oligonucleotide or a negative control has been delivered). A suitable control level of CFB mRNA expression can be a predetermined level or value so that the subject level does not need to be measured every time. The predetermined level or value can take a variety of forms, including a single cutoff value, such as a median or mean value. For example, the predetermined level or value can be at or about a level of 200 mg / dL of CFB protein, which corresponds to the level of CFB protein typically found in the serum of a healthy subject.

[0200] The expression level of CFB mRNA in a sample can be determined, for example, by detecting a transcribed polynucleotide or a portion thereof, such as mRNA. RNA can be extracted from cells using RNA extraction techniques, including, for example, the use of acid phenol / guanidine isothiocyanate extraction RNAZOL™ B; Biogenesis), RNEASY™ RNA preparation kit (Qiagen) or PAXGENE™ (PreAnalytix, Switzerland). CFB mRNA in a sample can also be measured using real-time PCR (RT-PCR). For example, RNA can be extracted by homogenizing tissue samples with QIAzo Lysis reagent using a TissueLyser II (Qiagen) followed by purification using MAGMAX® Technology (ThermoFisher Scientific) according to the manufacturer's instructions. cDNA can then be prepared using a High Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific). Specific primers and probes for CFB and housekeeping controls were used for PCR on a CFX384 Real-Time PCR Detection System (Bio-Rad Laboratories), and Ct values ​​were estimated using BioRad CFX Maestro software; expression levels were calculated in EXCEL® and plotted in Prism (GraphPad). Primers that can be used for RT-PCR include those listed in Table 2. Primers having any one of the nucleic acid sequences of SEQ ID NOs: 39-42 can be used to determine the level of CFB in human cells. Similarly, primers having the nucleic acid sequences of SEQ ID NOs: 43 and 44 can be used to determine the level of CFB in monkey cells. Furthermore, primers having the nucleic acid sequences of SEQ ID NOs: 43 and 44 can be used to determine the level of CFB in mouse cells.

[0201] [Table 2]

[0202] Typical assay formats using ribonucleic acid hybridization include nuclear run-on assay, RT-PCR, RNase protection assay, Northern blotting, in situ hybridization and microarray analysis. Circulating mRNA can be detected using the methods described in PCT Publication WO 2012 / 177906, the entire contents of which are incorporated herein by reference. The expression level of a gene of interest can also be determined using a nucleic acid probe.

[0203] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Northern or Southern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA of a gene of interest. For example, the isolated mRNA can be run on an agarose gel, and the mRNA can be immobilized on a solid surface by transferring the mRNA from the gel to a membrane such as nitrocellulose, and contacted with the probe. The probe can be immobilized on a solid surface, and the mRNA is contacted with the probe, for example, in an AFFYMETRIX® GENECHIP® array. Methods for detecting mRNA known in the art can be adapted for use in determining the level of mRNA of a gene of interest.

[0204] Alternative methods for determining the expression level of a gene of interest in a sample include, for example, RT-PCR (Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Qβ replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al. (1989) Bio / Technology 6:1197), and the like. The process includes nucleic acid amplification of, for example, mRNA in a sample by PCR (U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, and / or reverse transcriptase (to prepare cDNA), followed by detection of the amplified molecules using techniques known in the art. These detection schemes are useful for detection of nucleic acid molecules when such molecules are present in very low numbers. In some embodiments of the present disclosure, the expression level of a gene of interest (e.g., CFB) is determined by quantitative fluorescent RT-PCR (i.e., TAQMAN™ System) or DUAL-GLO® luciferase assay.

[0205] The expression level of mRNA of the gene of interest can be monitored using membrane blots (such as those used in hybridization analyses such as Northern, Southern, dot, etc.) or microwells, sample tubes, gels, beads or fibers (or any solid support containing bound nucleic acid). See U.S. Patent Nos. 5,770,722; 5,874,219; 5,744,305; 5,677,195; and 5,445,934, which are incorporated herein by reference. Determining gene expression levels can also include using nucleic acid probes in solution.

[0206] The aforementioned assays can be used to determine the efficacy of treatment with the oligonucleotides described herein based on the reduction in CFB mRNA levels. The reduction in the level of CFB mRNA can be a reduction of 1% or less, 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 70% or less, 80% or less, or 90% or less compared to an appropriate control level of CFB mRNA or the level of CFB in a subject prior to treatment. An appropriate control level can be the level of CFB mRNA expression in a cell or cell population that has not been contacted with an oligonucleotide described herein. In some embodiments, the effect of delivery of an oligonucleotide to a cell by the methods disclosed herein is evaluated after a finite period of time. For example, the level of CFB mRNA can be analyzed in a cell for at least 8 hours, 12 hours, 18 hours, 24 hours; or for at least 3, 4, 5, 10, 15, 20, 30, 40, 50, 60, 70, or 80 days after introduction of the oligonucleotide into the cell.

[0207] Furthermore, inhibition of the CFB gene can result in inhibition of CFB protein expression, which can be manifested by a decrease in the level of CFB protein expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for assessment of mRNA suppression, inhibition of protein expression levels in a treated cell or group of cells can also be expressed as a percentage of protein levels in a control cell or group of cells.

[0208] The results of inhibiting CFB protein expression can be confirmed by a suitable assay to evaluate one or more characteristics of a cell or subject or a biochemical method to evaluate a molecule indicative of CFB protein expression. The extent to which the oligonucleotides provided herein reduce the expression level of a CFB protein is evaluated by comparing the expression level with a suitable control (e.g., the level of CFB protein expression in a cell or cell population to which no oligonucleotide or a negative control is delivered). A suitable control level of CFB protein expression can be a predetermined level or value, such as an amount of CFB protein determined to be in the normal range, for example, about 200 mg / dL in serum, so that it is not necessary to measure the control level every time. The predetermined level or value can take various forms, including a single cutoff value, such as a median or mean value.

[0209] The level of CFB protein produced by expression of the CFB gene can be determined using any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC / MS / MS), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), immunofluorescence assays, electrochemiluminescence assays, and the like. Such assays can also be used to detect proteins that indicate the presence or replication of a protein produced by a gene of interest. Additionally, the above assays can also be used to report changes in the mRNA sequence of interest that result in restoration or alteration of protein function, thereby providing a therapeutic effect and benefit to the subject, treating a disorder in the subject, and / or alleviating symptoms of a disorder in the subject.

[0210] The aforementioned assays can be used to determine the efficacy of treatment with the oligonucleotides described herein based on the reduction in the amount of CFB protein. The reduction in the level of CFB protein can be a reduction of 1% or less, 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 70% or less, 80% or less, or 90% or less compared to an appropriate control level of CFB (e.g., about 200 mg / gL). An appropriate control level can be the level of CFB3 expression in a cell or cell population that has not been contacted with the oligonucleotides described herein. In some embodiments, the effect of delivery of oligonucleotides to cells by the methods disclosed herein is evaluated after a finite period of time. For example, the level of CFB can be analyzed in cells for at least 8 hours, 12 hours, 18 hours, 24 hours; or at least 1, 2, 3, 4, 5, 6, 7, or 14 days after introduction of the oligonucleotide into the cells. The level of CFB can be determined to assess whether retreatment of the subject is required. For example, if the level of CFB rises to pre-treatment levels (or to a level that is at least about 20% or more (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) of the pre-treatment levels), the subject is considered to require re-treatment.

[0211] Furthermore, inhibition of the CFB gene using the methods described herein may result in reduced transcription of CFB mRNA in cells of a subject identified as having a disease mediated by complement pathway activation and dysregulation. The methods provided herein are useful in any suitable cell type (e.g., cells expressing CFB, such as hepatocytes). In some embodiments, the cells are primary cells obtained from a subject, which may have undergone a limited number of passages, such that the cells substantially maintain their native phenotypic characteristics. In some embodiments, the cells to which the oligonucleotides are delivered are ex vivo or in vitro (i.e., cells in culture or may be delivered to an organism in which the cells reside). In specific embodiments, methods are provided for delivering an effective amount of the oligonucleotides disclosed herein to cells for the purpose of reducing expression of CFB only in hepatocytes.

[0212] An effective amount of an oligonucleotide disclosed herein can be determined as the amount of oligonucleotide that results in the alleviation of a symptom of a disease or disorder mediated by complement pathway activation or dysregulation, such as one of the diseases or disorders described herein. The alleviation of 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% alleviation, as determined using clinical evaluations known to those skilled in the art. The amount of alleviation of a symptom of a disease or disorder mediated by complement pathway activation or dysregulation can be used to determine whether the subject needs to be treated again with the RNAi oligonucleotide, pharmaceutical composition, vector or cell described herein. Examples of assays for determining the alleviation of a disease or disorder mediated by complement pathway activation or dysregulation include, but are not limited to, the measurement and / or quantification of circulating CFB protein, functional assays (e.g., WEISLAB assay and hemolytic assay). Quantification of CFB deposition can be performed via IHC or immunofluorescence or via specific disease biomarkers.

[0213] Furthermore, the oligonucleotides described herein, including both sense and antisense strands as double-stranded oligonucleotides, can be introduced into cells of a subject using any suitable nucleic acid delivery. The double-stranded oligonucleotides can be delivered to cells by injection of a solution containing the oligonucleotide, bombardment with particles coated with the oligonucleotide, exposure of cells or organisms to a solution containing the oligonucleotide, or electroporation of cell membranes in the presence of the oligonucleotide. The double-stranded oligonucleotides can also be delivered to cells using lipid-mediated carrier delivery, chemical-mediated delivery, cationic liposome transfection such as calcium phosphate, and vectors encoding nucleic acids of single-stranded oligonucleotides. The vectors used to deliver the double-stranded oligonucleotides can be viral vectors, such as retroviral vectors (e.g., lentiviral vectors), adenoviral vectors (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), and adeno-associated viral vectors (AAV) (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9).

[0214] Treatment method Also disclosed herein is a method of treating a disease mediated by complement pathway activation or dysregulation, including, for example, one or more of the diseases associated with complement pathway activation or dysregulation disclosed herein, in a subject by administering a composition described herein (e.g., an oligonucleotide, a vector encoding the oligonucleotide, a cell comprising the vector, a pharmaceutical composition). The method may include treating a disease mediated by complement pathway activation or dysregulation in a subject by administering a pharma- ceutically acceptable salt (e.g., a sodium salt) of an RNAi oligonucleotide described herein. The methods described herein typically involve administering an effective amount of the oligonucleotide or a pharma- ceutically acceptable salt thereof, i.e., an amount that can produce a desired therapeutic result (e.g., knockdown of CFB expression). A therapeutically acceptable amount can be an amount that can treat a disease or disorder mediated by complement pathway activation or dysregulation (e.g., activation or dysregulation of CFB). The appropriate dosage for any one subject may depend on certain factors, including the subject's size, body surface area, age, the specific composition administered, the active ingredients in the composition, the time and route of administration, general health, and other drugs administered concomitantly. Such therapeutic agents can be used, for example, to slow, stop or prevent any type of disease or disorder mediated by complement pathway activation or dysregulation, and can be administered prophylactically or therapeutically. Administration of a prophylactic agent can be performed prior to detection or manifestation of symptoms characteristic of a disease or disorder mediated by complement pathway activation or dysregulation, such that the disease or disorder is prevented or its progression is delayed. Subjects at risk for a disease or disorder 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.

[0215] The compositions disclosed herein can be administered to a subject using any standard method. For example, any one of the compositions disclosed herein can be administered enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intraarterial injection or infusion, intraosseous injection, intramuscular injection, intracerebral injection, intraventricular injection, intrathecal injection), locally (e.g., transdermally, by inhalation, eye drops or via mucosa) or by direct injection into a target organ (e.g., the liver of a subject). Typically, the oligonucleotides disclosed herein are administered intravenously or subcutaneously. The most suitable route for administration in any given case may depend on the particular composition administered, the subject, the particular disease or disorder mediated by complement pathway activation or dysregulation to be treated, the pharmaceutical formulation method, the method of administration (e.g., time and route of administration), the age, weight, sex of the subject, the severity of the disease to be treated, the diet of the subject, and the excretion rate of the subject.

[0216] To a subject suffering from a disease or disorder mediated by complement pathway activation or dysregulation, the oligonucleotides described herein can be administered, for example, annually (e.g., once every 12 months), semi-annually (e.g., once every 6 months), quarterly (e.g., once every 3 months), bimonthly (e.g., once every 2 months), monthly, or weekly. In other examples, the oligonucleotides can be administered once or more per week, 2 weeks, or 3 weeks, once or more per month, bimonthly, once or more per 3 months, once or more per quarter, once or more per 6 months, or once or more per year. In certain embodiments, the oligonucleotides can be administered daily.

[0217] The subject to be treated for a disease or disorder mediated by complement pathway activation or dysregulation may be a human or non-human primate or another mammalian subject. Other exemplary subjects that may be treated with the oligonucleotides described herein include domestic animals such as dogs and cats; livestock animals such as horses, cows, pigs, sheep, goats and chickens; and animals such as mice, rats, guinea pigs and hamsters.

[0218] Dosage The dosage of the compositions of the present disclosure (e.g., compositions comprising an RNAi oligonucleotide or a pharma- ceutically acceptable salt thereof as described herein) may vary depending on many factors, such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and extent of symptoms, the frequency of treatment and / or type of concurrent treatment (if any), and the clearance rate of the compound being treated. One of skill in the art can determine the appropriate dosage based on the above factors.

[0219] The oligonucleotides or pharma- ceutically acceptable salts thereof of the present disclosure may be administered in an amount and for a time effective to result in one or more (e.g., two or more, three or more, four or more) of: (a) reducing expression of a CFB protein in cells of a subject; (b) reducing transcription of CFB in cells of a subject; (c) reducing the level of a CFB protein in cells of a subject; (d) reducing activity of a CFB protein in cells of a subject; and / or (e) alleviating one or more symptoms of a disease or disorder mediated by activation or dysregulation of the complement pathway.

[0220] Thus, the present disclosure relates to a method of treating a disease or disorder mediated by complement pathway activation or dysregulation in a subject in need thereof, comprising administering an effective amount of an oligonucleotide described herein that specifically binds to CFB mRNA and inhibits expression of CFB protein in the subject. For example, the present disclosure provides a method of treating a disease or disorder mediated by alternative complement pathway dysregulation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an oligonucleotide, pharmaceutical composition, vector, or cell disclosed herein.

[0221] The disease or disorder mediated by complement pathway activation or dysregulation to be treated using the disclosed methods and compositions can be, for example, a skin disorder, a neurological disorder, a nephrological disorder, an acute care, a rheumatic disorder, a pulmonary disorder, a dermatological disorder, a hematological disorder, and an ophthalmological disorder.

[0222] Treatment of diseases mediated by complement pathway activation or dysregulation may be achieved by administration of an oligonucleotide or a pharma- ceutical acceptable salt thereof that inhibits expression and / or translation of CFB mRNA (e.g., expression of CFB protein) as described herein.

[0223] The disclosed compositions can be administered in amounts determined to be appropriate by one of skill in the art. In some embodiments, the oligonucleotides or pharma- ceutically acceptable salts thereof described herein can be administered initially at an appropriate dosage, which can be adjusted, if necessary, depending on the clinical response.

[0224] In some cases, the oligonucleotide or a pharma- ceutically acceptable salt thereof is administered at a dose of 0.01-100 mg / kg (e.g., 0.01-1 mg / kg, 1-5 mg / kg, 5-20 mg / kg, 20-50 mg / kg, 50-100 mg / kg) of the subject's body weight. In certain examples, the oligonucleotide is administered at a concentration of 0.01 mg-50 mg per kg of the subject's body weight (e.g., 0.01-1 mg / kg, 1-5 mg / kg, 5-10 mg / kg, 10-20 mg / kg, 20-30 mg / kg, 30-40 mg / kg, 40-50 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.01 mg-20 mg per kg of the subject's body weight (e.g., 0.01-1 mg / kg, 1-5 mg / kg, 5-10 mg / kg, 10-15 mg / kg, 15-20 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 (e.g., 0.01-1 mg / kg, 1-2 mg / kg, 2-5 mg / kg, 5-8 mg / kg, 8-10 mg / kg, 10-12 mg / kg, 12-15 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.01 mg to 10 mg per kg of the subject's body weight (e.g., 0.01-1 mg / kg, 1-2 mg / kg, 2-5 mg / kg, 5-8 mg / kg, 8-10 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.01 mg to 5 mg per kg of the subject's body weight (e.g., 0.01-1 mg / kg, 1-2 mg / kg, 2-3 mg / kg, 3-4 mg / kg, 4-5 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.1 mg to 20 mg per kg of the subject's body weight (e.g., 0.1-1 mg / kg, 1-5 mg / kg, 5-10 mg / kg, 10-15 mg / kg, and 15-20 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.1 mg to 10 mg per kg of the subject's body weight (e.g., 0.1-1 mg / kg, 1-2 mg / kg, 2-5 mg / kg, 5-7 mg / kg, and 7-10 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 0.1 mg to 5 mg per kg of the subject's body weight (e.g., 0.1-1 mg / kg, 2-3 mg / kg, 3-4 mg / kg, and 4-5 mg / kg).In other instances, the oligonucleotide is administered at a concentration of 1 mg to 50 mg per kg of the subject's body weight (e.g., 1-10 mg / kg, 10-20 mg / kg, 20-30 mg / kg, 30-40 mg / kg, and 40-50 mg / kg). In other instances, the oligonucleotide is administered at a concentration of 1 mg to 20 mg per kg of the subject's body weight (e.g., 1-5 mg / kg, 5-10 mg / kg, 10-15 mg / kg, and 15-20 mg / kg). In other instances, the oligonucleotide is administered at a concentration of 1 mg to 10 mg per kg of the subject's body weight (e.g., 1-2 mg / kg, 2-5 mg / kg, 5-7 mg / kg, and 7-10 mg / kg). In other instances, the oligonucleotide is administered at a concentration of 1 mg to 5 mg per kg of the subject's body weight (e.g., 1-2 mg / kg, 2-3 mg / kg, 3-4 mg / kg, and 4-5 mg / kg). In other examples, the oligonucleotide is administered at a concentration of 30 mg / kg to 300 mg / kg (e.g., 30 to 200 mg / kg, 30 to 100 mg / kg, 30 to 50 mg / kg, 50 to 300 mg / kg, 100 to 300 mg / kg, 200 to 300 mg / kg, and 250 to 300 mg / kg).

[0225] In certain embodiments, the oligonucleotide or a pharma- ceutically acceptable salt thereof is administered at a dose 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 dose of about 10 mg / kg or less. In another embodiment, the oligonucleotide is administered at a dose of about 9 mg / kg or less (e.g., 8.9 mg / kg, 8 mg / kg, 7 mg / kg, 5 mg / kg, 3 mg / kg, and 1 mg / kg or less). In other embodiments, the oligonucleotide is administered at a dose of about 8 mg / kg or less (e.g., 7.9 mg / kg, 7 mg / kg, 5 mg / kg, 3 mg / kg, and 1 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dose of about 7 mg / kg or less (e.g., 6.9 mg / kg, 6 mg / kg, 4 mg / kg, 2 mg / kg, and 1 mg / kg or less). In another embodiment, the oligonucleotide (e.g., RNAi oligonucleotide) is administered at a dose of about 6 mg / kg or less (e.g., 5.9 mg / kg, 5 mg / kg, 3 mg / kg, and 1 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dose of about 5 mg / kg or less (e.g., 4.9 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, and 1 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dose of about 4 mg / kg or less (e.g., 3.9 mg / kg, 3 mg / kg (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 dose of about 2 mg / kg or less (e.g., 1.9 mg / kg, 1.5 mg / kg, 1 mg / kg, and 0.5 mg / kg or less). In another embodiment, the oligonucleotide is administered at a dose of about 1 mg / kg or less (e.g., 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, and 0.1 mg / kg or less).

[0226] In another embodiment, the oligonucleotide or a pharma- ceutically acceptable salt thereof is administered at a dose of about 0.1-10 mg / kg, about 0.2-10 mg / kg, about 0.3-10 mg / kg, about 0.4-10 mg / kg, about 0.5-10 mg / kg, about 1-10 mg / kg, about 2-10 mg / kg, about 3-10 mg / kg, about 4-10 mg / kg, about 5-10 mg / kg, about 6-10 mg / kg, about 7-10 mg / kg, about 8-10 mg / kg, or about 9 mg / kg of the subject's body weight.

[0227] In other cases, the composition (e.g., a composition comprising an oligonucleotide described herein) is administered in an amount that is effective for prophylactic or therapeutic purposes. In some cases, a viral vector (e.g., an rAAV vector) comprising an oligonucleotide described herein is administered in an amount of 10 or more per subject. 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 In some embodiments, the viral vector (e.g., an rAAV vector) is administered at a dose of 10 genome copies (GC). 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 or 10 14 In another example, the oligonucleotide is administered at a dose of 0.1 mg / kg to about 150 mg / kg (e.g., 0.5 mg / kg, 1 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg).

[0228] Optionally, the disclosed oligonucleotides can be administered as part of a pharma- ceutically acceptable composition suitable for delivery to a subject, as described herein. The disclosed agents are included in the composition in an amount sufficient to provide the desired dosage and / or elicit a therapeutically beneficial effect, as can be readily determined by one of skill in the art.

[0229] The disclosed compositions described herein can be administered in an amount (e.g., an effective amount) sufficient and for a sufficient time to treat a subject or to produce one of the aforementioned outcomes (e.g., reduction of one or more symptoms of a disease in a subject). The disclosed compositions can be administered one or more times. The disclosed compositions can be administered once a day, twice a day, three times a day, every other day, once a week, twice a week, three times a week, once every two weeks, once a month, once every two months, twice a year, or once a year. Treatment can be discrete (e.g., injections) or continuous (e.g., treatment via an implant or infusion pump). Subjects can be evaluated for therapeutic efficacy one week, two weeks, one month, two months, three months, four months, five months, six months, or more after administration of the disclosed compositions, depending on the composition and route of administration used for treatment. A subject may be treated for a discrete period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months) or until the disease or condition is alleviated, or, depending on the severity and nature of the disease or condition being treated, treatment may continue long term (e.g., for the life of the subject). For example, a subject diagnosed with PNH and treated with a composition disclosed herein may be administered one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additional therapeutic agents if an initial round or subsequent rounds of treatment do not elicit a therapeutic benefit, including reduction in any one of the symptoms associated with PNH, such as fatigue, weakness, shortness of breath, easy bruising or bleeding, recurrent infections, severe headaches, difficulty controlling blood clots and bleeding, or a reduction in the level of CFB RNA or CFB protein levels in the subject's cells or serum.

[0230] kit The present disclosure also features a kit comprising (a) a pharmaceutical composition comprising an oligonucleotide (e.g., RNAi oligonucleotide) agent that reduces the level and / or activity of CFB in a cell or subject as described herein, or a pharma- ceutically acceptable salt thereof, and optionally a pharma- ceutically acceptable carrier, excipient, or diluent. The kit may contain a vector encoding an oligonucleotide (e.g., RNAi oligonucleotide) as described herein, or a cell comprising a vector encoding an oligonucleotide (e.g., RNAi oligonucleotide) as described herein. The kit may also include a package insert containing instructions for carrying out any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition comprising an oligonucleotide (e.g., RNAi oligonucleotide) agent that reduces the level and / or activity of CFB in a cell or subject as described herein, (b) an additional therapeutic agent, and (c) a package insert containing instructions for carrying out any of the methods described herein. EXAMPLES

[0231] The following examples are for illustrative purposes only and are not intended to limit the present disclosure in any way.

[0232] Example 1: Preparation of RNAi oligonucleotides Oligonucleotide synthesis and purification The RNAi oligonucleotides described in this Example and the following Examples were chemically synthesized using methods described herein. Generally, RNAi oligonucleotides can be prepared using known phosphoramidite synthesis (see, e.g., Hughes and Ellington (2017) COLD SPRING HARB PERSPECT BIOL. 9(1):a023812; Beaucage SL, Caruthers MH Studies on Nucleotide Chemistry V: Deoxy nucleoside Phosphoramidites-A New Class of Key Intermediates for Deoxypolynucleotide Synthesis. Tetrahedron Lett. 22:1859-1862, 1981; doi:10.1016 / S0040-4039(01)90461-7), as well as solid-phase oligonucleotide synthesis methods such as those described for 19-23mer siRNAs (see, e.g., Scaringe et al. (1990) Nucleic Acids Res. 18:5433-5441 and Usman et al. 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).

[0233] RNAi oligonucleotides with a 19-mer core sequence were formatted into constructs with a 25-mer sense strand and a 27-mer antisense strand to allow processing by the RNAi machinery. The 19-mer core sequence was complementary to a region of the CFB mRNA.

[0234] Individual RNA strands were synthesized and HPLC purified according to standard methods (Integrated DNA Technologies; Coralville, IA). For example, RNA oligonucleotides were synthesized using solid-phase phosphoramidite chemistry, deprotected, and then desalted on a NAP-5 column (Amersham Pharmacia Biotech; Piscataway, NJ) using standard techniques (Damha & Olgivie (1993) METHODS MOL. BIOL. 20:81-114; Wincott et al. (1995) NUCLEIC ACID RES. 23:2677-2684). Oligomers were purified using ion-exchange high-performance liquid chromatography (IE-HPC) on an Amersham Source 15Q column (1.0 cm x 25 cm; Amersham Pharmacia Biotech) using a 15 min step linear gradient. The gradient varied from 90:10 buffer A:B to 52:48 buffer A:B, where buffer A was 100 mM Tris pH 8.5 and buffer B was 100 mM Tris pH 8.5, 1 M NaCl. Samples were monitored at 260 nm and peaks corresponding to full-length oligonucleotide species were collected, pooled, desalted on a NAP-5 column and then lyophilized.

[0235] The purity of each oligomer was determined by capillary electrophoresis on a Beckman PACE 5000 (Beckman Coulter, Inc.; Fullerton, CA). The CE capillary had an inner diameter of 100 μm and contained ssDNA 100 R gel (Beckman-Coulter). Typically, approximately 0.6 nmoles of oligonucleotide were injected into the capillary, run at an electric field of 444 V / cm, and detected by UV absorbance at 260 nm. Denaturing Tris-borate-7M-urea running buffer was purchased from Beckman-Coulter. Oligoribonucleotides that were at least 90% pure, as assessed by CE, were obtained for use in the experiments described below. The identity of the compounds was verified by matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry on a VOYAGER DE™ Biospectrometry Work Station (Applied Biosystems; Foster City, CA) following the manufacturer's recommended protocol. Relative molecular weights of all oligomers were obtained and were often within 0.2% of the predicted molecular weights.

[0236] Preparation of the double helix Single-stranded RNA oligomers were resuspended (e.g., at a concentration of 100 μM) in duplex buffer consisting of 100 mM potassium acetate, 30 mM HEPES, pH 7.5. Complementary sense and antisense strands were mixed in equimolar amounts to give a final solution of, e.g., 50 μM duplex. Samples were heated to 100°C in 5'RNA buffer (IDT) and allowed to cool to room temperature before use. RNAi oligonucleotides were stored at -20°C. Single-stranded RNA oligomers were lyophilized or stored in nuclease-free water at -80°C.

[0237] Example 2: Generation of CFB-targeting RNAi oligonucleotides Identification of CFB mRNA target sequences Complement factor B (CFB) is a protein involved in the alternative pathway of complement. To generate RNAi oligonucleotide inhibitors of CFB expression, a computer-based algorithm was used to computationally identify CFB mRNA target sequences suitable for assaying inhibition of CFB expression by the RNAi pathway. Over 300 RNAi oligonucleotide guide (antisense) strand sequences (see Table 3), each with a region of complementarity to the appropriate CFB target sequence in human CFB mRNA, were prepared and assayed in vitro for CFB expression inhibition. From these RNAi oligonucleotides, a subset of nine (see Table 4) were selected for further study. A subset of the nine guide sequences identified by the algorithm was also complementary to the corresponding CFB target sequence in monkey CFB mRNA (SEQ ID NO: 51; Table 3). CFB RNAi oligonucleotides containing a region of complementarity to a homologous CFB mRNA target sequence with nucleotide sequence similarity are predicted to have the ability to target the homologous CFB mRNA.

[0238] [Table 3]

[0239] Example 3: Identification of RNAi oligonucleotides that inhibit CFB expression in vivo RNAi oligonucleotides (formatted as DsiRNA oligonucleotides) designed to inhibit CFB expression were individually evaluated in vitro using a cell-based assay. The methods used to prepare the oligonucleotides are described in Example 1. The methods used to design and generate the CFB mRNA target sequence are described in Example 2.

[0240] In vitro cell-based assays The ability of each engineered RNAi oligonucleotide to reduce CFB mRNA was measured using an in vitro cell-based assay. Briefly, human hepatocyte (Huh7) cells expressing the endogenous human CFB gene were transfected with 1 nM of each RNAi oligonucleotide (compounds A-I) in separate wells of a multi-well cell culture plate. After transfection with the modified RNAi oligonucleotides, the cells were maintained for 24 hours, after which the amount of residual CFB mRNA from the transfected cells was measured using a TAQMAN®-based qPCR assay. Two qPCR assays, the 3' assay and the 5' assay, were used to determine CFB mRNA levels, measured using a PCR probe conjugated with 6-carboxyfluorescein (FAM), and normalized to the HPRT housekeeping gene. Each primer pair was assayed for residual CFB mRNA (%), as shown in Figure 3A. RNAi oligonucleotides that resulted in 8% or less CFB mRNA remaining in RNAi oligonucleotide-transfected cells compared to mock-transfected cells were considered RNAi oligonucleotide "hits."

[0241] Dose-response studies were performed by transfecting HuH-7 human hepatocytes expressing endogenous CFB with RNAi oligonucleotides at three different concentrations as indicated (0.03 nM, 0.1 nM, 1 nM; see FIG. 3B) in separate wells of a multi-well cell culture plate. Cells were maintained for 24 hours post-transfection and the levels of CFB mRNA remaining from the transfected cells were measured using a TAQMAN®-based qPCR assay. Two qPCR assays (3' and 5' assays) were used to determine mRNA levels as measured by HEX and FAM probes, respectively. Nine RNAi oligonucleotides (compounds A-I) were further tested in an in vivo screening assay.

[0242] Taken together, these results demonstrate that RNAi oligonucleotides designed to target human CFB mRNA inhibit expression of CFB in cells, as determined by a reduction in the amount of CFB mRNA in RNAi oligonucleotide-transfected cells relative to target cells. These results demonstrate that nucleotide sequences, including RNAi oligonucleotides, are useful for generating RNAi oligonucleotides that inhibit CFB expression. Furthermore, these results demonstrate that multiple CFB mRNA target sequences are suitable for RNAi-mediated inhibition of CFB expression (Table 4).

[0243] [Table 4]

[0244] Example 4: Screening of RNAi oligonucleotides in mice expressing human CFB cDNA (HDI mice) The ability of CFB targeting oligonucleotides to knockdown target mRNA was verified by the in vitro screening assay of Example 3. To confirm the ability of RNAi oligonucleotides to knockdown CFB in vivo, a HDI mouse model was used.

[0245] The oligonucleotides shown in Table 4 were evaluated in mice engineered to transiently express human CFB mRNA in hepatocytes of the mouse liver. Briefly, 6-8 week old female CD-1 mice (n=4-5) were subcutaneously administered the indicated RNAi oligonucleotides at doses of 0.25 mg / kg, 0.3 mg / kg, 0.5 mg / kg, 1 mg / kg, or 3 mg / kg formulated in PBS. Control mice (n=5) received PBS only. Three days (72 hours) later, mice were hydrodynamically injected (HDI) with a DNA plasmid (25 μg) encoding the entire human CFB gene (SEQ ID NO: 12) under the control of the ubiquitous cytomegalovirus (CMV) promoter sequence. One day after DNA plasmid transfer, liver samples were collected from HDI mice. Total RNA from these HDI mice was subjected to qRT-PCR analysis to measure CFB mRNA levels as described in Example 3. mRNA levels were measured for human mRNA. Values ​​were normalized for transfection efficiency using the NeoR gene contained in the DNA plasmid.

[0246] The results in Figures 4A-4C demonstrate that RNAi oligonucleotides designed to target human CFB mRNA inhibited human CFB mRNA expression in HDI mice, as determined by reduced levels of human CFB mRNA expression in liver samples from HDI mice treated with RNAi oligonucleotides compared to control HDI mice treated with PBS only. All RNAi oligonucleotides tested were able to reduce CFB expression. Overall, the HDI studies identified a number of potential RNAi oligonucleotides for inhibiting CFB expression in the liver.

[0247] Example 5: Screening of RNAi oligonucleotides in Cynomolgus Macaques All compounds A-I preselected during mouse screening were tested in cynomolgus macaques (NHPs) for duration of CFB mRNA silencing after administration of a single subcutaneous dose of 4 mg / kg as described in Example 4. Liver biopsies (n=5 / compound) of all animals tested were collected pre-dose and at days 28 and 56 post-injection. As shown in Figure 5, most compounds tested reduced hepatic CFB mRNA levels by at least 50% compared to normalized baseline levels and time-matched PBS controls as determined by RT-qPCR. Two lead compounds (compounds A and B) were selected for testing in a multiple dose study based on knockdown levels of CFB mRNA in cynomolgus macaque livers after a single dose.

[0248] Compounds A and B were selected from the single dose study for further evaluation in a multiple dose NHP study. Cynomolgus macaques were administered a total of four doses at 1 mg / kg or 2 mg / kg subcutaneously on days 0, 28, 56, and 84. Liver biopsies were collected pre-dose and 28, 56, and 112 days after the first treatment for evaluation of liver CFB mRNA levels by RT-qPCR (Figure 6A). Serum samples were collected pre-dose, 1, 14, 28, 42, 56, 70, 84, 98, and 112 days after the first treatment for evaluation of CFB protein levels by immunoblot kit (Figure 6B), complement activity by WIESLAB® AP assay (Figure 8), and rabbit red blood cell hemolysis (Figure 9; only compound B was tested in the hemolysis assay). PBS-treated animals were used as controls from the CFB liver mRNA, CFB serum protein, and function assays. Multiple treatment of cynomolgus macaques with Compounds A or B resulted in sustained maintenance of CFB mRNA silencing in the liver, a significant reduction in circulating CFB in serum, a >95% reduction in alternative pathway complement activity, and complete inhibition of rabbit red blood cell lysis in a hemolysis assay after multiple doses of Compounds A and B, as shown in Figures 6A, 6B, 8, and 9, respectively.

[0249] The potencies of Compounds A and B were calculated by combining the results at Day 28 for both single and multiple dose NHP studies. The approximate EDs for Compound A (0.65 mg / kg) and Compound B (0.65 mg / kg) were 50 was calculated from dose-response curves generated for both compounds (Figure 7).

[0250] Example 6: Pharmacokinetic and pharmacodynamic study of compound J on CFB expression in CD-1 mice CD-1 mice were administered Compound J to assess the percent knockdown of CFB mRNA in the mouse liver and the amount of CFB protein in the mouse serum upon Compound J administration. Compound J is an RNAi oligonucleotide that targets mouse CFB expression and serves as an alternative to RNAi oligonucleotides that target human CFB expression, such as Compound A and Compound B. The percent knockdown of liver CFB mRNA was measured using RT-qPCR as a result of Compound J administration. The amount of CFB in serum was instead measured by immunoblot. Mice received a single subcutaneous dose of Compound J at 0.25 mg / kg, 0.5 mg / kg, or 3 mg / kg.

[0251] A single dose of Compound J demonstrated a dose-dependent percentage knockdown of hepatic CFB mRNA, with a greater than 90% reduction in CFB mRNA in livers from animals receiving the 3 mg / kg dose (n=5 mice / time point). The nadir of mRNA knockdown was 3-21 days from the 3 mg / Kg dose, as shown in Figure 10A. The percentage of CFB protein in the serum of CD-1 mice was measured over the study period and was correspondingly suppressed (Figure 10B).

[0252] The amount of Compound J in plasma, spleen, liver and kidney tissues of CD-1 mice administered a single subcutaneous dose of 3 mg / kg of Compound J was measured using stem-loop qPCR for 672 hours after receiving the dose (Figure 11). Pharmacokinetic analysis showed that the highest exposure of Compound J was in the liver, followed by the spleen, kidney and plasma (Figure 11).

[0253] The percentage of hepatic CFB was also measured using RT-qPCR, and the amount of CFB protein in serum was quantitatively assessed by immunoblot over a 70-day period in CD-1 mice receiving four doses of Compound J at either 0.5 mg / kg or 3 mg / kg on days 0, 14, 28, and 42, as shown in Figures 12A and 12B, respectively. Liver biopsies and serum collections were performed on days 3, 14, 17, 28, 31, 42, 45, 56, and 70 after the first dose for animals receiving the high dose (3 mg / kg) of Compound J, and at the same time points (except collected on day 63 instead of day 70) for animals receiving the low dose (0.5 mg / kg) of Compound J. Liver and plasma concentrations of Compound J after four doses of 0.5 mg / kg were analyzed from liver biopsies and plasma samples using stem-loop qPCR (SL-qPCR), as shown in Figures 13A and 13B, respectively. PBS-treated CD-1 mice were used as controls for both CFB liver mRNA and CFB serum protein levels.

[0254] This multiple dose study revealed that Compound J (mouse surrogate) showed a dose-dependent knockdown of hepatic CFB mRNA that was sustained over a 70 day period. The reduction in circulating CFB protein levels corresponded to the observed reduction in CFB mRNA in the liver. Furthermore, plasma and liver concentrations of Compound J from treated animals showed no accumulation of Compound J when administered every other week (0.5 mg / kg) (see Figures 13A and 13B, respectively).

[0255] Additionally, a single-dose absorption, distribution, metabolism, and excretion (ADME) study was conducted in male CD-1 mice to characterize the pharmacokinetics of Compound B following a single subcutaneous dose of 3, 10, or 100 mg / kg or a single intravenous dose of 3 mg / kg. Plasma Compound B concentrations following single subcutaneous administration were T 100 / 100 / 100 / 200 / 100 at 1 hour for all three subcutaneous dose groups. max After a subcutaneous and intravenous dose of 3 mg / kg, the area under the plasma concentration-time curve from zero to the last measurable concentration (AUC last Bioavailability was approximately 22%, based on a comparison of the C (C ) and C (C ) concentrations. Plasma exposure increased approximately dose-proportionally in the 10 mg / kg group and more than dose-proportionally in the 100 mg / kg group compared to the 3 mg / kg dose group. Liver and kidney exposure was approximately 1.5 times higher than the maximum concentration observed after dosing (C ). max ) and AUC last Based on the results, the 10 mg / kg group showed an almost dose-proportional increase compared with the 3 mg / kg group, and the 100 mg / kg group showed a less-than-dose-proportional increase. The hepatic half-life was 3.94-4.98 days.

[0256] Example 7: Effect of Compound J on CFB expression in CAIA-induced arthritis mouse model The effect of compound J on the treatment of symptoms related to arthritis was examined using a collagen antibody-induced arthritis (CAIA)-induced arthritis mouse model, a simple model of rheumatoid arthritis. The CAIA-induced arthritis mouse model was generated by administering collagen antibodies to mice on day 0, followed by an LPS booster on day 3. Compound J was tested in both preventive and therapeutic studies. Animals were administered 1.5 or 3 mg / kg of compound J on day -7 for preventive studies (Figure 14A) and after disease onset on day 5 for therapeutic studies (Figure 14B). Hind paw inflammation was visually analyzed on day 10, and the results of both preventive and therapeutic studies are shown in Figures 15A and 15B, respectively. Preventive treatment with compound J prevented hind paw swelling, a unique feature of this model (Figure 15A). Therapeutic treatment with compound J completely reversed clinical disease expression after a single dose when compared to PBS-treated control animals (Figure 15B).

[0257] Hematoxylin and eosin (H&E) staining was performed on hind paw and knee biopsies, showing a reduction in local mononuclear cell infiltration in mice treated prophylactically with three doses of 3 mg / kg of Compound J (Figures 16 and 18). In addition, to demonstrate a reduction in local inflammation as a result of therapeutic treatment with a single dose of Compound J at 6 mg / kg, lymphocyte (CD45 positive cells), leukocyte (CD11b positive cells) and macrophage (F4 / 80 positive cells) marker staining was performed on biopsy samples as shown in Figures 19, 20 and 21, respectively. Biopsy samples were also stained with Safranin O to visualize knee cartilage in the CAIA-induced arthritis mouse model. Animals treated with 3 mg / kg Compound J showed a significant reduction in cartilage erosion compared to PBS-treated mice when treated prophylactically (Figures 17 and 18). To assess complement expression at the local inflammatory site in CAIA-induced arthritic mice with or without treatment with 3 mg / kg Compound J, experiments using in situ hybridization to CFB and CD45 mRNA were performed on biopsy samples, as shown in Figure 22. Hepatic knockdown of CFB by Compound J reduced lymphocyte (CD45 positive cell) infiltration and local CFB mRNA expression in therapeutic treatment with Compound J compared to PBS-treated animals as a control group.

[0258] Example 8: Effect of Compound J on CFB expression in a mouse model of multiple sclerosis. Myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mouse model can be used to investigate immune-mediated mechanisms of neuroinflammation and demyelination. MOG-induced EAE mice were prophylactically treated with a 3 mg / kg dose of compound J (n=2 experiments). Liver CFB mRNA levels and CFB protein in serum after treatment with compound J were assessed using RT-qPCR and immunoblot, respectively, as shown in Figures 25A and 25B. Similarly, the percentage of CFB mRNA remaining after treatment with compound J and the amount of CFB in serum of MOG-induced EAE mice after treatment with a 3 mg / kg dose of compound J were assessed compared to PBS-treated animals. Liver knockdown of CFB by compound J reduced disease severity (Figure 23).

[0259] Lumbar spinal cord samples were also obtained from MOG-induced EAE mice treated with Compound J. Luxol fast blue staining was performed on spinal cord samples along with H&E staining to visualize myelination as well as mononuclear cell infiltration, as shown in Figure 24. Luxol fast blue spinal cord samples were compared between diseased animals treated with 3 mg / kg Compound J, PBS, as shown in Figure 24. Compound J-treated MOG-induced animals showed a moderate reduction in demyelination and prevention of immune cell infiltration.

[0260] Example 9: Treatment of multiple sclerosis with Compound B in humans A subject suffering from multiple sclerosis may be treated with a pharmaceutical composition containing compound B (e.g., at a dose of about 3 mg / kg). The subject may be administered the composition, for example, by intramuscular injection, once a week for a period of about 12 months or more (e.g., until symptoms resolve or stabilize). The subject's symptoms and serum CFB level may be evaluated by a clinician about once a month to assess the effectiveness of compound B. The subject's serum CFB may be quantified using a serum sample and compared to the amount of CFB protein found in the subject's serum before administration of compound B or a control amount of CFB protein or the amount of CFB protein present in a serum sample from a normal subject (e.g., disease-free subject). Treatment with compound B may be determined to be effective if the amount of CFB protein in serum is reduced by at least 10% compared to the amount of CFB protein in serum before treatment with compound B. In addition, the subject's symptoms associated with multiple sclerosis, such as blurred vision, slurred speech, dizziness, percussion pain, lack of coordination and unsteady gait, can be evaluated by a clinician to assess whether there is a reduction in any or all of the symptoms experienced by the subject compared to the symptoms experienced by the subject before administration of Compound B.

[0261] Example 10: Treatment of Arthritis with Compound B in Humans A subject diagnosed with arthritis may be treated with a pharmaceutical composition containing compound B (e.g., at a dose of about 1.5 mg / kg). The subject may be administered the composition, for example, by intramuscular injection, at a frequency of about once a month for a period of about 6 months or more (e.g., until symptoms resolve or stabilize). For example, every one or two months, the subject may be evaluated by a clinician (e.g., by evaluating the subject's symptoms and / or serum CFB level) to assess the effectiveness of compound B. The subject's serum CFB may be quantified using a serum sample and compared to the amount of CFB protein found in the subject's serum before compound B is administered or a control amount of CFB protein or the amount of CFB protein present in a serum sample from a normal subject (e.g., disease-free subject). Treatment with compound B may be determined to be effective if the amount of CFB protein in serum is reduced, i.e., reduced by at least 10%, compared to the amount of CFB protein in serum before treatment with compound B. In addition, the clinician can assess the subject's symptoms associated with arthritis, such as pain, stiffness, swelling, redness, and reduced range of motion, to assess whether there is a reduction in any or all of the symptoms experienced by the subject compared to the symptoms experienced by the subject before administration of Compound B.

[0262] Example 11: Evaluation of Toxicity in Cynomolgus Monkeys and Mice The safety and tolerability of subcutaneously administered Compound B was evaluated in cynomolgus monkeys and mice.

[0263] In a safety pharmacology study, increasing doses of Compound B (30, 100, and 300 mg / kg) were administered to the same cynomolgus monkeys once every 7 days after subcutaneous administration of 0.9% sodium chloride. No cardiovascular or neurological effects were observed at any dose level. At 300 mg / kg, minimal minute volume and tidal volume reductions were observed during respiratory evaluation. These respiratory findings were minimal in severity and were considered non-toxic as all animals maintained good health throughout the study. Therefore, the no observed adverse effect level (NOAEL) in this study was determined to be 300 mg / kg.

[0264] Genotoxicity assessments, including in vitro micronucleus assays and in vitro bacterial reverse mutation assays, were negative for induction of micronuclei and mutagen activation, respectively.

[0265] A 6-month mouse study assessed potential toxicity of repeated subcutaneous administration of Compound B (0, 30, 100, 300 mg / kg every 4 weeks for a total of 7 doses) and the reversibility, persistence, or delayed onset of any effects after an 8-week recovery period. The range of dose levels in this study was selected to achieve an exposure multiple of at least 10-fold greater than the expected exposure at the highest intended clinical dose. The toxicokinetic properties of Compound B were also determined. Non-toxic clinical pathology findings included increases in neutrophil (2.08-fold and 1.39-fold, respectively), monocyte (2.90-fold and 2.38-fold, respectively), and lymphocyte (1.92-fold and 1.35-fold, respectively) counts, which led to increases in total white blood cell counts (1.94-fold and 1.38-fold, respectively) in males and females in the 300 mg / kg / day group at day 171, and decreases in cholesterol concentrations (62.9-fold) in males in the 300 mg / kg / day group at day 171. Complete reversibility of all clinical pathology findings was evident at the end of the recovery period. Non-toxic microscopic findings included: hepatocellular karyocytic hypertrophy with associated increase in liver / gallbladder weight, mixed cell inflammation in the liver, intracytoplasmic basophilic granules and subcutaneous infiltration of histiocytes in the kidney, mixed cell inflammation in the dermis, and degeneration, necrosis, and / or regeneration of the muscularis carnosus at the injection site at terminal euthanasia due to hepatocellular karyocytic hypertrophy, mixed cell inflammation in the liver, subcutaneous infiltration of histiocytes, and degeneration, necrosis, and / or regeneration of the muscularis carnosus at the injection site that was still present at the time of recovery euthanasia.

[0266] Hepatic findings in these animals included microvesicular fatty changes, single cell necrosis, and karyocytic hypertrophy. As the liver was considered the target tissue during terminal euthanasia, the hepatic changes seen in these deceased mice were considered potentially related to Compound B. In addition to the deaths in the two main studies, there were two early deaths in the toxicokinetic group with unknown relevance to Compound B. These animals were either found dead (female in the 300 mg / kg group on day 14) or euthanized due to moribundity (male in the 30 mg / kg group on day 99). The only macroscopic finding from these animals that may be related was a pale discolored liver in a male in the 30 mg / kg group. Based on the mortality observed in females in the 100 mg / kg and 300 mg / kg groups in the main study, the NOAEL was considered to be 30 mg / kg for females and 300 mg / kg for males. These doses corresponded to mean plasma AUClast values ​​of 17,400 and 609,000 hr*ng / mL and mean Cmax values ​​of 6490 and 140,000 ng / mL for females in the 30 mg / kg / day group and males in the 300 mg / kg group, respectively, on Day 169.

[0267] A 9-month monkey study evaluated potential toxicity from repeated subcutaneous dosing (10 doses of Compound B at 0, 30, 100, or 300 mg / kg every 4 weeks) and reversibility, persistence, or delayed onset of any effects after an 8-week recovery period. The range of dose levels in the study was selected to achieve an exposure multiple of at least 10-fold greater than the expected exposure at the highest intended clinical dose. The toxicokinetic and pharmacodynamic effects of Compound B were evaluated. Minimal to moderate non-toxic microscopic findings of eosinophilic hepatocytes in the liver and vacuolated / granular macrophages in multiple tissues (gastrointestinal system, genitourinary system, lymphatic system, brain (choroid plexus), eye (choroid or ciliary body), heart, adrenal gland, thyroid, skin, skeletal muscle, femoro-tibial joint, and / or subcutaneous dosing site) were observed at the end of the dosing phase of the study at ≥30 mg / kg. These findings persisted at low incidence and / or severity after the end of the recovery phase, indicating that incomplete recovery is still ongoing. Exposure at all dose levels produced expected and correlated pharmacodynamic effects, as demonstrated by a >95% reduction in serum CFB protein concentration and a >95% reduction in alternative pathway complement function activity by day 28, and a >89% reduction in hepatic CFB mRNA expression after euthanasia. As no toxic findings were observed after the dosing period, the NOAEL was determined to be 300 mg / kg, with associated AUC last The mean HR was 1,360,000 hr*ng / mL and the Cmax was 66,200 ng / mL (for both males and females, day 253).

[0268] Example 12. CFB evaluation assay A variety of assays can be used to characterize the effect of Compound B on CFB levels, including assessment of Compound B in plasma or tissues, the WIESLAB® complement function activity assay, assessment of circulating CFB, CFB mRNA expression levels and pharmacokinetic assays, as described herein.

[0269] Compound B in plasma Plasma concentrations of compound B in mice and monkeys were measured by high performance liquid chromatography-fluorescence detection (HPLC-FD) analysis. The analyte (compound B) in 30 μL of plasma sample was enzymatically treated with proteinase K, hybridized with a fluorescent probe (peptide nucleic acid; 22-mer peptide nucleic acid PNA probe) having sequence complementarity with the antisense strand of compound B, and 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™ PA200 analytical column. The mobile phases were 30% acetonitrile (25 mM Tris HCl, 1 mM EDTA, 2 M urea) for mobile phase A and 1 M NaClO4 in mobile phase A for mobile phase B. The FL detector monitored the signal from 436 nm (Ex) to 484 nm (Em). The concentrations of Compound B were calculated using LabSolutions 6.70 with linear regression using least squares (1 / c2 weighting) over a quantification range of 2.00 ng / mL to 2000 ng / mL, with the lower and upper limits of these ranges defined as the lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ), respectively. This assay was used, for example, in Examples 5 and 6 above.

[0270] Compound B in tissue Compound B concentrations in the liver and kidney of both monkeys and mice were measured by HPLC-FD analysis. Compound B in the tissue samples was enzymatically treated with proteinase K using 2.5 mg tissue samples in 50 μL aliquots (tissue homogenate), followed by hybridization with a 22-mer PNA probe having a sequence complementary to the antisense strand of compound B. The treated samples were injected into an HPLC equipped with a fluorescence detector. Chromatographic separation was performed using a gradient system on a Shimadzu Prominence system using a DNAPAC™ PA200 analytical column. The mobile phases were: mobile phase A was 30% acetonitrile (25 mM Tris HCl, 1 mM EDTA, 2 M urea), and mobile phase B was 1 M NaClO4 in mobile phase A. The concentrations of Compound B were calculated using LabSolutions 6.89 with linear regression using the least squares method (including 1 / c2 weighting) over a quantification range of 30.0 ng / mL to 20,000 ng / mL, with the lower and upper limits of these ranges defined as the LLOQ and ULOQ, respectively. This assay was used, for example, in Examples 5 and 6 above.

[0271] WIESLAB® Complement Function Activity Assays (CCP, CAP, CLP) Classical pathway (CCP), CAP and lectin pathway activity were assessed using WIESLAB® Complement System Screening Assays using labeled antibodies specific for neoantigens to detect human terminal complement complex (C5b 9) complexes resulting from complement activation. These assays can also detect cynomolgus monkey C5b-9. The amount of neoantigen generated was proportional to the level of functional activity of the individual pathways. The wells of the assay microtiter strips were coated with specific activators of the classical or alternative or lectin pathways. Monkey serum samples were diluted in diluents containing blockers to ensure that only the respective pathway was activated. The wells were washed and C5b-9 was detected using specific alkaline phosphatase labeled antibodies against the expressed neoantigens. The amount of complement activation correlated with the intensity of color development measured by absorbance at 405 nm. The value of the positive control provided in the test kit was defined as 100% complement activation. All measurements were expressed as percent (%) complement activity and were determined as follows: [(sample-negative control) / (positive control-negative control)]*100 This assay was used, for example, in Example 5 above.

[0272] Protein levels The amount of factor B protein levels in circulating cynomolgus monkeys was assessed by Western blot using measurements of relative CFB serum protein concentration normalized to transferrin (TF) levels in monkey samples. Diluted serum samples mixed in sample buffer were combined with the fluorescent master mix. Samples were boiled at 95°C for 5 minutes, vortexed, and spun down. Samples were placed on ice and run on Western blots. Quantification was performed using ProteinSimple software according to the manufacturer's instructions. The degree of CFB protein reduction in treatment groups was calculated as a percentage of expression relative to the mean level of the PBS-treated control group on the same test day, with the monkey CFB level in the PBS-treated control group set as 100%. Graphs of mean ± standard deviation were generated and data were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA). Unpaired t-tests were performed to compare monkey CFB protein levels in compound B treatment groups with the PBS-treated control group on the same test day.

[0273] Assessment of circulating factor B protein levels was also assessed using a factor B enzyme-linked immunosorbent assay (ELISA) kit designed for the quantitative measurement of complement factor B concentrations in humans. Complement factor B specific antibodies were precoated on 96-well plates and blocked. Standards or test samples were added to the wells, followed by addition of complement factor B specific biotinylated detection antibodies, followed by subsequent washing with wash buffer. Streptavidin-peroxidase conjugate was added, and unbound conjugate was washed away with wash buffer. Tetramethylbenzidine (TMB) was then used to visualize the streptavidin-peroxidase enzymatic reaction. TMB was catalyzed by streptavidin-peroxidase to produce a blue product that turned yellow after the addition of an acidic stop solution. The density of the yellow coloration was directly proportional to the amount of complement factor B captured in the plate. The back-calculated concentrations of the samples were determined by a curve-fitting regression program generated by calibration standards. This assay was used, for example, in Example 5 above.

[0274] CFB mRNA expression level The amount of factor B in cynomolgus monkey liver was determined by measuring factor B mRNA expression relative to peptidyl-prolyl-cis-trans isomerase B (PPIB) mRNA expression in RNA isolated from cynomolgus monkey liver using a duplex real-time quantitative polymerase chain reaction (qPCR) assay after reverse transcription. First, mRNA was isolated from frozen liver tissue and quantified. The mRNA was then transcribed into complementary DNA (cDNA). This cDNA was then used as a template for a qPCR reaction to measure factor B mRNA levels by normalization to PPIB. The amount of factor B mRNA expression in the treatment group was calculated as a percentage of expression (normalized to PPIB mRNA levels) relative to the untreated or pre-treatment group, with factor B mRNA expression in the control group set to 100%. This assay was used, for example, in Examples 5 and 11 above.

[0275] Pharmacokinetic assays The concentration of compound B in human plasma is measured by HPLC-FD analysis. The analyte (compound B) in 30 μL of plasma sample is enzymatically treated with proteinase K, then hybridized with a fluorescent probe (peptide nucleic acid; 22-mer PNA probe) having sequence complementarity with the antisense strand of compound B, and injected into an HPLC equipped with a fluorescence detector. Chromatographic separation is performed using a gradient system on a Shimadzu Prominence system using a DNAPAC™ PA200 analytical column. The mobile phases were: mobile phase A was 30% acetonitrile (25 mM Tris HCl, 1 mM EDTA, 2 M urea), and mobile phase B was 1 M NaClO4 in mobile phase A. The FL detector monitored the signal from 436 nm (Ex) to 484 nm (Em). Compound B concentrations were calculated using LabSolutions 6.70 with linear regression using least squares (1 / c2 weighting) over a quantification range of 2.00 ng / mL to 2000 ng / mL, with the lower and upper limits of these ranges defined as the LLOQ and ULOQ, respectively.

[0276] Anti-drug antibody assay Anti-drug antibody (ADA) assays for Compound B can be performed using human serum and an electrochemiluminescence (ECL) bridging assay. Positive controls (PC) are made from rabbits immunized against an immunogenic cocktail consisting of keyhole limpet hemocyanin (KLH)-conjugated Compound B and KLH-conjugated oligonucleotides of various lengths corresponding to modified Compound B sequences. PCs, negative controls (NCs) and test samples can be subjected to an acid dissociation step at ambient room temperature and then added to a plate containing TRIS, biotin-Compound B and ruthenium-labeled Compound B to allow the formation of a crosslinked complex between the labeled Compound B present in the sample and the Compound B antibody. After incubation, the NCs, PCs 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 crosslinked complex. The plate may then be washed and Meso Scale Discovery® (MSD®) Read Buffer added to generate an ECL signal that is directly proportional to the amount of ADA present in the sample.

[0277] Example 13: Effect of Compound J in a rat model of membranous nephropathy The effect of Compound J on symptoms associated with membranous nephropathy was tested using a passive Heymann nephritis (PHN) rat model, a simple model of membranous nephropathy. The PHN rat model was generated by administering a single dose of sheep anti-rat FX1a antibody to rats on day 0.

[0278] Compound J was tested in a prophylactic study in which animals were administered 12 mg / kg of Compound J on days -14, -7 and 0 (Figure 26). Renal function was assessed as the ratio of protein:creatinine levels measured from urine samples collected daily from the animals, as shown in Figure 26. Prophylactic treatment with Compound J prevented the development of proteinuria, a hallmark of this model, compared to PBS-treated PHN control animals (Figure 26).

[0279] Serum samples were collected on day -1 before disease induction and on day 6 after disease onset to assess complement activity by hemolysis of rabbit red blood cells (Figure 27). Healthy and PBS-treated PHN animals served as controls for the CFB functional assay. PHN rats after administration of 2 doses (day 1 serum) or 3 doses (day 6) of Compound J showed a >95% reduction in alternative pathway complement activity, as measured by hemolysis of rabbit red blood cells in a hemolysis assay, compared to the hemolysis levels observed from both healthy or PBS-treated PHN control animals (Figure 27). PBS was administered in the same multiple dose regimen to constitute the disease control group, and sham animals served as healthy controls.

[0280] Other embodiments All publications, patents, and patent applications mentioned in this specification are incorporated 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 some embodiments have been described herein, those skilled in the art will recognize that further modifications and embodiments are encompassed and may be applied to the essential features described above and set forth in the following claims, including variations, uses, or modifications that generally follow the principles described herein, and including such departures from the present disclosure that are within the scope of known or customary practice in the art.

Claims

1. 1. An RNAi oligonucleotide or a pharmaceutically acceptable salt thereof for reducing complement factor B (CFB) expression, wherein the oligonucleotide comprises a sense strand and an antisense strand, the sense strand and the antisense strand form a double helix region, the antisense strand comprises a region of complementarity to a CFB mRNA target sequence of SEQ ID NO: 13 or 14, and the region of complementarity is at least 15 contiguous nucleotides in length.

2. 2. The RNAi oligonucleotide of claim 1, or a pharmaceutically acceptable salt thereof, wherein the region of complementarity is at least 19 contiguous nucleotides in length, optionally at least 20 nucleotides in length.

3. 2. The RNAi oligonucleotide of claim 1, or a pharmaceutically acceptable salt thereof, wherein the 3'-end of the sense strand comprises a stem-loop described as S1-L-S2, wherein S1 is complementary to S2, and L forms a loop of 3 to 5 nucleotides in length between S1 and S2, e.g., L is a triloop or tetraloop, e.g., L is a tetraloop.

4. 2. The RNAi oligonucleotide of claim 1, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises at least one modified nucleotide, for example, the oligonucleotide comprises 20 to 50 modified nucleotides, or all of the nucleotides of the oligonucleotide are modified.

5. 5. The RNAi oligonucleotide or a pharmaceutically acceptable salt thereof of claim 4, wherein the modified nucleotides comprise a 2'-modification, e.g., all of nucleotides 1-7, 12-27, and 31-36 of the sense strand and all of nucleotides 1, 6, 8, 9, 11-13, and 15-22 of the antisense strand are modified with 2'-O-methyl, e.g., 2'-O-methyl ribonucleosides.

6. The RNAi oligonucleotide of claim 5, or a pharmaceutically acceptable salt thereof, wherein the RNAi oligonucleotide comprises 5 to 15 2'-fluoro modifications, e.g., all of nucleotides 8, 9, 10, and 11 of the sense strand and all of nucleotides 2, 3, 4, 5, 7, 10, and 14 of the antisense strand are modified with 2'-fluoro, e.g., 2'-fluorodeoxyribonucleosides.

7. 2. The RNAi oligonucleotide of claim 1, or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide comprises at least one modified internucleotide bond, e.g., the at least one modified internucleotide bond is a phosphorothioate bond, e.g., the RNAi oligonucleotide has a phosphorothioate bond between nucleotides 1 and 2 of the sense strand and nucleotides 1 and 2, 2 and 3, 20 and 21, and 21 and 22 of the antisense strand.

8. 2. The RNAi oligonucleotide or a pharmaceutically acceptable salt thereof of claim 1, wherein there is no internucleotide bond between the sense strand and the antisense strand.

9. 2. The RNAi oligonucleotide or a pharmaceutically acceptable salt thereof of claim 1, wherein the 4'-carbon of the sugar of the 5'-nucleotide of the antisense strand comprises a phosphate analog.

10. The RNAi oligonucleotide comprises a uridine at position 1 of the 5' end of the antisense strand, e.g., the uridine comprises a phosphate analog, e.g., the phosphate analog is 4'-O-monomethylphosphonate, e.g., the uridine comprising the phosphate analog has the following structure: 【Chemistry 1】 The RNAi oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof, comprising:

11. 2. The RNAi oligonucleotide of claim 1, or a pharmaceutically acceptable salt thereof, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands, e.g., each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

12. 12. The RNAi oligonucleotide of claim 11, or a pharmaceutically acceptable salt thereof, wherein the RNAi oligonucleotide comprises one to five 2'-O-N-acetylgalactosamine (GalNAc) moieties conjugated to the sense strand, e.g., up to four nucleotides of L of the stem-loop are conjugated to monovalent GalNAc moieties, e.g., one or more of the nucleotides at nucleotide positions 28-30 of the sense strand are conjugated to monovalent GalNAc moieties, e.g., each of the nucleotides at positions 28-30 of the sense strand is conjugated to a monovalent GalNAc moiety.

13. The nucleotides at positions 27 to 30 of the sense strand have the following structure: 【Chemistry 2】 13. The RNAi oligonucleotide of claim 12, or a pharmaceutically acceptable salt thereof, comprising:

14. The RNAi oligonucleotide of claim 1 or a pharmaceutically acceptable salt thereof, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 4 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:

6.

15. 2. The RNAi oligonucleotide of claim 1, or a pharmaceutically acceptable salt thereof, wherein the sense strand comprises the nucleotide sequence set forth in SEQ ID NO: 37 and the antisense strand comprises the nucleotide sequence set forth in SEQ ID NO:

38.

16. A pharmaceutical composition comprising the RNAi oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 15, and a pharmaceutically acceptable carrier, excipient or diluent.

17. Paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), immunoglobulin A nephropathy (IgAN), membranous nephropathy (MN), Escherichia coli (E. coli)-induced or typical hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration, geographic atrophy, diabetic retinopathy, uveitis, intermediate uveitis, Behçet's uveitis, retinitis pigmentosa, macular edema, multifocal choroiditis, Vogt-Koyanagi-Harada syndrome, shotgun choroidoretinopathy, sympathetic ophthalmia, ocular scars Cicatricial pemphigoid, ocular pemphigoid, non-arthritic ischemic optic neuropathy, postoperative inflammation, retinal vein occlusion, neuropathy, multiple sclerosis, stroke, Guillain-Barré syndrome, traumatic brain injury, Parkinson's disease, hemodialysis complications, hyperacute graft rejection, xenograft rejection, interleukin-2-induced toxicity during IL-2 therapy, inflammatory diseases, inflammation in autoimmune diseases, Crohn's disease, adult respiratory distress syndrome, myocarditis, post-ischemic reperfusion states, myocardial infarction, balloon angioplasty, cardiopulmonary bypass or renal bypass Post-pump syndrome, atherosclerosis, hemodialysis, renal ischemia, mesenteric artery reperfusion after aortic reconstruction, infection or sepsis, immune complex disorders and autoimmune diseases, rheumatoid arthritis, systemic lupus erythematosus (SLE), SLE nephritis, proliferative nephritis, liver fibrosis, hemolytic anemia, myasthenia gravis, tissue regeneration, nerve regeneration, dyspnea, hemoptysis, acute respiratory distress syndrome (ARDS), asthma, chronic obstructive pulmonary disease (COPD), emphysema, pulmonary embolism and infarction, pneumonia, fibrous dust diseases, pulmonary fibrosis, allergies , bronchoconstriction, hypersensitivity pneumonitis, parasitic diseases, Goodpasture's syndrome, pulmonary vasculitis, microimmune vasculitis, immune complex-associated inflammation, antiphospholipid syndrome, glomerulonephritis, obesity, arthritis, autoimmune heart disease, inflammatory bowel disease, ischemia-reperfusion injury, Baraker-Simons syndrome, antineutrophil cytoplasmic antibody (ANCA) vasculitis, cryoglobulinemia, psoriasis, transplantation, central nervous system diseases such as Alzheimer's disease and other neurodegenerative conditions, dense deposition diseases, bullous skin diseases, membranoproliferative glomerulonephritis type II (MPGN)II), chronic graft-versus-host disease, Felty's syndrome, pyoderma gangrenosum (PG), hidradenitis suppurativa (HS), pulmonary arterial hypertension, primary Sjogren's syndrome, primary biliary cholangitis, autosomal dominant polycystic kidney disease and myelin oligodendrocyte glycoprotein antibody disease (MOGAD), for example, for the prevention or treatment of rheumatoid arthritis.

18. The RNAi oligonucleotides include pharmaceutically acceptable salts, for example, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfonate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, and oxalate.

16. The RNAi oligonucleotide of any one of claims 1 to 15, wherein the pharmaceutically acceptable salt is or comprises a salt of: palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, methylamine, dimethylamine, trimethylamine, triethylamine, or ethylamine; or an alkali or alkaline earth metal salt, e.g., the alkali or alkaline earth metal salt is selected from the group consisting of sodium, lithium, potassium, calcium, magnesium, and ammonium (e.g., quaternary ammonium and tetramethylammonium), e.g., the pharmaceutically acceptable salt is a sodium salt.

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