Molecules and compositions for inhibiting expression of complement factor b

Abstract

Disclosed herein is a double stranded RNA (dsRM A) molecule for RNA interference. A method for inhibiting the expression of complement factor B (CFB) and a method for treating a complement-related disease are also provided.

Description

MOLECULES AND COMPOSITIONS FOR INHIBITING EXPRESSION OF COMPLEMENT FACTOR BPRIORITY INFORMATION

[0001] The subject application claims priority to and benefit of U. S. Provisional Patent Application No. 63 / 738,910, filed December 26.2024, the content of which is incorporated herein by reference in its entirety’.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is submitted electronically in.xml format and is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure in general relates to inhibition of gene expression; more particularly, to molecules and compositions for inhibiting expression of complement factor B (CFB).BACKGROUND OF THE INVENTION

[0004] IgA nephropathy (IgAN) is the most common cause of chronic glomerulonephritis, predominantly affecting young and middle-aged adults. Approximately one-third of those diagnosed with the condition progress to end-stage renal disease within two decades. Currently, there are no effective treatments that address the root causes of IgA nephropathy.

[0005] Recently, there has been growing interest in the role of the complement system in IgA nephropathy. Kidney' biopsies from patients with IgAN have revealed the presence of various complement proteins, including mannose-binding lectin (MBL) from the lectin pathway and properdin from the alternative pathway. It is thought that complement factor D hydrolyzes factor B into two fragments: the smaller factor Ba and the larger factor Bb. Factor Bb then associates with factor C3b to form C3 convertase, which further hydrolyzes factor C3 into factor C3a and factor C3b. The binding of factor C3b with factor Bb leads to additional hydrolysis of factor C3, creating an amplification loop. However, the development of drugs targeting complement system proteins is complicated by challenges such as high protein concentrations and rapid metabolism, which necessitate that traditional small molecules or antibody drugs achieve high concentrations in the body to maintaineffective dosing. This presents limitations for the development of drugs related to the complement system.SUMMARY OF THE INVENTION

[0006] As embodied and broadly described herein, a double stranded RNA (dsRNA) molecule for RNA interference is provided. A method for inhibiting the expression of complement factor B and a method for treating a complement-related disease are also provided.

[0007] In certain embodiments, siRNA therapies may be utilized, providing prolonged therapeutic effects in comparison to small-molecule or antisense oligonucleotide (ASO) drugs, which could greatly improve patient adherence to treatment.

[0008] In one aspect, the present disclosure provides a double stranded RNA molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleic acid sequence of any one of SEQ ID NOs: 79, 153, 184, 227, 39, 121, 145. 171.182, 186, 225, 1, 3, 5. 7, 9, 11, 13, 15, 17, 19, 21, 23, 25. 27. 29, 31, 33, 35, 37, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 123, 125, 127, 129, 131, 133, 135, 137, 139. 141, 143, 147, 149, 151. 155, 157, 159, 161, 163. 165, 167. 169. 173, 175, 177, 179, 183, 185, 187, 188, 194. 195. 209, 210, 226, and 227 or a substantially similar sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0009] In some embodiments of the disclosure, the sense strand comprises a nucleic acid sequence of any one of SEQ ID NOs: 79, 153, 184. 227, 39, 121, 145, 171, 182, 186, and 225 or a substantially similar sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0010] In some further embodiments of the disclosure, the sense strand comprises a nucleic acid sequence of any one of SEQ ID NOs: 79, 153. 184 and 227 or a substantially similar sequence having at least 90%, 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, or 99% sequence identity.

[0011] In one aspect, the present disclosure provides a dsRNA molecule comprising a sense strand and an antisense strand, wherein the antisense strand comprises a nucleic acid sequence of any one of SEQ ID NOs: 80. 154, 216. 40. 122, 146. 172, 2, 4. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36. 38, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108. 110, 112, 114. 116, 118, 120, 124, 126. 128, 130, 132, 134, 136. 138, 140, 142, 144, 148, 150,152, 156, 158, 160, 162, 164, 166, 168, 170, 174, 176, 178, 180, 189, 190, 191, 192, 193, 196, 197. 198, 199, 200, 201, 202. 203, 204, 205, 206, 207. 208, 211, 212. 213, 214, 215, 217, 218, 219, 220, 221, 222, 223, and 224 or a substantially similar sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0012] In some embodiments of the disclosure, the antisense strand comprises a nucleic acid sequence of any one of SEQ ID NOs: 80, 154, 216, 40, 122, 146, and 172 or a substantially similar sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%, or 99% sequence identity

[0013] in some further embodiments of the disclosure, the antisense strand comprises a nucleic acid sequence of any one of SEQ ID NOs: 80, 154 and 216 or a substantially similar sequence having at least 90%, 91%, 92%. 93%. 94%, 95%, 96%, 97%. 98%, or 99% sequence identity.

[0014] In some embodiments, the dsRNA molecule comprises a combination of the sense strand and antisense strand comprising nucleic acid sequences of SEQ ID NOs: 79 and 80, 153 and 154, 184 and 154, 184 and 216, 227 and 154, 227 and 216, 1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10. 11 and 12, 13 and 14, 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70. 71 and 72. 73 and 74. 75 and 76, 77 and 78, 81 and 82, 83 and 84, 85 and 86. 87 and 88. 89 and 90, 91 and 92, 93 and 94. 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112, 113 and 114. 115 and 116. 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130, 131 and 132, 133 and 134, 135 and 136, 137 and 138, 139 and 140, 141 and 142, 143 and 144, 145 and 146, 147 and 148, 149 and 150, 151 and 152. 155 and 156, 157 and 158, 159 and 160, 161 and 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, 171 and 172, 173 and 174, 175 and 176, 177 and 178, 179 and 180, 182 and 146, 183 and 150, 185 and 170, 186 and 172, 187 and 174, 188 and 176. 182 and 189, 182 and 190, 182 and 191, 182 and 192, 182 and 193, 194 and 189, 195 and 189, 182 and 196, 182 and 197, 182 and 198, 182 and 199, 182 and 200, 182 and 201, 182 and 202, 182 and 203, 182 and 204, 182 and 205, 184 and 206, 184 and 207, 184 and 208, 209 and 207, 210 and 207, 184 and 211, 184 and 212, 184 and 213, 184 and 214, 184 and 215, 184 and 217, 184 and 218, 186 and 219, 186 and 220, 186 and 221, 186 and 222, 186 and 223, or 186 and 224 or a substantially similarsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0015] In some embodiments of the disclosure, the dsRNA comprises a combination of the sense strand and antisense strand comprising nucleic acid sequences of SEQ ID NOs: 79 and 80, 153 and 154, 184 and 154, 184 and 216, 227 and 154, 227 and 216, 39 and 40, 121 and 122, 145 and 146, 171 and 172, 182 and 146, 186 and 172, or 225 and 146. or a substantially similar sequence having at least 90%. 91%. 92%. 93%. 94%. 95%, 96%. 97%, 98%, or 99% sequence identity’.10016] In some embodiments, the dsRNA molecule is modified. In some embodiments, at least one nucleotide of the sense strand and / or the antisense strand is modified. Examples of the at least one of the modified nucleotides include, but are not limited to, a deoxy-nucleotide, a 3'-terminal deoxythimidine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'- fluoro modified nucleotide, a 2'-deoxy -modified nucleotide, a locked nucleotide (LNA), an unlocked nucleotide, hexitol nucleotide (HNA), a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-ammo-modified nucleotide, a 2'-O- allyl-modified nucleotide. 2'-C-alkyl-modified nucleotide, a 2' -methoxy ethyl modified nucleotide, a 2'-C-allyl-modified nucleotide, a 2'-hydroxyl-modified nucleotide, a 2'-O- alkyl -modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, a nucleotide comprising a 5'-phosphate mimic, a nucleotide comprising a 2'-phosphate group, e.g., cytidine-2'-phosphate (C2p); guanosine-2'- phosphate (G2p); uridine-2'-phosphate (U2p); adenosine-2'-phosphate (A2p); a thermally- destabilizing nucleotide e.g., an abasic modification; a mismatch with the opposing nucleotide in the duplex; and destabilizing sugar modification, a 2'-deoxy modification, an acyclic nucleotide, an unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA), a glycol modified nucleotide (GNA), e.g., Ggn, Cgn, Tgn, or Agn, and a 2-O-(N- methylacetamide) modified nucleotide; and combinations thereof.

[0017] In some embodiments, the antisense strand comprises a 3' overhang of at least 2 to 5 nucleotides.

[0018] In some embodiments, a duplex region between the sense strand and the antisense strand comprises 15 to 25, 16 to 24, 17 to 23, 18 to 22, 19 to 21, or 20 nucleotides.

[0019] In some embodiments, a duplex region between the sense strand and the antisense strand comprises 1, 2, or 3 mismatches.

[0020] In some embodiments, the antisense strand is complementary to the mRNA of complement factor B

[0021] In some embodiments, the antisense strand is complementary to the mRNA of CFB and comprises 1, 2, or 3 mismatches.

[0022] In some embodiments, the first nucleotide at the 5’ end of the antisense strand is adenine or uridine, or analogs thereof.

[0023] in some embodiments, the dsRNA molecule further comprises a ligand conjugated to the 3' end or the 5’ end of the sense strand.. An example of the ligand is a lectin, glycoprotein, lipid or protein or a derivative thereof. In some embodiments of the disclosure, the ligand is an N-acetylgalactosamine (GalN Ac) or a derivative thereof.

[0024] In some embodiments, the ligand is a trivalent sequential GalNAc linker. In some further embodiments, the ligand is shown as Formula (I),Formula (I);wherein X is O or S.

[0025] In some embodiments, the dsRNA molecule further comprises at least one phosphorothioate or methylphosphonate intemucleotide linkage.

[0026] In one aspect, the present disclosure provides a pharmaceutical composition comprising the dsRNA molecule as disclosed herein and optionally a delivery vehicle.

[0027] Examples of the delivery vehicle include, but are not limited to, lipid nanoparticles, polymers, or micelles.

[0028] In one aspect, the present disclosure provides a method for inhibiting the expression of CFB in a cell comprising contacting the dsRNA molecule or pharmaceutical composition as disclosed herein with the cell. In some embodiments, the method is an in vitro method.

[0029] In some embodiments, the method is RN A interference

[0030] In some embodiments, the cell is derived from the liver.

[0031] In one aspect, the present disclosure provides a method for treating a complement- related disease in a subject in need of such treatment comprising administering the dsRNA molecule or pharmaceutical composition as disclosed herein to the subject. Alternatively, the present disclosure provides use of the dsRNA molecule or pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating a complement-related disease in a subject in need of such treatment. Alternatively, the present disclosure provides the dsRNA molecule or pharmaceutical composition as disclosed herein for use in treating a complement-related disease in a subject in need of such treatment.

[0032] Examples of the complement-related disease or disorder include, but are not limited to, atypical hemolytic uremic syndrome (aHUS), lupus nephritis (LN), primary membranous nephropathy (1MN), C3 glomerulopathy (C3G), coronary’ artery' disease (CAD), or idiopathic thrombocytopenic purpura (ITP).

[0033] In one aspect, the present disclosure provides a method of treating a subject having a disorder that would benefit from reduction in complement factor B expression comprising administering the dsRNA molecule or pharmaceutical composition as disclosed herein to the subject. Alternatively, the present disclosure provides use of the dsRNA molecule or pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating a subject having a disorder that would benefit from reduction in complement factor B expression. Alternatively, the present disclosure provides the dsRNA molecule or pharmaceutical composition as disclosed herein for use in treating a subject having a disorder that would benefit from reduction in complement factor B expression.

[0034] In another aspect, the present disclosure provides a method of preventing development of a disorder that would benefit from reduction in complement factor B expression in a subject having at least one sign or symptom of a disorder who does not yet meet the diagnostic criteria for that disorder comprising administering the dsRNA molecule or pharmaceutical composition as disclosed herein to the subject, thereby preventing the subject progressing to meet the diagnostic criteria of the disorder that would benefit from reduction in CFB expression. Alternatively, the present disclosure provides use of the dsRNA molecule or pharmaceutical composition as disclosed herein in the manufacture ofa medicament for preventing development of a disorder that would benefit from reduction in complement factor B expression in a subject having at least one sign or symptom of a disorder who does not yet meet the diagnostic criteria for that disorder, thereby preventing the subject progressing to meet the diagnostic criteria of the disorder that would benefit from reduction in CFB expression. Alternatively, the present disclosure provides the dsRNA molecule or pharmaceutical composition as disclosed herein for use in preventing development of a disorder that would benefit from reduction in complement factor B expression in a subject having at least one sign or symptom of a disorder who does not yet meet the diagnostic criteria for that disorder, thereby preventing the subject progressing to meet the diagnostic criteria of the disorder that would benefit from reduction in CFB expression.BRIEF DESCRIPTION OF DRAWINGS

[0035] FIG. 1 shows that screening of CFB siRNA activities was conducted in comparison to A-CFB siRNA, an unmodified siRNA based on Alnylam™ AD560018. The relative expression of CFB mRNA was normalized to the CFB mRNA levels observed after transfection of 10 nM A-CFB siRNA into three different cell lines: HepG2. Hep3B. and Huh7.

[0036] FIG. 2 shows results of siRNA in vivo screening based on human CFB mRNA inhibition in the liver of hCFB-Kl mice.

[0037] FIG. 3 shows results of siRNA in vivo screening based on plasma human CFB protein inhibition in the plasma of hCFB-KI mice.

[0038] FIG. 4 shows PBMC immunotoxicity' evaluation by IL-6 production.

[0039] FIG. 5 shows PBMC immunotoxicity evaluation by TNFa production.

[0040] FIG. 6 shows sequential GalNAc-siRNA in vivo screening based on human CFB mRNA inhibition in the liver of hCFB-KI mice,

[0041] FIG. 7 shows sequential GalNAc-siRNA in vivo screening based on plasma human CFB protein inhibition in the plasma of hCFB-KI mice.

[0042] FIG. 8 shows structure of 3S03 sequential GalNAc siRNA, X = S or O

[0043] FIG. 9 shows triantennary GalNAc-siRNA in vivo screening based on human CFB mRNA inhibition in the liver of hCFB-KI mice.

[0044] FIG. 10 shows triantennary GalNAc-siRNA in vivo screening based on plasma human CFB protein inhibition in the plasma of hCFB-KI mice.DETAILED DESCRIPTION OF THE INVEN TION

[0045] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art.

[0046] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: The term "and / or" as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example " A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0047] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

[0048] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. Additional exemplary nucleosides include inosine, 1 -methyl inosine, pseudouridine, 5,6-dihydrouridine. ribothymidine, 2N- methylguanosine and N2, N2-dimethylguanosine (also referred to as "rare" nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester or phosphorothioate linkage between 5' and 3' carbon atoms.

[0049] The term " RNA" or " RNAmolecule" or "ribonucleic acid molecule" refers to a polymer of ribonucleotides (e.g., 2. 3, 4, 5, 10, 15, 20. 25. 30. or more ribonucleotides). The term " DNA" or " DNA molecule" or "deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi -stranded (e.g., double stranded,i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" is single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.

[0050] As used herein, the term "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNAs") refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs), which is capable of directing or mediating RNA interference. In certain embodiments, a siRNA comprises between about 15-30 nucleotides or nucleotide analogs, or between about 16-25 nucleotides (or nucleotide analogs), or between about 18-23 nucleotides (or nucleotide analogs), or between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to a siRNA comprising about 21 nucleotides (or nucleotide analogs), for example, 19. 20, 21 or 22 nucleotides. The term "long" siRN A refers to a siRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRN A retains the ability to mediate RNAi. Likewise, long siRNAs may. in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, e.g., enzymatic processing, to a short siRNA.

[0051] The term "nucleotide analog" or "altered nucleotide" or "modified nucleotide" refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function. Examples of positions of the nucleotide that may be derivatized include: the 5 position, e.g.. 5-(2-amino)propyl uridine, 5-bromo uridine, 5- propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyl uridine: and the 8-position for adenosine and / or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza- adenosine; O- and N-modified nucleotides (e.g., alkylated, e.g,, N6-methyl adenosine, or as otherwise known in the art).

[0052] Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotide. For example, the 2' OH-group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NFb, NHR, NR₂, or COOR, wherein R is substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc.

[0053] The phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g.. phosphorothioates), or by making other substitutions, which allow the nucleotide to perform its intended function. Certain of the modifications (e g., phosphate group modifications) decrease the rate of hydrolysis of, for example, polynucleotides comprising said analogs in vivo or in vitro.

[0054] The term " RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA, but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA. As discussed above, the oligonucleotides may be linked with linkages, which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages. For example, the nucleotides of the analog may comprise methylenediol, ethylene diol, oxy methyl thio, oxyethylthio, oxycarbonyloxy, phosphorodi ami date, phosphoroamidate, and / or phosphorothioate linkages. Some RNA analogues include sugar- and / or backbone- modified ribonucleotides and / or deoxyribonucleotides. Such alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). An RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate RNA interference.

[0055] As used herein, the term " RNA interference" (" RNAi”) refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g.. viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA, which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.

[0056] An RNAi agent, e.g., an RNA silencing agent, having a strand, which has sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi). The strand has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.

[0057] As used herein, the term " RNA silencing" refers to a group of sequence-specific regulatory mechanisms (e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS). quelling, co-suppression, and translational repression) mediated by RNA molecules, which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0058] The term "in vitro" has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term "in vivo" also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and / or cells in an organism.

[0059] As used herein, the term "target gene" is a gene whose expression is to be substantially inhibited or "silenced." This silencing can be achieved by RNA silencing, e.g., by cleaving the mRNA of the target gene or translational repression of the target gene. The term "nontarget gene" is a gene whose expression is not to be substantially silenced. In one embodiment, the polynucleotide sequences of the target and non-target gene (e.g. mRNA encoded by the target and non-target genes) can differ by one or more nucleotides. In another embodiment, the target and non-target genes can differ by one or more polymorphisms (e.g., Single Nucleotide Polymorphisms or SNPs). In another embodiment, the target and non-target genes can share less than 100% sequence identity. In another embodiment, the non-target gene may be a homologue (e.g. an orthologue or paralogue) of the target gene.

[0060] As used herein, the term "antisense strand" of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of the gene targeted for silencing. The antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process (RNAi interference) or complementarity sufficient to trigger translational repression of the desired target mRNA.

[0061] The term "sense strand" or "second strand” of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is complementary to the antisense strand or first strand. Antisense and sense strands can also be referred to as first or second strands, the first or second strand having complementarity to the target sequence and the respective second or first strand having complementarity to said first or second strand. miRNA duplex intermediates or siRNA-like duplexes include a miRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a miRNA strand having sufficient complementarity to form a duplex with the miRNA strand.

[0062] As used herein, the term "guide strand" refers to a strand of an RNA silencing agent, e.g., an antisense strand of an siRNA duplex or siRNA sequence, that enters the RISC complex and directs cleavage of the target mRNA.

[0063] As used herein, the "5' end," as in the 5' end of an antisense strand, refers to the 5' terminal nucleotides, e.g., between one and about 5 nucleotides at the 5' terminus of the antisense strand. As used herein, the "3' end," as in the 3' end of a sense strand, refers to the region, e.g., a region of between one and about 5 nucleotides, that is complementary to the nucleotides of the 5' end of the complementary antisense strand.

[0064] As used herein, the term "base pair" refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of a RNA silencing agent and a target mRNA sequence), due primarily to H-bonding, van der Waals interactions, and the like between said nucleotides (or nucleotide analogs). As used herein, the term "bond strength" or "base pair strength" refers to the strength of the base pair.

[0065] As used herein, the term "mismatched base pair" refers to a base pair consisting of non- complementary or non- Watson-Crick base pairs, for example, not normal complementary G: C, A: T or A: U base pairs As used herein the term "ambiguous base pair" (also known as a non-discriminatory base pair) refers to a base pair formed by a universal nucleotide.

[0066] As used in the present disclosure, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent administered to a mammal, for example a human, for preventing, treating, or eliminating a particular disease or pathological condition that the mammal suffers.10067] As used herein, the terms "treatment," "treating," and the like, cover any treatment of a disease in a mammal, particularly in a human, and include: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0068] As interchangeably used herein, the terms "subject" and "patient," refer to a mammal, including, but not limited to. murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc.10069] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in therealm of a care giver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the compounds of the present disclosure.

[0070] The present disclosure provides a double-stranded RNA molecule for inhibiting the expression of complement factor B. The dsRNA molecule reduces the expression of CFB via RNA interference. The present disclosure also provides a double-strand RNA composition that modulates the RNA-induced silencing complex (RISC)-mediated interaction by cleavage of RNA transcripts encoding complement factor B or by translational suppression. Tire complement factor B may be located within a cell, such as a cell in a subject, including a human subject.

[0071] In some embodiments, compared to ASO drugs, chemically modified siRNA drugs have significantly improved stability, and with the addition of the GalNAc liver-targeting molecule, siRNA can achieve much higher concentrations in hepatocytes, enhancing its inhibitory efficacy. Current siRNA drugs have the potential to be administered as infrequently as once every’ six months or even once a year. This is expected to provide longer-lasting therapeutic effects compared to small-molecule or ASO drugs, greatly improving patient adherence to treatment.

[0072] Complement factor B is a vital element of the alternative pathway of complement activation, which plays a key role in the immune defense system. Produced in the liver, CFB is found in the bloodstream as a single-chain polypeptide. When the alternative pathway is activated, CFB is cleaved by complement factor D into tw’o fragments: Ba, which is non-catalytic, and Bb, an active serine protease. The Bb fragment pairs with C3b to create C3 convertase, which then generates C5 convertase. This enzyme triggers the assembly of the membrane attack complex (MAC), a structure that disrupts the membranes of target cells, resulting in cell lysis. However, improper regulation or overactivation of CFB has been associated with various diseases, including paroxysmal nocturnal hemoglobinuria (PNH), multiple sclerosis, and rheumatoid arthritis. This connection highlights the necessity for therapeutic strategies aimed at inhibiting or silencing CFB in conditions where the activation of the complement pathway is detrimental. There is a demand for compositions and methods that can effectively inhibit or silence CFB in individuals suffering from diseases linked to complement pathway activation or dysregulation.

[0073] In another embodiment, the dsRNA molecule is a pharmaceutically acceptable salt thereof. “Pharmaceutically acceptable salts” of the dsRNA molecule herein include, but arenot limited to, a sodium salt, a calcium salt, a lithium salt, a potassium salt, an ammonium salt, a magnesium salt, a mixture thereof. One skilled in the art will appreciate that the dsRNA molecule, when provided as a polycationic salt having one cation per free acid group of the optionally modified phosphodiester backbone and / or any other acidic modifications (e.g., 5'-terminal phosphonate groups). For example, an oligonucleotide of ''n" nucleotides in length contains n-1 optionally modified phosphodiesters, so that an oligonucleotide of 21 nt in length may be provided as a salt having up to 20 cations (e.g, 20 sodium cations). Similarly, a dsRNA molecule having a sense strand of 21 nt in length and an antisense strand of 23 nt in length may be provided as a salt having up to 42 cations (e.g, 42 sodium cations). In the preceding example, where the dsRNA molecule also includes a 5'-terminal phosphate or a 5'-terminal vinylphosphonate group, the dsRNA molecule may be provided as a salt having up to 44 cations (e.g. 44 sodium cations).

[0074] In some embodiments, the dsRNA molecule comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15, e.g., 15. 16, 17. 18. 19. 20, 21, 22, or 23. contiguous nucleotides differing by no more than 0, 1, 2. or 3 nucleotides from the nucleotide sequence of SEQ ID NO: 79, 153, 184, 227, 39, 121, 145, 171, 182, 186, 225, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 123, 125, 127, 129, 131. 133, 135. 137. 139, 141. 143, 147, 149. 151, 155. 157, 159, 161. 163, 165, 167, 169, 173, 175, 177, 179, 183. 185, 187, 188. 194, 195, 209, 210, 226, and 227; particularly 79, 153, 184, 227, 39, 121, 145, 171, 182, 186, and 225; more particularly 79, 153, 184 and 227.

[0075] and the antisense strand comprises at least 15, e.g., 15, 16, 17. 18. 19, 20, 21, 22, or 23, contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the corresponding portion of the nucleotide sequence of SEQ ID NO: 80, 154, 216, 40, 122, 146, 172, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 82. 84. 86. 88. 90, 92, 94, 96, 98, 100, 102. 104, 106, 108. 110. 112. 114, 116, 118. 120, 124, 126, 128, 130. 132, 134, 136.138, 140, 142, 144, 148, 150, 152, 156, 158, 160, 162, 164, 166, 168. 170, 174, 176, 178, 180, 189, 190, 191, 192, 193, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 211, 212, 213, 214, 215, 217, 218, 219, 220, 221, 222. 223, and 224; particularly 80, 154, 216. 40. 122, 146. and 172: more particularly 80. 154 and 216.

[0076] In some embodiments, the dsRNA molecule comprises a combination of the sense strand and antisense strand comprising nucleic acid sequences of SEQ ID NOs: 79 and 80, 153 and 154, 184 and 154, 184 and 216, 227 and 154, 227 and 216, I and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12, 13 and 14. 15 and 16, 17 and 18, 19 and 20, 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56. 57 and 58, 59 and 60. 61 and 62, 63 and 64, 65 and 66, 67 and 68. 69 and 70. 71 and 72, 73 and 74, 75 and 76, 77 and 78, 81 and 82, 83 and 84, 85 and 86, 87 and 88. 89 and 90, 91 and 92, 93 and 94, 95 and 96, 97 and 98, 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112. 113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122. 123 and 124. 125 and 126, 127 and 128, 129 and 130, 131 and 132, 133 and 134, 135 and 136, 137 and 138, 139 and 140, 141 and 142, 143 and 144, 145 and 146, 147 and 148, 149 and 150, 151 and 152. 155 and 156, 157 and 158, 159 and 160, 161 and 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, 171 and 172, 173 and 174, 175 and 176, 177 and 178, 179 and 180, 182 and 146, 183 and 150.185 and 170, 186 and 172. 187 and 174, 188 and 176. 182 and 189, 182 and 190, 182 and 191, 182 and 192, 182 and 193, 194 and 189, 195 and 189, 182 and 196, 182 and 197, 182 and 198, 182 and 199, 182 and 200, 182 and 201, 182 and 202, 182 and 203, 182 and 204, 182 and 205, 184 and 206, 184 and 207, 184 and 208, 209 and 207. 210 and 207, 184 and 211, 184 and 212, 184 and 213, 184 and214, 184 and 215, 184 and 217, 184 and 218, 186 and 219, 186 and 220, 186 and 221, 186 and 222, 186 and 223, or 186 and 224. In some embodiments, the dsRNA molecule comprises a combination of the sense strand and antisense strand comprising nucleic acid sequences of SEQ ID NOs: 79 and 80, 153 and 154, 184 and 154, 184 and 216, 227 and 154, or 227 and 216.

[0077] In one embodiment, the dsRNA molecule comprises at least one modified nucleotide.

[0078] In one embodiment, substantially all of the nucleotides of the sense strand and / or the antisense strand comprise a modification.

[0079] In one embodiment, at least one nucleotide of the sense strand and / or the antisense strand is modified with a 2'-O-methyl group, a 2'-fluoro group, a 2’-hydroxyl group, a locked nucleic acid, or a phosphorothioate linkage.

[0080] In some embodiments, the duplex region between the sense strand and the antisense strand may be 19-30 nucleotide pairs in length; 19-25 nucleotide pairs in length; 19-23 nucleotide pairs in length; 19-21 nucleotide pairs in length: 23-27 nucleotide pairs in length; or 21-23 nucleotide pairs in length

[0081] In some embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0082] In some embodiments, the region of complementarity may be at least 17 nucleotides in length; 19-23 nucleotides in length; or 19 nucleotides in length.

[0083] In one embodiment, at least one strand comprises a 3' overhang of at least 1 nucleotide.In another embodiment, at least one strand comprises a 3' overhang of at least 2 nucleotides.

[0084] In some embodiments, the dsRNA molecule further comprises a ligand.

[0085] In one embodiment, the ligand is conjugated to the 3' end of the sense strand of the dsRNA molecule. In another embodiment, the ligand is conjugated to the 5' end of the sense strand of the dsRNA molecule. In still another embodiment, the ligand is conjugated to the internucleotide of the sense strand of the dsRNA molecule.

[0086] In certain embodiments, the functional moieties may comprise one or more ligands tethered to an RNA silencing agent to improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism. Ligands and associated modifications can also increase sequence specificity and consequently decrease off-site targeting. A tethered ligand can include one or more modified bases or sugars that can function as intercalators. These can be located in an internal region, such as in a bulge of RNA silencing agent / target duplex. The intercalator can be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can have stacking capabilities, and can include systems with 2. 3, or 4 fused rings. The universal bases described herein can be included on a ligand. In one embodiment, the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid. The cleaving group can be, for example, a bleomycin (e.g., bleomycin-A5, bleomycin- A2, or bleomycin-B2). pyrene, phenanthroline (e.g., O-phenanthroline), a poly amine, a tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group The metal ion chelating group can include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II) 2,9- dimethylphenanthroline derivative, a Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge by free metal ions, such as Lu(III). In some embodiments, a peptide ligand can be tethered to a RNA silencing agent to promote cleavage of the target RNA, e.g., at the bulge region. For example, 1,8-dimethyl- 1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage. A tethered ligand can be an aminoglycoside ligand, which can cause an RNA silencing agent to have improvedhybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog can increase sequence specificity’. For example, neomycin B has a high affinity for RNA as compared to DNA, but low sequence-specificity. An acridine analog, neo-5-acridine, has an increased affinity for the HIV Rev-response element (RRE). In some embodiments, the guanidine analog (the guanidinogly coside) of an aminoglycoside ligand is tethered to an RNA silencing agent. In a guanidinoglycoside, the amine group on the amino acid is exchanged for a guanidine group. Attachment of a guanidine analog can enhance cell permeability’ of an RNA silencing agent. A tethered ligand can be a poly- arginme peptide, peptoid or peptidomimetic, which can enhance the cellular uptake of an oligonucleotide agent.

[0087] Exemplary ligands are coupled, either directly or indirectly, via an intervening tether, to a ligand-conjugated carrier. In certain embodiments, the coupling is through a covalent bond. In certain embodiments, the ligand is attached to the carrier via an intervening tether. In certain embodiments, a ligand alters the distribution, targeting or lifetime of an RNA silencing agent into which it is incorporated. In certain embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g.. compared to a species absent such a ligand

[0088] Exemplary ligands can improve transport, hybridization, and specificity’ properties and may also improve nuclease resistance of the resultant natural or modified RNA silencing agent, or a polymeric molecule comprising any combination of monomers described herein and / or natural or modified ribonucleotides. Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake: diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics. Ligands can include a naturally occurring substance, (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrinor hyaluronic acid); amino acid, or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic poly amino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspaitic acid, poly L -glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamme, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a poly amine, or an alpha helical peptide.

[0089] Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine (GalNAc) or derivatives thereof. N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated poly aminoacids, multivalent galactose, transferrin, bisphosphonate, poly glutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin Bl 2, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g. acridines and substituted acridines), cross-linkers (e.g. psoralene. mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphynn), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lys tripeptide, aminoglycosides, guanidinium aminoglycosides, artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol (and thio analogs thereof), cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., CIO, Cll, C12, C13, C14, C15, C16, C17, C18, C19, or C20 fatty acids) and ethers thereof, e.g., C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl: e.g., l,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG. [MPEG]2,polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport / absorption facilitators (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g.. imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu³⁺ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof. In some embodiments, the ligand targets to asialoglycoprotein receptor (ASGPR).

[0090] In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) or a derivative thereof. In one embodiment, the ligand is one or more GalNAc derivatives attached through a monovalent, bivalent, or trivalent branched linker. In one another embodiment, the ligand is one or more GalNAc derivatives attached through a trivalent branched linker as a trivalent sequential GalNAc linker.

[0091] In one embodiment, the ligand is shown as Formula (I),Formula (I);wherein X is O or S,

[0092] In one embodiment, the dsRNA molecule further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage. In one embodiment, the phosphorothioate or methylphosphonate intemucleotide linkage is at the 3'-end of one strand, e.g., the antisense strand or the sense strand. In another embodiment, the phosphorothioate or methylphosphonate intemucleotide linkage is at the 5'-end of one strand, e.g., the antisense strand or the sense strand. In one embodiment, the phosphorothioate or methylphosphonate intemucleotide linkage is at the both the 5'-and 3'- end of one strand. In one embodiment, the strand is the antisense strand.

[0093] In one embodiment, the first nucleotide at the 5’ end of the antisense strand is adenine or uridine, or analogs thereof. The base pair at the first position of the 5'-end of the antisense strand of the duplex region is an AU base pair.

[0094] In one aspect, the present disclosure provides a pharmaceutical composition comprising the dsRNA molecule as disclosed herein and optionally a delivery vehicle.

[0095] Examples of the delivery vehicle include, but are not limited to, lipid nanoparticles, polymers, or micelles.

[0096] Compositions formed via the methods described herein may be particularly useful for administering an agent to a subject in need thereof. In some embodiments, the compositions are used to deliver a pharmaceutically active agent. The compositions may be administered in any way known in the art of drug delivery’, for example, orally, parenterally, intravenously, intramuscularly, subcutaneously, intradermally, transdermally, intrathecally, submucosally, sublingually, rectally, vaginally, etc.

[0097] Once the compositions have been prepared, they may be combined with pharmaceutically acceptable excipients to form a pharmaceutical composition. As w’ould be appreciated by one of skill in this art, the excipients may be chosen based on the route of administration as described below, the agent being delivered, and the time course of delivery’ of the agent.

[0098] Pharmaceutical compositions described herein and for use in accordance with the embodiments described herein may include a pharmaceutically acceptable excipient. As used herein, the term "pharmaceutically acceptable excipient" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary’ of any type. Some examples of materials which can serve as pharmaceutically acceptable excipients are sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch: cellulose and its derivatives such as sodium carboxymethyl cellulose, methylcellulose, hydroxypropylmethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil: glycols such as propylene glycol: esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; citric acid, acetate salts, Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. The pharmaceutical compositions of this invention can be administered to humans and / or to animals, orally, rectally, parenterally, intracisternally, intravaginally. intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), bucally, or as an oral or nasal spray.

[0099] In some embodiments, the pharmaceutical composition may include the dsRNA molecule in an unbuffered solution, e.g., saline or water, or the pharmaceutical composition may include the dsRNA molecule in a buffer solution, e.g., a buffer solution comprising acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof; or phosphate buffered saline (PBS).

[0100] In one aspect, the present disclosure provides a method for inhibiting the expression of CFB in a cell comprising contacting the dsRNA molecule or pharmaceutical composition as disclosed herein with the cell. In some embodiments, the method is an in vitro method.

[0101] In some embodiments, the method is RNA interference,

[0102] In some embodiments, the cell is derived from the liver.

[0103] In one embodiment, the expression of CFB is inhibited by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0104] In one aspect, the present disclosure provides a cell containing the dsRNA molecule as disclosed herein.

[0105] In one aspect, the present disclosure provides a method for treating a complement- related disease in a subject in need of such treatment comprising administering the dsRNA molecule or pharmaceutical composition as disclosed herein to the subject Alternatively, the present disclosure provides use of the dsRNA molecule or pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating a complement-related disease in a subject in need of such treatment. Alternatively, the present disclosure provides the dsRNA molecule or pharmaceutical composition as disclosed herein for use in treating a complement-related disease in a subject in need of such treatment. In one aspect, the present disclosure provides a method of treating a subject having a disorder that would benefit from reduction in complement factor B expression comprising administering the dsRNA molecule or pharmaceutical composition as disclosed herein to the subject. Alternatively, the present disclosure provides use of the dsRNA molecule or pharmaceutical composition as disclosed herein in the manufacture of a medicament for treating a subject having a disorder that w ould benefit from reduction in complement factor B expression. Alternatively, the present disclosure provides the dsRNA molecule orpharmaceutical composition as disclosed herein for use in treating a subject having a disorder that would benefit from reduction in complement factor B expression.

[0106] In another aspect, the present disclosure provides a method of preventing development of a disorder that would benefit from reduction in complement factor B expression in a subject having at least one sign or symptom of a disorder who does not yet meet the diagnostic criteria for that disorder. The method includes administering to the subject a prophylactically effective amount of the dsRNAs or pharmaceutical compositions as disclosed herein, thereby preventing the subject progressing to meet the diagnostic criteria of the disorder that would benefit from reduction in CFB expression. Alternatively, the present disclosure provides use of the dsRNA molecule or pharmaceutical composition as disclosed herein in the manufacture of a medicament for preventing development of a disorder that would benefit from reduction in complement factor B expression in a subject having at least one sign or symptom of a disorder who does not yet meet the diagnostic criteria for that disorder, thereby preventing the subject progressing to meet the diagnostic criteria of the disorder that would benefit from reduction in CFB expression. Alternatively, the present disclosure provides the dsRNA molecule or pharmaceutical composition as disclosed herein for use in preventing development of a disorder that would benefit from reduction in complement factor B expression in a subject having at least one sign or symptom of a disorder who does not yet meet the diagnostic criteria for that disorder, thereby preventing the subject progressing to meet the diagnostic criteria of the disorder that would benefit from reduction in CFB expression.

[0107] In another embodiment, a CFB protein level in serum of the subject is decreased by at least 50%, 60%, 70%, 80%, 90%, or 95%.

[0108] Examples of the complement-related disease or disorder include, but are not limited to, atypical hemolytic uremic syndrome (aHUS), lupus nephritis (LN), primary membranous nephropathy (iMN), C3 glomerulopathy (C3G), coronary artery disease (CAD), or idiopathic thrombocytopenic purpura (ITP).

[0109] In one embodiment, the dsRNA molecule is administered to the subject at a dose of about 0.01 mg / kg to about 50 mg / kg, about 0.05 mg / kg to about 45 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 35 mg / kg, about 1 mg / kg to about 30 mg / kg, about 5 mg / kg to about 25 mg / kg, about 10 mg / kg to about 20 mg / kg, or about 12 mg / kg to about 15 mg / kg.

[0110] The following examples are provided to aid those skilled in the art in practicing the present disclosure.

[0111] EXAMPLES

[0112] Example 1 siRNA design

[0113] siRNAs targeting the human complement factor B (CFB) gene, (human: Ensembl ID:ENSG00000243649; NCBI GenelD: 629; HGNC:1037) was designed using custom Rand Python™ scripts. The human mRNA (Ensembl ID: ENST00000425368.7; NCBI refseqlD: NM 001710.6) has a length of 2476 bases. Detailed lists of the unmodified CFB sense and antisense strand nucleotide sequences are shown in Table 1.

[0114] Table 1. unmodified siRNA sequence tableDCB ID Sense_2l Seq Antisense_23 Seq ID IDC-siRNA- GGGA AUGUGACC AGGUC 001 CCUAGACCUGGUCACAUUC 002 001 LI AGG CCUUC-siRNA- GUUUC AGC UUGGAC ACL 003 GCUCAGUGUCCAAGCUGAA 004 002 GAGC ACUCC-siRNA- GCCUGAUGCCCUUUAUC 005 CCAAGAUAAAGGGCAUCAG 006 003 UUGG GCAGC -siRNA- ACACGUACCUGCAGAUC 007 CGU AGAU CUGC AGGU ACGU 008 004 UACG GUCUC-siRNA- CCCUGAAGACUCAAGAC 009 UUUGGUCUUGAGUCUUCA 010 005 CAAA GGGUGC-siRNA- AGAGCAAUCCACUGUCC Oil UCUUGGACAGUGGAUUGC 012 006 AAGA UCLJGCC-siRNA- GGCCCAGGGGAACAACA 013 CUUCUGUUGUUCCCCUGGG 014 007 GAAG CCGUC-siRNA- CAGGCUCCAUGAACAUC 015 GGUAGAUGUUCAUGGAGC 016 008 LJACC CUGAAC -siRNA- ACAGCAUUGGGGCCAGC 017 AGUUGCUGGCCCCAAUGCU 018 009 AACU GUCUC-siRNA- LT UGAGA AGGU GGC A AGU 019 CAUAACUUGCCACCUUCUC 020 010 UAUG AAUUC-siRNA- GCCACAUACCCC AAAAU 021 CCAAAUUUUGGGGUAUGU 022 011 UUGG GGCAUC-siRNA- GGGUC ACGAAGC AGC U C 023 CAUUGAGCUGCUUCGUGAC 024 012 A AUG CCAGC-siRNA- CG A AGC AGC U C A A LT GA A 025 UGAUUUCAUUGAGCUGCU 026 013 AUCA UCGUGC-siRNA- AGCUCAAUGAAAUCAAU 027 C AUAAUUG AUUUC AUUGA 028 014 UAUG GCUGCC-siRNA- GC UCAAUGAAAUC AAUU 029 UC AU AAUUGAUUU C AUUG 030 015 AUGA AGCUGC-siRNA- CUCAAUGAAAUCAAUUA 031 UUCAUAAUUGAUUUCAUU 032 016 UGAA GAGCUC -siRNA- AGGAUUAUCUGGAUGUC 033 CAUAGACAUCCAGAUAAUC 034017 UAUG CUCCC-siRNA- GUGUUCAAAGUCAAGGA 035 CAUAUCCUUGACUUUGAAC 036 018 UAUG ACAUC-siRNA- UUUUCUACCAAAUGAUC 037 CAUCGAUCAUUUGGUAGA 038 019 GAUG AAACAC-siRNA- GAUGACAAGGAACACUC 039 GAUUGAGUGUUCCUUGUC 040 020 AAUC AUCCAC-siRNA- ACAAGGAACACUCAAUC 041 CCUUGAUUGAGUGUUCCUU 042 021 AAGG GUCAC -siRNA- AGCUCAAGAAUAAGCUG 043 AUUUCAGCUUAUUCUUGA 044 022 AAAU GCUUGC-siRNA- CU C AAGAAUAAGCUGAA 045 AUAU UUCAGCUUAU UC UU 046 023 AUAU GAGCUC-siRNA- UCAAGAAUAAGCUGAAA 047 CAUAUUUCAGCUUAUUCUU 048 024 UAUG GAGCC-siRNA- CAAGAAUAAGCUGAAAU 049 CCAUAUUUCAGCUUAUUCU 050 025 AUGG UGAGC -siRNA- CUGAAAUAUGGCCAGAC 051 GAUAGUCUGGCCAUAUUUC 052 026 UAUC AGCUC-siRNA- GAGGG A AC AAC U C GAGC 053 CAAAGC UCGAGUUGUUCCC 054 027 UUUG UCGGC-siRNA- GUGAGAGAGAUGCU C AA 055 C AU AU UGAGC AUC UCU CU C 056 028 UAUG ACAGC-siRNA- CC A AU ACUUGC AGAGGU 057 AAUCACCUCUGCAAGUAUU 058 029 GAUU GGGGC -siRNA- AGAGAAGUCGUUUCAUU 059 CUU GAAU GAAACGACUUCU 060 030 CAAG CUUGC-siRNA- GCCCAGGGGAACAACAG 061 GCUUCUGUUGUUCCCCUGG 062 031 AAGC GCCGC-siRNA- U C AACU U AAUUGAGAAG 063 CCACCUUCUCAAUUAAGUU 064 032 GUGG GACUC-siRNA- GAAGCAGCUCAAUGAAA 065 UUGAUUUCAUUGAGCUGC 066 033 UCAA UUCGUC-siRNA- AUCCGGGACUUGCUAUA 067 AAUGUAUAGCAAGUCCCGG 068 034 CAUU AUCUC-siRNA- CCGGGACUUGCU AU AC A 069 CC A AUGU AUAGC A AGUCCC 070 035 UUGG GGAUC-siRNA- GC A A A A AC C C A AGGG AG 071 AAUCCUCCCU UGGGUUUUU 072 036 GAUU GCGAC-siRNA- UCAAGGAUAUGGAAAAC 073 CC AGGUUUUCC AUAUCCUU 074 037 CUGG GACUC-siRNA- AAAUGAU CGAUGAAAGC 075 ACUGGCUUUC AUCGAUC AU 076 038 CAGU UUGGC-siRNA- GGGCUGUGGUGUCUGAG 077 AGUACUCAGACACCACAGC 078 039 UACU ccccC-siRNA- GAC A AGGA AC ACU C A AU 079 CUUGAUUGAGUGUUCCUU 080 040 CAAG GUCAUC-siRNA- GGCCAGACUAUCAGGCC 081 AAUGGGCCUGAUAGUC UG 082041 CAUU GCCAUC-siRNA- GCCAGACUAUCAGGCCC 083 AAAUGGGCCUGAUAGUCU 084 042 AUUU GGCCAC-siRNA- GGAAC AAC UCGAGC UUU 085 CC UC AAAGC UCGAGUUGUU 086 043 GAGG CCCUC-siRNA- GAAGCGGCAAAAGCAGG 087 GGUACCUGCUUUUGCCGCU 088 044 UACC UCUGC-siRNA- UCAC AUCAACCUCUU UC 089 ACUUGAAAGAGGUUGAUG 090 045 AAGU UGAAAC -siRNA- GCUCCUUCCGACUUCUC 091 CUUGGAGAAGUCGGAAGG 092 046 CAAG AGCCGC-siRNA- CCCUCAAGAGGUGGCCG 093 GCUUCGGCCACCUCUUGAG 094 047 AAGC GGGUC-siRNA- ACGAAGCAGCUCAAUGA 095 GAUUUCAUUGAGCUGCUUC 096 048 A AUC GUGAC-siRNA- CC UGAAGGCUGGAACCG 097 GGUGCGGUUCCAGCCUUCA 098 049 CACC GGAGC -siRNA- AAUACUUGCAGAGGUGA 099 AGAAUCACCUCUGCAAGUA 100 050 UUCU UUGGC-siRNA- AGUGUCUAGUCAACUUA 101 CAAUUAAGUUGACUAGAC 102 051 AUUG ACUUUC-siRNA- CAACAUGUGUUCAAAGU 103 C UUGAC UUUGAAC AC AU GU 104 052 CAAG UGCUC-siRNA- GG AAGG AGGUCUAC AUC 105 UCUUGAUGUAGACCUCCUU 106 053 AAGA CCGAC -siRNA- AGGAGGU CUACAUC AAG 107 CAUUCUUGAUGUAGACCUC 108 054 AAUG CUUCC-siRNA- GGAGGUCUACAUCAAGA 109 CCAUUCUUGAUGUAGACCU 110 055 AUGG CCUUC-siRNA- U CC AC UGCU AUGACGGU 111 UGUAACCGUCAUAGCAGUG 112 056 UACA GAAAC-siRNA- ACAACUCGAGCUUUGAG 113 AAGCCUCAAAGCUCGAGUU 114 057 GCUU GUUCC-siRNA- GAC U C GGAAGGAGGUC U 115 AUGU AGACC UCC UU CCGAG 116 058 ACAU UCAGC-siRNA- CAAUACUUGCAGAGGUG 117 GAAUC ACCUCUGCA AGUAU 118 059 AUUC UGGGC-siRNA- GGUC ACGAAGCAGC UCA 119 UCAUUGAGCUGCUUCGUGA 120 060 AUGA CCCAC-siRNA- AAGC AGCUC A AUGA A AU 121 AUUGAUUUCAUUGAGCUG 122 061 CAAU CUUCGC-siRNA- AUGACAAGGAACACUCA 123 UGAUU GAGU GUUCCUU GU 124 062 AUCA CAUCCC-siRNA- UGACAAGGAACACUCAA 125 UUGAUUGAGUGUUCCUUG 126 063 UCAA UCAUCC-siRNA- UGGAGGUGUGACCACCA 127 GG AGU GGU GGU C AC AC C U C 128 064 CUCC CAGAC-siRNA- CACC CU GAAGAC UC AAG 129 UGGUC UUGAGUCUUC AGG 130065 ACCA GUGCUC-siRNA- AGUGACAUAUGCCACAU 131 GGGUAUGUGGCAUAUGUC 132 066 ACCC ACUAGC-siRNA- GAUCGAUGAAAGCCAGU 133 AGAGACUGGCUUUCAUCGA 134 067 CUCU UCAUC-siRNA- CCCUGCACCGAGGGAAC 135 AGUUGUUCCCUCGGUGCAG 136 068 AACU GGGAC-siRNA- GAGUGCAGAGCAAUCCA 137 ACAGUGGAUUGCUCUGCAC 138 069 CUGU UCUGC -siRNA- UAGUGACAUAUGCCACA 139 GGUAUGUGGCAUAUGUCA 140 070 UACC CUAGAC-siRNA- CAAGCUCAAGAAUAAGC 141 UUCAGCUUAUUC UUGAGCU 142071 UGAA UGAU

[0115] Example 2 The Primary In Vitro Screening Methods

[0116] HepG2 were cultured and transfected using Lipofectamine RNAiMAX™ reagent. The transfection mix was prepared by combining 3 pL of Lipofectamine, 1 pL of siRNA, and 6 pL of MEM to create the Mix buffer, which was then added to a 96-well dish at a volume of 10 pL per well. Subsequently, 10,000 HepG2 cells were seeded in each well with 90 pL of culture medium The cells were incubated at 37°C in a 5% CO2 atmosphere. After 24 hours, samples were collected, and the wells were washed with 100 pL of PBS(-). To each well, 50 L of Lysis Solution (containing 0.3 pL of gDNA Remover mixed with 49.7 pL of Lysis Solution) was added, followed by gentle mixing for 30 seconds and incubation at room temperature for 4.5 minutes. Next, 10 pL of Stop Solution (comprising 0.5 pL of RNase Inhibitor in 9.5 pL of Stop Solution) was added, mixed gently for another 30 seconds, and incubated at room temperature for an additional 1.5 minutes before transferring the plate to ice. For qPCR analysis, the Master mix was prepared by combining 5 pL of 4x TaqMan Master Mix, 1 pL of TaqMan CFB Probe, 1 pL of GAPDH TaqMan assay (20X), and 12 pL of RNase-free water, totaling 19 pL per PCR tube. Finally, 1 pL of the sample from each well was added to the respective PCR tubes before proceeding with the qPCR analysis.

[0117] Hep3B and Huh7 cells were seeded at a density of 20,000 cells per well in a 96-well plate and incubated overnight at 37°C in a 5% CO2 atmosphere. Following incubation, siRNA was transfected using Lipofectamine RNAiMAX™ reagent, and the cells were maintained at 37°C m a 5% CO2 environment for an additional 24 hours. After the transfection period, the medium was removed, and the cells were washed once with PBS RNA was then collected using the SuperPrep™ Cell Lysis for qPCR kit. For the quantitative PCR (qPCR) analysis, a Master mix was prepared for each PCR tube, totaling 19 pL, which included 5 pL of 4x TaqMan Master Mix, 0.3 pL of 60x CFB probe. 0.3 Lof 60x GAPDH probe, and 13.33 µL of ddH₂O. To this mixture, 1 pL of the RNA sample was added.

[0118] The results of single dose (10 nM) transfection screens in HepG2, Hep3B. and Huh 7 of the siRNA agents in Table 1 are shown in FIG. 1.

[0119] Example 3 The Secondary In Vitro Screening Methods

[0120] A subset of chemically modified siRNAs based on Table 1 is presented. Detailed lists of the chemically modified CFB sense and antisense strand nucleotide sequences are provided in Table 2

[0121] Table 2. Full chemically modified siRNA sequence tableSense 21(5'— >3') Seq Antisense 23(5’— >3’) Seq ID ID AD560 [A*A*GAGA] / A / [G] / UCG / 143 [A*] / U* / [GAA] / U / [G] / AA / [ACGA] / 144 018 [UUUCAUUCAU] C / [U] / U / [CUCUU*G*U]m20U [G*A*UGAC].9V[A] / GG / V 145 [U]* / A / *[UUG]AV[G] / UG / [UUCC] / 146 [ACACUCAAUA] U / [U] / G / [UCAUC*C*A]m29 [C*C*AAUA] / C / [U] / UGC / [ 147 [A] * / A / * [UCA] / C / [C] / UC / [UGC A] / 148 AGAGGUGAUU] A / [G] / U / [AUUGG*G*G]m29U | C*C*AAUA] / C / [ U] / UGC /

[0149] U]* / A / *[UCA] / C / | C] / UC / [UGG A] / 150 AGAGGUGAUA] A'[G] / U / [AUUGG*G*G]m40 [G*A*CAAG] / G / [A] / ACA-'[ 151 [C] * / U / * [UGA] / U / [U] / GA / [GUGU] / 1.52 CUCAAUCAAG] U / [C] / C / [UUGUC*A*U]m40U [G*A*CAAG| / G / [A] / ACA / [ 153 [U|* / U / *[UGA| / U / [U] / GA / [GUGU] / 154 CUCAAUCAAA] U / [C] / C / [UUGUC*A*U]m48 [A*G*GAAG] / C / [A] / GCU / [ 155 [G] NA / * [UUU] / C / [ A] / UU / [GAGC] / 156 C A AUG A A AUG] U / [G] / C / [UUCGU*G*A]m48U [A*C*GAAG] / C / [A] / GCU / [ 157 [U]* / A / *[UUU] / C / [A| / UU / [GAGC] / 158 CAAUGAAAUA] U / [G] / C / [UUCGU*G*A]m56 [U*C*CACU] / G / [C] / UAU / [ 159 [U] * / G / * [U AA] / C / [C] / GU / [CAU A] / 160 GACGGUUACA] G / [C] / A / [ GUGGA* A* A]m58 [G*A*CUCG] / G / [A] / AGG / [ 161 [A] * / U / * | GU A ] / G / [ A] / CC / [UCCU] / 162 AGGUCUACAU] U / [C] / C / [GAGUC*A*G]m58U [G* A*C UCG] / G / [A] / AGG / [ 163 [U]* / U / *[GUA] / G / [A] / CC / [UCCU] / 164 AGGUCUACAA] U / [C] / C / [GAGUC*A*G]m.59 [C*A*AUAC] / U / [U] / GCA / [ 165 [G] * / A / * [AUC] / A''[C] / CU / [CUGC] / 166 GAGGUGAUUC] A / [ A] / G / [U AUUG* G* G]m59U [C*A*AUAC] / U / [U] / GCA / [ 167 [U] * / A / * [ AUG ] / A / [C] / C U / [CUGC] / 168 GAGGUGAUUA] Az[ A] / G / [U AUUG* G* G]m60 [G*G*UCAC] / G / [A] / AGC / [ 169 [U]* / C / *[AUU] / G / [A] / GC / [UGCU] / 170 AGCUCAAUGA] U / [C] / G / [UGACC*C*A]m61 [A*A*GCAG| / C / [U] / CAA / [ 171 [A]* / U / *[UGA] / U / [U] / UC / [AUUG] / 172 UGAAAUCAAU] A / [G] / C / [UGCUU*C*G]m63 [U*G*ACAA] / G / [G] / AAC / [ 173 [U] * / UZ* [G AU] / U / [G] / AG / [UGUU] / 174 ACUCAAUCAA] C / [C] / U / [UGUCA*U*C[m67 ]G*A*UCGA] / U / [GJ / AAA / 175 [A] * / G / * [ AGA] / C / [U ] / GG / [CUUU] / 176[GCCAGUCUCU] C / [A] / U / [CGAUC*A*U]m69 [G*A*GUGC] / A / [G] / AGC / | 177 [A] * / C / * [ AGU] / G / [G] / AU / [UGCU] / 178 AAUCCACUGU] C / [U] / G / [CACUC*U*G]m71 [C*A*AGCU] / C / [A] / AGA / [ 179 [U] * / U / * [C AG] / C / [L] / U A / [UUC U] / 180AUAAGCUGAA] U / [G] / A / [GCUUG*A*U]

[0122] [ ]= 2'0-Me; / / = 2 ‘-Fluoro; *= phosphorothioate linkage; dA= DNA

[0123] Hep3B cells were seeded at a density of 20,000 cells per well in a 96-well plate and incubated overnight at 37°C in a 5% CO2 atmosphere. Following incubation, siRNA was transfected using Lipofectamine RN AiMAX™ reagent at varying concentrations of 0.1 nM.1 nM, 10 nM, and 100 nM to evaluate the effects on gene silencing. The cells were maintained at 37°C in a 5% CO2 environment for an additional 24 hours. After the transfection period, the medium was removed, and the cells were washed once with PBS. RNA was then collected using the SuperPrep™ Cell Lysis for qPCR kit. For the quantitative PCR (qPCR) analysis, a Master mix was prepared for each PCR tube, totaling 19 pL, which included 5 pL of 4x TaqMan™ Master Mix, 0.3 pL of 60x CFB probe, 0.3 pL of 60x GAPDH probe, and 13.33 pL of ddH₂O. To this mixture, 1 pL of the RNA sample was added. The resulting mixture was used for one-step RNA real-time PCR to quantify gene expression levels.

[0124] The results of transfection screens in Hep3B of the siRNA agents in Table 2 are show n in Table 3 and Table 4.

[0125] Table 3. IC50 of chemical modified siRNA in Hep3B% of RemainingSO nM 16.7 nM 5.6 nM 1.9 nM 0.6 nM 0.2 nM Max.Inhib ID MeaniSD MeaniSD MeaniSD MeaniSD MeaniSD MeaniSD 1C5Oition (nM) (%) NC I00.0il.6 100.0i0.7 100. Oil 8 1 100.0i2.2 lOO. OiO. O 100.0+6.6 NAAD28.8±6.9 32.5i2.5 46.7i3.6 75.1i7.3 93.5il8.5 78.7+8.5 6.43 71 560018m20U 32.7i8.7 28.2.0.4 36.8i0.6 70. HO 9 86.0i0.1 82.6+3.5 4.19 67 m29 44.2±23.5 34.6i3.2 46,5±8.6 75.3i3.5 101.3i2.8 85.8i8.1 4.68 56 m29U 29.0±l.7 41.9il.l 47.8i0.3 76.5i3.5 105.4il.5 90.0i25.2 5.19 71 m40 37.4±na. 36.2+3.3 54.4i0.2 73.0i8.6 96.6i31.7 90.5+30.6 7.17 63 m40U 23.9±3.7 27.5il.6 39.5il2.3 70.08.3 88.4i2.3 96.7i3.5 3.95 76 m48 44.7±3.5 38.7+4.9 42.6il.2 80.5i8.7 107.8i28.9 97.9+18.7 2.38 55 m48U 29.6±3.7 33.6i0.1 48.8i6.3 86.1±12.4 105.9i22.3 98.0+23.3 5.15 70 m56 64.0±l 5.4 63.1±1.6 70.9ill.8 96.0i28.3 121.2i37.6 95.0±4.3 NA 36 m58 37.9i7.9 35.9±3.5 44.8i9.1 81.3il3.0 106.9il7.7 97.6+9.3 4.02 62 m58U 31.6il.4 41.1i5.8 62.8i0.3 93. Iil5.8 121.7il6.7 80.3i2.7 8.24 68m59 33.3i4.2 31.8±7.4 46.8±8.1 90.8il8.8 90.8il.l 113.6i37.6 4.92 67m59U 31.3*1.1 103.4*19.1 108.2*1.6 127.1*25.1131,9*4.3 86.4*18.1 40.72 69

[0126] Table 4. IC50 of chemical modified siRNA in Hep3B% of Remaining50 nM 16.7 nM 5.6 nM 1.9 nM 0.6 nM 0.2 nM Max.Inhib ic50ID Mean*SD Mean*SD Mean±SD Mean±SD Mean±SD Mean±SD ition (nM)(%) NC 100.0*14,8 100.0*6.5 100.0*23.8 100,0*17.5 100.0*2.2 100.0*19, 1 NAAD28.8*5.7 56.9*10.3 86.8*1.1 92.6*23.8 99.2*16.5 99.0*17.5 21.61 71 560018m6l 29.7*0.3 55.8*3.7 78.5±17.9 101.8*28.0 82.2*45 93.2*12.7 22.44 70 m63 45.9-4.6 78.0*7.3 88.1*12.0 94.6*0.5 101.6*50.5 89.7*5.1 47.69 54 m67 34.6*9.0 72.7*9.1 89.8* 13.1 96.9*4.1 101.0*5.0 102.9*36.3 33.54 65 m69 52.1*3.0 91.6*11.9 106.3*14.2 104.6*9.8 99.0*18.3 95.6*18.8 NA 48m71 28.6*4.4 49.3*4.4 81.7*3.7 98.4*8.2 95.4*4.0 99.3*9.5 16.49 71

[0127] Example 4 The Primary In Vivo Screening of siRNA

[0128] A subset of chemically modified siRNAs based on Table 1 is presented. Detailed lists of the chemically modified CFB sense and antisense strand nucleotide sequences are provided in Table 5.

[0129] Table 5. Full chemically modified siRNA sequence tableSense_21(5'-->3') Seq ID Antisense_23(5'— >3') Seq ID CFB [A*A*GAGA] / A / [G] / UCG / [ 181 [A] * / U* / [ GAA] / U / [G] / AA / | AC 144 siRNA UUUCAUUC*A*U] GA] / C / [U] / U / [CUCUU*G*U]C-020U [G*A*UGAC] / A / '[A] / GGA / [ 182 [U]* / A / *[UUG] / A / [G] / UG / [UU 146 ACACUCAA*U*A] CC] / U / [U] / G / [ UCAUC*C*A] C-029U [C*C*AALiA| / C / [U] / UGC / [ 183 [ U] * / A / * [D C A] / C / [C] / D C / [UGC 150 AGAGGUGA*U*A] A] / A / [G] / U / [AIJUGG*G*G]C-040U [G*A*CAAG] / G / [A] / ACA'[ 184 [U]* / U / *[UGA] / U / [U] / GA / [GU 154 CUCAAUCA*A*A] GU] / U / [C] / C / [UUGUC*A*U] C-060 [G*G*UCAC] / G / [A] / AGC / [ 185 [U]* / C / *[AUU] / G / [A] / GC / [UGC 170 AGCUCAAU*G*A] U] / Li / [C] / G / [LiGACC*C*A]C-061 [A*A*GCAG] / C / [U] / CAA / [ 186 [A]* / U / *[UGA] / U / [U] / UC / [ALf 172 UGAAAUCA*A*U] UG] / A / [G] / C / [UGCUU*C*G] C-063 |U*G*ACAA] / G / [G] / AAC / [ 187 [U]* / U / *[GAU] / U / [G] / AG / [UG 174 ACUCAAUC*A*A] UU] / C / [C] / U / [UGUCA*U*C] C-071 [C*A*AGCU] / C / [A] / AGA / [ 188 [ U] * / U / * [C AG] / C / [ U] / UA / [ LJUC 176AUAAGCUG*A*A] U] / U / [G] / A / [GCUUG*A! HU]

[0130] [ ]= 2'0-Me; / / =2’-Fluoro: *= phosphorothioate linkage; dA= DNA

[0131] In this in vivo experiment, hepatocyte-specific human CFB knock-in mice (ROSA26- pTBG-hCFB KI / +) were utilized to evaluate the efficacy of siRNA screening using the in vivo-jetPEI transfection reagent. Mice received intravenous injections of siRNA complexes at a concentration of 2 mg / kg (mpk) for a total of five doses administered over two weeks. The siRNA was formulated with in vivo-jetPEI according to the manufacturer’s protocol, ensuring optimal delivery to the liver. After the two-week treatment period, mouse livers were harvested for mRNA analysis to assess gene expression levels, while plasma samples were collected for the measurement of human Complement Factor B (CFB) protein concentration using an ELISA kit (abl37973, Abeam).

[0132] The results of in vivo screens in human CFB knock-in mice (ROSA26-pTBG-hCFB KI / +) are shown in FIG. 2, for liver human CFB mRNA expression assay, and FIG. 3 for plasma CFB protein expression assay.

[0133] Examples Immunotoxicity Assay / w Vitro

[0134] A subset of chemically modified siRNAs based on Table 1 is presented. Detailed lists of the chemically modified CFB sense and antisense strand nucleotide sequences are provided in Table 5.

[0135] Human PBMC were isolated from whole blood from healthy donors by a standard Ficoll-Hypaque density centrifugation technique. For immune stimulation assays, 500000 PBMC were seeded in duplicate in 96-welI plates and cultured in RPMI 1640 medium with 10% FBS, 2 mM glutamine. A total of 10 pM of siRNA w'ere added to cells and culture supernatants were collected after 24h and assayed for IL-6 and IFN-α by sandwich ELISA (abeam IL-6 ELISA Kit ab229434, abeam TNFa ELISA Kit ab229399). R848 5pg / ml was a positive control; ISIS 104838 10 pM was a negative control.

[0136] The results of immunotoxicity assay are shown in FIG. 4 and FIG. 5.

[0137] Example 6 Determination of mRNA Inhibition ICso in HepG2 Cells

[0138] HepG2 cells (2 × 104per well) were electroporated with siRNA at various concentrations (1.5, 0.3, 0.06, 0.012, 0.0024, and 0.00048 pM) using the Neon™ Transfection System. Following a 16-hour incubation, total RNA was extracted, and mRNA expression levels were quantified by qPCR. Dose-response curves were generated, and IC₅₀ values were calculated by nonlinear regression analysis using GraphPad Prism software.

[0139] The results of IC50 are shown in Table 6.

[0140] Table 6. IC50 of chemical modified siRNA in HepG2#ID IC50±SD (nM) Max Inh. (%)@1.5 uM siRNACFB ASO-696844 4035±1071 95CFB siRNA 1.7±1.7 81C-020U 7.5±3.6 89C-029U 13.6±10.1 84C-040U 9.0±1.1 84C-060 59.3±57.0 77C-061 1.8±5.0 90C-063 328.2±99.8 84C-071 13.9±11.5 83

[0141] Example 7 Primary screen of chemical modified siRNA

[0142] HepG2 cells (2 x 104per well) were electroporated with siRNA at various concentrations (50, 10, and 2 nM) using the Neon™ Transfection System. Following a 16- hour incubation, total RNA was extracted, and mRNA expression levels were quantified by qPCR The chemical modified siRNA are listed in Table 7. The results of transfection screens in HepG2 of the siRNA agents in Table 7 are shown in Table 8. Table 9 and Table 10.

[0143] Table 7. Full chemically modified siRNA sequence tableDCB Sense 21(5 '->3') Seq Antisense 23(5'->3') Seq ID ID IDC020U [G*A*UGAC] / A / [A] / GGA / 182 [U] * / AJ* [UUG] d A / G / [UGU] / U / [C 189 -M001 [ACACUCAA*U*A] CUU] / G / [UC AUG *C * A]C020U [G* A* UGAC] / A / | A] / GGA / 182 [U] * / A7* [UUGA] / G / [UGU] / U / [CC 190 -M002 [ACACUCAA*U*A] UU] / G / [UCAUC*C*A]C020U [G*A*UGAC] / A''[A] / GGA / 182 [U] * / A / * [UUG] d A[GUGU] / U / [CC 191 -M003 |ACACUCAA*U*A] UU| / G / [UCAUC*C*A|C020U [G*A*UGAC] / A-'[A| / GGA / 182 [U] * / A / * / UUG / d A| G] dT[ G] / UUC 192 -M004 [ACACUCAA*U*A] CU7[U] / G / [UCAUC*C*A]C020U [G*A*UGAC] / A / [A] / GGA / 182 [U]* / A / * / UU / [G]dA[GU]dG[U] / U 193 -M005 [ACACUCAA*U*A] CC / [UU] / G / [UCAUC *C * A]C020U [G* A*UGAC | / A / 1 A] / GG / [ 194 [ U] * / A- * | UUGj d A / G / [UGU | / U / [ C 189 -M006 AACACUCAA*U*A] CUU] / G / [UCAUC*C*A]C020U [G* A* UGAU] / A / [ A] / GG / [ 195 [U] * / A / * [UUG]dA / G / [UGU] / U / [C 189 -M007 AACACUCAA*U*A] CUU] / G / [UCAUC*C*A]C020U [G*A*UGAC] / A-'[A] / GGA / 182 [U] * / A / * [ U] / UG / d A[G] dT[G] / UU 196 -M008 [ACACUCAA*U*A] CCU / [U] / G / [UCAUC*C*A]C020U [G*A*UGAC] / A / [A] / GGA / 182 [U] * / A-* [U] / UG / dA[G] dT[G] / U U / 197 -M009 [ACACUCAA*U*A] [CC] / U / [U] / G / [UCAUC*C*A] C020U [G*A*UGAC] / A / [A] / GGA / 182 [U] * / AJ* [U] / UG / dA[G] dT[G] / U / [ 198 -M010 [ACACUCAA*U*A] UCC] / U / [U] / G / [UC AU C * C * A] C020U [G* A* UGAC] / A / [A] / GGA / 182 [U]* / A / *[U] / U / |G] / dA[G]dT[G] / U 199-M011 [ACACUCAA*U*A] UCCU / [U] / G / [UCAUC*C*A]C020U [G*A*UGAC] / A'[A] / GGA7 182 [U] * / A / * [ U] / U / [G] dA[ G]dT[G] / U / | 200 -MO 12 [ACACUCAA*U*A] UC] / CU / [U] / G / [UCAUC*C*A] C020U [G*A*UGAC] / A / [A] / GGA / 182 [U] * / A'* [U] / U / dGdA'G / [U] dG[UU 201 -MO 13 [ACACUCAA*U*A] ] dC [ C]. / U / | U] / G / [UC AUC *C * A] C020U [G*A*UGAC] / A / [A] / GGA / 182 [ U] * / A'* [U] / U / dGdA / G / [U] / G / dT| 202 -MO 14 [ACACUCAA*U*A] UCC] / U / [U] / G / [UC AU C * C * A] C020U [G* A* UGAC] / A / |A] / GGA / 182 [ U] * / 'A'* [UU j dGdA| G] / UG / | UUC 203 -MO 15 [ACACUCA A*U*A] C] / U / [U] / G / [UCAUC*C*A] C020U [G*A*UGAC] / A'[A] / GGA 182 [U] * / A / * [UU] dGdA [G] / U / [G] dT[U 204 -MO 16 |ACACUCAA*U*A] 1 dC [C] / U / [U] / G / [UC AUC *C * A] C020U | G* A* U GAC ] / A'[ Aj / GGA / 182 [U|* / A / *[UU]dGdA[G| / U / [GUU]d 205 -MO 17 [ACACUCAA*U*A] C [C] / U / [U] / G / [UC AUC *C* A] C040U [G* A* C A AG] / G / [ A] / AC A / 184 [U] * / U / * [UGAU] dT[GAGUGUUC 206 -MOO1 [CUCAAUCA*A*A] ] / C / [UUGUC*A*U]C040U [G^A*CAAG] / G / [A] / ACA / 184 [U]* / U7* / U / [GAU]dT[GAGU] / G / [ 207 -M002 [CUCAAUCA*A*A] UUC] / C / [UUGUC * A*U]C040U [G*A*CAAG] / G / [A] / ACA' 184 [U] * / U / * dT / G / [ AU] dT[GA] dG [UG 208 -MOO3 [CUCAAUCA*A*A] UUC ] / C / [ UUGUC * A* U]C040U [G*A*CAAG] / G / [A] / A / |C] / 209 [U]* / U / * / U / [GAU]dT[GAGU] / G / [ 207 -M004 A7[CUCAAUCA*A*A] UUC] / C / [UUGUC*A*U]C040U [G*A*UAAG] / G / [A] / A'[C] 210 [U]* / U / * / U / [GAU]dT[GAGU] / G / [ 207 -MOO5 / A / [CUC A AUC A* A* A] UUC] / C / [UUGUC*A*U]C040U [ G* A* C A AG] / G / 1 A] / AC A / 184 [U]* / U / * / U / [GA]dT[U|dG[AGU| / 211 -M006 [CUCAAUCA*A*A] G / [UUC] / C / [UUGUC*A*U] C040U [G*A*CAAG] / G / [A] / ACA7 184 [U]* / U / *7U / [GA]dT[U]dG[AGU] / 212 -M007 | CUC A AUC A* A* A] G / [U] / U / [C] / C / [UUGUC*A*U] C040U |G*A*CAAG] / G / [A]7ACA' 184 [U]* / U / * / U / [GAU]dT[G]dA[GU] / 213 -MOO8 [CUCAAUCA*A*A] G / [UUC] / C / [UUGUC* A*U] C040U [G*A*CAAG] / G / [A] / ACA / 184 [U]* / U / *[UGAU]dT[GAGUGU] / U 214 -M009 [CUCAAUCA*A*A] / [C] / C / [UUGUC*A*U|C040U I G* A* C A AG | ZGZ | A j / AC A / 184 [ U] * / U / * / U / [ GA |dT[ U ] / GA / dG[ U] 215 -MO 10 [CUCAAUCA*A*A] dG[U] / U / [C] / C / [UUGUC*A*U] C040U [G*A*CAAG] / G / [A] / ACA / 184 [U]* / U / * / U / [GAU]dT[GAGUGU] / 216 -MO 11 [CUCAAUCA*A*A] U / [C] / C / [UUGUC*A*U]C040U [G*A*CAAG] / G / [A] / ACA' 184 [U] * / U / * / U / [GAU] / GU A / dG[U] dG 217 -MO 12 [CUCAAUCA*A*A] [U] / U / [C ] / C / [UUGUC* A* U] C040U |G*A*CAAG] / G / [A] / ACA / 184 [U] * / U / * [ U] / G / [ AU] dT[GAGUGU 218 -MO 13 [CUCAAUCA*A*A] ] / U / [C] / C / [UUGUC*A*U]C06le- [A*A*GCAG] / C / [U] / CAA7 186 [A] * / U / * [UGAUU] / U / [C A] / UU / [G 219 M001 [UGAAAUCA*A*U] ]dA[G] / C7[UGCUU*C*G]C061e- [A*A*GCAG] / C / [U] / CAA / 186 [A] * / U7* [U] dG[ AUU] / U / [ CA] / UU / 220 M002 [UGAAAUCA*A*U] [G] d A[G] / C / [UGCUU* C * G] C061e- [ A* A * GC AG] / C / [U] ZC A A / 186 [A] * / U / * [UGAU] dT / U / [C A] / UU / [ 221 M003 |UGAAAUCA*A*U] G|dA[G] / C / | UGCUU*C*G] C061e- [A*A*GCAG] / C / [U] / CAA / 186 [A] * / U / * [ UGA] / U / [U] / UC / [ AUUG 222 M004 [UGAAAUCA*A*U] ] / A7[G] / U / [UGCUU*C*G]C061e- [A*A*GCAG] / C / [U] / CAA / 186 [A] * / U / * [UGA] / U / [U] / UC / [AUU]d 223M005 [UGAAAUCA*A*U] G / A / dG / U / [UGCUU*C*G]C061e- [A*A*GCAG] / C / [U] / CAA / 186 [A] * / U / * ]UGAUU] / U / [C A] / UU / d 224M006 [UGAAAUCA*A*U] GdAdG / L / [UGCUU*C*G]

[0144] [ ]== 2'0-Me; / / :::2’-Fluoro; phosphorothioate linkage; dA::= DNA

[0145] Table 8. Summary of primary screening for C-020U series siRNA in HepG2 Cells 2 nM 10 nM 50 nM C-020U 81.6 ± 11.1 42.3 ± 11.6 21.0 ± 7.1C-020U M001 106.9 ± 2.2 118.6 ± 5.1 52.9 ± 0.9C-020U M002 100 ± 2.5 120.7 ± 7.8 66.4 ± 7.2C-020U M003 100.2 ± 14.9 103.0 ± 2.9 79.7 ± 10C-020U M004 88.8 ± 17.2 61.2 ± 31.4 29.9 ± 8.4C-020U M005 96.7 ± 7.5 86.3 ± 4.8 98.5 ± 4.7C-020U M006 101.6 ± 1.0 97.6 ± 8.5 83.9 ± 3.8C-020U M007 108.3 ± 9.3 86.3 ± 4.5 56.5 ± 2.0C-020U M008 100.6 ± 3.7 46.6 ± 3.9 22.1 ± 0.4C-020U M009 80.1 ± 5.6 34.1 ± 3.2 6.9 ± 0.3C-020U M010 64.7 ± 5.9 16.7 ± 0.4 6.7 ± 0.4C-020U M011 98.9 ± 11.5 40.6 ± 4.8 11.2 ± 1.2C-020U M012 57.6 ± 4.7 15.3 ± 0.0 7.0 ± 0.1C-020U M013 57.6 ± 0.2 9.8 ± 0.7 3.7 ± 0.2C-020U M014 48.7 ± 1.7 19.0 ± 1.3 5.6 ± 0.1C-020U M015 73.9 ± 2.6 34.4 ± 0.5 21.3 ± 0.7C-020U M016 56.7 ± 1.8 22.5 ± 0.6 13.2 ± 0.0C-020U M017 54.9 ± 4.4 29.1 ± 3.4 10.4 ± 0.7

[0146] Table 9. Summary of primary screening for C-040U series siRNA in HepG2 Cells 2 nM 10 nM 50 nM C-040U 52.2 ± 22.0 26.1 ± 8.2 15.9 ± 6.3C-040U M001 84.1 ± 21.3 71.5 ± 10.9 44.2 ± 6.8C-040U M002 68.8 ± 16.2 64.9 ± 6.3 28.6 ± 0.5C-040U M003 89.4 ± 4.2 92.6 ± 0.0 67.5 ± 10.0C-040U M004 90.7 ± 5.4 99.2 ± 10.0 82.2 ± 3.4C-040U M005 95.2 ± 7.5 88.1 ± 8.0 57.8 ± 3.7C-040U M006 70.2 ± 1.8 34.8 ± 0.9 16.5 ± 0.9C-040U M007 25.5 ± 1.5 19.0 ± 1.5 10.1 ± 0.3C-040U M008 61.0 ± 7.2 24.5 ± 1.8 8.6 ± 0.3C-040U M009 23.3 ± 0.0 18.5 ± 0.1 9.4 ± 0.1C-040U M010 20.9 ± 1.2 10.7 ± 0.1 6.3 ± 0.0C-040U M011 24.5 ± 0.1 14.7 ± 0.6 10.0 ± 0.4C-040U MO 12 52.7 ± 3.8 37.4 ± 0.8 19.8 ± 0.6C-040U M013 21.1 ± 0.8 16.1 ± 0.6 12.1 ± 0.7

[0147] Table 10. Summary of primary screening for C-061 e series siRNA in HepG2 Cells 2 nM 10 nM 50 nMC-061e 33.5 ± 11.4 18.4 ± 4.1 12.6 ± 2.3 C-061e M001 38.6 ± 6.2 28.3 ± 11.8 13.2 ± 0.3C-061e M002 35.8 ± 5.2 18.5 ± 7.9 7.9 ± 0.0C-061e M003 39.5 ± 16.1 7.5 ± 1.1 6.2 ± 0.8C-061e M004 34.2 ± 3.7 21.1 ± 1.3 7.4 ± 2.1C-061e M005 22.0 ± 6.3 25.3 ±12.0 9.7 ± 1.161.9 ± 4.0 60.3 ± 1.6 32.7 ± 0.5C-061e M006

[0148] Example 8 Determination of mRNA Inhibition IC50in HepG2 Cells

[0149] HepG2 cells (2 × 104per well) were electroporated with siRNA at various concentrations (1500, 300, 60, 12, 2.4, 0.48, 0.096 and 0.0192 nM) or (150, 30, 6, 1.2, 0.24, 0.048, 0.0096 and 0.00192 nM) using the Neon™ Transfection System. Following a 16- hour incubation, total RNA was extracted, and mRNA expression levels were quantified by qPCR Dose-response curves were generated, and IC₅₀ values were calculated by nonlinear regression analysis using GraphPad Prism software.

[0150] The results of IC50are shown in Table 11.

[0151] Table 11. IC50of chemically modified siRNA in HepG2#ID IC50± SD (nM) Max Inh. (%) Max Inh. (%)at 1500 nM at 150 nMC-020U 3.88 ± 1.23 96C-020U M010 2.36 ± 0.81 97C-020U M012 1.02 ± 0.43 97C-020U M013 1.22 ± 0.85 96C-020U M014 3.95 ± 1.15 93C-020U M016 3.57 ± 1.86 94C-020U M017 5.09 ± 1.30 94C-040U 2.21 ± 0.40 90C-040U M007 1.69 ± 0.69 94C-040U M009 0.92 ± 0.41 97C-040U M010 2.24 ± 0.67 93C-040U M011 1.54 ± 0.47 90C-040U M013 2.47 ± 1.03 86C-061e 0.66 ± 0.23 95C-061e M001 1.41 ± 0.47 95C-061e M002 2.02 ± 0.46 93C-061e M003 1.23 ± 0.29 94C-061e M004 0.50 ± 0.20 94C-061e M005 7.31 ± 2.15 92

[0152] Example 9 The Primary In Vivo Screening of Sequential GalNAc-siRNA

[0153] A subset of chemically modified siRNAs based on Table 1 is presented. Detailed lists of the chemically modified CFB sense and antisense strand nucleotide sequences with Sequential GalNAc conj ugation are provided m Table 12.

[0154] Table 12. Sequential GalNAc conjugated siRNADCB ID Sense_21(5'->3') Seq Antisense_23(5'— >3') Seq ID ID3S03- [G* A* UGAC] / A / [A] / GGA / [ 225 [U]* / A / * [UUG] / A / [G] / UG / [UU 146 C020U ACACUC AA*U* A]-3S03 CC] / U / [U ] / G / [UC AUC *C* A] 3S03- [G*A*UGAC] / A / [A] / GGA / [ 225 [U| * / A / * [ U] / UG / dA[ G] dT[ G] / U 198 C020U- ACACUCAA*U*A]-3S03 / [UCC] / U7[U] / G / [UCAUC*C*A M0103S03- [G* A*UGAC] / A / | A] / GGA / | 225 [ U] * / A7* [U] / U / |G] dA[G]dT[G] / 200 C020U- AC AC UC AA*U * A] -3 S 03 U / [UC] / CU / [U] / G / [UCAUC*C* M012 A]3S03- [G*A*UGAC] / A-'[A] / GGA / [ 225 [U] * / A / * [U] / U / dGd A / G / [U]dG[ 201 C020U-[ACACUCAA*U*A]-3S03 U U ] dC [ C] / U / [U] / G / [ UCAUC*C MO 13 *A]3S03- [G*A*UGAU] / A / [A] / GGA / [ 226 [U]* / A / * [UUG] / A / [G] / UG / [UU 146 C020U ACACUCAA*U*A]-3S03 CC] / U / [U] / G / [UCAUC*C*A] (CtoU)3S03- [G*A*UGAU] / A / [A] / GGA / [ 226 [U] * / A / * [U] / UG / dA[G] dT[G] / U 198 C020U(Ct AC AC UC AA* U * A] -3 S 03 / [UCC] / U / [U] / G / [UCAUC*C*A oU)-M0103S03- [G*A*UGAU] / A / [A] / GGA / [ 226 [U]* / A / * [U] / U / [G]dA[G]dT[G] / 200 C020U(Ct ACACUCAA*U*A]-3S03 U / [UC] / CU / [U1 / G / [UCAUC*C* oU)-M012 A]3SO3- [G* A* UGAU] / A / [A] / GGA / [ 226 [U] * / A / * [U] / U / dGdA / G / [U]dG[ 201 C020U(Ct ACACUC AA*U*AJ-3S03 UU] dC [ C] / U / | U] / G / [UC AUC* C oU)-M013 *A]3S03- [G*A*CAAG] / G / [A] / ACA / [ 227 [U] * / U / * [UGA] / U / [U] / GA / [GU 154 C040U CUCAAUCA*A*A] -3S03 GU] / U / [ C| / C / [ UUGUC * A*U] 3S03- [G* A*C AAG] / G / | A] / AC A / [ 227 [ U] * / U / * / U / [ GA]dT[U] dG| AGU 212 C040U- CUCAAUCA*A*A] -3S03 ] / G / [U] / U / [C] / C / [UUGU C * A* U M0073S03- [G*A*CAAG] / G / [A] / ACA / [ 227 [U ] * / U / * [UGAU] dT[G AGUGU 214 C040U- CUCAAUCA*A*A] -3S03 ] / U / [C] / C / [UUGUC*A*U]M0093S03- [G* A*C AAG] / G / | A] / AC A / [ 227 [ U] * / U / * / U / [ GAU] dT| GAGUG 216 C040U- CUCAAUCA*A*A] -3S03 U] / U7[C] / C / [UUGUC*A*U]M011

[0155] [ |= 2'0-Me; / / = 2’-Fluoro; *= phosphorothioate linkage; dA= DNA; 3S03= S03 Sequential GalNAc conjugated at the 3’ of sense strand

[0156] Sequential GalNAc structure of S03 is shown in Formula (II)Formula (II)

[0157] In this in vivo experiment, hepatocyte-specific human CFB knock-in mice (ROSA26- pTBG-hCFB KI / +) were utilized to evaluate the efficacy of GalNAc conjugated siRNA. Mice received a single subcutaneously injection of GalNAc-siRNA at a concentration of 1 mg / kg (mpk) for two weeks. After the two-week treatment period, mouse livers were harvested for mRNA analysis to assess gene expression levels, while plasma samples were collected for the measurement of human Complement Factor B (CFB) protein concentration using an ELISA kit (abl37973, Abeam™).

[0158] Tire results of in vivo screens in human CFB knock-in mice (ROSA26-pTBG-hCFB KI / +) are shown in FIG. 6, for liver human CFB mRN A expression assay, and FIG. 7 for plasma CFB protein expression assay.

[0159] Example 10 Preparation S03 Sequential GalNAc phosphoramidite

[0160] Synthesis of perfluorophenyl 2,2,5-trimethyl-1,3-dioxane-5-carboxylate (1) is shown in Scheme (1):FO p FxJrz.FyVYCF3I? £ £d ) i II £ •. > / S0rFVO F'' "'Y ^F DMF °> / F' \11 r Yr0'1 Scheme (I). 10161 ] A dry DMF solution (20 mL) of 2,2,5-trimethyl-l,3-dioxane-5-carboxylic acid (5.8 g, 1 0 eq ), and TEA (5.0 g. 1.5 eq.) was stirred for 10 mm at room temperature, andperfluorophenyl 2,2,2-trifluoroacetate (11.6 g, 1.25 eq.) was dropwise added. After 3 h, the reaction mixture was taken into 200 mL of EA and 200 mL H2O mixture. The organic layer was washed with H2O and saturated aqueous solutions of NaCI. Silica gel column chromatography (EA: Hex. = 1: 50) gave 7.5 g of 1 (66 % yield); *H NMR (600 MHz, CDCI3) 84.34 (d, J = 12.0 Hz, 2H), 3.81 (d, J = 12.0 Hz, 2H), 1.50 (s, 3H), 1.46 (s, 3H), 1.36 (s, 3H).

[0162] Synthesis of perfluorophenyl 3-hydroxy-2-(hydroxymethyl)-2-methyIpropanoate (2) is shown in Scheme (II):F F~ & Scheme (II).

[0163] Perfluorophenyl 2,2,5-trimethyl-1,3-dioxane-5-carboxylate 1 (7.5 g, 22.4 mmol) was dissolved in a mixture of 50 mL of THF and 50 mL of 1 M HC1. The solution was stirred for 6 h at room temperature The reaction mixture was extracted with ethyl acetate. The organic phase was dried with Na2SO4 and the solvent was evaporated under reduced pressure to give 5.7 g perfluorophenyl diol 2 (white solid) (86 % yield). 'H NMR (600 MHz, CDCk) 84.09 (dd, J - 11.1, 6.8 Hz, 2H), 3.90 (dd, J = 11.4, 6.0 Hz, 2H), 2.67 (t, J - 6.5 Hz, 2H), 1.33 (s, 3H).

[0164] Synthesis of perfluorophenyl 3-(bis(4-methoxyphenyl)(phenyl)methoxy)-2- (hydroxymethyl)-2-methylpropanoate (3) is shown in Scheme (III):DCMScheme (III).

[0165] A dry DCM solution (40 mL) of 2 (3.13 g,l.l eq.) and DMAP (0.21 g,0.2 eq.) was cooled on ice under nitrogen, and Et3N (1.44 g, 1.5 eq.) was dropwise added. After 10 min.DMT-C1 (3.2 g,1.0 eq.) in 40 mL of DCM was added dropwise. After 3 h. the reaction mixture was taken into 200 mL of DCM and 200 mL H2O mixture. The organic layer was washed with saturated aqueous solutions of NaCl, dried over anhydrous Na? S()4 and concentrated under reduced pressure Silica gel column chromatography (EA: Hex. = 1: 4) gave 1.1 g of 3 (18 % yield).]H NMR (600 MHz, CDC13) 87.47 (d, J = 7.6 Hz, 2H), 7.37 (dd, J = 8.8, 3.2 Hz, 4H). 7.32 (t, J = 7.6 Hz, 2H), 7.25 (t, J = 7.2 Hz, 1H), 6.87 (d, J = 8.7 Hz. 4H), 4.04 - 3.91 (m, IH), 3.87 - 3.74 (m, 7H). 3.64 (d, J = 9.1 Hz, IH). 3.30 (d. J = 9.1 Hz, IH), 1.93 (t, J = 6.7 Hz, IH), 1 34 (d, J = 18.8 Hz. 3H)

[0166] Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-(3-(bis(4- methoxyphenyl)(phenyl)methoxy)-2-(hydroxymethyl)-2-methylpropanamido) hexyl)oxy)tetrahydro-2H-pyran-3.4-diyl diacetate (5) is shown in Scheme (IV):Scheme (IV).

[0167] To a solution of3 (1.1 eq.) and 4 (1.13 g, 1.0 eq.) in dry THF (13 mL) under nitrogen, DIPEA (0.6 g,2.5 eq.) was added dropwise After stirring at room temperature for 2 h, the reaction mixture was extracted with DCM (150 mL) and H2O (150 mL). The organic layer was washed with saturated aqueous solutions of NaCl, dried over anhydrous Na2SO4and concentrated under reduced pressure. Silica gel column chromatography (DCM: MeOH = 97: 3) gave 1.0 g of 5 (59 % yield). ’H NMR (600 MHz, DMSO-d6) 57.82 (d, J = 9.3 Hz, IH), 7.41 - 7.34 (m, 3H), 7.29 (t, J === 7.8 Hz, 2H), 7.25 - 7.17 (m, 5H), 6.88 (d, J - 8.6 Hz, 4H), 5.21 (d, J = 3.4 Hz, 1H), 4.96 (dd, J= 11.3, 3.4 Hz, IH), 4.77 (dd, J = 5.0, 4.1 Hz, 1H), 4.44 (dd, J - 43.6, 8.2 Hz, 1H), 4.08 - 3.96 (m, 3H), 3.87 (dl, J = 11.1, 8.9 Hz, 1H). 3.74 (d, J = 6.7 Hz, 6H), 3.67 (dt, J = 10.1. 6.3 Hz, IH), 3.52 (dd, J === 104, 4.9 Hz, IH), 3.42 (dd. J = 10.4, 4.9 Hz, 1H), 3.10 - 3.02 (m, 3H), 2. 10 (s, 3H), 2.00 ( d, J = 5.6 Hz, 3H), 1.90 (s, 3H), 1.76 (s, 3H), 1.39 (dt, J = 21.3, 6.8 Hz, 4H), 1.26 - 1.18 (m, 5H), 1.05 (s, 3H).

[0168] Synthesis of (2R,3R,4R,5R,6R)-5-acetamido-2-(acetoxymethyl)-6-((6-(3-(bis(4- methoxyphenyl)(phenyl)methoxy)-2-((((2- cyanoethoxy)(diisopropylamino)phosphaneyl)oxy)methyl)-2-methylpropanamido)hexyl)oxy)tetrahydro-2H-pyran-3,4-diyl diacetate (S03 phosphoramidite) is shown in Scheme (V):Scheme (V).

[0169] To a solution of 5 (1.81 g, 1.0 eq.) and DCI (0.17 g, 0.7 eq.) in dry DCM (20 mL) under nitrogen. 3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile (0.95 g, 1.5 eq.) was added dropwise. After 1.5 h. 150 mL of DCM was poured into the mixture The organic solution was washed with 100 mL of saturated aqueous solution of NaHCOs and of NaCl, dried over Na2SO4and concentrated under reduced pressure. Silica gel column chromatography (EA: Hex.with TEA=7:3 and 2 %) gave 1.12 g of S03 phosphoramidite (47 % yield). 'HNMR (500 MHz, DMSO-d6) 87.82 (d, J = 9.2 Hz, 1H). 7.43 (dt, J = 19.8, 5.5 Hz, 1H), 7.36 (d, J === 7.5 Hz, 2H), 7.29 (t, J === 7.6 Hz, 2H), 7.22 (d, J = 8.8 Hz, 5H), 6.87 (d, J = 8.9 Hz, 4H), 5.21 (d, J = 3.3 Hz, 1H), 4.96 (dd, J = 11.2, 3.3 Hz, HI), 4.48 (d, J = 8.5 Hz, 1H), 4.03 (q, J = 6.9 Hz, 4H), 3.87 (dd, J = 19.9, 9.0 Hz. 1H), 3.81 - 3.71 (m, 7H), 3.71 - 3.56 (m, 5H). 3.54 - 3.44 (m, 3H). 3.07 (ddd, J = 14.0, 12.3, 6.9 Hz, 4H), 2.66 (dt, J = 5.3, 4.2 Hz, 2H), 2.11 (d, J = 8.5 Hz, 3H), 1.99 (s, 3H), 1.90 (d, J - 8.1 Hz, 3H), 1.78 - 1.72 (m, 3H), 1.46 - 1.34 (m, 4H), 1.23 (d, J = 9.5 Hz, 5H), 1.14 - 1.07 (m, 9H), 1.03 (dd, J = 6.7, 2.8 Hz. 6H), 3 IP NMR (202 MHz, DMSO-d6) 5 146.83 (s), 146.44 (s).

[0170] Example 11 Preparation of sequential G alNAc-siRNA

[0171] Solid-phase nucleic acid synthesis begins with the deprotection of the dimethoxytrityl (DMT) protecting group on the solid support (Unylinker™ support) using an acidic reagent such as dichloroacetic acid (DCA). This is followed by a coupling reaction between the trivalent phosphoramidite and the free hydroxyl group, catalyzed by an activator such as 5- (benzylthio)-lH-tetrazole (BTT), 4.5-Di cyanoimidazole (DCI), or IH-tetrazole, to form the specified nucleic acid sequence. The trivalent phosphorus is then converted to pentavalent phosphorus as a phosphate ester or phosphorothioate ester via an oxidation reaction or sulfurization reaction. A capping reaction is then performed, and the cycle is repeated sequentially to synthesize the target nucleic acid sequence. Finally, the cyanoethyl protecting group on the phosphate ester is cleared using an organic base, such as DEA orTEA, via a P-elimination mechanism. All remaining protecting groups on the nucleobases are removed, and the target nucleic acid is cleaved from the solid support using concentrated aqueous ammonia. The final product is obtained after subsequent procedures including analysis, purification, desalting, and lyophilization (freeze-drying).

[0172] Equipment and Reagent Preparation

[0173] The solid-phase nucleic acid synthesis is performed using a Cytiva Äkta™ synthesizer model. Due to the high reactivity of the phosphorami dite raw materials in solid-phase nucleic acid synthesis, all ami dite raw materials are removed from the -20°C refrigerator, allowed to warm up, and opened inside an anhydrous glove box. Solutions are prepared with ACS grade anhydrous MeCN and used after adding an appropriate amount of 3 A molecular sieves. (MeCN and toluene are both checked for water content by Karl Fischer titration). The activating reagents, 5-(benzylthio)-lH-tetrazole (BIT) or 4,5- dicyanoimidazole (4,5-DCI), are also opened inside the anhydrous glove box, dissolved in ACS grade anhydrous MeCN, and an appropriate amount of 3 A molecular sieves is added. Under a positive-pressure nitrogen hood, dichloroacetic acid is prepared as a 3%-10% deprotection reagent in diy toluene, with no need to add molecular sieves. The oxidation reagent is prepared by mixing iodine with a dH2O / pyridine (10 / 90%, v / v) solution The sulfurization reagent is prepared using: ADTT (xanthane hydride, XH) mixed with pyridine / MeCN (3 / 2, v / v) solution.

[0174] PADS mixed with 2.6-lutidine / MeCN (1 / 1. v / v) or 3-picoline / MeCN to form a 0.2M solution, which is allowed to mature for at least 12 hours before use. Cap A is a 20% 1- methylimidazole in MeCN solution or a 20% 1 -methylimidazole, 30% pyridine in 50% dry toluene solution. Cap B is a 20% acetic anhydride, 30% 2,6-lutidme, 50% MeCN solution or acetic anhydride in dry toluene (1 / 4, v / v) The phosphorus deprotection step uses DEA mixed with MeCN to form a 20% mixed solution; or TEA mixed with MeCN to form a 50% solution The established solid-phase short-chain nucleic acid synthesis system has been optimized for suitable reaction reagents, equivalents, reaction times, and concentrations. It can successfully synthesize the sequential GalNAc sense strand (GalNAc ssRNA) and antisense strand (asRNA) required for the manufacturing of small interfering RNA (siRN A), with a crude yield of the solid-phase synthesis product ranging from 60%- 80% per batch.

[0175] Oligonucleotide Sequences and Modifications

[0176] As shown in Table 12, the GalNAc ssRNA is a 21 mer sequence consisting of modified nucleic acids. Tri S03 GalNAc monomers (3 mer, 1+1) are sequentially introduced fromthe 5’ and 3’ ends and are linked to the ssRNA backbone via a phosphorothioate bond (PS bond) or phosphodiester bond (PO bond). Within the ssRNA sequence, the two nucleosides at each end are linked by phosphorothioate bond linkages, and the remainder are linked by PO bond. The asRNA is a 23 mer sequence consisting of modified nucleic acids The two nucleosides at the 5’ end and the two nucleosides at the 3’ end are linked by phosphorothioate bond linkages, totaling four PS bonds, while the rest are linked by PO bonds,

[0177] Final siRNA Production

[0178] siRNA consists of one sense strand and one antisense strand, so each complete siRNA sequence requires the independent synthesis of the sense and antisense strands followed by annealing. Reagents used in the synthesis process include DNA phosphoramidite, 2’-OMe RNA phosphoramidite, 2 -F RNA phosphoramidite and S03 GalNAc phosphoramidite. After the solid-phase nucleic acid synthesis reaction is complete, 28% concentrated aqueous ammonia is added, and the mixture is heated to remove all protecting groups. The mixture is filtered under vacuum, and the solvent is removed by rotary evaporation. The remaining crude product is purified by anion exchange chromatography to obtain the desired nucleic acid sequence. The 3S03 GalNAc ssRNA and asRNA raw materials are mixed in a specific molar ratio in a specific buffer solution, typically phosphate-buffered saline or w ater, and heated for annealing to form the corresponding siRNA product. Finally, the product is concentrated to a specific volume and dialyzed a specific number of times (monitored by conductivity measurement) using a Molecular Weight Cut-Off (MWCO) 3000 dialysis membrane. The final 3S03 GalNAc siRNA product is obtained after lyophilization to remove water.

[0179] Example 12 The Primary In Vivo Screening of Triantennary GalNAc-siRNA

[0180] A subset of chemically modified siRNAs based on Table 1 is presented. Detailed lists of the chemically modified CFB sense and antisense strand nucleotide sequences with Triantennary GalNAc conjugation are provided in Table 13.

[0181] Table 13. Triantennary-GalNAc conjugated siRNASense_21(5’->3') Seq Antisense_23(5'-->3') Seq ID ID20U- [G* A*UGAC] / A''[A] / GGA / [A 228 [U] * / A / * [UUG] / A / [G] / UG / [UUCC 146 3GN CACUCAA*U*A]-L96 ] / U / [U] / G / [UCAUC*C*A]40U- |G*A*CAAG] / G / [A| / ACA / [C 229 [U] * / U / * [UG A] / U / [U] / G A / [ GUGU 1543 GN UCAAUCA*A*A]-L96 ] / LV[C] / C / [UUGUC*A*U]61e- [A*A*GCAG] / C / [U] / CAA / [U 230 [A] * / U / * [UG A] / U / [U] / UC / [ AUUG 1723 GN GAAAUCA*A*U]-L96 ] / A / [G] / C / [UGCUU*C*G]

[0182] [ ]== 2'0-Me; / 2’-Fluoro;phosphorothioate linkage; dA= DNA DNA; L96= Triantennary GalNAc conjugated at the 3’ of sense strand

[0183] Sequential GalNAc structure of L96 is shown in Formula (III)Formula (III)

[0184] In this in vivo experiment, hepatocyte-specific human CFB knock-in mice (ROSA26- pTBG-hCFB KI / +) were utilized to evaluate the efficacy of GalNAc conjugated siRNA Mice received a single subcutaneously injection of GalNAc-siRNA at a concentration of 1 mg / kg (mpk) for two weeks. After the two-week treatment period, mouse livers were harvested for mRNA analysis to assess gene expression levels, while plasma samples were collected for the measurement of human Complement Factor B (CFB) protein concentration using an ELISA kit (ab137973, Abcam™).

[0185] The results of in vivo screens in human CFB knock-in mice (ROSA26-pTBG-hCFB KI / +) are shown in FIG. 9, for liver human CFB mRNA expression assay, and FIG. 10 for plasma CFB protein expression assay.

[0186] While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives thereto and modifications and variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are regarded as falling within the scope of the present invention

Claims

Claims1. A double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand, wherein the sense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 79, 153. 184, 227, 39, 121, 145, 171, 182, 186, 225, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 81, 83.

85.

87. 89, 91, 93, 95, 97, 99, 101, 103. 105, 107, 109. I l l, 113, 115. 117, 119.

123. 125, 127, 129. 131, 133, 135.137, 139, 141, 143, 147, 149, 151. 155, 157, 159. 161, 163, 165, 167, 169, 173, 175, 177, 179, 183, 185, 187, 188, 194, 195, 209, 210, 226, and 227 or a substantially similar sequence having at least 95% sequence identity.

2. The dsRNA molecule of claim 1, wherein the sense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 79, 153, 184, 227, 39, 121, 145, 171, 182, 186, and 225 or a substantially similar sequence having at least 95% sequence identity.

3. The dsRNA molecule of claim 1, wherein the sense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 79, 153, 184 and 227 or a substantially similar sequence having at least 95% sequence identity.4 The dsRNA molecule of claim 1, wherein the antisense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 80, 154, 216.

40. 122, 146, 172. 2, 4, 6, 8.

10.

12.

14. 16, 18, 20, 22, 24, 26, 28, 30. 32, 34, 36.

38.

42.

44. 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78.

82. 84, 86, 88, 90, 92, 94, 96, 98, 100. 102, 104, 106, 108, 110, 112, 114. 116, 118, 120, 124, 126, 128, 130. 132, 134, 136, 138, 140, 142, 144, 148, 150, 152, 156, 158, 160, 162, 164, 166, 168. 170, 174, 176. 178, 180, 189. 190, 191. 192, 193, 196, 197, 198, 199, 200, 201, 202. 203, 204.

205. 206, 207. 208, 211, 212, 213, 214, 215, 217, 218, 219, 220, 221. 222, 223, and 224 or a substantially similar sequence having at least 95% sequence identity.

5. The dsRNA molecule of claim 1, wherein the antisense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 80, 154, 216.

40. 122, 146, and 172 or a substantially similar sequence having at least 95% sequence identity.

6. The dsRNA molecule of claim 1, wherein the antisense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 80, 154 and 216, or a substantially similar sequence having at least 95% sequence identity,7. The dsRNA molecule of 1, which comprises a combination of the sense strand and antisense strand comprising nucleic acid sequences of SEQ ID NOs: 79 and 80, 153and 154, 184 and 154, 184 and 216, 227 and 154, 227 and 216, 1 and 2, 3 and 4, 5 and 6. 7 and 8, 9 and 10, 11 and 12, 13 and 14. 15 and 16, 17 and 18, 19 and 20. 21 and 22, 23 and 24, 25 and 26, 27 and 28, 29 and 30, 31 and 32, 33 and 34, 35 and 36, 37 and 38, 39 and 40, 41 and 42, 43 and 44, 45 and 46, 47 and 48, 49 and 50, 51 and 52, 53 and 54, 55 and 56, 57 and 58, 59 and 60, 61 and 62, 63 and 64, 65 and 66, 67 and 68, 69 and 70, 71 and 72, 73 and 74, 75 and 76, 77 and 78. 81 and 82, 83 and 84, 85 and 86, 87 and 88, 89 and 90, 91 and 92. 93 and 94, 95 and 96. 97 and 98. 99 and 100, 101 and 102, 103 and 104, 105 and 106, 107 and 108, 109 and 110, 111 and 112. 113 and 114, 115 and 116, 117 and 118, 119 and 120, 121 and 122, 123 and 124, 125 and 126, 127 and 128, 129 and 130, 131 and 132, 133 and 134, 135 and 136, 137 and 138, 139 and 140, 141 and 142, 143 and 144. 145 and 146. 147 and 148. 149 and 150, 151 and 152. 155 and 156. 157 and 158, 159 and 160, 161 and 162, 163 and 164, 165 and 166, 167 and 168, 169 and 170, 171 and 172, 173 and 174, 175 and 176, 177 and 178, 179 and 180, 182 and 146, 183 and 150, 185 and 170, 186 and 172, 187 and 174, 188 and 176, 182 and 189, 182 and 190, 182 and 191, 182 and 192, 182 and 193, 194 and 189, 195 and 189. 182 and 196. 182 and 197. 182 and 198, 182 and 199. 182 and 200, 182 and 201, 182 and 202, 182 and 203, 182 and 204, 182 and 205, 184 and 206, 184 and 207, 184 and 208, 209 and 207, 210 and 207, 184 and 211, 184 and 212, 184 and 213, 184 and 214, 184 and 215, 184 and 217. 184 and 218, 186 and 219, 186 and 220, 186 and 221, 186 and 222, 186 and 223, or 186 and 224 or a substantially similar sequence having at least 95% sequence identity.

8. The dsRNA molecule of 1, which comprises a combination of the sense strand and antisense strand comprising nucleic acid sequences of SEQ ID NOs: 79 and 80, 153 and 154, 184 and 154, 184 and 216, 227 and 154. 227 and 216 or a substantially similar sequence having at least 95% sequence identity.

9. The dsRNA molecule of claim 1, which comprises at least one modified nucleotides selected from the group consisting of a deoxy -nucleotide, a 3 ’-terminal deoxy thimi dine (dT) nucleotide, a 2’-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide (LNA), an unlocked nucleotide, hexitol nucleotide (HNA), a confomiationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2’ -amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-carbyl-modified nucleotide, a 2'-hydroxy 1 -modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate. a non-natural base comprisingnucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphoro thioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5'-phosphate, a nucleotide comprising a 5'-phosphate mimic, a nucleotide comprising a 2’-phosphate group, a thermally destabilizing nucleotide, a glycol modified nucleotide (GN A), and a 2-O-(N -methylacetamide) modified nucleotide; and combinations thereof,10. The dsRNA molecule of claim 1. wherein the antisense strand comprises a 3' overhang of at least 2-5 nucleotides.

11. The dsRNA molecule of claim 1, wherein a duplex region between the sense strand and the antisense strand comprises 15 to 25 nucleotides.

12. The dsRNA molecule of claim 1, wherein a duplex region between the sense strand and the antisense strand comprises 1 to 3 mismatches.

13. The dsRNA molecule of claim 1, wherein the antisense strand is complementary to the mRNA of complement factor B (CFB).

14. The dsRNA molecule of claim 1, wherein the antisense strand is complementary to the mRNA of CFB and comprises 1 to 3 mismatches.

15. The dsRNA molecule of claim 1, wherein the first nucleotide at the 5’ end of the antisense strand is adenine or uridine, or analogs thereof.

16. The dsRNA molecule of claim 1, which further comprises a ligand conjugated to the 3' end or the 5’ end of the sense strand.

17. The dsRNA molecule of claim 16, wherein the ligand is a lectin, glycoprotein, lipid or protein or a derivative thereof.

18. The dsRNA molecule of claim 16, wherein the ligand is an N-acetylgalactosamine (GalN Ac) or a derivative thereof.

19. The dsRNA agent of claims 16, wherein the ligand is a trivalent sequential GalNAc linker.

20. The dsRNA agent of claim 16, wherein the ligand is shown as Formula (I),HO ) — (HO OHFormula (I);wherein X is O or S.

21. The dsRNA molecule of claim 1, wherein the dsRNA molecule further comprises at least one phosphorothioate or methylphosphonate intemucleotide linkage.

22. A dsRNA molecule comprising a sense strand and an antisense strand, wherein the antisense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 80, 154. 216, 40, 122, 146, 172, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 82, 84, 86. 88, 90, 92, 94, 96, 98, 100, 102, 104. 106, 108, 110, 112, 114, 116, 118. 120, 124, 126. 128, 130, 132.

134.

136.

138. 140, 142.

144. 148, 150. 152, 156, 158, 160, 162, 164, 166, 168. 170, 174, 176. 178, 180, 189, 190, 191, 192, 193, 196, 197, 198, 199, 200, 201, 202. 203, 204, 205, 206, 207, 208, 211, 212, 213, 214, 215, 217. 218, 219, 220, 221, 222, 223, and 224 or a substantially similar sequence having at least 95% sequence identity.

23. The dsRNA molecule of claim 22, wherein the antisense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 80, 154. 216, 40, 122, 146, and 172 or a substantially similar sequence having at least 95% sequence identity’.

24. The dsRNA molecule of claim 23, wherein the antisense strand comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 80, 154 and 216, or a substantially similar sequence having at least 95% sequence identity.

25. A pharmaceutical composition comprising the dsRNA molecule of any one of claims 1 to 24 and optionally a delivery vehicle.

26. The pharmaceutical composition of claim 25, wherein the delivery vehicle is lipid nanoparticles, polymers, or micelles.

27. A method for inhibiting the expression of CFB in a cell comprising contacting the dsRNA molecule of any one of claims 1 to 24 or pharmaceutical composition of claim 25 or 26 with the cell.

28. The method of claim 27, which is RNA interference.

29. The method of claim 27. wherein the cell is derived from the liver.

30. Use of the dsRNA molecule of any one of claims 1 to 24 or pharmaceutical composition of claim 25 or 26 in the manufacture of a medicament for treating a complement-related disease in a subject in need of such treatment.

31. The use of claim 30. wherein the complement-related disease or disorder is selected from atypical hemolytic uremic syndrome (aHUS), lupus nephritis (LN), primary membranous nephropathy (iMN), C3 glomerulopathy (C3G), coronary artery' disease (CAD), or idiopathic thrombocytopenic purpura (ITP),32. Use of the dsRNA molecule of any one of claims 1 to 24 or pharmaceutical composition of claim 25 or 26 in the manufacture of a medicament for treating a subject having a disorder that would benefit from reduction in complement factor B expression.

33. Use of the dsRN molecule of any one of claims 1 to 24 or pharmaceutical composition of claim 25 or 26 in the manufacture of a medicament for preventing development of a disorder that would benefit from reduction in complement factor B expression in a subject having at least one sign or symptom of a disorder who does not yet meet the diagnostic criteria for that disorder, thereby preventing the subject progressing to meet the diagnostic criteria of the disorder that would benefit from reduction in CFB expression.

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