Treatment of Complement-Mediated Diseases

Abstract

Inappropriate or excessive complement activation is the underlying cause or contributing factor of many serious diseases and conditions, and over the past few decades, great efforts have been made to explore various complement inhibitors as therapeutic agents. We describe RNAs, such as miRNA and siRNA, and their use in the treatment of complement-mediated diseases.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 193,573, filed May 26, 2021, which is incorporated by reference in its entirety herein. [Background technology]

[0002] Complement is a system of more than 30 plasma and cell-bound proteins that play important roles in both innate and adaptive immunity. The proteins of the complement system act in a series of enzymatic cascades through various protein interactions and cleavage events. Complement activation occurs through three major pathways: the antibody-dependent classical pathway, the alternative pathway, and the mannose-binding lectin (MBL) pathway. Inappropriate or excessive complement activation is the underlying cause or contributing factor of many serious diseases and conditions, and over the past decades, considerable efforts have been made to explore various complement inhibitors as therapeutic agents. Summary of the Invention

[0003] In one aspect, the present disclosure relates to a method for treating complement-mediated eye disease, comprising reducing the level of C3 in the liver of a subject, thereby treating eye disorder.In some embodiments, the method comprises systemically administering to the subject siRNA that targets the C3 mRNA of the subject.In some embodiments, the siRNA comprises a liver targeting moiety.In some embodiments, the liver targeting moiety is a GalNAc site.

[0004] In some embodiments, the siRNA comprises an antisense strand comprising a sequence set forth in Table 2A, 2B, 4, 5, 6, 10, 15, 16, 17, 18, 24-70, or 72-73, and / or a sense strand comprising a sequence set forth in Table 1, 3A, 3B, 10, 15, 16, 17, 18, or 21-71.

[0005] In some embodiments, the siRNA comprises an antisense strand and a sense strand, wherein the antisense strand is complementary to a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 76-100, and / or the sense strand comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 76-100. In some embodiments, the antisense strand is complementary to a nucleotide sequence that comprises a sequence that differs from any one of SEQ ID NOs: 76-100 by 1, 2, 3, or 4 nucleotides or less. In some embodiments, the antisense strand is complementary to a nucleotide sequence that comprises any one of SEQ ID NOs: 76-100. In some embodiments, the antisense strand comprises a nucleotide sequence that comprises any one of SEQ ID NOs: 101-125.

[0006] In some embodiments, one or both of the sense and antisense strands have at least one overhang region. In some embodiments, the at least one overhang region comprises an overhang of 1, 2, 3, 4 or 5 nucleotides. In some embodiments, the at least one overhang region comprises a 3' overhang. In some embodiments, the at least one overhang region is complementary to a fragment of SEQ ID NO: 75. In some embodiments, the 3' overhang comprises an overhang of 2 nucleotides.

[0007] In some embodiments, one or both of the sense and antisense strands contain at least one additional nucleotide at the 5' end, the 3' end, or both the 5' and 3' ends that is not complementary to a fragment of SEQ ID NO:75.

[0008] In some embodiments, one or both of the sense strand and the antisense strand comprises at least one modified nucleotide. In some embodiments, the at least one modified nucleotide comprises a nucleotide comprising a 2'-O-methyl group, a nucleotide comprising a 2'-fluoro group, and / or a phosphorothioate bond with an adjacent nucleotide. In some embodiments, the at least one modified nucleotide comprises a phosphorothioate bond between the last 2, 3 or 4 nucleotides at (i) the 5' end of the sense strand, (ii) the 3' end of the sense strand, (iii) the 5' end of the antisense strand, and / or (iv) the 3' end of the antisense strand. In some embodiments, the at least one modified nucleotide comprises a phosphorothioate bond between the last 3 nucleotides at (i) the 5' end of the sense strand, (ii) the 3' end of the sense strand, (iii) the 5' end of the antisense strand, and / or (iv) the 3' end of the antisense strand. In some embodiments, at least one modified nucleotide comprises a phosphorothioate bond between the last 2, 3, or 4 nucleotides of: (i) the 5' end of the sense strand, (ii) the 3' end of the sense strand, (iii) the 5' end of the antisense strand, and (iv) the 3' end of the antisense strand.

[0009] In some embodiments, the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 76-100, 126-150, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 255, 259, 264, 268, 272, 276, 325, 326, and 327. In some embodiments, the antisense strand comprises any one of the nucleotide sequences of SEQ ID NOs: 101-125, 151-200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 257, 258, 260, 261, 262, 263, 265, 266, 267, 269, 270, 271, 273, 274, 275, 277, 278, and 300-324.In some embodiments, the siRNA comprises the following sense / antisense sequence pairs: 201 / 202, 203 / 204, 205 / 206, 207 / 208, 209 / 210, 211 / 212, 213 / 214, 215 / 216, 217 / 218, 219 / 220, 221 / 222, 223 / 224, 225 / 226, 227 / 228, 229 / 230, 231 / 232, 233 / 234, 235 / 236, 237 / 238, 239 / 240, 241 / 242, 243 / 244, 245 / 246, 247 / 248, 249 / 250, 251 / 252, 253 26, 227 / 228, 229 / 230, 231 / 232, 233 / 234, 235 / 236, 237 / 238, 239 / 240, 241 / 242, 243 / 244, 245 / 246, 247 / 248, 249 / 250, 251 / 252, 253 / 254, 201 / 256, 255 / 256, 255 / 257, 2 01 / 258, 255 / 258, 207 / 260, 259 / 260, 259 / 261, 207 / 262, 259 / 262, 217 / 263, 264 / 263, 264 / 265, 217 / 266, 264 / 266, 219 / 267, 268 / 267, 268 / 269, 219 / 270, 268 / 270, 231 / 2 The sense strand nucleotide sequence / antisense strand nucleotide sequence of any one of the following: 71, 272 / 271, 272 / 273, 231 / 274, 272 / 274, 243 / 275, 276 / 275, 276 / 277, 243 / 278, 276 / 278, 325 / 275, 326 / 260, and 327 / 258.

[0010] In some embodiments, the siRNA comprises at least one ligand attached to one or more of the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and the 3' end of the antisense strand. In some embodiments, the ligand comprises at least one GalNAc moiety. In some embodiments, the ligand comprises three GalNAc moieties.

[0011] In some embodiments, the methods include administering to a subject a composition comprising a nucleic acid encoding the siRNA.

[0012] In some embodiments, after administration of siRNA or composition, the level of C3 transcript or C3 protein in the subject or in the biological sample from the subject is reduced relative to the level before administration of siRNA or composition.In some embodiments, the level of C3 transcript or C3 protein is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% relative to the level before administration.

[0013] In some embodiments, the siRNA or composition is administered intravenously or subcutaneously to the subject.In some embodiments, the siRNA or composition is administered to the subject's hepatocytes.In some embodiments, the siRNA or composition is administered to the hepatocytes ex vivo.In some embodiments, the siRNA or composition is administered to the hepatocytes in vivo.

[0014] In some embodiments, the method further comprises systemically administering to the subject a second agent, hi some embodiments, the second agent is an anti-C3 antibody or a compstatin analog.

[0015] In some embodiments, the eye disease is geographic atrophy or intermediate AMD.

[0016] In another aspect, the present disclosure relates to a method for inhibiting or reducing the level of complement C3 in the eye of a subject relative to a control, comprising reducing the level of C3 in the liver of the subject, thereby reducing the level of C3 in the eye. In some embodiments, the method comprises systemically administering to the subject siRNA that targets the C3 mRNA of the subject. In some embodiments, the siRNA comprises a liver targeting moiety. In some embodiments, the liver targeting moiety is a GalNAc site.

[0017] In some embodiments, the siRNA comprises an antisense strand comprising a sequence set forth in Table 2A, 2B, 4, 5, 6, 10, 15, 16, 17, 18, 24-70, or 72-73, and / or a sense strand comprising a sequence set forth in Table 1, 3A, 3B, 10, 15, 16, 17, 18, or 21-71.

[0018] In some embodiments, the siRNA comprises an antisense strand and a sense strand, wherein the antisense strand is complementary to a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 76-100, and / or the sense strand comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 76-100. In some embodiments, the antisense strand is complementary to a nucleotide sequence that comprises a sequence that differs from any one of SEQ ID NOs: 76-100 by 1, 2, 3, or 4 nucleotides or less. In some embodiments, the antisense strand is complementary to a nucleotide sequence that comprises any one of SEQ ID NOs: 76-100. In some embodiments, the antisense strand comprises a nucleotide sequence that comprises any one of SEQ ID NOs: 101-125.

[0019] In some embodiments, one or both of the sense and antisense strands have at least one overhang region. In some embodiments, the at least one overhang region comprises an overhang of 1, 2, 3, 4 or 5 nucleotides. In some embodiments, the at least one overhang region comprises a 3' overhang. In some embodiments, the at least one overhang region is complementary to a fragment of SEQ ID NO: 75. In some embodiments, the 3' overhang comprises an overhang of 2 nucleotides.

[0020] In some embodiments, one or both of the sense and antisense strands contain at least one additional nucleotide at the 5' end, the 3' end, or both the 5' and 3' ends that is not complementary to a fragment of SEQ ID NO:75.

[0021] In some embodiments, one or both of the sense strand and the antisense strand comprises at least one modified nucleotide. In some embodiments, the at least one modified nucleotide comprises a nucleotide comprising a 2'-O-methyl group, a nucleotide comprising a 2'-fluoro group, and / or a phosphorothioate bond with an adjacent nucleotide. In some embodiments, the at least one modified nucleotide comprises a phosphorothioate bond between the last 2, 3 or 4 nucleotides at (i) the 5' end of the sense strand, (ii) the 3' end of the sense strand, (iii) the 5' end of the antisense strand, and / or (iv) the 3' end of the antisense strand. In some embodiments, the at least one modified nucleotide comprises a phosphorothioate bond between the last 3 nucleotides at (i) the 5' end of the sense strand, (ii) the 3' end of the sense strand, (iii) the 5' end of the antisense strand, and / or (iv) the 3' end of the antisense strand. In some embodiments, at least one modified nucleotide comprises a phosphorothioate bond between the last 2, 3, or 4 nucleotides of (i) the 5' end of the sense strand, (ii) the 3' end of the sense strand, (iii) the 5' end of the antisense strand, and (iv) the 3' end of the antisense strand. In some embodiments, the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 76-100, 126-150, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 255, 259, 264, 268, 272, 276, 325, 326, and 327. In some embodiments, the antisense strand comprises any one of the nucleotide sequences of SEQ ID NOs: 101-125, 151-200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 257, 258, 260, 261, 262, 263, 265, 266, 267, 269, 270, 271, 273, 274, 275, 277, 278, and 300-324.In some embodiments, the siRNA comprises the following sense / antisense sequence pairs: 201 / 202, 203 / 204, 205 / 206, 207 / 208, 209 / 210, 211 / 212, 213 / 214, 215 / 216, 217 / 218, 219 / 220, 221 / 222, 223 / 224, 225 / 226, 227 / 228, 229 / 230, 231 / 232, 233 / 234, 235 / 236, 237 / 238, 239 / 240, 241 / 242, 243 / 244, 245 / 246, 247 / 248, 249 / 250, 251 / 252, 253 26, 227 / 228, 229 / 230, 231 / 232, 233 / 234, 235 / 236, 237 / 238, 239 / 240, 241 / 242, 243 / 244, 245 / 246, 247 / 248, 249 / 250, 251 / 252, 253 / 254, 201 / 256, 255 / 256, 255 / 257, 2 01 / 258, 255 / 258, 207 / 260, 259 / 260, 259 / 261, 207 / 262, 259 / 262, 217 / 263, 264 / 263, 264 / 265, 217 / 266, 264 / 266, 219 / 267, 268 / 267, 268 / 269, 219 / 270, 268 / 270, 231 / 2 The sense strand nucleotide sequence / antisense strand nucleotide sequence of any one of the following: 71, 272 / 271, 272 / 273, 231 / 274, 272 / 274, 243 / 275, 276 / 275, 276 / 277, 243 / 278, 276 / 278, 325 / 275, 326 / 260, and 327 / 258.

[0022] In some embodiments, the siRNA further comprises at least one ligand attached to one or more of the 5' end of the sense strand, the 3' end of the sense strand, the 5' end of the antisense strand, and the 3' end of the antisense strand. In some embodiments, the ligand comprises at least one GalNAc moiety. In some embodiments, the ligand comprises three GalNAc moieties.

[0023] In some embodiments, the methods include administering to a subject a composition comprising a nucleic acid encoding the siRNA.

[0024] In some embodiments, after the administering step, the level of C3 transcript or C3 protein is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% relative to the subject's control level of C3 prior to the administering step.

[0025] In some embodiments, the method further comprises systemically administering to the subject a second agent, hi some embodiments, the second agent is an anti-C3 antibody or a compstatin analog.

[0026] In some embodiments, the subject is afflicted with a complement-mediated disease. In some embodiments, the eye disease is geographic atrophy or intermediate AMD.

[0027] In some embodiments of any of the aspects described herein, the siRNA, composition, or second agent is not administered locally to the eye of the subject.

[0028] definition Antibody: As used herein, the term "antibody" refers to an immunoglobulin or derivative thereof that contains an immunoglobulin domain capable of binding to an antigen. The antibody may be of any species, e.g., human, rodent, rabbit, goat, chicken, etc. The antibody may be a member of any immunoglobulin class, including the human classes of IgG, IgM, IgA, IgD, and IgE, or subclasses thereof, such as IgG1, IgG2, etc. In various embodiments of the invention, the antibody is a fragment, such as a Fab', F(ab')2, scFv (single chain variable) or other fragment that retains the antigen binding site, or a recombinantly produced scFv fragment, including a recombinantly produced fragment. See, e.g., Allen, T., Nature Reviews Cancer, Vol. 2, 750-765, 2002, and references therein. The antibody may be monovalent, bivalent, or multivalent. The antibody may be, for example, a chimeric or "humanized" antibody in which a variable domain of rodent origin is fused to a constant domain of human origin, thus retaining the specificity of rodent antibodies. The domain of human origin need not be directly derived from a human, in the sense that it is first synthesized in a human. Alternatively, the "human" domain may have been generated in a rodent that has integrated human immunoglobulin genes into its genome. See, for example, Vaughan, et al., (1998), Nature Biotechnology, 16:535-539. The antibody may be partially or fully humanized. The antibody may be polyclonal or monoclonal, although for the purposes of the present invention, monoclonal antibodies are generally preferred. Methods for producing antibodies that specifically bind to virtually any molecule of interest are known in the art. For example, monoclonal or polyclonal antibodies can be purified from the blood or ascites fluid of an animal that produces the antibody (e.g., following natural exposure to or immunization with the molecule or an antigenic fragment thereof), can be produced using recombinant techniques in cell culture or transgenic organisms, or can be made at least in part by chemical synthesis.

[0029] Approximately: As used herein, the terms "approximately" or "about" in reference to numbers are generally interpreted to include numbers within 5%, 10%, 15%, or 20% in either direction (greater or less) of the number unless specifically stated otherwise or otherwise apparent from the context (except where such number is less than 0% or greater than 100% of a possible value).

[0030] Complementary: As used herein, "complementary" refers to the capacity for precise pairing between specific bases, nucleosides, nucleotides, or nucleic acids, in accordance with its art-accepted meaning. For example, adenine (A) is complementary to uridine (U), adenine (A) is complementary to thymidine (T), and guanine (G) is complementary to cytosine (C), which is referred to in the art as Watson-Crick base pairing. If a nucleotide at a particular position in a first nucleic acid sequence is complementary to an oppositely positioned nucleotide in a second nucleic acid sequence when the strands are aligned in an antiparallel orientation, then the nucleotides form a complementary base pair and the nucleic acids are complementary at that position. The percent complementarity of a first nucleic acid to a second nucleic acid can be evaluated by aligning these nucleic acid sequences in an antiparallel orientation to maximize complementarity over an evaluation window, determining the total number of nucleotides (nt) of both strands that form complementary base pairs within the window, dividing by the total number of nt in the window, and multiplying by 100. For example, AAAAAAAA and TTTGTTAT are 75% complementary because there are 12 nt of complementary base pairs out of a total of 16 nt. When calculating the number of complementarities required to obtain a particular percent complementarity, decimals are rounded to the nearest integer. A position occupied by a non-complementary nucleotide constitutes a mismatch. That is, the position is occupied by a non-complementary base pair. In certain embodiments, the evaluation window has a length as described herein in the duplex portion or the target portion. Complementary sequences include base pairing between a polynucleotide comprising a first nucleotide sequence and a polynucleotide comprising a second nucleotide sequence over the entire length of the first and second nucleotide sequences (if they are the same length) or over the entire length of the shorter sequence (if they are different lengths). Such sequences can be referred to herein as "fully complementary" (100% complementary) to each other. Nucleic acids that are at least 70% complementary over an evaluation window are considered to be "substantially complementary" over that window.In certain embodiments, complementary nucleic acids are at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% complementary over the evaluation window.When a first sequence is said to be "substantially complementary" with respect to a second sequence herein, the two sequences may be fully complementary or may contain one or more unmatched bases upon hybridization while maintaining the ability to hybridize under the conditions most relevant to their intended use, for example, may contain up to about 5%, 10%, 15%, 20%, or 25% unmatched bases upon hybridization, for example, 1, 2, 3, 4, 5, or 6 mismatched base pairs upon hybridization in a duplex of 30 base pairs or less.It should be understood that when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered as mismatched or unpaired nucleotides for determining the percentage of complementarity (%). For example, two strands of dsRNA containing one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length may be referred to herein as "fully complementary" if the longer oligonucleotide contains a 21 nucleotide sequence that is fully complementary to the shorter oligonucleotide, with a 2 nucleotide overhang. As used herein, "complementary" sequences may contain one or more non-Watson-Crick base pairs and / or base pairs formed from non-natural and other modified nucleotides, so long as the requirements for their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairs.Those skilled in the art will recognize that guanine, cytosine, adenine and uracil can be substituted for other bases without substantially changing the base-pairing properties of polynucleotides containing nucleotides with these bases, according to the so-called "wobble" rules (e.g., Murphy, FV IV & V Ramakrishnan, V., Nature Structural and Molecular Biology 11:1251-1252 (2004)). For example, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, in the nucleotide sequences of the inhibitory RNAs described herein, nucleotides containing uracil, guanine, or adenine can be substituted for nucleotides containing, for example, inosine. It is understood that the terms "complementary", "fully complementary" and "substantially complementary" can be used in reference to base matching between any two nucleic acids, such as base matching between the sense and antisense strands of a dsRNA, or base matching between the antisense strand of a ds inhibitory RNA (e.g., siRNA) and a target sequence, or base matching between an antisense oligonucleotide and a target sequence, as will be understood by those skilled in the art. As used herein, "hybridize" refers to the interaction between two nucleic acid sequences that contain or consist of complementary portions, such that a stable duplex structure is formed under certain conditions of interest.

[0031] Complement components: As used herein, the term "complement components" or "complement proteins" refers to molecules that are involved in the activation of the complement system or participate in one or more complement-mediated activities. Classical complement pathway components include, for example, C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9, and the C5b-9 complex, also called the membrane attack complex (MAC), as well as active fragments or enzymatic cleavage products of any of the foregoing (e.g., C3a, C3b, C4a, C4b, C5a, etc.). Alternative pathway components include, for example, factors B, D, H, and I, and properdin, with factor H being a negative regulator of the pathway. Lectin pathway components include, for example, MBL2, MASP-1, and MASP-2. Complement components also include cell-bound receptors for soluble complement components. Such receptors include, for example, C5a receptor (C5aR), C3a receptor (C3aR), complement receptor 1 (CR1), complement receptor 2 (CR2), complement receptor 3 (CR3), etc. The term "complement components" is not intended to include molecules and molecular structures that act as "triggers" for complement activation, such as antigen-antibody complexes, foreign structures found on microorganisms or artificial surfaces, etc.

[0032] Host cell: As used herein, the term "host cell" refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Those skilled in the art will understand upon reading this disclosure that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations, either due to mutations or environmental influences, such progeny may not in fact be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life suitable for expressing exogenous DNA (e.g., recombinant nucleic acid sequences). Exemplary cells include prokaryotic and eukaryotic (unicellular or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions, e.g., hybridomas or quadromas. In some embodiments, the cells are human, monkey, ape, hamster, rat, or mouse cells. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-1 1CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293EBNA, MSR293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC5, Colo205, HB8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT060562, Sertoli cells, BRL3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the aforementioned cells. In some embodiments, the cell comprises one or more viral genes.

[0033] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, multiple polymer molecules are considered to be "substantially identical" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. For example, the calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison (e.g., gaps may be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). In certain embodiments, the length of the aligned sequence for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the reference sequence. The nucleotides at the corresponding positions are then compared. If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and the determination of the percent identity between two sequences can be achieved using a mathematical algorithm. For example, the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated in the ALIGN program (version 2.0) can be used to determine the percent identity between two nucleotide sequences. In some exemplary embodiments, comparison of nucleic acid sequences using the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.The percent identity between two nucleotide sequences can alternatively be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

[0034] Linked: As used herein, the term "linked" when used in reference to two or more moieties means that the moieties are physically bound or connected to each other to form a molecular structure that is sufficiently stable so that the moieties remain linked under the conditions under which the bond is formed, preferably under the conditions under which the new molecular structure is used, e.g., physiological conditions. In certain preferred embodiments of the invention, the linkage is a covalent bond. In other embodiments, the linkage is non-covalent. The moieties may be linked directly or indirectly. When two moieties are directly linked, they are covalently bonded to each other or are in sufficient proximity that intermolecular forces between the two moieties maintain their association. When two moieties are indirectly linked, they are each linked, either covalently or non-covalently, to a third moiety, which maintains the association between the two moieties. In general, when two moieties are referred to as being linked by a "linker" or "linking moiety" or "linking moiety", the linkage between the two linking moieties is indirect, and typically each of the linking moieties is covalently bonded to the linker. The linker can be any suitable moiety that will react with the two moieties being linked in sufficient quantity and within a reasonable period of time to produce a reasonable yield, under conditions consistent with the stability of the moieties (which can be appropriately protected depending on the conditions).

[0035] MicroRNA (miRNA): As used herein, the term "microRNA" or "miRNA" refers to a small non-coding RNA molecule that may function in the transcriptional and / or post-transcriptional regulation of target gene expression. The term encompasses mature miRNA sequences or precursor miRNA sequences, including primary transcripts (pri-miRNAs) and stem-loop precursors (pre-miRNAs). Naturally occurring miRNA biogenesis begins in the nucleus with transcription by RNA polymerase II to generate primary transcripts (pri-miRNAs). The primary transcripts are cleaved by the Drosha ribonuclease III enzyme to generate approximately 70 nt stem-loop precursor miRNAs (pre-miRNAs). The pre-miRNAs are then actively exported to the cytoplasm, where they are cleaved by the Dicer ribonuclease to form mature miRNAs, including an "antisense strand" or "guide strand" (containing a region substantially complementary to the target sequence) and a "sense strand" or "passenger strand" (containing a region substantially complementary to the region of the antisense strand). Those skilled in the art will understand that the guide strand may be fully complementary to the target region of the target RNA, or may not be fully complementary to the target region of the target RNA. The guide strand of the miRNA is incorporated into an RNA-induced silencing complex (RISC) that recognizes the target mRNA by base pairing with the miRNA, generally resulting in the inhibition or destabilization of translation of the target mRNA. As is understood in the art, in the case of naturally occurring miRNAs, recognition of the target mRNA occurs by imperfect base pairing with the mRNA. In some embodiments, the miRNA is synthetic or engineered, and recognition of the target mRNA occurs by perfect base pairing with the mRNA. Generally, the target mRNA contains a sequence complementary to the "seed" sequence of the miRNA, which usually corresponds to nucleotides 2-8 of the miRNA.Information about miRNAs and associated pri-miRNA and pre-miRNA sequences is available in miRNA databases such as miRBase (Grifiths-Jones et al., 2008 Nucl Acids Res 36, (Database Issue: D154-D158) and the NCBI Human Genome Database.

[0036] Operably linked: As used herein, the term "operably linked" refers to a positional relationship in which each described component is in a relationship that allows them to function in their intended manner. A control element "operably linked" to a functional element is associated such that expression and / or activity of the functional element is obtained under conditions compatible with the control element. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest. In some embodiments, the control element acts in trans or remotely on the functional element of interest.

[0037] Recombinant: As used herein, the term "recombinant" is intended to mean a polypeptide that is designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic for or has been otherwise engineered to express a gene(s) or genetic component(s) encoding and / or directing the expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or a polypeptide prepared, expressed, created, or isolated by any other means, including splicing or ligation of selected nucleic acid sequence elements together, chemical synthesis of selected sequence elements, and / or alternative generation of a nucleic acid encoding and / or directing the expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of the selected sequence elements are found in nature. In some embodiments, one or more of the selected sequence elements are designed in silico. In some embodiments, one or more of the selected sequence elements result from, for example, mutagenesis (e.g., in vivo or ex vivo) of known sequence elements from natural or synthetic sources, such as, for example, the germline of a source organism of interest (e.g., human, mouse, etc.).

[0038] RNA interference: RNA interference: As used herein, the term "RNA interference" or "RNAi" generally refers to a process in which a double-stranded or short hairpin RNA molecule reduces or inhibits expression of a nucleic acid sequence with which the double-stranded or short hairpin RNA molecule shares substantial or complete homology. Without wishing to be bound by any theory, in nature, the RNAi pathway is believed to be initiated by a type III endonuclease known as Dicer, which cleaves long double-stranded RNA (dsRNA) into double-stranded fragments called "short interfering RNAs" ("siRNAs"), usually consisting of 21-23 base pairs, with a 2-base 3' overhang (although variations in length and overhang are contemplated). Such siRNAs comprise two single-stranded RNAs (ssRNAs) comprising an "antisense strand" or "guide strand" that contains a region substantially complementary to the target sequence, and a "sense strand" or "passenger strand" that contains a region substantially complementary to a region of the antisense strand. One of skill in the art will understand that the guide strand can be perfectly complementary to the target region of the target RNA, or may not be perfectly complementary to the target region of the target RNA.

[0039] Subject: As used herein, the term "subject" or "subject" refers to any organism to which a provided compound or composition is administered in accordance with the present invention, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Exemplary subjects include animals (e.g., mice, rats, rabbits, non-human primates, and humans, insects, worms, etc.). In some embodiments, the subject may be suffering from a relevant disease, disorder, and / or condition.

[0040] Substantially: As used herein, the term "substantially" refers to a qualitative condition exhibiting the entire or nearly entire extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena, if they exist at all, rarely go to completion and / or rarely proceed completely or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0041] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of the disease, disorder, and / or condition.

[0042] Target gene: As used herein, a "target gene" refers to a gene whose expression is to be modulated, e.g., inhibited. As used herein, the term "target RNA" refers to an RNA that is to be degraded, or whose translation is to be repressed or inhibited, using one or more miRNAs. A target RNA may also be referred to as a target sequence or a target transcript. The RNA may be a primary RNA transcript (e.g., pre-mRNA) or a processed transcript (e.g., an mRNA encoding a polypeptide) transcribed from a target gene. As used herein, the term "target portion" or "target region" refers to a contiguous portion of a nucleotide sequence of a target RNA. In some embodiments, a target portion of an mRNA is at least long enough to serve as a substrate for RNA interference (RNAi)-mediated cleavage within that portion in the presence of an appropriate inhibitory RNA. The target portion may be about 8-36 nucleotides in length, e.g., about 10-20 or about 15-30 nucleotides in length. The length of the target portion may have a particular value or subrange within the aforementioned ranges. For example, in certain embodiments, the targeting moiety may be about 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19- 28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length.

[0043] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0044] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that induces a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be appreciated by those skilled in the art, the effective amount of a substance may vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, improves, relieves, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms or signs of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0045] Treating: As used herein, the term "treating" refers to providing therapy, i.e., providing any type of medical or surgical management of a subject. Treatment may be provided to reverse, alleviate, inhibit, prevent or reduce the progression of a disease, disorder, or condition, or to reverse, alleviate, prevent the progression of, or reduce the likelihood of, one or more symptoms or signs of a disease, disorder, or condition. "Preventing" refers to preventing such a disease, disorder, condition, or symptom or sign from occurring in at least some individuals, at least for a period of time. Treating can include administering an agent to a subject after the onset of one or more symptoms or signs indicative of a complement-mediated condition, for example, to reverse, alleviate, reduce the severity, and / or inhibit or prevent the progression of the condition, and / or to reverse, alleviate, reduce the severity, and / or inhibit one or more symptoms or signs of the condition. The compositions of the present disclosure can be administered to subjects who have a complement-mediated disease or who are at high risk of developing such a disease compared to members of the general population. The compositions of the present disclosure can be administered prophylactically, i.e., before the onset of a symptom or sign of a condition. In this case, the subject is typically at risk of developing the condition.

[0046] Nucleic acid: The term "nucleic acid" includes any nucleotide, its analogs, and polymers thereof. As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, double- and single-stranded RNA. These terms include, as equivalents, analogs of RNA or DNA made from nucleotide analogs and modified polynucleotides, such as, but not limited to, methylated, protected, and / or capped nucleotides or polynucleotides. The term includes poly- or oligoribonucleotides (RNA) and poly- or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N- or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus atom bridges (also referred to herein as "internucleotide linkages"). The term includes nucleic acids that contain any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, but are not limited to, nucleic acids that contain a ribose moiety, nucleic acids that contain a deoxyribose moiety, nucleic acids that contain both a ribose moiety and a deoxyribose moiety, and nucleic acids that contain a ribose moiety and a modified ribose moiety. In some embodiments, the prefix "poly" refers to a nucleic acid that contains from 2 to about 10,000, from 2 to about 50,000, or from 2 to about 100,000 nucleotide monomer units. In some embodiments, the prefix "oligo" refers to a nucleic acid that contains from 2 to about 200 nucleotide monomer units.

[0047] Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule linked to it. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of inducing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0048] Standard techniques of recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation are used (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly practiced in the art or as described herein. The techniques and procedures described above can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for all purposes. [Brief description of the drawings]

[0049] [Figure 1]1 shows a chart disclosing exemplary modification patterns 1-5 for the sense and antisense strands of an inhibitory RNA (e.g., siRNA) duplex. "2OM" represents a 2'-O-methyl modification, "2F" represents a 2'-fluoro modification, and "PS" represents a phosphorothioate linkage to the adjacent 3' nucleotide. [Diagram 2] 1 shows the structure of pegcetacoplan ("APL-2"), assuming n is about 800 to about 1100 and PEG is about 40 kD. [Diagram 3] Figure 1 shows the results of an in vivo study in non-human primates. siRNA58 was administered at doses of 3mg / kg, 10mg / kg, 30mg / kg, or vehicle by subcutaneous injection. The graph shows the time course of serum C3 protein levels for each group up to 67 days after administration. Serum C3 protein levels were measured using an ELISA assay. Values ​​on day -1 were used as baseline. [Figure 4] Data from an in vivo study in non-human primates are shown. siRNA58 at doses of 3 mg / kg, 10 mg / kg, 30 mg / kg, or vehicle was administered by subcutaneous injection. The graph shows C3 mRNA expression in liver biopsies taken from non-human primates 15 days after injection. The levels of C3 mRNA in the samples were measured using a quantitative PCR assay. In these experiments, C3 mRNA levels were normalized to the levels of ActB mRNA. [Diagram 5] Data from an in vivo study in non-human primates are shown. siRNA58 at doses of 3 mg / kg, 10 mg / kg, 30 mg / kg, or vehicle was administered by subcutaneous injection. The graph shows C3 mRNA expression in liver biopsies taken from non-human primates 46 days after injection. The levels of C3 mRNA in the samples were measured using a quantitative PCR assay. In these experiments, C3 mRNA levels were normalized to the levels of ActB mRNA. [Figure 6]Figure 1 shows the time course of the levels of alternative pathway (AH50) activity in serum from non-human primates injected with different doses of siRNA58 (3 mg / kg, 10 mg / kg, 30 mg / kg, or vehicle) collected through day 29. Alternative pathway activity (AH50) was determined using an ELISA assay. Day -1 values ​​were used as baseline. [Figure 7] The percent change in plasma C3 concentration from baseline following subcutaneous administration of siRNA59 as a single bolus (A) or three times daily bolus (B) is shown. Values ​​shown as zero were below the LLOQ of the assay. Data represent the mean ± SEM (n=3). [Figure 8] Measurement of alternative pathway activity by detection and quantification of soluble C5b-9 complexes using ELISA (optical density readings = OD) in serum of animals treated subcutaneously with vehicle (A), siRNA (-) control (B), 3 mg / kg siRNA59 (C), 10 mg / kg siRNA59 (D), and (E) 30 mg / kg siRNA59. Data represent mean ± SEM (n=3). [Figure 9] Shown are levels of C3 mRNA in liver tissue 3 days (A) and 30 days (B and C) after a single dose (A and B) or three daily doses (C) of siRNA59. Data represent the mean ± SEM (n=3). [Figure 10] Figure 1 shows serum C3 levels from non-human primates at various times after administration of siRNA (100mg / kg) or vehicle. Panel A shows the mean serum C3 concentration (ng / ml). Panel B shows the mean serum C3 reduction as a percentage decrease from the level before siRNA or vehicle was administered (i.e., baseline level). [Figure 11] Figure 1 shows plasma C3a levels from non-human primates at various times after administration of siRNA (100 mg / kg) or vehicle. Panel A shows the mean plasma C3a concentration (ng / ml). Panel B shows the mean plasma C3a reduction as a percentage decrease from the level before siRNA or vehicle was administered (i.e., baseline level). [Figure 12]C3 (Panel A) and C3a (Panel B) levels (ng / ml) in the vitreous humor (VH) of non-human primates 44 days after administration of siRNA (100 mg / kg) or vehicle are shown. [Figure 13] C3 (Panel A) and C3a (Panel B) levels (ng / ml) in the aqueous humor (AH) of non-human primates 44 days after administration of siRNA (100 mg / kg) or vehicle are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] The present disclosure is based in part on the discovery that ocular diseases (e.g., complement-mediated ocular diseases) can be treated by targeted reduction of complement in the liver without local administration of a complement inhibitor to the eye. In some embodiments, a complement-mediated ocular disorder can be treated by systemic administration of one or more complement inhibitors (e.g., a complement inhibitor described herein, e.g., a complement inhibitor targeted to the liver) without local administration of a complement inhibitor, e.g., a complement inhibitor described herein, to the eye.

[0051] I. Complement system To facilitate understanding of the present disclosure, without intending to limit the invention in any way, this section provides an overview of complement and its activation pathways. Further details can be found, for example, in Kuby Immunology, 6th ed., 2006; Paul, WE, Fundamental Immunology, Lippincott Williams & Wilkins; 6th ed., 2008; and Walport MJ., Complement. First of two parts. N Engl J Med., 344(14):1058-66, 2001.

[0052] Complement is a weapon of the innate immune system that plays a key role in defending the body against infectious pathogens. The complement system is composed of over 30 serum and cellular proteins that participate in three major pathways, known as the classical, alternative, and lectin pathways. The classical pathway is usually triggered by the binding of a complex of antigen and IgM or IgG antibodies to C1 (although certain other activators can also initiate the pathway). Activated C1 cleaves C4 and C2 to generate C4a and C4b, in addition to C2a and C2b. C4b and C2a combine to form C3 convertase, which cleaves C3 to form C3a and C3b. Binding of C3b to C3 convertase generates C5 convertase, which cleaves C5 to C5a and C5b. C3a, C4a, and C5a are anaphylatoxins that mediate multiple responses in acute inflammatory responses. C3a and C5a are also chemotactic factors that attract immune system cells such as neutrophils. It will be appreciated that the earlier designations "C2a" and "C2b" have later been reversed in the scientific literature.

[0053] The alternative pathway is initiated and amplified, for example, by microbial surfaces and various complex polysaccharides. In this pathway, the naturally occurring low-level hydrolysis of C3 to C3(H2O) leads to the binding of factor B, which is cleaved by factor D to generate the fluid-phase C3 convertase that activates complement by cleaving C3 into C3a and C3b. C3b binds to targets such as cell surfaces and forms a complex with factor B, which is subsequently cleaved by factor D to become the C3 convertase. The surface-bound C3 convertase cleaves and activates additional C3 molecules, leading to rapid deposition of C3b in close proximity to the activation site, forming additional C3 convertase, which in turn generates additional C3b. This process results in a cycle of C3 cleavage and C3 convertase formation, greatly amplifying the response. Cleavage of C3 and binding of another molecule of C3b to the C3 convertase gives rise to the C5 convertase. The C3 and C5 convertases of this pathway are regulated by the cellular molecules CR1, DAF, MCP, CD59, and fH. The mechanisms of action of these proteins include decay-accelerating activity (i.e., the ability to dissociate the convertases), the ability to function as a cofactor in the degradation of C3b or C4b by factor I, or both. Normally, the presence of complement regulatory proteins on the cell surface prevents significant complement activation from occurring thereon.

[0054] The C5 convertase produced by both pathways cleaves C5 to generate C5a and C5b. C5b then binds to C6, C7, and C8 to form C5b-8, and catalyzes the polymerization of C9 to form the C5b-9 membrane attack complex (MAC). The MAC inserts itself into the target cell membrane, causing cell lysis. Small amounts of MAC on the cell membrane can have a variety of consequences other than cell death.

[0055] The lectin complement pathway is initiated by the binding of mannose-binding lectin (MBL) and MBL-associated serine proteases (MASPs) to carbohydrates. The MB1-1 gene (known as LMAN-1 in humans) encodes a type I integral membrane protein that is localized in the intermediate region between the endoplasmic reticulum and the Golgi apparatus. The MBL-2 gene encodes a soluble mannose-binding protein found in serum. In the human lectin pathway, MASP-1 and MASP-2 are involved in the proteolysis of C4 and C2, leading to the C3 convertases mentioned above.

[0056] Complement activity is regulated by a variety of mammalian proteins called complement control proteins (CCPs) or regulators of complement activation (RCA) proteins (US Pat. No. 6,897,290). These proteins differ with respect to their ligand specificity and mechanism(s) of complement inhibition. They may accelerate the normal decay of convertases and / or function as cofactors for factor I, enzymatically cleaving C3b and / or C4b into smaller fragments. CCPs are characterized by the presence of multiple (usually 4-56) homologous motifs of approximately 50-70 amino acids in length, including a conserved motif containing four disulfide-linked cysteines (two disulfide bonds), proline, tryptophan, and many hydrophobic residues, known as short consensus repeats (SCRs), complement control protein (CCP) modules, or SUSHI domains. The CCP family includes complement receptor type 1 (CR1; C3b:C4b receptor), complement receptor type 2 (CR2), membrane complement protein (MCP; CD46), decay accelerating factor (DAF), complement factor H (fH), and C4b binding protein (C4bp). CD59 is a membrane-bound complement regulatory protein that is structurally unrelated to the CCPs. Complement regulatory proteins normally function to limit complement activation that can occur in mammalian cells or tissues, such as the human host. Thus, "self" cells are normally protected from the deleterious effects that would result from complement activation proceeding on these cells. Deficiencies or defects in complement regulatory protein(s) have been implicated in the pathogenesis of various complement-mediated diseases, such as those described herein.

[0057] Complement components (including C3 protein or C3 mRNA) have been reported to be expressed in ocular tissues (including retina, RPE, and choroid) and various cell types (including microglia, astrocytes, myeloid cells, and vascular cells) (see, e.g., Jong et al., Prog. Retinal and Eye Research, https: / / doi.org / 10.1016 / j.preteyeres.2021.100952 (2021)). Expression of C3 mRNA by microglia / monocytes in the retina has been reported to contribute to complement activation in the aging retina of rats (see, e.g., Rutar et al., PLoS ONE PLoS ONE 9(4):e93343 doi:10.1371 / journal.pone.0093343 (2014)). Furthermore, local complement regulatory defects have been reported in neovascular age-related macular degeneration (see, e.g., Schick et al., Eye 31:810-813 (2017)). Using a mouse model of retinal degeneration, it has been reported that intravitreal injection of C3 siRNA inhibits complement activation and deposition and reduces cell death, whereas systemic reduction of serum complement was ineffective (see, e.g., Natoli et al., Invest. Am. Ophthalmol. Vis. Sci. 58:2977-2990 (2017)).

[0058] II. Inhibitory RNA for C3 The present disclosure includes compositions and methods relating to one or more nucleotide sequences that are, contain, or encode an inhibitory RNA that binds to and inhibits expression of messenger RNA (mRNA) produced by a target gene (e.g., C3). The inhibitory RNA can be a single-stranded (e.g., antisense oligonucleotide) or double-stranded nucleic acid. In some embodiments, the inhibitory RNA comprises a double-stranded RNA duplex, such as a microRNA (miRNA) or a small interfering RNA (siRNA). In some embodiments, the inhibitory RNA is an siRNA or miRNA, or a vector that comprises a nucleotide sequence encoding an siRNA or miRNA.

[0059] In some embodiments, the inhibitory RNA can inhibit expression of one or more non-human species of C3, such as non-human primate C3, such as cynomolgus C3, or green monkey C3, in addition to human C3. The cynomolgus C3 gene is designated as NCBI Gene ID: 102131458, and the predicted amino acid and nucleotide sequences of cynomolgus C3 are listed as NCBI Reference Sequence Accession Nos. XP_005587776.1 and XM_005587719.2, respectively. In some embodiments, the inhibitory RNA comprises an antisense strand that is complementary to a target portion of the human and cynomolgus C3 transcripts that is identical. In some embodiments, the inhibitory RNA comprises an antisense strand that is complementary to a target portion of the human C3 transcript that differs from the sequence in the cynomolgus C3 transcript by 1, 2, or 3 nucleotides. It will be appreciated that inhibitory RNA that inhibits expression of human C3 may also inhibit expression of non-primate C3, such as rat or mouse C3, particularly if a conserved region of the C3 transcript is targeted.

[0060] The amino acid and nucleotide sequences of human C3 are well known in the art and can be found in publicly available databases, such as the NCBI (National Center for Biotechnology Information) Reference Sequence (RefSeq) database, and are set forth in Reference Sequence Accession Nos. NP_000055 (Accession Version No. NP_000055.2) and NM_000064 (Accession Version No. NM_000064.4), respectively ("amino acid sequence" in this context refers to the sequence of the C3 polypeptide, and "nucleotide sequence" refers to the mRNA sequence of C3 as represented in genomic DNA, although it is understood that the nucleotide sequence of the actual mRNA contains U rather than T). Those skilled in the art will recognize that the foregoing sequences are those of the complement C3 preproprotein, including the signal sequence that is cleaved and therefore not present in the mature protein. The human C3 gene has been designated as NCBI gene ID: 718, and the genomic C3 sequence has reference sequence accession number NG_009557 (accession version number NG_009557.1). The nucleotide sequence of human C3 mRNA is shown below (reference sequence accession number NM_000064.3 with T replaced by U; AUG start codon is underlined starting at position 94):

[0061] GUUAUAUCUCAAAAAAAAAAAAAAAA (SEQ ID NO: 75)

[0062] In some embodiments, the inhibitory RNA comprises a nucleic acid strand complementary to a target portion of a C3 transcript, e.g., C3 mRNA (e.g., complementary to a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a target portion of SEQ ID NO: 75). The target portion can be 15-30 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, although shorter and longer target portions are also contemplated. In some embodiments, the target portion comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the sequences set forth in Table 1 below. [Table 1]

[0063] Administration of inhibitory RNA can reduce the level of C3 transcript or C3 protein in subject or biological sample (e.g., blood, serum or plasma sample, sample containing liver cells) compared to the level before administration of composition.In some embodiments, the level of C3 transcript or C3 protein is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the level before administration.The level of C3 protein can be measured, for example, in blood (serum or plasma) sample.

[0064] MicroRNA The present disclosure also includes compositions and methods related to one or more oligonucleotides that are, comprise, or encode microRNAs. MicroRNAs (miRNAs) are a class of highly conserved small RNA molecules that are transcribed from the DNA of plant and animal genomes but are not translated into proteins. Naturally occurring miRNAs are initially transcribed as a primary transcript (pri-miRNA) that contains a long hairpin. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to generate a ~70 nt stem-loop precursor miRNA (pre-miRNA), which contains an "antisense strand" or "guide strand" (containing a region substantially complementary to the target sequence) and a "sense strand" or "passenger strand" (containing a region substantially complementary to a region of the antisense strand). The pre-miRNA is then actively exported to the cytoplasm where it is cleaved by the Dicer ribonuclease to form a mature miRNA. The processed microRNA is incorporated into the RNA-induced silencing complex (RISC) to form a mature gene silencing complex, which induces target mRNA degradation and / or translational repression. Many miRNA sequences have been identified so far, and examples can be found in, for example, "miRBase: microRIVA sequences, targets and gene nomenclature" Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A, Enright AJ.NAR, 2006, 34, Database Issue, D140-D144; "The microRNA Registry" Griffiths-Jones S.NAR, 2004, 32, Database Issue, D109-D111.

[0065] miRNAs can be designed and / or synthesized as mature molecules or precursors (e.g., pri- or pre-miRNAs). In some embodiments, the pre-miRNA comprises a guide strand and a passenger strand of the same length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides). In some embodiments, the pre-miRNA comprises a guide strand and a passenger strand of different lengths (e.g., one strand is about 19 nucleotides and the other is about 21 nucleotides). In some embodiments, the miRNA can target the coding region, the 5' untranslated region, and / or the 3' untranslated region of an endogenous mRNA. In some embodiments, the miRNA comprises a guide strand that comprises a nucleotide sequence having a sequence sufficiently complementary to an endogenous mRNA of interest to hybridize with and inhibit expression of the endogenous mRNA.

[0066] In some embodiments, the miRNA comprises a nucleic acid strand that includes a region that is fully complementary to at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides of SEQ ID NO: 75 (e.g., any one of SEQ ID NOs: 76-100). In some embodiments, the miRNA comprises a mature guide strand that has a nucleotide sequence that is fully complementary to a target portion that includes a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 76-100.

[0067] siRNA In some embodiments, the inhibitory RNA is double-stranded RNA (dsRNA) and inhibits C3 expression by RNA interference (RNAi). RNAi is a process of sequence-specific post-transcriptional gene silencing, in which double-stranded RNA (dsRNA) that is homologous to a target locus can specifically inactivate gene function (Hammond et al., Nature Genet. 2001; 2: 110-119; Sharp, Genes Dev. 1999; 13: 139-141). This dsRNA-induced gene silencing can be mediated by short double-stranded small interfering RNA (siRNA) that is generated from longer dsRNA by RNase III cleavage (Bernstein et al., Nature 2001; 409: 363-366 and Elbashir et al., Genes Dev. 2001; 15: 188-200). RNAi-mediated gene silencing is thought to occur through sequence-specific RNA degradation, with sequence specificity determined by the interaction of the siRNA with its complementary sequence within the target RNA (eg, Tuschl, Chem. Biochem. 2001;2:239-245). RNAi can involve, for example, the use of siRNAs with a foldback stem-loop structure (Elbashir, et al., Nature 2001;411:494-498) or short hairpin RNAs (shRNAs) (Paddison et al., Genes Dev. 2002;16: 948-958; Sui et al., Proc. Natl. Acad. Sci. USA 2002;99:5515-5520; Brummelkamp et al., Science 2002;296:550-553; Paul et al., Nature Biotechnol. 2002;20:505-508).

[0068] The present disclosure includes siRNA molecules targeting C3 transcripts, such as C3 mRNA (SEQ ID NO: 75). In some embodiments, the siRNA molecule includes a sequence complementary to a target region including any one of SEQ ID NOs: 76-100. In some embodiments, the siRNA molecule includes (i) a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 76-100 (or a portion thereof), and / or (ii) a nucleotide sequence complementary to a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 76-100 (or a portion thereof).

[0069] In some embodiments, the siRNA of the present disclosure is a double-stranded nucleic acid duplex (e.g., of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 base pairs) that comprises an annealed complementary single-stranded nucleic acid molecule. In some embodiments, the siRNA is a short dsRNA that comprises an annealed complementary single-stranded RNA. In some embodiments, the siRNA comprises an annealed RNA:DNA duplex, in which the sense strand of the duplex is a DNA molecule and the antisense strand of the duplex is an RNA molecule. In some embodiments, the siRNA comprises a sense strand having a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 76-100 (or a portion thereof).

[0070] In some embodiments, the siRNA comprises an antisense strand having a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 101-125 in Table 2A below. [Table 2]

[0071] In some embodiments, the siRNA comprises an antisense strand having a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the sequences in Table 2B below. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16]

[0072] In some embodiments, the siRNA comprises an antisense strand comprising any one of the sequences disclosed in WO2020 / 104669, WO2021 / 037941, WO2015 / 089368, WO2019 / 089922, WO2021 / 081026, WO2021 / 178607, US7,582,746, WO2007 / 089375, or WO2003 / 066805, each of which is incorporated by reference in its entirety, whether modified or unmodified.

[0073] In some embodiments, the siRNA comprises mismatch(es) with the target, mismatches within the duplex, or a combination thereof. Mismatches can occur in overhang regions and / or duplex portions. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pair. The simplest approach is to examine each pair on an individual pair basis, but the following adjacent pair analysis or similar analysis can also be used). From the perspective of promoting dissociation, A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I=inosine).

[0074] In some embodiments, the siRNA comprises at least one of the first 1, 2, 3, 4 or 5 base pairs in the duplex portion from the 5' end of the antisense strand independently selected from the group of A:U, G:U, I:C, and mismatch pairs. In some embodiments, the nucleotide at position 1 in the duplex portion from the 5' end of the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Additionally or alternatively, at least one of the first 1, 2 or 3 base pairs in the duplex portion from the 5' end of the antisense strand is an AU base pair. Additionally or alternatively, the first base pair in the duplex portion from the 5' end of the antisense strand is an AU base pair.

[0075] In some embodiments, the sense strand may include one or more (e.g., 2, 3, 4 or 5) nucleotides at the 3' and / or 5' end that are not identical to the target sequence, and / or the antisense strand may include one or more (e.g., 2, 3, 4 or 5) nucleotides at the 3' and / or 5' end that are not complementary to the target sequence. For example, in some embodiments, the double-stranded siRNA includes a sense strand that includes a sequence listed in Table 3 below. Each sequence in Table 3 includes an adenine (A) nucleotide at the 3' end, which is complementary to the target sequence in some sequences (e.g., complementary to the next adjacent nucleotide of the target sequence). In some sequences in Table 3A, the adenine (A) nucleotide at the 3' end is not complementary to the target sequence (e.g., not complementary to the next adjacent nucleotide of the target sequence). [Table 4]

[0076] In some embodiments, the siRNA comprises a sense strand having a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of the sequences in Table 3B below. [Table 5-1]

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

[0077] In some embodiments, the siRNA comprises a sense strand comprising any one of the sequences disclosed in WO2020 / 104669, WO2021 / 037941, WO2015 / 089368, WO2019 / 089922, WO2021 / 081026, WO2021 / 178607, US7,582,746, WO2007 / 089375, or WO2003 / 066805, each of which is incorporated by reference in its entirety, whether modified or unmodified.

[0078] In some embodiments, the duplexed siRNA comprises blunt ends at both ends. In some embodiments, the duplexed siRNA comprises at least one overhang region. In some embodiments, the duplexed siRNA comprises a 3' overhang of 1, 2, 3, 4, 5 or 6 nucleotides on the sense and / or antisense strand of the duplex. In some embodiments, the duplexed siRNA comprises a 5' overhang of 1, 2, 3, 4, 5 or 6 nucleotides on the sense and / or antisense strand of the duplex.

[0079] In some embodiments, the antisense strand comprises an overhang comprising one or more nucleotides complementary to the C3 mRNA transcript (SEQ ID NO: 75). In some embodiments, the antisense strand comprises an overhang comprising 1, 2, 3, 4, 5, or 6 or more nucleotides complementary to the C3 mRNA transcript (SEQ ID NO: 75). For example, in some embodiments, the duplex siRNA comprises an antisense strand comprising any one of the sequences of SEQ ID NOs: 300-324. [Table 6]

[0080] In some embodiments, the double-stranded siRNA comprises an antisense strand that comprises the sequence of any one of SEQ ID NOs: 300 to 324, but lacks the "U" at the 5' end.

[0081] In some embodiments, the antisense strand comprises an overhang comprising one or more nucleotides that are not complementary to the C3 mRNA transcript (SEQ ID NO: 75). In some embodiments, the antisense strand comprises an overhang comprising 1, 2, 3, 4, 5, or 6 or more nucleotides that are not complementary to the C3 mRNA transcript (SEQ ID NO: 75). In one example, the overhang comprises a 3' overhang comprising 1, 2, or 3 uracil nucleotides on the antisense strand and / or the sense strand. In one example, the overhang comprises a 3' overhang comprising 1, 2, or 3 uracil nucleotides on the antisense strand and / or the sense strand.

[0082] In some embodiments, the duplexed siRNA comprises an antisense strand comprising a sequence set forth in Table 5 below. [Table 7]

[0083] In some embodiments, the duplexed siRNA comprises an antisense strand comprising a sequence set forth in Table 6 below. [Table 8]

[0084] In some embodiments, the siRNA comprises a 5'-phosphate group and / or a 3'-hydroxyl group (e.g., at one or both ends of the sense strand and / or one or both ends of the antisense strand) and / or can comprise one or more further modifications described herein.

[0085] qualification In some embodiments, the inhibitory RNA (e.g., siRNA or miRNA) of the present disclosure comprises one or more naturally occurring nucleobases and / or one or more modified nucleobases derived from naturally occurring nucleobases. Examples include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine, each of whose amino groups are protected by an acyl protecting group; 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouracil, and other modified nucleobases such as 8-substituted purines, xanthines, or hypoxanthines (the last two being natural degradation products). Exemplary modified nucleobases are disclosed in Chiu and Rana, RNA, 2003, 9, 1034-1048, Limbach et al. Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313.

[0086] Modified nucleobases also include the nucleobases that one or more aryl rings, such as phenyl rings, are expanded in size.The nucleobase substitutions described in Glen Research catalogue (www.glenresearch.com);Krueger AT et al,Acc.Chem.Res.,2007,40,141-150;Kool,ET,Acc.Chem.Res.,2002,35,936-943;Benner SA,et al.,Nat.Rev.Genet.,2005,6,553-543;Romesberg,FE,et al.,Curr.Opin.Chem.Biol.,2003,7,723-733;Hirao,I.,Curr.Opin.Chem.Biol.,2006,10,622-627 are contemplated as useful in the siRNA described herein. Modified nucleobases also include structures that are not considered nucleobases, but are other moieties, such as, but not limited to, corrin-derived rings or porphyrin-derived rings. Porphyrin-derived base substitutions are described in Morales-Rojas, H and Kool, ET, Org. Lett., 2002, 4, 4377-4380.

[0087] In some embodiments, a modified nucleobase is any one of the following structures, optionally substituted: [ka]

[0088] In some embodiments, the modified nucleobase is fluorescent. Exemplary such fluorescent modified nucleobases include phenanthrene, pyrene, stilbene, isoxanthine, isozanthopterin, terphenyl, terthiophene, benzoterthiophene, coumarin, lumazine, tethered stilbene, benzouracil, and naphthouracil, as shown below: [ka]

[0089] In some embodiments, modified nucleobase is unsubstituted. In some embodiments, modified nucleobase is substituted. In some embodiments, modified nucleobase is substituted, for example, to include heteroatoms, alkyl groups, or linker moieties that are linked to fluorescent moieties, biotin or avidin moieties, or other proteins or peptides. In some embodiments, modified nucleobase is not a nucleobase in the most classical sense, but a "universal base" that functions similarly to a nucleobase. One representative example of such a universal base is 3-nitropyrrole.

[0090] In some embodiments, the siRNAs described herein include nucleosides incorporating modified nucleobases and / or nucleobases covalently linked to modified sugars. Examples of nucleosides incorporating modified nucleobases include 4-acetylcytidine; 5-(carboxyhydroxymethyl)uridine; 2'-O-methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; dihydrouridine; 2'-O-methylpseudouridine; β,D-galactosylqueosine; 2'-O-methylguanosine; N-acetylglucosyl ... 6 -Isopentenyl adenosine;1-Methyl adenosine;1-Methyl pseudouridine;1-Methyl guanosine;1-Methyl inosine;2,2-Dimethyl guanosine;2-Methyl adenosine;2-Methyl guanosine;N 7 -Methylguanosine;3-Methylcytidine;5-Methylcytidine;5-Hydroxymethylcytidine;5-Formylcytosine;5-Carboxylcytosine;N 6 -Methyladenosine;7-Methylguanosine;5-Methylaminoethyluridine;5-Methoxyaminomethyl-2-thiouridine;β,D-Mannosylqueuosine;5-Methoxycarbonylmethyluridine;5-Methoxyuridine;2-Methylthio-N 6-isopentenyladenosine; N-((9-β,D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine; N-((9-β,D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl)threonine; uridine-5-oxyacetic acid methyl ester; uridine-5-oxyacetic acid(v); pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine; 2-thiouridine; 4-thiouridine; 5-methyluridine; 2'-O-methyl-5-methyluridine; and 2'-O-methyluridine.

[0091] In some embodiments, the nucleoside comprises a 6'-modified bicyclic nucleoside analog having either (R) or (S) chirality at the 6'-position, including analogs described in U.S. Patent No. 7,399,845. In other embodiments, the nucleoside comprises a 5'-modified bicyclic nucleoside analog having either (R) or (S) chirality at the 5'-position, including analogs described in U.S. Patent Application Publication No. 20070287831. In some embodiments, the nucleobase or modified nucleobase is 5-bromouracil, 5-iodouracil, or 2,6-diaminopurine. In some embodiments, the nucleobase or modified nucleobase is modified by substitution with a fluorescent moiety.

[0092] Methods for preparing modified nucleobases are described, for example, in U.S. Pat. Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,457,191, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594, ,121,5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088.

[0093] In some embodiments, siRNA described herein comprises one or more modified nucleotides, in which phosphate group or linking phosphorus in nucleotide is linked to various positions of sugar or modified sugar.As a non-limiting example, phosphate group or linking phosphorus can be linked to 2', 3', 4' or 5' hydroxyl moiety of sugar or modified sugar.Nucleotides incorporating modified nucleobases described herein are also contemplated in this context.

[0094] Other modified sugars can also be incorporated into the siRNA molecules. In some embodiments, the modified sugar has one or more substituents at the 2' position, including one of the following: -F; -CF3, -CN, -N3, -NO, -NO2, -OR', -SR', or -N(R')2, where each R' is independently defined above and as described herein; -O-(C1-C2 10 Alkyl), -S-(C1-C 10 Alkyl), -NH-(C1-C 10 alkyl), or -N(C1-C 10 Alkyl)2;-O-(C2-C 10alkenyl), -S-(C2-C 10 alkenyl), -NH-(C2-C 10 alkenyl), or -N(C2-C 10 alkenyl)2;-O-(C2-C 10 alkynyl), -S-(C2-C 10 alkynyl), -NH-(C2-C 10 alkynyl), or -N(C2-C 10 alkynyl)2; or -O-(C1-C 10 Alkylene)-O-(C1-C 10 Alkyl), -O-(C1-C 10 Alkylene)-NH-(C1-C 10 Alkyl) or -O-(C1-C 10 Alkylene)-NH(C1-C 10 Alkyl)2, -NH-(C1-C 10 Alkylene)-O-(C1-C 10 alkyl), or -N(C1-C 10 Alkyl)-(C1-C 10 Alkylene)-O-(C1-C 10 alkyl), where alkyl, alkylene, alkenyl, and alkynyl may be substituted or unsubstituted. Examples of substituents include, but are not limited to, -O(CH2) n OCH3 and -O(CH2) n NH2 (wherein the aryl group is from 1 to about 10), MOE, DMAOE, DMAEOE. Also contemplated herein are modified sugars described in WO2001 / 088198 and Martin et al., Helv. Chim. Acta, 1995, 78, 486-504. In some embodiments, the modified sugar comprises one or more groups selected from a substituted silyl group, an RNA cleaving group, a reporter group, a fluorescent label, an intercalator, a group for improving the pharmacokinetic properties of a nucleic acid, a group for improving the pharmacodynamic properties of a nucleic acid, or other substituents with similar properties. In some embodiments, the modification is at one or more of the 2', 3', 4', 5', or 6' positions of the sugar or modified sugar, including the 3' position of the 3' terminal nucleotide or the 5' position of the 5' terminal nucleotide.

[0095] In some embodiments, the 2'-OH of the ribose is substituted with a substituent that includes one of the following: -H, -F, -CF3, -CN, -N3, -NO, -NO2, -OR', -SR', or -N(R')2 (wherein each R' is independently defined above and as described herein); -O-(C1-C2 10 Alkyl), -S-(C1-C 10 Alkyl), -NH-(C1-C 10 alkyl), or -N(C1-C 10 Alkyl)2;-O-(C2-C 10 alkenyl), -S-(C2-C 10 alkenyl), -NH-(C2-C 10 alkenyl), or -N(C2-C 10 alkenyl)2;-O-(C2-C 10 alkynyl), -S-(C2-C 10 alkynyl), -NH-(C2-C 10 alkynyl), or -N(C2-C 10 alkynyl)2; or -O-(C1-C 10 Alkylene)-O-(C1-C 10 Alkyl), -O-(C1-C 10 Alkylene)-NH-(C1-C 10 alkyl), or -O-(C1-C 10 Alkylene)-NH(C1-C 10 Alkyl)2, -NH-(C1-C 10 Alkylene)-O-(C1-C 10 alkyl), or -N(C1-C 10 Alkyl)-(C1-C 10 Alkylene)-O-(C1-C 10alkyl), where alkyl, alkylene, alkenyl, and alkynyl may be substituted or unsubstituted. In some embodiments, 2'-OH is replaced with -H (deoxyribose). In some embodiments, 2'-OH is replaced with -F. In some embodiments, 2'-OH is replaced with -OR'. In some embodiments, 2'-OH is replaced with -OMe. In some embodiments, 2'-OH is replaced with -OCH2CH2OMe.

[0096] Modified sugars also include locked nucleic acids (LNAs). In some embodiments, locked nucleic acids have the structure shown below: A locked nucleic acid of the following structure is shown, where Ba represents a nucleobase or modified nucleobase as described herein, and R 2s is -OCH2C4'-. [ka]

[0097] In some embodiments, the modified sugar is an ENA, e.g., as described in Seth et al., J Am Chem Soc. 2010 October 27;132(42):14942-14950. In some embodiments, the modified sugar is an XNA (xenonucleic acid), e.g., any of those found in arabinose, anhydrohexitol, threose, 2'fluoroarabinose, or cyclohexene.

[0098] Modified sugars include sugar mimetics such as cyclobutyl or cyclopentyl moieties in place of the pentofuranosyl sugar (see, e.g., U.S. Pat. Nos. 4,981,957, 5,118,800, 5,319,080, and 5,359,044). Some modified sugars contemplated include sugars in which the oxygen atom in the ribose ring has been replaced with nitrogen, sulfur, selenium, or carbon. In some embodiments, the modified sugar is ribose in which the oxygen atom in the ribose ring has been replaced with nitrogen, which is optionally substituted with an alkyl group (e.g., methyl, ethyl, isopropyl, etc.).

[0099] A non-limiting example of modified sugar includes glycerol to form glycerol nucleic acid (GNA) analogs. One example of GNA analogs is described in Zhang, R et al., J.Am.Chem.Soc., 2008, 130, 5846-5847; Zhang L, et al., J.Am.Chem.Soc., 2005, 127, 4174-4175 and Tsai CH et al., PNAS, 2007, 14598-14603. Another example of GNA-derived analogs is flexible nucleic acid (FNA) based on mixed acetal aminals of formylglycerol, described in Joyce GF et al., PNAS, 1987, 84, 4398-4402 and Heuberger BD and Switzer C, J.Am.Chem.Soc., 2008, 130, 412-413. Further non-limiting examples of modified sugars include hexapyranosyl (6' to 4'), pentopyranosyl (4' to 2'), pentopyranosyl (4' to 3'), or tetrafluoronosyl (3' to 2') sugars.

[0100] Modified sugars and glycomimetics can be prepared by methods well known in the art, including, but not limited to, the following: A. Eschenmoser, Science (1999), 284:2118; M. Bohringer et al, Helv. Chim. Acta (1992), 75:1416-1477; M. Egli et al, J. Am. Chem. Soc. (2006), 128(33):10847-56; A. Eschenmoser in Chemical Synthesis: Gnosis to Prognosis, C. Chatgilialoglu and V. Sneekus, Ed., (Kluwer Academic, Netherlands, 1996), p. 293; K.-U. Schoning et al, Science (2000), 290:1347-1351; A. Eschenmoser et al. al, Helv. Chim. Acta (1992), 75:218; J. Hunziker et al, Helv. Chim. Acta (1993), 76:259; G. Otting et al, Helv. Chim. Acta (1993), 76:2701; K. Groebke et al, Helv. Chim. Acta (1998), 81:375; and A. Eschenmoser, Science (1999), 284:2118. Modifications at the 2' position can be found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein. Specific modifications to ribose can be found in the following references: 2'-fluoro (Kawasaki et al., J. Med. Chem., 1993, 36, 831-841), 2'-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938), "LNA" (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310); PCT Publication No. WO2012 / 030683).

[0101] According to certain embodiments, various nucleotide modifications or patterns of nucleotide modifications can be used selectively in either the sense or antisense strand of the inhibitory RNA (e.g., siRNA) described herein. For example, in some embodiments, unmodified ribonucleotides can be used in the antisense strand (at least in the duplex portion thereof), while modified nucleotides and / or modified or unmodified deoxyribonucleotides can be used at some or all positions in the sense strand. In some embodiments, a specific pattern of modification is used throughout some or all of one or both strands of the siRNA. The nucleotide modifications can occur in any of a variety of patterns. For example, alternating patterns can be used. For example, the antisense, sense strand, or both, can have 2'-O-methyl or 2'-fluoro modifications at every other nucleotide. In some embodiments, the inhibitory RNA (e.g., siRNA) comprises a sense and / or antisense strand with at least one unmodified nucleotide.

[0102] In some embodiments, the sense and / or antisense strand comprises one or more motifs of three identical modifications in three consecutive nucleotides.For example, in some embodiments, the double-stranded siRNA comprises one or more motifs of three identical modifications in three consecutive nucleotides in the sense strand, the antisense strand, or both.In some embodiments, such motifs can be at or near the cleavage site of one or both strands.Examples of such motifs are described in US Patent Application Publication Nos. 20150197746, 20150247143, and 20160298124.

[0103] In some embodiments, the inhibitory RNA (e.g., siRNA) is a bluntmer 19 nucleotides in length, the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end. In some embodiments, the inhibitory RNA (e.g., siRNA) is a double ended bluntmer 20 nucleotides in length, the sense strand contains at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 8, 9, and 10 from the 5' end, and the antisense strand contains at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end. In some embodiments, the inhibitory RNA (e.g., siRNA) is a double-sided bluntmer 21 nucleotides in length, with the sense strand containing at least one motif of three 2'-F modifications at three consecutive nucleotides, positions 9, 10, and 11 from the 5' end, and the antisense strand containing at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides, positions 11, 12, and 13 from the 5' end.

[0104] In some embodiments, the inhibitory RNA (e.g., siRNA) comprises a 19-nucleotide sense strand and a 21-nucleotide antisense strand, the sense strand comprises at least one motif of three 2'-F modifications at three consecutive nucleotides at positions 7, 8, and 9 from the 5' end, and the antisense strand comprises at least one motif of three 2'-O-methyl modifications at three consecutive nucleotides at positions 11, 12, and 13 from the 5' end, and one end of the inhibitory RNA (e.g., siRNA) is blunt-ended, and the other end comprises a two-nucleotide overhang. Preferably, the two-nucleotide overhang is at the 3' end of the antisense strand. When the two-nucleotide overhang is at the 3' end of the antisense strand, there may be two phosphorothioate internucleotide bonds between the three terminal nucleotides, two of the three nucleotides being overhang nucleotides, and the third nucleotide being the paired nucleotide next to the overhang nucleotide. In some embodiments, the inhibitory RNA (e.g., siRNA) further comprises two phosphorothioate internucleotide bonds between the three terminal nucleotides at both the 5'-end of the sense strand and the 5'-end of the antisense strand. In some embodiments, all nucleotides of the sense strand and the antisense strand of the inhibitory RNA (e.g., siRNA), including nucleotides that are part of a motif, are modified nucleotides. In some embodiments, each residue is independently modified, for example, with 2'-O-methyl or 3'-fluoro in an alternating motif.

[0105] In some embodiments, the inhibitory RNA (e.g., siRNA) comprises a 19 nucleotide sense strand and a 21 nucleotide antisense strand, where (i) the sense strand comprises 2'-F modifications at positions 3, 7, 8, 9, 12, and 17 from the 5' end, (ii) the sense strand comprises 2'-O-methyl modifications at positions 1, 2, 4, 5, 6, 10, 11, 13, 14, 15, 16, 18, and 19 from the 5' end, and (iii) the antisense strand comprises 2'-O-methyl modifications at positions 1, 2, 4, 5, 6, 10, 11, 13, 14, 15, 16, 18, and 19 from the 5' end, and (iv) the antisense strand comprises 2'-F modifications at positions 2 and 14 from the 5' end, and (iv) the antisense strand comprises 2'-O-methyl modifications at positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, and 21 from the 5' end, and one end of the inhibitory RNA (e.g., siRNA) is blunt-ended and the other end comprises a two-nucleotide overhang at the 3' end of the antisense strand. In some embodiments, the inhibitory RNA (e.g., siRNA) comprises an antisense strand comprising two phosphorothioate internucleotide linkages between the three nucleotides at the 3' end, two of the three nucleotides being overhanging nucleotides, and the third nucleotide being the paired nucleotide next to the overhanging nucleotide. In some embodiments, the inhibitory RNA (e.g., siRNA) further comprises two phosphorothioate internucleotide linkages between the three terminal nucleotides at both the 5' end of the sense strand and the 5' end of the antisense strand.

[0106] In some embodiments, all nucleotides of the sense and antisense strands of an inhibitory RNA (e.g., siRNA), including those that are part of a motif, may be modified. Each nucleotide may be modified with the same or different modifications, which may include alteration of one or both of the non-linked phosphate oxygens and / or one or both of the linked phosphate oxygens, alteration of components of the ribose sugar, such as the 2' hydroxyl group of the ribose sugar, replacement of the phosphate moiety entirely with a "dephosphorylated" linker, modification or replacement of naturally occurring bases, and replacement or modification of the ribose-phosphate backbone.

[0107] In some embodiments, at least 50%, 60%, 70%, 80%, 90% or more, e.g., 100%, of the residues in the sense and antisense strands are independently modified with LNA, CRN, cET, UNA, HNA (1,5-anhydrohexitol nucleic acid), CeNA (cyclohexenyl nucleic acid, a DNA mimic in which deoxyribose is replaced with a 6-membered cyclohexene ring), 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy, 2'-hydroxyl, or 2'-fluoro. Each strand may have multiple modifications. In some embodiments, at least 50%, 60%, 70%, 80%, 90% or more, e.g., 100%, of the residues in the sense and antisense strands are independently modified with 2'-O-methyl or 2'-fluoro. In some embodiments, at least two different modifications are present in the sense and antisense strands. These two modifications may be 2'-O-methyl or 2'-fluoro modifications, or others.

[0108] In some embodiments, the sense and antisense strands of an inhibitory RNA (e.g., siRNA) duplex comprise any one of the modification patterns depicted as patterns 1-5 in FIG. 1. In FIG. 1, "2OM" represents a 2'-O-methyl modification and "2F" represents a 2'-fluoro modification at any particular position. "PS" represents a phosphorothioate linkage between the nucleotide at the position designated "PS" and the adjacent nucleotide 3' to the position designated "PS". In some embodiments, any one of the antisense strands disclosed in SEQ ID NOs: 176-200 and 300-324 may be modified according to any one of the antisense strand ("AS") modification patterns 1-5 depicted in FIG. 1. In some embodiments, any one of the sense strands disclosed in SEQ ID NOs: 126-150 may be modified according to any one of the sense strand ("SS") modification patterns 1-5 disclosed in Figure 1. In some embodiments, the sense and / or antisense strand of an inhibitory RNA (e.g., siRNA) duplex comprises any one of the modification patterns shown as patterns 1-5 in Figure 1, but any of positions 1, 2, 3 or 4 of the sense strand and / or antisense strand does not comprise a modification shown at position 1, 2, 3 or 4 in one of patterns 1-5.

[0109] In some embodiments, the siRNA comprises any one of modification patterns 1-5 (shown in FIG. 1 ) and further comprises phosphorothioate linkages between the last 2, 3, or 4 nucleotides of (i) the 5' end of the sense strand, (ii) the 3' end of the sense strand, (iii) the 5' end of the antisense strand, and / or (iv) the 3' end of the antisense strand. For example, in some embodiments, the siRNA comprises: (i) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5'-end and between the 2nd and 3rd nucleotides from the 5'-end; (ii) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 3'-end and between the 2nd and 3rd nucleotides from the 3'-end; (iii) an antisense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5'-end and between the 2nd and 3rd nucleotides from the 5'-end; and / or (iv) an antisense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 3'-end and between the 2nd and 3rd nucleotides from the 3'-end.

[0110] In some embodiments, the siRNA may be modified according to any one of modification patterns 1-5 in FIG. 1 and may be conjugated to a ligand, for example, as described herein. In some such cases, the ligand may be conjugated to either the 3' or 5' end of the sense or antisense strand. In some embodiments, the siRNA (e.g., any of siRNAs 1-57 in Tables 10 and 15, e.g., siRNAs 22, 32, and 53) comprises a ligand (e.g., GalNAc of formula XD or XE described herein) conjugated to a terminus (e.g., a GalNAc ligand, e.g., the 3' or 5' end of the sense or antisense strand), and the siRNA does not comprise a phosphorothioate bond between the two, three, or four nucleotides of the terminus conjugated to the ligand. For example, in some embodiments, an siRNA (e.g., any of siRNAs 1-57 described in Tables 10 and 15, e.g., siRNAs 22, 32, and 53) comprises a ligand (e.g., a GalNAc ligand, e.g., GalNAc of formula XD or XE described herein) attached to the 5' end of the sense strand, and the siRNA comprises: (i) a sense strand that does not have a phosphorothioate bond between the 1st, 2nd, 3rd, or 4th nucleotide from the 5' end; (ii) a sense strand that has phosphorothioate bonds between the 1st and 2nd nucleotide from the 3' end and between the 2nd and 3rd nucleotides from the 3' end; (iii) an antisense strand that has phosphorothioate bonds between the 1st and 2nd nucleotide from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; and (iv) an antisense strand that has phosphorothioate bonds between the 1st and 2nd nucleotide from the 3' end and between the 2nd and 3rd nucleotides from the 3' end.

[0111] In some embodiments, an siRNA (e.g., any of siRNAs 1-57 listed in Tables 10 and 15, e.g., siRNAs 22, 32, and 53) comprises a ligand (e.g., a GalNAc ligand, e.g., GalNAc of formula XD or XE described herein) attached to the 3' end of the sense strand, and the siRNA comprises: (i) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; (ii) a sense strand having no phosphorothioate bonds between the 1st, 2nd, 3rd, or 4th nucleotides from the 3' end; (iii) an antisense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; and (iv) an antisense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 3' end and between the 2nd and 3rd nucleotides from the 3' end.

[0112] In some embodiments, an siRNA (e.g., any of siRNAs 1-57 described in Tables 10 and 15, e.g., siRNAs 22, 32, and 53) comprises a ligand (e.g., a GalNAc ligand, e.g., GalNAc of formula XD or XE described herein) attached to the 5' end of the antisense strand, and the siRNA comprises: (i) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; (ii) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 3' end and between the 2nd and 3rd nucleotides from the 3' end; (iii) an antisense strand that does not have phosphorothioate bonds between the 1st, 2nd, 3rd, or 4th nucleotides from the 5' end; and (iv) an antisense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 3' end and between the 2nd and 3rd nucleotides from the 3' end.

[0113] In some embodiments, an siRNA (e.g., any of siRNAs 1-57 listed in Tables 10 and 15, e.g., siRNAs 22, 32, and 53) comprises a ligand (e.g., a GalNAc ligand, e.g., GalNAc of formula XD or XE described herein) attached to the 3' end of the antisense strand, and the siRNA comprises: (i) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; (ii) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 3' end and between the 2nd and 3rd nucleotides from the 3' end; (iii) an antisense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; and (iv) an antisense strand that does not have phosphorothioate bonds between the 1st, 2nd, 3rd, or 4th nucleotides from the 3' end.

[0114] In some embodiments, an siRNA (e.g., any of siRNAs 1-57 listed in Tables 10 and 15, e.g., siRNAs 22, 32, and 53) comprises a ligand (e.g., GalNAc of formula XD or XE described herein) attached to its terminus (e.g., a GalNAc ligand, e.g., the 3' or 5' terminus of the sense or antisense strand), and the siRNA comprises phosphorothioate linkages between two, three, or four nucleotides of the terminus attached to the ligand.

[0115] For example, in some embodiments, an siRNA (e.g., any of siRNAs 1-57 described in Tables 10 and 15, e.g., siRNAs 22, 32, and 53) comprises a ligand (e.g., a GalNAc ligand, e.g., GalNAc of formula XD or XE described herein) attached to the 5' end of the sense strand, and the siRNA comprises: (i) a sense strand having a phosphorothioate bond between the 1st, 2nd, 3rd, or 4th nucleotide from the 5' end; (ii) a sense strand having a phosphorothioate bond between the 1st and 2nd nucleotide from the 3' end and between the 2nd and 3rd nucleotide from the 3' end; (iii) an antisense strand having a phosphorothioate bond between the 1st and 2nd nucleotide from the 5' end and between the 2nd and 3rd nucleotide from the 5' end; and (iv) an antisense strand having a phosphorothioate bond between the 1st and 2nd nucleotide from the 3' end and between the 2nd and 3rd nucleotides from the 3' end.

[0116] In some embodiments, an siRNA (e.g., any of siRNAs 1-57 listed in Tables 10 and 15, e.g., siRNAs 22, 32, and 53) comprises a ligand (e.g., a GalNAc ligand, e.g., GalNAc of formula XD or XE described herein) attached to the 3' end of the sense strand, and the siRNA comprises: (i) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; (ii) a sense strand having phosphorothioate bonds between the 1st, 2nd, 3rd, or 4th nucleotides from the 3' end; (iii) an antisense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; and (iv) an antisense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 3' end and between the 2nd and 3rd nucleotides from the 3' end.

[0117] In some embodiments, an siRNA (e.g., any of siRNAs 1-57 described in Tables 10 and 15, e.g., siRNAs 22, 32, and 53) comprises a ligand (e.g., a GalNAc ligand, e.g., GalNAc of formula XD or XE described herein) attached to the 5' end of the antisense strand, and the siRNA comprises: (i) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; (ii) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 3' end and between the 2nd and 3rd nucleotides from the 3' end; (iii) an antisense strand having phosphorothioate bonds between the 1st, 2nd, 3rd, or 4th nucleotides from the 5' end; and (iv) an antisense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 3' end and between the 2nd and 3rd nucleotides from the 3' end.

[0118] In some embodiments, an siRNA (e.g., any of siRNAs 1-57 listed in Tables 10 and 15, e.g., siRNAs 22, 32, and 53) comprises a ligand (e.g., a GalNAc ligand, e.g., GalNAc of formula XD or XE described herein) attached to the 3' end of the antisense strand, and the siRNA comprises: (i) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; (ii) a sense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 3' end and between the 2nd and 3rd nucleotides from the 3' end; (iii) an antisense strand having phosphorothioate bonds between the 1st and 2nd nucleotides from the 5' end and between the 2nd and 3rd nucleotides from the 5' end; and (iv) an antisense strand having phosphorothioate bonds between the 1st, 2nd, 3rd, or 4th nucleotides from the 3' end.

[0119] In some embodiments, the sense strand and / or the antisense strand comprise an alternating pattern of modifications. As used herein, the term "alternating motif" refers to a motif having one or more modifications, each modification occurring in alternating groups of one or more nucleotides of a strand. For example, alternating nucleotides can refer to every other nucleotide, or every third nucleotide, or a similar pattern. For example, where A, B, and C each represent one type of modification to a nucleotide, the alternating motif can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB...", or "ABCABCABCABC...", etc.

[0120] The types of modifications within the alternating motif can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternation pattern, i.e., the modifications at every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from among several modification possibilities within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD...", or "CDCDCD...".

[0121] In some embodiments, an inhibitory RNA (e.g., an siRNA) comprises a modification pattern of an alternating motif on the sense strand that is shifted relative to the modification pattern of the alternating motif on the antisense strand. The shift may be such that a modified group of nucleotides in the sense strand corresponds to a group of nucleotides in the antisense strand having a different modification (or vice versa). For example, when paired with the antisense strand of a dsRNA duplex, the alternating motif of the sense strand may begin with "ABABAB" in the 5' to 3' direction of the strand within the duplex portion, and the alternating motif of the antisense strand may begin with "BABABA" in the 5' to 3' direction of the strand. As another example, the alternating motif of the sense strand may begin with "AABBAABB" in the 5' to 3' direction of the strand within the duplex portion, and the alternating motif of the antisense strand may begin with "BBAABBAA" in the 5' to 3' direction of the strand, such that a complete or partial shift in the modification pattern occurs between the sense and antisense strands.

[0122] In some embodiments, an inhibitory RNA (e.g., an siRNA) comprises a pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the sense strand, with a shift relative to the pattern of alternating motifs of 2'-O-methyl and 2'-F modifications on the antisense strand (i.e., 2'-O-methyl modified nucleotides on the sense strand base pair with 2'-F modified nucleotides on the antisense strand (or vice versa)). Position 1 of the sense strand may start with a 2'-F modification, and position 1 of the antisense strand may start with a 2'-O-methyl modification.

[0123] In some embodiments, one or more motifs of three identical modifications can be introduced into three consecutive nucleotides of the sense and / or antisense strands to interrupt the initial modification pattern present in the sense and / or antisense strands. In some embodiments, when a motif of three identical modifications on three consecutive nucleotides is introduced into either strand, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif is "...NaYYYNb...", where "Y" represents a modification of the motif of three identical modifications on three consecutive nucleotides, "Na" and "Nb" represent modifications of the nucleotides next to the motif "YYY" that are different from the modification of Y, and Na and Nb can be the same or different modifications.

[0124] The inhibitory RNA (e.g., siRNA) can further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the internucleotide linkage modification may be made to every nucleotide on the sense strand and / or antisense strand, each internucleotide linkage modification may be made in an alternating pattern on the sense strand and / or antisense strand, or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of internucleotide linkage modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of internucleotide linkage modifications on the sense strand may be shifted relative to the alternating pattern of internucleotide linkage modifications on the antisense strand. In some embodiments, the inhibitory RNA (e.g., siRNA) comprises 6-8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand contains two phosphorothioate internucleotide linkages at the 5' end and two phosphorothioate internucleotide linkages at the 3' end, and the sense strand contains at least two phosphorothioate internucleotide linkages at either the 5' end or the 3' end.

[0125] In certain embodiments, the inhibitory RNA (e.g., siRNA) can have any of the configurations and / or modification patterns described in WO2015 / 089368, pages 59 (line 20) to 65 (line 15), or U.S. Patent Application Publication No. 20160298124, paragraphs

[0469] to

[0537] , or any of the claims of any or both of the above publications. For example, in some embodiments, the inhibitory RNA (e.g., siRNA) comprises a sense strand and an antisense strand, the sense strand being complementary to the antisense strand, and the antisense strand comprising a region complementary to a portion of an mRNA encoding C3 (e.g., a target region described herein), each strand being about 14 to about 30 nucleotides in length, and the agent is a compound represented by Formula (III): Sense:5'n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a -n q 3' Antisense:3'n p’ -N a’ -(X'X'X') k -N b’ -Y'Y'Y'-N b’ -(Z'Z'Z') l -N a’ -n q’ 5' (In the formula, i, j, k, and 1 each independently represent 0 or 1; p, p', q, and q' each independently represent 0 to 6; each N a and N a ' independently represent oligonucleotide sequences each containing 0-25 nucleotides that are modified or unmodified or a combination thereof, each having at least two different substitutions, each sequence being b and N b ' independently represents an oligonucleotide sequence containing 0 to 10 nucleotides that are modified or unmodified or a combination thereof, and each n p , n p ',n q , and n qXXX, YYY, ZZZ, X'X'X', Y'Y'Y', and Z'Z'Z' represent one motif of three identical modifications on three consecutive nucleotides; N b The modification of N is different from the modification of Y. b ' modification is different from the Y' modification, and the sense strand is conjugated to at least one ligand) In some embodiments, i is 0, j is 0, i is 1, j is 1, i and j are both 0, or i and j are both 1. In some embodiments, XXX is complementary to X'X'X', YYY is complementary to Y'Y'Y', and ZZZ is complementary to Z'Z'Z'. It should be understood that each X may contain different bases, so long as each X contains the same modification. For example, XXX can represent AGC, with the proviso that each nucleotide contains a 2-F modification. Similarly, each X', each Y, each Y', each Z, and each Z may be different.

[0126] In some embodiments, formula (III) has formula (IIIa): Sense:5'n p -N a -YYY-N a -n q 3' Antisense:3'n p’ -N a’ -Y'Y'Y'-N a’ -n q’ 5' or formula (III) is represented by formula (IIIb): Sense:5'n p -N a -YYY-N b -ZZZ-N a -n q 3' Antisense:3'n p’ -N a’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5' (In the formula, each Nb and N b’ each independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides) or formula (III) is represented by formula (IIIc): Sense:5'n p -N a -XXX-N b -YYY-N a -n q 3' Antisense:3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N a’ -n q’ 5' (In the formula, each N b and N b’ each independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides) or formula (III) is represented by formula (IIId): Sense:5'n p -N a -XXX-N b -YYY-N b -ZZZ-N a -n q 3' Antisense:3'n p’ -N a’ -X'X'X'-N b’ -Y'Y'Y'-N b’ -Z'Z'Z'-N a’ -n q’ 5 (In the formula, N b and N b’ each independently represents an oligonucleotide sequence containing 1 to 5 modified nucleotides, a and N a’ each independently represents an oligonucleotide sequence containing 2 to 10 modified nucleotides. It is represented by:

[0127] In some embodiments, the modification of the nucleotide is selected from the group consisting of LNA, CRN, cET, UNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'-hydroxyl, and combinations thereof.

[0128] In some embodiments, the modification of the nucleotide is 2'-O-methyl or 2'-fluoro modification. In some embodiments, the ligand is one or more GalNAc derivatives linked via a bivalent or trivalent branched linker. In some embodiments, the ligand is represented by formula XA, XB or XC, or another GalNAc structure described herein.

[0129] In some embodiments, the linker is attached to the 3' end of the sense strand. In some embodiments, the linkage is as shown in formula XD shown below.

[0130] In some embodiments, the inhibitory RNA (eg, siRNA) further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0131] In some embodiments, p'>0, or p'=2.

[0132] In some embodiments, q'=0, p=0, q=0, and the nucleotides of the p' overhang are complementary to C3 mRNA. In some embodiments, q'=0, p=0, q=0, and the nucleotides of the p' overhang are non-complementary to C3 mRNA.

[0133] In some embodiments, at least one of n p’ is linked to the adjacent nucleotide via a phosphorothioate bond

[0134] In some embodiments, the ligand targets the nucleic acid molecule to hepatocytes. For example, in some embodiments, the ligand binds to hepatocyte-specific asialoglycoprotein receptor (ASGPR), for example, the ligand comprises a galactose derivative, such as GalNAc.

[0135] In some embodiments, the inhibitory RNA (e.g., siRNA) is bound to or physically associated with one or more moieties that modulate (e.g., increase) the activity, stability, cellular distribution and / or cellular uptake of the inhibitory RNA (e.g., siRNA) and / or change one or more physical properties of the inhibitory RNA (e.g., siRNA), such as charge or solubility. In some embodiments, the moiety can include an antibody or a ligand. The ligand can be a carbohydrate, lectin, protein, glycoprotein, lipid, cholesterol, steroid, bile acid, nucleic acid hormone, growth factor, or receptor. In some embodiments, biologically inactive variants of naturally occurring hormones, growth factors, or other ligands can also be used. In some embodiments, the moiety includes a targeting moiety that targets the inhibitory RNA (e.g., siRNA) to a particular cell type, such as hepatocytes. In some embodiments, the targeting moiety binds to the hepatocyte-specific asialoglycoprotein receptor (ASGPR).

[0136] In some embodiments, the moiety is attached to the inhibitory RNA (e.g., siRNA) via a reversible bond. A "reversible bond" is a linkage that includes a reversible bond. A "reversible bond" (also called a labile or cleavable bond) is a covalent bond other than a covalent bond with a hydrogen atom that can be selectively cleaved or cleaved under selected conditions more quickly than other bonds in the molecule, and that can be selectively cleaved or cleaved under conditions in which other covalent bonds in the same molecule are not substantially cleaved or cleaved. The cleavage or instability of a bond can be measured by the half-life (t 1 / 2) (the time required for half of the bonds to be cleaved). Unless otherwise specified, a reversible bond for purposes herein is a "physiologically reversible bond", meaning that the bond is cleavable under conditions that normally occur or are similar to those that occur in a mammalian body. A physiologically reversible bond is a linkage that includes at least one physiologically reversible bond. In some embodiments, a physiologically reversible bond is reversible under mammalian intracellular conditions, including, for example, pH, temperature, oxidative or reductive conditions or agents, and chemical conditions such as salt concentration, or similar chemical conditions found in a mammalian cell. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell, for example, by proteolytic or hydrolytic enzymes. Enzymatically labile bonds are cleaved by endogenous enzymes, such as intracellular enzymes. pH-labile bonds are cleaved at pH 7.0 or below. Examples of reversible bonds and linkages and their use to attach moieties to inhibitory RNA (e.g., siRNA) are described, for example, in U.S. Patent Application Publication Nos. 20130281685 and 20150273081.

[0137] In some embodiments, the moiety comprises a protein transduction domain (PTD). A protein transduction domain is a polypeptide or portion thereof that facilitates uptake of a heterologous molecule bound to the domain (such heterologous molecules may be referred to as "cargo"). Protein transduction domains that are peptides may be referred to as cell penetrating peptides (CPPs). Many protein transduction domains / peptides are known in the art. PTDs include a variety of natural or synthetic peptides that are arginine-rich. Arginine-rich peptides are peptides that contain at least 30% arginine residues, such as at least 40%, 50%, 60% or more. Examples of PTDs include TAT (at least amino acids 49-56), antennapedia homeodomain, HSV VP22, and polyarginine. Such peptides may be cationic, hydrophobic, or amphipathic peptides, and may include the use of non-standard amino acids and / or various modifications or alterations, such as circular permutations, inverso, retro, retro-inverso, or peptidomimetics. The association of the PTD and cargo may be covalent or non-covalent.

[0138] Exemplary PTDs that can be used are described in U.S. Patent Application Publication Nos. 20090093026, 20090093425, 20120142763, 20150238516, and 20160215022. The PTD can include two or more PTDs (e.g., 2-10 PTDs), which can be the same or different. The PTDs can be directly attached to each other or separated by a linker moiety, which can include one or more amino acids and / or one or more non-amino acid moieties, such as an alkyl chain or an oligoethylene glycol moiety.

[0139] In some embodiments, the inhibitory RNA (e.g., siRNA) comprises or is physically bound to an anionic charge neutralizing moiety. An anionic charge neutralizing moiety refers to a molecule or chemical group that can reduce the overall net anionic charge of the nucleic acid to which it is physically bound. One or more anionic charge neutralizing molecules or groups can be bound to a nucleic acid, each independently contributing to a reduction in anionic charge and / or an increase in cationic charge. "Charge neutralized" means that the anionic charge of the nucleic acid is reduced, neutralized, or more cationic than the same nucleic acid in the absence of the anionic charge neutralizing molecule or group. Examples of anionic charge neutralizing groups include phosphodiester and / or phosphothioate protecting groups. In some embodiments, the inhibitory RNA (e.g., siRNA) comprises a protecting group at one or more positions that reduces the net anionic charge of the backbone (e.g., phosphodiester or phosphorothioate backbone) that contains negatively charged groups. In some embodiments, the negatively charged phosphodiester backbone is neutralized by synthesis with bioreversible phosphotriester protecting groups that are converted to charged phosphodiester bonds intracellularly by the action of cytoplasmic thioesterases, resulting in biologically active agents in inhibiting expression, such as inhibitory RNAs (e.g., siRNAs) that can mediate RNAi. Such agents are therefore also referred to as short interfering ribonucleic acid neutrals (siRNNs) and can function as siRNA prodrugs. It should be understood that the backbone need not be completely neutralized (i.e., lose charge). In some embodiments, 5% to 100%, e.g., 25% to 50%, or 50% to 75%, or 75% to 100%, of the phosphate groups are protected. In certain embodiments, at least 5, 6, 7, 8, 9, or 10 phosphate groups on one or both strands are protected. Examples of useful phosphodiester and / or phosphothioate protecting groups, their methods of preparation, and their use in nucleic acids (e.g., to generate RNAi drug prodrugs) are described in U.S. Patent Application Publication Nos. 20110294869, 20090093425, 20120142763, and 20150238516.In various embodiments, the siRNA may include any of the modifications described herein. For example, in some embodiments, the siRNA may include 2' sugar modifications (e.g., 2'-F, 2'-O-Me). Furthermore, the siRNN may have any of the configurations or modification patterns described herein.

[0140] In some embodiments, the moiety attached to the inhibitory RNA (e.g., siRNA) comprises a carbohydrate. Exemplary carbohydrates include monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4, 5, 6, 7, 8, or 9 monosaccharide units. In certain embodiments, the carbohydrate comprises galactose or a galactose derivative, such as galactosamine, N-formylgalactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine, and N-isobutanoylgalactosamine. In certain embodiments of particular interest, the galactose derivative comprises N-acetylgalactosamine (GalNAc). In certain embodiments, the moiety comprises multiple galactose or galactose derivatives, such as multiple N-acetylgalactosamine moieties, e.g., three GalNAc moieties. As used herein, the term "galactose derivative" includes both galactose and derivatives of galactose that have affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. The term "galactose cluster" refers to a structure that includes at least two galactose derivatives that are physically linked to each other, typically by being covalently attached to another moiety. In some embodiments, the galactose cluster has 2-10 (e.g., 6), or 2-4 (e.g., 3) terminal galactose derivatives. The terminal galactose derivative may be attached to another moiety through the C-1 carbon of the galactose derivative. In some embodiments, two or more, e.g., three, galactose derivatives are attached to a moiety that functions as a branch point and can be attached to an inhibitory RNA (e.g., siRNA). In some embodiments, the galactose derivative is attached to the moiety that functions as a branch point via a linker or spacer. In some embodiments, the moiety that functions as a branch point can be attached to an inhibitory RNA (e.g., siRNA) via a linker or spacer. For example, in some embodiments, the galactose derivative is attached to the branch point via a linker or spacer that includes an amide, carbonyl, alkyl, oligoethylene glycol moiety, or a combination thereof.In some embodiments, the linker or spacer attached to each galactose derivative is the same. In some embodiments, the galactose cluster has three terminal galactosamines or galactosamine derivatives (e.g., GalNAc), each of which has affinity for the asialoglycoprotein receptor. A structure in which three terminal GalNAc moieties are attached (e.g., via the C-1 carbon of the sugar) to a moiety that serves as a branch point can be referred to as a triantennary N-acetylgalactosamine (GalNAc3). In some embodiments, one or more monomer units containing a galactose derivative can be site-specifically incorporated into an inhibitory RNA (e.g., siRNA). Such a galactose derivative-containing monomer unit can include a galactose derivative (e.g., GalNAc) attached to a nucleoside or non-nucleoside moiety. In some embodiments, at least three nucleoside-GalNAc monomers or at least three non-nucleoside-GalNAc monomers are site-specifically incorporated into an inhibitory RNA (e.g., siRNA). In some embodiments, such incorporation can be performed during solid phase synthesis using phosphoramidite chemistry or during post-synthesis conjugation. In some embodiments, the galactose derivative-containing monomer units are linked to each other and / or to nucleosides of the inhibitory RNA (e.g., siRNA) that do not have a galactose derivative attached via phosphodiester bonds. In some embodiments, two, three, or more galactose derivative-containing monomer units are arranged consecutively (i.e., without an intervening unit that does not have a galactose derivative). In some embodiments, a carbohydrate, such as a galactose cluster, e.g., triantennary N-acetylgalactosamine, or two or more GalNAc-containing monomer units are present at the ends of the strands, e.g., at the 3'-end of the sense strand or the 5'-end of the antisense strand.Exemplary carbohydrates (e.g., galactose clusters), galactose derivative-containing monomer units, carbohydrate-modified inhibitory RNAs, and methods of making and using same are described in U.S. Patent Application Publication Nos. 20090203135, 20090239814, 20110207799, 20120157509, 20150247143, U.S. Patent Application Publication No. "124", Nair, JK, et al., J. Am. Chem. Soc. 136, 16958-16961 (2014); Matsuda, S., et al., ACS Chem. Biol. 10, 1181-1187 (2015); Rajeev, K., et al., ChemBioChem 16, 903-908 (2015); Migawa, M. T., et al. al., Bioorg Med Chem Lett. 26(9):2194-7 (2016);Prakash, TP, et al., J Med Chem. 59(6):2718-33 (2016). Exemplary galactose clusters are shown below. [ka]

[0141] Additional GalNAc structures are shown below (and can be synthesized as described in Sharma et al., Bioconjug. Chem. 29:2478-2488 (2018)): [ka] In some embodiments, m=0 and n=2. In some embodiments, m=1 and n=1. In some embodiments, m=1 and n=2. In some embodiments, m=1 and n=3. [ka]

[0142] One of skill in the art will recognize that the structure of each linker moiety connecting each GalNAc to a branch point can vary. In some embodiments, an inhibitory RNA (e.g., an siRNA) is attached to the GalNAc shown below: [ka]

[0143] In some embodiments, an inhibitory RNA (eg, an siRNA, such as any of siRNAs 1-57 listed in Tables 10 and 15, such as siRNAs 22, 32 and 53) is conjugated to a GalNAc ligand (eg, GalNAc of formula XD or XE).

[0144] In some embodiments, a GalNAc ligand (e.g., shown in formula XD or XE) is attached to the 3'-terminal nucleotide of the sense or antisense strand of an siRNA (e.g., any one of siRNAs 1-57, such as siRNAs 22, 32, and 53). In some embodiments, a GalNAc ligand (e.g., shown in formula XD or XE) is attached to the 3' position of the sugar of the 3'-terminal nucleotide of the sense or antisense strand of an siRNA.

[0145] In some embodiments, a GalNAc ligand (e.g., as shown in formula XD or XE) is attached to the 5'-terminal nucleotide of the sense or antisense strand of an siRNA (e.g., any one of siRNAs 1-57, such as siRNAs 22, 32, and 53). In some embodiments, a GalNAc ligand (e.g., as shown in formula XD or XE) is attached to the 5' position of the 5'-terminal nucleotide of the sense or antisense strand of an siRNA.

[0146] In some embodiments, when an inhibitory RNA (e.g., an siRNA of SEQ ID NOs: 1-57, e.g., siRNAs 22, 32 and 53) is conjugated to a ligand (e.g., a GalNAc ligand), the inhibitory RNA may not have a modification (e.g., a phosphorothioate linkage "PS") in the nucleotide(s) conjugated to the ligand.

[0147] In some embodiments, siRNAs (e.g., siRNAs 1-57, e.g., siRNAs 22, 32, and 53) are conjugated to a GalNAc ligand (e.g., as shown in Formula XD or XE) at one end of either the sense or antisense strand. In some embodiments, the other three ends not conjugated to a GalNAc ligand include a modification such as a phosphorothioate linkage ("PS"). In some embodiments, the modification has a PS linkage between the 2, 3, or 4 most 5' or 3' nucleotides. In some embodiments, the end conjugated to a GalNAc ligand does not include a phosphorothioate linkage between the 2, 3, or 4 most 5' or 3' nucleotides.

[0148] In some embodiments, the siRNA described herein can be conjugated to the galactose structure shown below: [ka]

[0149] In some embodiments, the linker comprises an amide, carbonyl, alkyl, oligoethylene glycol moiety, or a combination thereof.

[0150] In some embodiments, the siRNA described herein can be conjugated to the galactose structure shown below: [ka]

[0151] In some embodiments, the linker comprises an amide, carbonyl, alkyl, oligoethylene glycol moiety, or a combination thereof.

[0152] Methods for synthesizing GalNAc ligands, methods for attaching GalNAc ligands to inhibitory RNAs, and further GalNAc ligands are well known in the art, for example, those described in WO2017 / 021385, WO2017 / 178656, WO2018 / 215391, WO2019 / 145543, WO2017 / 084987, WO2017 / 055423, and WO2012 / 083046, the entireties of which are incorporated by reference herein.

[0153] In some embodiments, the inhibitory RNA (e.g., siRNA) is conjugated to a ligand as shown below: [ka] where X is O or S. In most embodiments, X is O. Those skilled in the art will recognize that the structure of the linker moiety connecting the galactose cluster to the phosphate group can vary.

[0154] In certain embodiments, the moiety comprises a lipophilic moiety. In some embodiments, the lipophilic moiety comprises a tocopherol, e.g., α-tocopherol. In some embodiments, the lipophilic moiety comprises cholesterol. In some embodiments, the lipophilic compound comprises an alkyl or heteroalkyl group. In some embodiments, the lipophilic compound comprises palmitoyl, hexadeca-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, or C16-C20 acyl. In some embodiments, the lipophilic moiety comprises at least 16 carbon atoms. In some embodiments, the lipophilic moiety comprises -(CH) n -NH-(C=O)-(CH m In some embodiments, n and m are each independently 1 to 20. In some embodiments, n+m is at least 10, 12, 14, or 16. In some embodiments, the lipophilic moiety is as shown below and / or is attached to the sugar moiety as shown below. [ka]

[0155] Generally, the moiety can be attached to the end or internal subunit of the inhibitory RNA (e.g., siRNA). In some embodiments, the moiety is attached to a modified subunit of the inhibitory RNA (e.g., siRNA). Those skilled in the art will recognize suitable methods for producing nucleic acids with attached moieties. A nucleic acid strand containing a modified nucleotide containing a reactive functional group can be reacted with a moiety containing a second reactive functional group, and the first and second reactive functional groups can react with each other under conditions compatible with maintaining the structure of the nucleic acid strand. In some embodiments, the moiety can be attached to the sense strand or antisense strand before the sense strand or antisense strand is hybridized with the complementary antisense strand or sense strand, respectively. In some embodiments, the strands can be hybridized to form a duplex before incorporating the moiety. In general, the various conjugation methods described herein can be used. See, for example, Hermanson, G., Bioconjugate Techniques, 2nd ed., Academic Press, San Diego, 2008.

[0156] In some embodiments, the inhibitory RNA (e.g., siRNA) is a chimeric siRNA. As used herein, a "chimeric" siRNA is an siRNA that contains two or more chemically distinct regions, each composed of at least one monomer unit, and each region confers a different property to the compound. In some embodiments, at least one region is modified to confer high resistance to nuclease degradation, high uptake into cells, and / or high binding affinity to the target nucleic acid to the siRNA, and at least one additional region of the siRNA can function as a substrate for an enzyme (e.g., RNase H) that can cleave RNA:DNA or RNA:RNA hybrids. In some embodiments, at least one region of the siRNA can function as a substrate for an enzyme (e.g., RNase H) that can cleave RNA:DNA or RNA:RNA hybrids, and at least one region can inhibit translation by steric hindrance.

[0157] In some embodiments, the inhibitory RNA (e.g., siRNA) described herein can be introduced into a target cell as an annealed duplex siRNA. In some embodiments, the inhibitory RNA (e.g., siRNA) described herein can be introduced into a target cell as single-stranded sense and antisense nucleic acid sequences that anneal in the target cell to form an inhibitory RNA (e.g., siRNA) duplex. In some embodiments, the sense and antisense strands of the inhibitory RNA (e.g., siRNA) can be encoded by an expression vector (e.g., an expression vector described herein) that is introduced into the target cell. When expressed in the target cell, the transcribed sense and antisense strands can anneal to form an inhibitory RNA (e.g., siRNA).

[0158] The inhibitory RNAs described herein (e.g., siRNAs or miRNAs, or vectors containing nucleotide sequences encoding siRNAs or miRNAs) can be synthesized by standard methods well known in the art, for example, using an automated synthesizer. RNAs produced by such methods tend to be highly pure and efficiently anneal to form inhibitory RNA (e.g., siRNA) duplexes. After chemical synthesis, single-stranded RNA molecules can be deprotected, annealed to form siRNAs, and purified (e.g., by gel electrophoresis or HPLC). Alternatively, standard techniques can be used, for example, to in vitro transcribe RNA from a DNA template having one or more RNA polymerase promoter sequences (e.g., T7 or SP6 RNA polymerase promoter sequences). Protocols for preparing siRNAs using T7 RNA polymerase are well known in the art (e.g., Donze and Picard, Nucleic Acids Res. 2002; 30: e46; and Yu et al., Proc. Natl. Acad. Sci. USA 2002; 99: 6047-6052). The sense and antisense transcripts can be synthesized in two separate reactions and then annealed, or they can be synthesized simultaneously in a single reaction.

[0159] Inhibitory RNA (e.g., siRNA or miRNA) can also be formed in a cell by transcription of RNA from an expression construct introduced into the cell (see, e.g., Yu et al., Proc. Natl. Acad. Sci. USA 2002;99:6047-6052). Expression constructs for producing inhibitory RNA (e.g., siRNA) molecules in vivo can include one or more siRNA coding sequences operably linked to elements required for proper transcription of the siRNA coding sequence(s), including, for example, promoter elements and transcription termination signals. Preferred promoters for use in such expression constructs include polymerase III HI-RNA promoter (see, for example, Brummelkamp et al., Science 2002;296:550-553) and U6 polymerase III promoter (see, for example, Sui et al., Proc. Natl. Acad. Sci. USA 2002; Paul et al., Nature Biotechnol. 2002;20:505-508; and Yu et al., Proc. Natl. Acad. Sci. USA 2002;99:6047-6052). The siRNA expression construct may further comprise one or more vector sequences that facilitate cloning of the expression construct. Standard vectors that can be used include, for example, pSilencer2.0-U6 vector (Ambion Inc., Austin, Tex.).

[0160] III. Expression Vectors In some embodiments, the inhibitory RNA described herein is delivered to a subject (e.g., to a cell of a subject, such as a liver cell of a subject) using an expression vector.Many forms of vectors can be used to deliver the inhibitory RNA described herein.Non-limiting examples of expression vectors include viral vectors (e.g., vectors suitable for gene therapy), plasmid vectors, bacteriophage vectors, cosmids, phagemids, artificial chromosomes, etc.

[0161] In some embodiments, the nucleotide sequences encoding the inhibitory RNAs described herein are incorporated into viral vectors, non-limiting examples of which include retroviruses (e.g., Moloney Murine Leukemia Virus (MMLV), Harvey Murine Sarcoma Virus, Mouse Mammary Tumor Virus, Rous Sarcoma Virus), adenoviruses, adeno-associated viruses, SV40 viruses, polyoma viruses, Epstein-Barr viruses, papilloma viruses, herpes viruses, vaccinia viruses, and polio viruses.

[0162] In vivo, many complement proteins, including C3, are synthesized primarily in the liver, and therefore, in some embodiments, hepatocytes are targeted for delivery of the inhibitory RNAs described herein. Several classes of viral vectors have been described, including retroviral vectors (see, e.g., Axelrod et al., PNAS 87:5173-5177 (1990); Kay et al., Hum. Gene Ther. 3:641-647 (1992); Van den Driessche et al., PNAS 96:10379-10384 (1999); Xu et al., ASAIO J. 49:407-416 (2003); and Xu et al., PNAS 102:6080-6085 (2005)), lentiviral vectors (see, e.g., McKay et al., Curr. Pharm. Des. 17:2528-2541 (2011); Brown et al., Blood 109:2797-2805 (2007); and Matrai et al., PNAS 102:6080-6085 (2005)), and mitogenic vectors (see, e.g., McKay et al., Curr. Pharm. Des. 17:2528-2541 (2011); Brown et al., Blood 109:2797-2805 (2007)). vectors (see, e.g., Brown et al., Blood 103:804-810 (2004) and Ehrhardt et al., Blood 99:3923-3930 (2002)) have been shown to be suitable for liver-targeted delivery of gene therapy constructs, including hepatocyte-associated virus (HCV) vectors (see, e.g., Herzog et al., Blood 91:4600-4607 (1998)), adenovirus vectors (see, e.g., Brown et al., Blood 103:804-810 (2004) and Ehrhardt et al., Blood 99:3923-3930 (2002)).

[0163] Retroviruses are enveloped viruses that belong to the virus family Retroviridae. Once inside the host's cells, the virus replicates by transcribing its RNA into DNA using viral reverse transcriptase. Retroviral DNA replicates as part of the host genome and is called a provirus. A selected nucleic acid can be inserted into a vector and packaged into a retroviral particle using techniques well known in the art. Protocols for producing replication-defective retroviruses are well known in the art (e.g., Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E.J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, NJ 1991). The recombinant virus can then be isolated and delivered to the subject's cells in vitro or ex vivo. Many retroviral systems are known in the art (see, e.g., U.S. Patent Nos. 5,994,136, 6,165,782, and 6,428,953). Retroviruses include the genera Alpharetrovirus (e.g., avian leukosis virus), Betaretrovirus (e.g., mouse mammary tumor virus), Deltaretrovirus (e.g., bovine leukemia virus and human T-lymphotropic virus), Epsilonretrovirus (e.g., walleye cutaneous sarcoma virus), and Lentivirus.

[0164] In some embodiments, the retrovirus is a lentivirus of the Retroviridae family. In some examples, the lentivirus is, but is not limited to, human immunodeficiency virus (HIV-1 and HIV-2), simian immunodeficiency virus (S1V), feline immunodeficiency virus (FIV), equine infectious anemia (EIA), and visna virus.

[0165] In some embodiments, the vector is an adenovirus vector. Adenoviruses are a large family of viruses that contain double-stranded DNA. Adenoviruses replicate in the nucleus of the host cell, using the host's cellular machinery to synthesize viral RNA, DNA, and proteins.

[0166] In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. AAV systems are generally well known in the art (see, for example, Kelleher and Vos, Biotechniques, 17(6):1110-17(1994); Cotten et al., PNASUSA, 89(13):6094-98(1992); Curiel, Nat Immun, 13(2-3):141-64(1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129(1992); and Asokan A, et al., Mol. Ther., 20(4):699-708(2012)). Methods for generating and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0167] Several AAV serotypes have been characterized, including AAV1, AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as variants thereof. In general, any AAV serotype can be used to deliver the inhibitory RNA described herein. However, each serotype has a different tropism, e.g., each serotype selectively infects different tissues. In some embodiments, the AAV serotype is selected based on the hepatic tropism found in at least serotypes AAV2, AAV3 (e.g., AAV3B), AAV5, AAV7, AAV8, and AAV9 (see, e.g., Shaoyong et al., Mol. Ther. 23:1867-1876 (2015)).

[0168] The AAV sequences of rAAV vectors usually contain cis-acting 5' and 3' inverted terminal repeat sequences (see, e.g., BJ Carter, "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are approximately 145 bp in length. In some embodiments, substantially the entire ITR-encoding sequence can be used in the rAAV vector, although some modification of these sequences is tolerated. Modification of these ITR sequences is within the skill of the art (see, e.g., Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). Exemplary rAAV vectors of the present disclosure include "cis-acting" plasmids that contain a transgene (e.g., a nucleic acid encoding an inhibitory RNA described herein) in which a selected transgene sequence and associated regulatory elements are flanked 5' and 3' by AAV ITR sequences, which may be derived from any known AAV, including the mammalian AAV types identified herein.

[0169] In addition to the major elements identified above for rAAV vectors, the vector also includes conventional control elements operably linked to the transgene in a manner that allows transcription, translation, and / or expression of the transgene in cells transfected with the vector or infected with a virus produced according to the present disclosure. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequences); sequences that increase protein stability; and sequences that increase secretion of the encoded product, as appropriate. Many expression control sequences, including natural, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be included in the vectors described herein. In some embodiments, the operably linked coding sequence produces a functional RNA (e.g., miRNA or siRNA).

[0170] Examples of constitutive promoters include, but are not limited to, the retroviral Rous Sarcoma Virus (RSV) LTR promoter (optionally including the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally including the CMV enhancer), the SV40 promoter, and the dihydrofolate reductase promoter. Inducible promoters allow for the regulation of gene expression and can be regulated by the presence of exogenously supplied compounds, environmental factors such as temperature, or specific physiological conditions, such as acute phase, specific differentiation states of cells, or only in replicating cells. Inducible promoters and inducible systems are available from a wide range of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously provided promoters include zinc-inducible sheep metallothionine (MT) promoter, dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerase promoter system, ecdysone insect promoter, tetracycline repression system, tetracycline inducible system, RU486 inducible system and rapamycin inducible system. Still other types of inducible promoters that may be useful in this context are those that are regulated by specific physiological conditions, such as temperature, acute phase, specific differentiation state of cells, or only in replicating cells. In another embodiment, the native promoter of the transgene or a fragment thereof is used. In further embodiments, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences, can also be used to mimic native expression.

[0171] In some embodiments, the regulatory sequence confers tissue-specific gene expression. In some cases, the tissue-specific regulatory sequence binds to tissue-specific transcription factors that induce transcription in a tissue-specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers, etc.) are well known in the art. In some embodiments, the promoter is chicken β-actin promoter, pol II promoter, or pol III promoter.

[0172] In some embodiments, the rAAV is designed to express the inhibitory RNA described herein in hepatocytes, and the rAAV comprises one or more liver-specific regulatory elements that substantially restrict the expression of the inhibitory RNA to hepatocytes.In general, the liver-specific regulatory element can be derived from any gene that is known to be expressed only in the liver. WO2009 / 130208 identifies several genes that are specifically expressed in the liver, including serpin peptidase inhibitor, also known as alpha-antitrypsin, clade A member 1 (SERPINA1; gene ID 5265), apolipoprotein CI (APOC1; gene ID 341), apolipoprotein C-IV (APOC4; gene ID 346), apolipoprotein H (APOH; gene ID 350), transthyretin (TTR; gene ID 7276), albumin (ALB; gene ID 213), aldolase B (ALDOB; gene ID 229), cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1; gene ID 1571), fibrinogen alpha chain (FGA; gene ID 2243), transferrin (TF; gene ID 7018), and haptoglobin-related protein (HPR; gene ID 3250). In some embodiments, the viral vectors described herein contain liver-specific regulatory elements derived from the genomic loci of one or more of these proteins. In some embodiments, the promoter can be the liver-specific promoter thyroxine-binding globulin (TBG). Alternatively, other liver-specific promoters may be used (see, e.g., The Liver Specific Gene Promoter Database, Cold Spring Harbor, http: / / rulai.cshl.edu / LSPD / , e.g., alpha 1 antitrypsin (A1AT); human albumin (Miyatake et al., J. Virol. 71:5124 32 (1997)); humA1b; hepatitis B virus core promoter (Sandig et al., Gene Ther. 3:1002 9 (1996)); or LSP1.Further vectors and regulatory elements are described, for example, in Baruteau et al., J. Inherit. Metab. Dis. 40:497-517 (2017).

[0173] In some embodiments, a viral vector (e.g., a rAAV vector) comprises a DNA sequence encoding an inhibitory RNA described herein.

[0174] In some embodiments, a vector (e.g., a viral vector) comprises one or more nucleotide sequences encoding multiple (e.g., 2, 3, 4, 5, or more) miRNAs or siRNAs that comprise nucleic acid strands complementary to a target portion of a C3 transcript, e.g., C3 mRNA (SEQ ID NO: 75). In some embodiments, a vector comprises multiple nucleotide sequences, each nucleotide sequence encoding a different inhibitory RNA as described herein. In some embodiments, a vector comprises multiple nucleotide sequences encoding at least two different inhibitory RNAs, at least two of which are copies of the same inhibitory RNA as described herein.

[0175] In some embodiments, in addition to one or more sequences encoding one or more inhibitory RNAs described herein, the vector (e.g., a viral vector) comprises one or more additional nucleotide sequences encoding one or more C3 inhibitors, e.g., a C3 inhibitor described herein. For example, the C3 inhibitor can be a polypeptide inhibitor and / or a nucleic acid aptamer (see, e.g., U.S. Patent Application Publication No. 20030191084). Exemplary polypeptide inhibitors include compstatin analogs (e.g., compstatin analogs described herein that include genetically encodable amino acids), anti-C3 or anti-C3b antibodies (e.g., scFvs or single domain antibodies, e.g., nanobodies), enzymes that degrade C3 or C3b (see, e.g., U.S. Pat. No. 6,676,943), or mammalian complement regulatory proteins (e.g., CR1, DAF, MCP, CFH, CFI, C1 inhibitor (C1-INH), soluble form of complement receptor 1 (sCR1), TP10 or TP20 (Avant Therapeutics), or portions thereof. Additional polypeptide inhibitors include mini-factor H (see, e.g., U.S. Patent Publication No. 20150110766), Efb protein from Staphylococcus aureus or complement inhibitor (SCIN) protein, or variants or derivatives or mimetics thereof (see, e.g., U.S. Patent Application Publication No. 20140371133).

[0176] In some embodiments, the polypeptide inhibitor is linked to a secretory signal sequence for secretion of the expressed polypeptide inhibitor from the host cell.

[0177] IV. Production of Expression Vectors Methods for obtaining expression vectors, such as rAAV, are well known in the art. In general, the methods involve culturing a host cell that contains nucleic acid sequences encoding the AAV capsid protein or a fragment thereof, a functional rep gene, a recombinant AAV vector consisting of the AAV inverted terminal repeats (ITRs) and a transgene, and sufficient helper functions to allow packaging of the recombinant AAV vector into the AAV capsid protein.

[0178] The components that are cultured in the host cell to package the rAAV vector into an AAV vector can be provided in trans to the host cell. Alternatively, any one or more of the necessary components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) can be provided by a stable host cell that has been engineered to contain one or more of the necessary components using methods well known to those of skill in the art. In some embodiments, such a stable host cell contains the necessary component(s) under the control of an inducible promoter. In other embodiments, the necessary component(s) can be under the control of a constitutive promoter. In other embodiments, the selected stable host cell can contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell can be made that is derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contains rep and / or cap proteins under the control of an inducible promoter. Other stable host cells can be made using routine methods according to those of skill in the art.

[0179] The recombinant AAV vector, rep sequence, cap sequence, and helper functions required to generate the rAAV of the present disclosure can be delivered to the packaging host cell using any suitable genetic element (e.g., vector). The selected genetic element can be delivered by any suitable method in the art, including, for example, genetic engineering, recombinant engineering, and / or synthetic techniques well known to those skilled in the art of nucleic acid manipulation (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY). Similarly, methods for generating rAAV virions are well known, and any suitable method can be used in the present disclosure (see, for example, K. Fisher et al., J. Virol., 70:520-532 (1993), and U.S. Patent No. 5,478,745).

[0180] In some embodiments, recombinant AAV can be produced using a triple transfection method (e.g., as described in U.S. Pat. No. 6,001,650). In some embodiments, recombinant AAV is produced by transfecting a host cell with an AAV vector (including a transgene) for packaging into an AAV particle, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap) that function in trans to perform productive AAV replication and encapsidation. In some embodiments, the AAV helper function vector supports efficient AAV production without producing detectable wild-type AAV virions (i.e., AAV virions that contain functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present disclosure include pHLP19 (see, e.g., U.S. Pat. No. 6,001,650), and pRep6cap6 vectors (e.g., U.S. Pat. No. 6,156,303). Accessory function vectors encode nucleotide sequences for non-AAV derived viral and / or cellular functions (i.e., accessory functions) that AAV depends on to replicate. Accessory functions include functions required for AAV replication, including, but not limited to, those involved in transcriptional activation of AAV genes, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of the cap expression product, and assembly of the AAV capsid. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0181] In some embodiments, the present specification provides a transfected host cell. The term "transfection" refers to the uptake of foreign DNA by a cell, and a cell is "transfected" when exogenous DNA is introduced inside the cell membrane. Many transfection methods are generally known in the art (see, for example, Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.). Using these methods, one or more exogenous nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, can be introduced into a suitable host cell.

[0182] In some embodiments, the host cell is a mammalian cell. The host cell can be used as a recipient of AAV helper constructs, AAV minigene plasmids, accessory function vectors, and / or other transfer DNAs involved in the generation of recombinant AAV. The term includes the progeny of the original transfected cell. Thus, as used herein, "host cell" can refer to a cell transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical to the original parent in morphology or in genomic or total DNA complement due to natural, accidental, or deliberate mutations.

[0183] Further methods for generating and isolating AAV viral vectors suitable for delivery to a subject are described, for example, in U.S. Pat. No. 7,790,449, U.S. Pat. No. 7,282,199, WO2003 / 042397, WO2005 / 033321, WO2006 / 110689, and U.S. Pat. No. 7,588,772. In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct(s) encoding rep and cap. In another system, a packaging cell line that stably provides rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In each of these systems, AAV virions are produced upon infection with a helper adenovirus or herpesvirus, and rAAV is separated from contaminating viruses. Other systems do not require infection with a helper virus to recover AAV, and helper functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also provided in trans by the system. In such systems, helper functions are provided by transiently transfecting the cells with constructs encoding the helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level.

[0184] In yet another system, a transgene flanked by ITRs and rep / cap genes is introduced into an insect host cell by infection with a baculovirus-based vector, and such production systems are well known in the art (see, e.g., Zhang et al., 2009, Human Gene Therapy 20:922-929). Methods of making and using these and other AAV production systems are also described in U.S. Patent Nos. 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0185] The above methods for constructing recombinant vectors are not intended to be limiting, and other suitable methods will be apparent to those skilled in the art.

[0186] V. Compositions and Administration Inhibitory RNA (e.g., siRNA or miRNA as described herein), or a vector comprising a nucleotide sequence encoding an siRNA or miRNA as described herein, can be used to treat a subject suffering from or susceptible to a complement-mediated disease or disorder, e.g., a complement-mediated disease or disorder as described herein. The route and / or mode of administration of the inhibitory RNA can vary depending on the desired outcome. Those skilled in the art, i.e., physicians, will recognize that the administration regimen can be adjusted to provide a desired response, e.g., a therapeutic response. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intravaginal, transdermal, rectal, inhalation, or topical. The mode of administration is left to the discretion of the medical practitioner.

[0187] In some embodiments, an inhibitory RNA described herein is administered systemically and not locally to the eye (e.g., by suprachoroidal, subretinal, or intravitreal injection). In some embodiments, an inhibitory RNA described herein is administered systemically and not an additional complement inhibitor (e.g., systemically to the subject or locally to the subject's eye). In some embodiments, one or more additional inhibitory RNAs described herein are administered systemically and not locally to the eye (e.g., by suprachoroidal, subretinal, or intravitreal injection). In some embodiments, an inhibitory RNA described herein does not penetrate or cross Bruch's membrane (e.g., does not substantially penetrate or cross Bruch's membrane) after systemic administration. In some embodiments, an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA) does not include a moiety that targets the inhibitory RNA to the eye, increases uptake into the eye, and / or increases transport across Bruch's membrane.

[0188] In some embodiments, systemic administration of an inhibitory RNA described herein to a subject can result in, for example, a control level of C3, C3a, C3b, and / or C3d (e.g., a control level of C3, C3a, C3b, and / or C3d in the subject's eye (e.g., the vitreous humor, aqueous humor, retina, and / or retinal pigment epithelium of the eye) prior to administration of the inhibitory RNA, a control level of C3, C3a, C3b, and / or C3d in the eye (e.g., the vitreous humor, aqueous humor, retina, and / or retinal pigment epithelium of the eye) of a subject having a disease described herein, resulting in a decrease in the level of C3 expression or activity (e.g., a decrease in the level of one or more C3 activation products (e.g., C3a, C3b and / or C3d)) in the subject's eye (e.g., the vitreous humor, aqueous humor, retina and / or retinal pigment epithelium of the eye) relative to the levels of C3b and / or C3d, and / or the average levels of C3, C3a, C3b and / or C3d in the eyes (e.g., the vitreous humor, aqueous humor, retina and / or retinal pigment epithelium of the eye) of a population of subjects having a disease described herein. In some embodiments, systemic administration of an inhibitory RNA described herein to a subject can result in, for example, a reduction in a control level of C3 (and / or C3 activation products, e.g., C3a, C3b, and / or C3d) (e.g., the level of C3 (and / or C3 activation products, e.g., C3a, C3b, and / or C3d) in or on the surface of microglia, astrocytes, bone marrow cells, vascular cells, drusen, or plaque in the eye of a subject prior to administration of the inhibitory RNA, a reduction in a control level of C3 (and / or C3 activation products, e.g., C3a, C3b, and / or C3d) in or on the surface of microglia, astrocytes, bone marrow cells, vascular cells, drusen, or plaque in the eye of a subject having a disease described herein, or a reduction in a control level of C3 (and / or C3 activation products, e.g., C3a, C3b, and / or C3d) in or on the surface of microglia, astrocytes, bone marrow cells, vascular cells, drusen, or plaque in the eye of a subject having a disease described herein). (and / or C3 activation products, e.g., C3a, C3b and / or C3d) and / or the average levels of C3 (and / or C3 activation products, e.g., C3a, C3b and / or C3d) in or on the surface of ocular microglia, astrocytes, bone marrow cells, vascular cells, drusen or plaque of a population of subjects having a disease as described herein).In some embodiments, systemic administration of an inhibitory RNA described herein reduces the level of C3 (and / or C3 activation products, e.g., C3a, C3b, and / or C3d) in the subject's eye (e.g., in the vitreous humor, aqueous humor, retina, and / or retinal pigment epithelium of the subject's eye, and / or in microglia, astrocytes, bone marrow cells, vascular cells, drusen, or plaque of the subject's eye) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% compared to a control level of C3a, C3b, and / or C3d. In some embodiments, the level of C3 is C3 protein level. In some embodiments, the level of C3 is the mRNA level of C3.

[0189] Delivery of the inhibitory RNA (e.g., siRNA) described herein to cells can be accomplished in a number of different ways. In vivo delivery can be achieved by administering a composition comprising the inhibitory RNA to a subject, for example, by parenteral administration routes, such as subcutaneous or intravenous or intramuscular administration.

[0190] In some embodiments, the inhibitory RNA is associated with a delivery agent. A "delivery agent" refers to a substance or entity that is non-covalently or covalently associated with or co-administered with an inhibitory RNA and performs one or more functions to increase the stability and / or efficacy of the bioactive agent beyond the stability and / or efficacy that would result if the bioactive agent were delivered (e.g., administered to a subject) in the absence of the delivery agent. For example, the delivery agent can protect the inhibitory RNA from degradation (e.g., in the blood), facilitate the entry of the inhibitory RNA into a cell or a cellular compartment of interest (e.g., the cytoplasm), and / or enhance the binding of the inhibitory RNA to a particular cell that contains the molecular target to be regulated. Those skilled in the art will recognize many delivery agents that can be used to deliver inhibitory RNAs, such as siRNAs. For a review of some of these techniques, see Kanasty, R., et al. Nat Mater. 12(11):967-77 (2013). In some embodiments, the inhibitory RNA can be combined with a delivery agent, such as a nanoparticle, a dendrimer, a polymer, a liposome, or a cationic delivery system, for example, to administer the inhibitory RNA systemically. Without wishing to be bound by any theory, it is believed that the positively charged cationic delivery system promotes the binding of the negatively charged inhibitory RNA and promotes interactions at the negatively charged cell membrane, allowing efficient uptake of the inhibitory RNA by the cell. Lipids (e.g., cationic or neutral lipids), dendrimers, or polymers may be attached to the inhibitory RNA or form vesicles or micelles that encapsulate the inhibitory RNA. Methods for preparing and administering complexes that include cationic agents and inhibitory RNA are well known in the art. In some embodiments, it is specifically contemplated to use any of the delivery agents described in US Patent Publication No. 20160298124. In some embodiments, the inhibitory RNA is complexed with cyclodextrin for systemic administration. In some embodiments, the inhibitory RNA is administered in combination with lipids or lipid-containing particles.In some embodiments, the inhibitory RNA is administered in association with a cationic polymer (which may be a polypeptide or a non-polypeptide polymer), lipid, peptide, PEG, cyclodextrin, or combinations thereof, which may be in the form of nanoparticles or microparticles. The lipid or peptide may be cationic. "Nanoparticle" refers to a particle having two or three dimensions greater than 1 nanometer (nm) and less than about 150 nm, e.g., 20 nm to 50 nm or 50 nm to 100 nm. "Microparticle" refers to a particle having two or three dimensions greater than 150 nm and less than about 1000 nm. The nanoparticles may be covalently or non-covalently bound to a targeting moiety and / or a cell penetrating or membrane active moiety. Nanoparticles, such as lipid nanoparticles, are described, for example, in Tatiparti et al., Nanomaterials 7:77 (2017). Exemplary delivery agents, methods of manufacture and use in delivery of inhibitory RNA are described in U.S. Patent Nos. 7,427,605, 8,158,601, 9,012,498, 9,415,109, 9,062,021 and 9,402,816. In some embodiments, it is contemplated to use a delivery technology known in the art as "smart particles". In some embodiments, it is contemplated to use a delivery technology known in the art as "stable nucleic acid lipid particles" (SNALPs), in which the nucleic acid to be delivered is encapsulated in a lipid bilayer comprising a mixture of cationic lipids and fusogenic lipids, which are also coated with a diffusible polyethylene glycol-lipid (PEG-lipid) conjugate that provides a neutral hydrophilic outer surface.

[0191] In some embodiments, the delivery agent comprises one or more aminoalcohol cationic lipids, such as those described in US Pat. No. 9,044,512.

[0192] In some embodiments, the delivery agent comprises one or more amino acid lipids.Amino acid lipids are molecules that comprise amino acid residues (e.g., arginine, homoarginine, nor-norarginine, ornithine, lysine, homolysine, histidine, 1-methylhistidine, pyridylalanine, asparagine, N-ethylasparagine, glutamine, 4-aminophenylalanine, their N-methylated forms, and their side chain modified derivatives) and one or more lipophilic tails.Exemplary amino acid lipids and their use for delivering nucleic acids are described in US Patent Publication No. 20110117125, as well as US Patent Nos. 8,877,729, 9,139,554, and 9,339,461.In some embodiments, membrane-soluble poly(amidoamine) polymers and polyconjugates can be used, such as those described in US Patent Publication No. 20130289207. In some embodiments, the delivery agent comprises a lipopeptide compound comprising a central peptide and having a lipophilic group attached to each terminus. In some embodiments, the lipophilic group can be derived from a naturally occurring lipid. In some embodiments, the lipophilic group can comprise C(1-22) alkyl, C(6-12) cycloalkyl, C(6-12) cycloalkyl-alkyl, C(3-18) alkenyl, C(3-18) alkynyl, C(1-5) alkoxy-C(1-5) alkyl, or sphinganine, or (2R,3R)-2-amino-1,3-octadecanediol, icosasphinganine, sphingosine, phytosphingosine, or 4-sphingenine. The central peptide can comprise a cationic or amphipathic amino acid sequence. Examples of such lipopeptides and their use to deliver nucleic acids are described, for example, in U.S. Pat. No. 9,220,785.

[0193] "Masking moiety" refers to a molecule or group that, when physically bound to another agent (e.g., a polymer), blocks, inhibits or inactivates one or more properties (biophysical or biochemical properties) or activities of the agent. In some embodiments, the masking moiety can be covalently or non-covalently bound to the inhibitory RNA. The masking moiety can be reversible, meaning that the masking moiety is bound to the inhibitory RNA that it masks through a reversible bond. As will be appreciated by those skilled in the art, a sufficient number of masking moieties are bound to the inhibitory RNA to be masked to achieve the desired level of inactivation.

[0194] In some embodiments, the inhibitory RNA is attached to a delivery agent that is a polymer. Useful delivery polymers include, for example, poly(acrylate) polymers (see, for example, US Patent Publication No. 20150104408), poly(vinyl ester) polymers (see, for example, US Patent Publication No. 20150110732), and certain polypeptides. In some embodiments, the delivery polymer is a reversibly masked membrane active polymer. In some embodiments, a targeting moiety is attached to the inhibitory RNA or the polymer, or both. In some embodiments, the inhibitory RNA or the inhibitory RNA-targeting moiety conjugate is co-administered with the delivery polymer, but is not attached to the polymer. In this context, "co-administered" means that the inhibitory RNA and the delivery polymer are administered to a subject such that they are present in the subject at overlapping times. The inhibitory RNA-targeting moiety conjugate and the delivery polymer may be administered simultaneously or delivered sequentially. In the case of simultaneous administration, they may be mixed prior to administration. In the case of sequential administration, either the inhibitory RNA or the delivery polymer may be administered first. The inhibitory RNA and the delivery polymer may be administered in the same composition, or may be administered separately close enough in time that the cytoplasmic delivery of the inhibitory RNA to the cell is enhanced relative to the cytoplasmic delivery that would occur without administration of the polymer. In some embodiments, the inhibitory RNA and the delivery polymer are administered no more than 15 minutes, 30 minutes, 60 minutes, or 120 minutes apart. In some embodiments, the delivery polymer is a targeted, reversibly masked, membrane active polymer. The polymer is conjugated with a targeting moiety that targets the polypeptide to cells where enhanced cytoplasmic delivery of the inhibitory RNA is desired. The inhibitory RNA can be targeted to the same cells, optionally using the same targeting moiety. That is, the inhibitory RNA can be administered as an inhibitory RNA-targeting moiety conjugate.As used herein, a membrane active polymer is a surface active amphiphilic polymer that can induce one or more of the following effects on biological membranes: membrane denaturation or destruction, pore formation in the membrane, membrane disruption, or membrane destruction or lysis, allowing non-membrane permeable molecules to enter or pass through the cell. As used herein, a membrane or cell membrane includes a lipid bilayer. Membrane denaturation or destruction can be functionally defined by the activity of the polymer in at least one of the following assays: red blood cell lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis, and endosomal release. Membrane active polymers can enhance the delivery of polynucleotides to cells, for example, by disrupting or destabilizing plasma membranes or internal vesicular membranes (such as endosomes or lysosomes) by forming pores in the membrane, or by disrupting endosomal or lysosomal vesicles, thereby releasing the contents of the vesicles into the cytoplasm. In some embodiments, the targeted reversibly masked membrane active polymer is an endosomolytic polymer. Endosomolytic polymers are polymers that can cause endosome disruption or lysis in response to a change in pH, or release of compounds that are normally cell membrane impermeable, such as polynucleotides or proteins, from endosomes or lysosomes or other endomembrane-enclosed vesicles. In some embodiments, the polymer is a reversibly modified amphiphilic membrane-active polyamine, where the reversible modification inhibits membrane activity and neutralizes the polyamine to reduce the positive charge, forming a polymer with a near-neutral charge. The reversible modification can also provide cell-type specific targeting of the polymer and / or inhibit non-specific interactions. The polyamine can be reversibly modified by reversible modification of each amine of the polyamine. A reversibly masked membrane-active macromolecule is substantially non-membrane active when masked, and becomes membrane active when unmasked. The masking moiety is typically covalently attached to the membrane-active polymer via a physiologically reversible bond.By using a physiologically reversible bond, the masking moiety can be cleaved from the polymer in vivo, thereby unmasking the polymer and restoring the activity of the unmasked polymer. By selecting an appropriate reversible bond, the activity of the membrane active polymer is restored after the conjugate is delivered or targeted to a desired cell type or cellular location. The reversibility of the bond allows for selective activation of the membrane active polymer. A physiologically reversible bond is reversible under mammalian intracellular conditions, including chemical conditions found in mammalian cells or similar chemical conditions, such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration. In some embodiments, a targeting moiety, such as an ASGPR targeting moiety, can function as a masking moiety. In some embodiments, a lipophilic moiety is attached to the ASGPR targeting moiety. Exemplary targeting moieties (e.g., ASGPR targeting moieties), physiologically labile linkages (e.g., enzymatically labile linkages, pH labile linkages), masking moieties, membrane active polymers (e.g., endosomolytically active polymers), lipophilic moieties, RNAi agent-targeting moiety conjugates, delivery agent-targeting moiety conjugates, conjugates comprising an RNAi agent, a targeting moiety, and a delivery agent, and conjugates for delivering nucleic acids to cells (e.g., hepatocytes). Methods for achieving this are described in U.S. Patent Application Publication Nos. 20130245091, 20130317079, 20120157509, 20120165393, 20120172412, 20120230938, 20140135380, 20140135381, 20150104408, and 20150110732. In some embodiments, the inhibitory RNA is co-administered with a melittin peptide, e.g., as described in U.S. Patent Application No. 20120165393. The inhibitory RNA, the melittin peptide, or both may optionally be linked to a targeting moiety via a reversible bond. In some embodiments, the masking moiety comprises a dipeptide-amidobenzyl-carbonate or a disubstituted maleic anhydride masking moiety, for example as described in US Patent Application Publication No. 20150110732.

[0195] In some embodiments, the inhibitory RNA can be administered in a "naked" form (i.e., in the absence of a delivery agent). The naked inhibitory RNA can be in a suitable buffer. The buffer can include, for example, acetate, citrate, prolamin, carbonate, or phosphate, or any combination thereof. In some embodiments, the buffer is phosphate buffered saline (PBS). The pH and osmolality of the buffer can be adjusted to be suitable for administration to a subject. In some embodiments, the inhibitory RNA is administered not physically associated with a lipid or lipid-containing particle. In some embodiments, the inhibitory RNA is administered not physically associated with a nanoparticle or microparticle. In some embodiments, the inhibitory RNA is administered not physically associated with a cationic polymer. In some embodiments, the inhibitory RNA is administered not physically associated with a cyclodextrin. In some embodiments, the inhibitory RNA administered in a "naked" form includes a targeting moiety.

[0196] An inhibitory RNA (e.g., an siRNA or miRNA described herein) or a vector comprising a nucleotide sequence encoding an siRNA or miRNA described herein can be added to a pharmaceutical composition. Such pharmaceutical compositions are useful, among others, for administration and delivery to a subject in vivo or ex vivo. In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent, e.g., a pharmaceutical agent that does not itself induce a harmful immune response in an individual receiving the composition and that can be administered without undue toxicity. As used herein, the terms "pharmaceutical acceptable" and "physiologically acceptable" refer to a biologically acceptable formulation, gas, liquid or solid, or mixture thereof, that is suitable for one or more routes of administration, in vivo delivery or contact. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugar, and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, etc., salts of organic acids such as acetates, propionates, malonates, benzoates, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0197] The pharmaceutical compositions may be provided as salts and may be formed with a number of acids, including but not limited to hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc. Salts tend to be more soluble in aqueous or other protic solvents than the corresponding free base forms. In some embodiments, the pharmaceutical compositions may be lyophilized powders.

[0198] Pharmaceutical compositions may include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersions and suspension media, coatings, isotonicity and absorption enhancing or retarding agents that are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharma-ceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents and antifungal agents) may also be incorporated into the compositions.

[0199] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as described herein or as known to those skilled in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0200] Compositions suitable for parenteral administration can include aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non-limiting illustrative examples include water, buffered saline, Hank's solution, Ringer's solution, dextrose, fructose, ethanol, animal oils, vegetable oils, or synthetic oils. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the active compound can be prepared as suitable oil injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, such suspensions can also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agent, allowing for the preparation of highly concentrated solutions.

[0201] Cosolvents and adjuvants can be added to the formulation.Non-limiting examples of cosolvents include alcohols with hydroxyl groups or other polar groups, such as isopropyl alcohol; glycols such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.Adjuvants include surfactants such as soybean lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0202] After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment, such labeling can include the amount, frequency, and method of administration.

[0203] Pharmaceutical compositions and delivery systems suitable for use with the compositions, methods, and methods of the present disclosure are well known in the art (see, e.g., Remington: The Science and Practice of Pharmacy. 21st Edition. Philadelphia, PA. Lippincott Williams & Wilkins, 2005).

[0204] The disclosure also provides methods of introducing an inhibitory RNA (e.g., an siRNA or miRNA described herein), or a vector comprising a nucleotide sequence encoding an siRNA or miRNA described herein, into a cell or animal. In some embodiments, such methods include contacting or administering to a subject (e.g., a cell or tissue of a subject) an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein) to a subject (e.g., a subject such as a mammal) to cause the inhibitory RNA to be expressed in the subject (e.g., a cell or tissue of the subject). In another embodiment, the method includes expressing an inhibitory RNA in an individual (a patient or subject such as a mammal) by providing to a cell of the individual an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein).

[0205] The inhibitory RNA compositions described herein (or vectors (e.g., rAAV vectors) containing nucleotide sequences encoding the inhibitory RNAs described herein) may be administered in sufficient or effective amounts to a subject in need of administration of the inhibitory RNA compositions. Doses may vary and depend on the type, onset, progression, severity, frequency, duration, or probability of the disease being treated, the desired clinical endpoint, previous or concurrent treatments, the general health, age, sex, race, or immunocompetence of the subject, and other factors recognized by those of skill in the art. The amount, number, frequency, or duration of administration may be increased or decreased proportionately as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy, and the condition of the subject. Those of skill in the art will recognize factors that may affect the dosage and timing required to provide an amount sufficient to provide a therapeutic or prophylactic benefit.

[0206] The dosage to achieve a therapeutic effect, e.g., dosage of vector genome per kg body weight (vg / kg) (e.g., for vector delivery) or mg per kg body weight (mg / kg), will vary based on several factors, including, but not limited to, the route of administration, the expression level of inhibitory RNA required to achieve a therapeutic effect, the particular disease being treated, any host immune response to the viral vector, the host immune response to the heterologous inhibitory RNA, and the stability of the expressed inhibitory RNA. For vector delivery of inhibitory RNA, one of skill in the art can determine the dosage range of rAAV / vector genome to treat a patient with a particular disease or disorder based on the above factors as well as other factors. Generally, to achieve a therapeutic effect, the dosage should be at least 1×10 per kg of subject body weight. 8 More than, for example, 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 The range of vector genome (vg / kg) is as above.

[0207] In some embodiments, the inhibitory RNA composition is administered to the subject in an amount between 0.01 mg / kg and 50 mg / kg. In some embodiments, the inhibitory RNA composition is administered at a dose of about 0.01 mg / kg to about 10 mg / kg, or about 0.5 mg / kg to about 15 mg / kg. In some embodiments, the inhibitory RNA composition is administered at a dose of about 10 mg / kg to about 30 mg / kg. In some embodiments, the inhibitory RNA composition is administered at a dose of about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 3.5 mg / kg, about 4 mg / kg, about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, or about 50 mg / kg. In some embodiments, the amount is 0.01 mg / kg to 0.1 mg / kg, 0.01 mg / kg to 0.1 mg / kg, 0.1 mg / kg to 1.0 mg / kg, 1.0 mg / kg to 2.5 mg / kg, 2.5 mg / kg to 5.0 mg / kg, 5.0 mg / kg to 10 mg / kg, 10 mg / kg to 20 mg / kg, 20 mg / kg to 30 mg / kg, 30 mg / kg to 40 mg / kg, or 40 mg / kg to 50 mg / kg. In some embodiments, a fixed dose is administered. In some embodiments, the dose is 5 mg to 1.0 g, e.g., 5 mg to 10 mg, 10 mg to 20 mg, 20 mg to 40 mg, 40 mg to 80 mg, 80 mg to 160 mg, 160 mg to 320 mg, 320 mg to 640 mg, 640 mg to 1 g. In some embodiments, the dosage is about 1 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg. According to some embodiments, the dosage is a daily dosage. In some embodiments, the dosage is administered according to a dosing schedule with a dosing interval of at least 2 days, for example, at least 7 days, for example, about 2, 3, 4, 6, or 8 weeks. For example, in some embodiments, the inhibitory RNA composition is administered according to a dosing schedule with a dosing interval of at least 7 days.In some embodiments, the inhibitory RNA composition is administered daily, weekly, monthly, or every 2, 3, 4, 5, or 6 months or more. In some embodiments, any of the doses and / or dosing schedules described herein are administered subcutaneously. In some embodiments, the inhibitory RNA composition is administered once and the level of inhibition is measured, and when the level of inhibition has decreased to a certain level, the next dose of the inhibitory composition is administered.

[0208] In some embodiments, the subject exhibits sustained inhibition of C3, e.g., as measured by C3 mRNA expression (e.g., in liver tissue, e.g., liver biopsy), for a period of at least 2 days, e.g., at least 7 days, e.g., about 2, 3, 4, 6, 8, 10, 12, 16, or 20 weeks, after administration. In some embodiments, the subject exhibits a reduction in serum C3 levels, and the reduction in serum C3 levels is maintained, e.g., for a period of at least 2 days, e.g., at least 7 days, e.g., about 2, 3, 4, 6, 8, 10, 12, 16, or 20 weeks, after administration.

[0209] An effective or sufficient amount can be given in a single administration (although this is not required) or may require multiple administrations, and can be administered alone or in combination with another composition (e.g., another complement inhibitor described herein) (although this is not required). For example, the amount can be increased proportionately as indicated by the needs of the subject, type, condition and severity of the disease being treated or side effects of the treatment (if any). An amount considered effective also includes an amount that leads to the use of another treatment, treatment regimen or protocol, e.g., a reduction in the administration of another complement inhibitor described herein.

[0210] Thus, the pharmaceutical compositions of the present disclosure include compositions that contain an effective amount of active ingredient to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the skill of the artisan using the techniques and guidance provided in this disclosure. The therapeutic dose may depend on, among other factors, the age and general condition of the subject, the severity of the complement-mediated disease or disorder, and the strength of the regulatory sequence that regulates the expression level of the inhibitory RNA described herein. Thus, a therapeutically effective amount in humans will fall within a relatively broad range that can be determined by a medical practitioner based on an individual patient's response to treatment with the vector. The pharmaceutical compositions can be delivered to a subject by gene and / or cell-based therapy or by modifying the patient's or donor's cells ex vivo to enable the inhibitory RNA described herein to be produced in vivo.

[0211] The disclosed methods and uses include systemic delivery and administration by any route, such as injection or infusion. In vivo delivery of pharmaceutical compositions can generally be achieved via injection using a conventional syringe, but other delivery methods such as convection-enhanced delivery can also be used (see, for example, U.S. Pat. No. 5,720,720). For example, the compositions can be administered subcutaneously, epidermally, intradermally, intramucosally, intraperitoneally, intravenously, intrapleurally, intraarterially, orally, intrahepatically, via the portal vein, and intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal application. Clinicians who specialize in treating patients with complement-mediated diseases can determine the optimal route for administering inhibitory RNA (e.g., siRNA or miRNA described herein) or vectors containing nucleotide sequences encoding siRNA or miRNA described herein.

[0212] In some embodiments, the inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding the inhibitory RNA described herein) can be administered to a subject once a day, once a week, once every two, three or four weeks, or at longer intervals. In some embodiments, the inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding the inhibitory RNA described herein) can be administered according to a dosing regimen that includes (i) an initial administration once a day, once a week, once every two, three or four weeks, or at longer intervals, followed by (ii) a non-administration period of, for example, 1, 2, 3, 4, 5, 6, 8, 9 or 10 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years. In some embodiments, the vector comprising a nucleotide sequence encoding the inhibitory RNA described herein can be administered (i) one or more times during an initial period of 2 weeks, 4 weeks, or 6 weeks or less, followed by (ii) a non-administration period of, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years. In some embodiments, the subject is monitored before and / or after treatment for levels of C3 expression and / or activity, e.g., as measured using an alternative pathway assay, a classical pathway assay, or both. Suitable assays are well known in the art and include, e.g., hemolytic assays. In some embodiments, the subject is treated or re-treated when the measured level of C3 expression and / or activity is 10%, 20%, 30%, 40%, 50%, 100%, 200% or more relative to the measured level of C3 expression and / or activity in a control subject.

[0213] VI. Diseases, Disorders and Conditions In some embodiments, the inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding the inhibitory RNA described herein) is administered systemically (e.g., subcutaneously) to a subject, alone or in combination with one or more additional complement inhibitors described herein, to treat an ocular disease such as macular degeneration (e.g., age-related macular degeneration (AMD) and Stargardt's macular dystrophy), diabetic retinopathy, glaucoma, or uveitis. In some embodiments, the inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding the inhibitory RNA described herein) can be administered systemically (e.g., subcutaneously or intravenously) to treat a subject suffering from or at risk for AMD, alone or in combination with one or more additional complement inhibitors described herein. In some embodiments, the AMD is neovascular (wet) AMD. In some embodiments, the AMD is dry AMD. As will be appreciated by those skilled in the art, dry AMD includes geographic atrophy (GA), intermediate AMD, and early AMD. In some embodiments, a subject with GA is treated to slow or stop the progression of the disease. For example, in some embodiments, treating a subject with GA reduces the rate of retinal cell death. The reduction in the rate of retinal cell death can be shown by a reduction in the rate of GA lesions in patients treated with an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein), alone or in combination with one or more additional complement inhibitors described herein, compared to a control (e.g., a patient administered sham). In some embodiments, the subject has intermediate AMD. In some embodiments, the subject has early AMD. In some embodiments, a subject with intermediate or early GA is treated to slow or stop disease progression. For example, in some embodiments, treating a subject with intermediate AMD can slow or prevent progression to advanced AMD (neovascular AMD or GA). In some embodiments, treating a subject with intermediate AMD can slow or prevent progression to advanced AMD (neovascular AMD or GA).In some embodiments, the eye has both GA and neovascular AMD, hi some embodiments, the eye has GA but does not have wet AMD.

[0214] In some embodiments, the subject has an ocular disease characterized by macular degeneration, choroidal neovascularization (CNV), retinal neovascularization (RNV), ocular inflammation, or any combination of the above. Macular degeneration, CNV, RNV, and / or ocular inflammation may be defining and / or diagnostic features of the disease. Exemplary diseases characterized by one or more of these features include, but are not limited to, macular degeneration-related conditions, diabetic retinopathy, retinopathy of prematurity, proliferative vitreoretinopathy, uveitis, keratitis, conjunctivitis, and scleritis. In some embodiments, the subject is in need of treatment for ocular inflammation. Ocular inflammation may occur in many ocular structures, such as the conjunctiva (conjunctivitis), cornea (keratitis), episclera, sclera (scleritis), uveal tract, retina, vasculature, and / or optic nerve. Evidence of ocular inflammation may include one or more symptoms such as the presence of inflammation-related cells, such as white blood cells (e.g., neutrophils, macrophages) in the eye, the presence of endogenous inflammatory mediator(s), eye pain, redness, light sensitivity, blurred vision, and floaters. Uveitis is a general term that refers to inflammation in the uvea of ​​the eye, such as any of the uveal structures, including the iris, ciliary body, or choroid. Specific types of uveitis include iritis, iridocyclitis, cyclitis, pars planitis, and choroiditis. In some embodiments, the ocular disease is an ocular disease characterized by optic nerve damage (e.g., optic nerve degeneration), such as glaucoma.

[0215] In some embodiments, it is contemplated that an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein) alone or in combination with one or more additional complement inhibitors described herein for a relatively short period of time, e.g., 1-6 weeks, e.g., about 2-4 weeks, may provide a long-term effect. In some embodiments, remission is achieved over an extended period of time, e.g., 1-3 months, 3-6 months, 6-12 months, 12-24 months, or more. In some embodiments, the subject can be monitored and / or prophylactically treated before symptoms recur. For example, the subject can be treated before or after exposure to a triggering event. In some embodiments, the subject can be monitored for, e.g., an increase in a biomarker, e.g., a biomarker including an indicator of Th17 cells or Th17 cell activity, or complement activation, and can be treated in response to an increase in the level of such a biomarker. For further discussion, see, e.g., PCT / US2012 / 043845.

[0216] VII. Combination Therapy In some aspects, the method of the present disclosure comprises administering rAAV as described herein alone or in combination with one or more additional complement inhibitors.In some embodiments, the inhibitory RNA is administered to a subject who is already undergoing treatment with another complement inhibitor.In some embodiments, another complement inhibitor is administered to a subject who is undergoing treatment with the inhibitory RNA.In some embodiments, both the inhibitory RNA and another complement inhibitor are administered to a subject.

[0217] In some embodiments, administration of the inhibitory RNA allows the second complement inhibitor to be administered at a reduced dosing regimen (e.g., lower individual dose size, less frequent dosing, less frequent dosing, and / or reduced overall exposure) compared to administration of the second complement inhibitor as a monotherapy. Without wishing to be bound by any theory, in some embodiments, a reduced dosing regimen of the second complement inhibitor can avoid one or more undesirable side effects that may otherwise occur.

[0218] In some aspects, systemic administration of an inhibitory RNA in combination with a second complement inhibitor can sufficiently reduce the amount of C3 in a subject's blood such that a reduction in the dosing regimen of the inhibitory RNA and / or the second complement inhibitor is required to achieve the desired degree of complement inhibition.

[0219] In some aspects, systemic administration of an inhibitory RNA in combination with a second complement inhibitor can sufficiently reduce the amount of C3 in the subject's blood such that a reduction in the dosing regimen of the inhibitory RNA and / or the second complement inhibitor is required to achieve a desired amount or level of improvement in one or more signs, symptoms, biomarkers, or prognostic measures of a complement-mediated disease.

[0220] In some embodiments, such reduced doses can be administered in smaller amounts, or using lower concentrations, or using longer dosing intervals, or any combination of the above, compared to administration of the inhibitory RNA or second complement inhibitor as a monotherapy.

[0221] Any complement inhibitor, such as any complement inhibitor known in the art, can be administered in combination with the inhibitory RNA described herein. In some embodiments, the complement inhibitor is compstatin or a compstatin analog.

[0222] Compstatin is a cyclic peptide that binds to C3 and inhibits complement activation. US Patent No. 6,319,897 describes a peptide having the sequence Ile-[Cys-Val-Val-Gln-Asp-Trp-Gly-His-His-Arg-Cys]-Thr (SEQ ID NO: 1), with the disulfide bond between the two cysteines shown in brackets. It will be understood that although the name "compstatin" is not used in US Patent No. 6,319,897, it has been used subsequently in the scientific and patent literature (see, for example, Morikis, et al., Protein Sci., 7(3):619-27, 1998) to refer to a peptide having the same sequence as SEQ ID NO: 2 disclosed in US Patent No. 6,319,897, but amidated at the C-terminus. The term "compstatin" is used herein in accordance with such usage. Compstatin analogues have been developed that have greater complement inhibitory activity than compstatin. For example, WO2004 / 026328(PCT / US2003 / 029653), Morikis,D.,et al.,Biochem Soc Trans.32(Pt 1):28-32,2004,Mallik,B.,et al.,J.Med.Chem.,274-286,2005;Katragadda,M.,et See, e.g., et al. J. Med. Chem., 49:4616-4622, 2006; WO2007062249 (PCT / US2006 / 045539); WO2007044668 (PCT / US2006 / 039397), WO / 2009 / 046198 (PCT / US2008 / 078593); WO / 2010 / 127336 (PCT / US2010 / 033345).

[0223] As used herein, the term "compstatin analog" includes compstatin and any complement inhibitory analogs thereof. The term "compstatin analog" encompasses compstatin, as well as other compounds designed or identified based on compstatin, whose complement inhibitory activity is at least 50% as great as the complement inhibitory activity of compstatin, as measured, for example, using any art-recognized complement activation assay or a substantially similar or equivalent assay. Certain suitable assays are described in U.S. Patent No. 6,319,897, WO 2004 / 026328, Morikis, supra, Mallik, supra, Katragadda 2006, supra, WO 2007062249 (PCT / US2006 / 045539); WO 2007044668 (PCT / US2006 / 039397), WO / 2009 / 046198 (PCT / US2008 / 078593); and / or WO / 2010 / 127336 (PCT / US2010 / 033345). The assay may, for example, measure alternative or classical pathway-mediated erythrocyte lysis, or may be an ELISA assay. In some embodiments, the assays described in WO / 2010 / 135717 (PCT / US2010 / 035871) are used.

[0224] Table 8 provides a non-limiting list of compstatin analogs useful in the present disclosure. Analogs are abbreviated in the left column by indicating the specific modification at the designated positions (1-13) compared to the parent peptide compstatin. Consistent with usage in the art, "compstatin" as used herein, and the activity of the compstatin analogs described herein compared to the activity of compstatin, refers to the compstatin peptide amidated at the C-terminus. Unless otherwise indicated, the peptides in Table 8 are amidated at the C-terminus. Bold text is used to indicate the specific modification. Activity compared to compstatin is based on published data and assays described therein (WO2004 / 026328, WO2007044668, Mallik, 2005; Katragadda, 2006). In certain embodiments, the peptides listed in Table 8, when used in the therapeutic compositions and methods of the present disclosure, are cyclized via a disulfide bond between two Cys residues. Alternative means for cyclizing peptides are also within the scope of this disclosure. [Table 9-1] [Table 9-2]

[0225] In certain embodiments of the compositions and methods of the present disclosure, the compstatin analog has a sequence selected from sequences 9-36. In one embodiment, the compstatin analog has the sequence of SEQ ID NO: 28. As used herein, "L-amino acid" refers to any of the naturally occurring levorotatory alpha-amino acids normally present in proteins or alkyl esters of these alpha-amino acids. The term "D-amino acid" refers to dextrorotatory alpha-amino acids. Unless otherwise specified, all amino acids referred to herein are L-amino acids.

[0226] In some embodiments, one or more amino acid(s) of a compstatin analog (e.g., any of the compstatin analogs disclosed herein) can be an N-alkyl amino acid (e.g., an N-methyl amino acid). For example, without limitation, at least one amino acid within the cyclic portion of the peptide, at least one amino acid at the N-terminus of the cyclic portion, and / or at least one amino acid at the C-terminus of the cyclic portion can be an N-alkyl amino acid, e.g., an N-methyl amino acid. In some embodiments, for example, a compstatin analog includes an N-methyl glycine, e.g., at a position corresponding to position 8 of compstatin and / or at a position corresponding to position 13 of compstatin. In some embodiments, one or more of the compstatin analogs in Table 8 includes at least one N-methyl glycine, e.g., at a position corresponding to position 8 of compstatin and / or at a position corresponding to position 13 of compstatin. In some embodiments, one or more of the compstatin analogs in Table 8 includes at least one N-methyl isoleucine, e.g., at a position corresponding to position 13 of compstatin. For example, the Thr at or near the C-terminus of the peptides whose sequences are listed in Table 8, or any other compstatin analog sequence, can be replaced with N-methyl Ile. As will be appreciated, in some embodiments, the N-methylated amino acid comprises an N-methyl Gly at position 8 and an N-methyl Ile at position 13. In some embodiments, a compstatin analog (e.g., any one of the compstatin analogs listed in Table 8) comprises an isoleucine at a position corresponding to position 3 of SEQ ID NO:8, in place of or in addition to one or more of the substitutions described herein. For example, in some embodiments, a compstatin analog comprises or consists of the sequence of any one of SEQ ID NOs:8-36, except that position 3 is an isoleucine. In some embodiments, a compstatin analog comprises or consists of the sequence of any one of SEQ ID NOs:25, 33, or 36, except that position 4 is an isoleucine. Additional compstatin analogs are described, for example, in WO2019 / 166411.

[0227] Compstatin analogs can be prepared by various synthetic methods of peptide synthesis known in the art through condensation of amino acid residues, for example, from an appropriate nucleic acid sequence encoding the compstatin analog, by expression in vitro according to conventional peptide synthesis methods, or in living cells using methods known in the art. For example, peptides can be synthesized using standard solid phase methodologies such as those described in Malik, supra, Katragadda, supra, WO2004026328, and / or WO2007062249. Potentially reactive moieties, such as amino and carboxyl groups, reactive functional groups, can be protected and subsequently deprotected using various protecting groups and methodologies known in the art. See, for example, "Protective Groups in Organic Synthesis", 3 rd See, ed. Greene, TW and Wuts, PG, Eds., John Wiley & Sons, New York: 1999. Peptides can be purified using standard approaches such as reversed-phase HPLC. Separation of diastereomeric peptides can be performed, if desired, using known methods such as reversed-phase HPLC. Preparations can be lyophilized, if desired, and then dissolved in a suitable solvent, e.g., water. The pH of the resulting solution can be adjusted, e.g., to physiological pH, using a base such as NaOH. Peptide preparations can be characterized, if desired, by mass spectrometry, e.g., to confirm mass and / or disulfide bond formation. See, e.g., Mallik, 2005, and Katragadda, 2006.

[0228] Compstatin analogs can be modified by the addition of molecules such as polyethylene glycol (PEG) to stabilize the compound, reduce its immunogenicity, extend its lifetime in the body, increase and decrease its solubility, and / or increase its resistance to degradation. Methods of pegylation are well known in the art (Veronese, FM & Harris, Adv. Drug Deliv. Rev. 54, 453-456, 2002; Davis, FF, Adv. Drug Deliv. Rev. 54, 457-458, 2002); Hinds, KD & Kim, SW Adv. Drug Deliv. Rev. 54, 505-530 (2002; Roberts, MJ, Bentley, MD & Harris, JM Adv. Drug Deliv. Rev. 54, 459-476; 2002); Wang, YSet al. Adv. Drug Deliv. Rev. 54, 547-570, 2002). A wide variety of polymers, such as PEG and modified PEG, including derivatized PEG, that can be conveniently conjugated to polypeptides are described in the Nektar Advanced Pegylation 2005-2006 Product Catalog, Nektar Therapeutics, San Carlos, Calif., which also provides details of suitable conjugation procedures.

[0229] In some embodiments, a compstatin analog of any of SEQ ID NOs: 9-36 is extended at the N-terminus, C-terminus, or both, by one or more amino acids, where at least one of the amino acids has a side chain that includes a reactive functional group, such as a primary or secondary amine, a sulfhydryl group, a carboxyl group (which may be present as a carboxylate group), a guanidino group, a phenol group, an indole ring, a thioether, or an imidazole ring, that facilitates conjugation with a reactive functional group for attaching PEG to the compstatin analog. In some embodiments, a compstatin analog includes an amino acid that has a side chain that includes a primary or secondary amine, e.g., a Lys residue. For example, a Lys residue, or a sequence that includes a Lys residue, is added to the N-terminus and / or C-terminus of a compstatin analog described herein (e.g., a compstatin analog including any one of SEQ ID NOs: 9-36).

[0230] In some embodiments, the Lys residue is separated from the cyclic portion of the compstatin analog by a rigid or flexible spacer. The spacer may include, for example, a substituted or unsubstituted, saturated or unsaturated alkyl chain, an oligo(ethylene glycol) chain, and / or other moieties, for example, as described herein with respect to the linker. The length of the chain may be, for example, between 2 and 20 carbon atoms. In other embodiments, the spacer is a peptide. A peptide spacer may be, for example, between 1 and 20 amino acids in length, for example, between 4 and 20 amino acids in length. A suitable spacer may, for example, comprise or consist of multiple Gly residues, Ser residues, or both. Optionally, the amino acid having a side chain containing a primary or secondary amine and / or at least one amino acid in the spacer is a D-amino acid. Any of a variety of polymer backbones or scaffolds could be used. For example, the polymer backbone or scaffold may be a polyamide, polysaccharide, polyanhydride, polyacrylamide, polymethacrylate, polypeptide, polyethylene oxide, or dendrimer. Suitable methods and polymer backbones are described, for example, in WO98 / 46270 (PCT / US98 / 07171) or WO98 / 47002 (PCT / US98 / 06963). In one embodiment, the polymer backbone or scaffold contains multiple reactive functional groups, such as carboxylic acid, anhydride, or succinimide groups. The polymer backbone or scaffold is reacted with a compstatin analog. In one embodiment, the compstatin analog contains any of several different reactive functional groups, such as carboxylic acid, anhydride, or succinimide groups, that react with appropriate groups on the polymer backbone. Alternatively, monomer units that can be linked together to form a polymer backbone or scaffold are first reacted with a compstatin analog, and the resulting monomers are polymerized. In another embodiment, short chains are prepolymerized and functionalized, and then a mixture of short chains of different compositions is assembled into a longer polymer.

[0231] In some embodiments, a compstatin analog moiety is attached to each end of a linear PEG. Bifunctional PEGs with reactive functional groups at each end of the chain can be used, for example, as described herein. In some embodiments, the reactive functional groups are the same, while in some embodiments, different reactive functional groups are present at each end.

[0232] Generally, and for the compounds depicted herein, the polyethylene glycol moieties are depicted with the oxygen atom on the right side of the repeat unit or the left side of the repeat unit. Where only one orientation is depicted, the present disclosure contemplates both orientations of the polyethylene glycol moiety of a given compound or genus (i.e., (CH2CHO) n and (OCH2CH2) n Alternatively, if a compound or genus contains multiple polyethylene glycol moieties, all combinations of orientations are encompassed by the disclosure.

[0233] In some embodiments, a bifunctional linear PEG comprises a moiety containing a reactive functional group at each of its termini. The reactive functional groups can be the same (homobifunctional) or different (heterobifunctional). In some embodiments, the structure of the bifunctional PEG can be symmetrical, with the same moiety bearing the reactive functional group as -(CH2CH2O). n It is used to connect to the oxygen atoms at each end of the chain. In some embodiments, different moieties are used to connect the two reactive functional groups to the PEG portion of the molecule. An exemplary bifunctional PEG structure is shown below. For illustration purposes, a formula is shown in which the reactive functional group includes an NHS ester, although other reactive functional groups can be used.

[0234] In some embodiments, the bifunctional linear PEG is of formula A: [ka] wherein each T and “reactive functionality” is independently defined below and described in classes and subclasses herein, and n is defined above and described in classes and subclasses herein. Each T is independently a covalent bond or C 1-12 A linear or branched hydrocarbon chain, wherein one or more carbon units of T are optionally independently selected from -O-, -S-, -N(R x )-, -C(O)-, -C(O)O-, -OC(O)-, -N(R x )C(O)-, -C(O)N(R x )-, -S(O)-, -S(O)2-, -N(R x )SO2-, or -SO2N(R x )-replaced by; Each R x are independently hydrogen or C 1-6 It is aliphatic. The reactive functional group has the structure -COO-NHS.

[0235] Exemplary bifunctional PEGs of Formula A include the following: [ka]

[0236] In some embodiments, a functional group on a compstatin analog (e.g., an amine, hydroxyl, or thiol group) can be reacted with a PEG-containing compound having a "reactive functional group" as described herein to produce such a conjugate. As an example, Formula I can form a compstatin analog conjugate having the following structure: [ka] During the ceremony, [ka] represents the point of attachment of an amine group on a compstatin analog. In certain embodiments, the amine group is a lysine side chain group.

[0237] In certain embodiments, the PEG component of such conjugates has an average molecular weight of about 5 kD, about 10 kD, about 15 kD, about 20 kD, about 30 kD, or about 40 kD. In certain embodiments, the PEG component of such conjugates has an average molecular weight of about 40 kD.

[0238] The term "bifunctional" or "bifunctionalized" may be used herein to refer to a compound that includes two compstatin analog moieties linked to PEG. Such compounds may be designated with the letters "BF". In some embodiments, the bifunctionalized compound is symmetrical. In some embodiments, the bonds between the PEG and each compstatin analog moiety of the bifunctionalized compound are the same. In some embodiments, each bond between the PEG and the compstatin analog of the bifunctionalized compound includes a carbamate. In some embodiments, each bond between the PEG and the compstatin analog of the bifunctionalized compound includes a carbamate and does not include an ester. In some embodiments, each compstatin analog of the bifunctionalized compound is directly linked to the PEG via a carbamate. In some embodiments, each compstatin analog of the bifunctionalized compound is directly linked to the PEG via a carbamate, and the bifunctionalized compound has the following structure: [ka] .

[0239] In some embodiments of the formulas and embodiments described herein, [ka] represents the point of attachment of the lysine side chain group in a compstatin analog having the following structure: [ka] In the formula, the symbol [ka] represents the point of attachment of the chemical moiety to the remainder of the molecule or chemical formula.

[0240] PEG containing one or more reactive functional groups, in some embodiments, can be obtained from, for example, NOF America Corp. White Plains, NY or BOC Sciences 45-16 Ramsey Road Shirley, NY 11967, USA, among others, or can be prepared using methods known in the art.

[0241] In some embodiments, a linker is used to connect the compstatin analog described herein to the PEG described herein. Suitable linkers for connecting the compstatin analog to the PEG are broadly described above and in the classes and subclasses herein. In some embodiments, the linker has multiple functional groups, one functional group connected to the compstatin analog and another functional group connected to the PEG moiety. In some embodiments, the linker is a bifunctional compound. In some embodiments, the linker has the structure NH2(CH2CHO)nCH2C(=O)OH, where n is 1 to 1000. In some embodiments, the linker is 8-amino-3,6-dioxaoctanoic acid (AEEAc). In some embodiments, the linker is activated for conjugation to a polymer moiety or functional group of the compstatin analog. For example, in some embodiments, the carboxyl group of AEEAc is activated prior to conjugation with the amine group of the side chain of the lysine group.

[0242] In some embodiments, a suitable functional group on the compstatin analog (e.g., an amine, hydroxyl, thiol, or carboxylic acid group) is used for conjugation to the PEG moiety, either directly or via a linker. In some embodiments, the compstatin analog is conjugated to the PEG moiety via a linker through an amine group. In some embodiments, the amine group is an α-amino group of an amino acid residue. In some embodiments, the amine group is an amine group of a lysine side chain. In some embodiments, the compstatin analog is conjugated to the PEG moiety through an amino group of a lysine side chain (ε-amino group) via a linker having the structure NH2(CH2CHO)nCH2C(=O)OH, where n is 1-1000. In some embodiments, the compstatin analog is conjugated to the PEG moiety through an amino group of a lysine side chain via an AEEAc linker. In some embodiments, the NH2(CH2CH2O)nCH2C(=O)OH linker introduces a -NH(CH2CH2O)nCH2C(=O)- moiety onto the lysine side chain of compstatin after conjugation. In some embodiments, the AEEAc linker introduces a -NH(CH2CH2O)2CH2C(=O)- moiety onto the lysine side chain of compstatin after conjugation.

[0243] In some embodiments, the compstatin analog is conjugated to the PEG moiety via a linker, the linker comprising an AEEAc moiety and an amino acid residue. In some embodiments, the compstatin analog is conjugated to the PEG moiety via a linker, the linker comprising an AEEAc moiety and a lysine residue. In some embodiments, the C-terminus of the compstatin analog is connected to the amino group of AEEAc, and the C-terminus of AEEAc is connected to a lysine residue. In some embodiments, the C-terminus of the compstatin analog is connected to the amino group of AEEAc, and the C-terminus of AEEAc is connected to the α-amino acid of a lysine residue. In some embodiments, the C-terminus of the compstatin analog is connected to the amino group of AEEAc, and the C-terminus of AEEAc is connected to the α-amino group of a lysine residue, and the PEG moiety is conjugated via the ε-amino group of the lysine residue. In some embodiments, the C-terminus of the lysine residue is modified. In some embodiments, the C-terminus of the lysine residue is modified by amidation. In some embodiments, the N-terminus of the compstatin analog is modified. In some embodiments, the N-terminus of a compstatin analog is acetylated.

[0244] In certain embodiments, a compstatin analog may be represented as M-AEEAc-Lys-B2, where B2 is a blocking moiety, e.g., NH2, and M represents any of SEQ ID NOs: 9-36, with the proviso that the C-terminal amino acid of any of SEQ ID NOs: 9-36 is linked to AEEAc-Lys-B2 via a peptide bond. The NHS moiety of a mono- or multi-functional (e.g., bi-functional) PEG reacts with the free amine of a lysine side chain to produce a mono-functionalized (one compstatin analog moiety) or multi-functionalized (multiple compstatin analog moieties) PEGylated compstatin analog. In various embodiments, any amino acid containing a side chain containing a reactive functional group may be used in place of (or in addition to) Lys. Mono- or multi-functional PEGs containing suitable reactive functional groups may be reacted with such side chains in a manner similar to the reaction of NHS-ester activated PEG with Lys.

[0245] With respect to any of the above formulas and structures, it should be understood that embodiments are expressly disclosed in which the compstatin analog moiety comprises any of the compstatin analogs described herein, e.g., any of SEQ ID NOS: 9-36. For example, and without limitation, the compstatin analog may comprise the amino acid sequence of SEQ ID NO: 28. An exemplary PEGylated compstatin analog in which the compstatin analog moiety comprises the amino acid sequence of SEQ ID NO: 28 is shown in FIG. 2. It will be understood that the PEG moiety may have a variety of different molecular weights or average molecular weights in various embodiments, as described herein. In certain embodiments, the compstatin analog is pegcetacoplan ("APL-2"), having the structure of the compound of FIG. 2 with an n of about 800 to about 1100 and a PEG with an average molecular weight of about 40 kD. Pegcetacoplan is a compound consisting of poly(oxy-1,2-ethanediyl), α-hydro-ω-hydroxy-, N-acetyl-L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophyl-L-glutaminyl-L-α-aspartyl-L-tryptophyl-glycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-L-threonyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-N 6 -Carboxy-L-lysine amide cyclic (2-->12)-(disulfide) containing 15,15'-diester; or O,O'-bis[(S 2 ,S 12 -Cyclo{N-acetyl-L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophyl-L-glutaminyl-L-α-aspartyl-L-tryptophyl-glycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteine-L-threonyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-L-lysineamide})-N 6.15 -carbonyl]polyethylene glycol (n=800-1100). Additional compstatin analogs are described, for example, in WO2012 / 155107 and WO2014 / 078731.

[0246] In some embodiments, a compstatin analog described herein is administered to a subject at a dosage of about 800 mg to about 1200 mg, e.g., about 1060 mg to about 1100 mg, e.g., about 1070 mg to about 1090 mg, e.g., about 1075 mg to about 1085 mg, e.g., about 1080 mg, twice weekly or every three days for about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52 weeks, about 1.2 years, 1.4 years, 1.6 years, 1.8 years, 2 years, 3 years, 4 years, 5 years, or more.

[0247] In some embodiments, a composition comprising one or more inhibitory RNAs (e.g., siRNAs or miRNAs described herein) or a vector comprising a nucleotide sequence encoding an siRNA or miRNA described herein is administered to a subject in combination with a compstatin analog, whereby the compstatin analog and / or inhibitory RNA composition is administered less frequently and / or at a lower dosage. In some embodiments, a composition comprising one or more inhibitory RNAs (e.g., siRNAs or miRNAs described herein) or a vector comprising a nucleotide sequence encoding an siRNA or miRNA described herein is administered to a subject in combination with a compstatin analog, whereby the compstatin analog is administered once a week, once every two weeks, once a month, once every two months, three months, four months, five months, or longer at a dosage of about 800 mg to about 1200 mg, e.g., about 1060 mg to about 1100 mg, e.g., about 1070 mg to about 1090 mg, e.g., about 1075 mg to about 1085 mg, e.g., about 1080 mg.

[0248] In some embodiments, the complement inhibitor is an antibody, e.g., an anti-C3 antibody and / or an anti-C5 antibody, or a fragment thereof. In some embodiments, an antibody fragment can be used to inhibit activation of C3 or C5. The fragmented anti-C3 or anti-C5 antibody can be a Fab', Fab'(2), Fv, or single chain Fv. In some embodiments, the anti-C3 or anti-C5 antibody is monoclonal. In some embodiments, the anti-C3 or anti-C5 antibody is polyclonal. In some embodiments, the anti-C3 or anti-C5 antibody is deimmunized. In one embodiment, the anti-C3 or anti-C5 antibody is a fully human monoclonal antibody. In some embodiments, the anti-C5 antibody is eculizumab. In some embodiments, the complement inhibitor is an antibody, e.g., an anti-C3 antibody and / or an anti-C5 antibody, or a fragment thereof.

[0249] In some embodiments, the complement inhibitor is a polypeptide inhibitor and / or a nucleic acid aptamer (see, e.g., US Patent Publication No. 20030191084). Exemplary polypeptide inhibitors include enzymes that degrade C3 or C3b (see, e.g., US Patent Publication No. 6,676,943). Additional polypeptide inhibitors include mini-factor H (see, e.g., US Patent Publication No. 20150110766), Efb protein from Staphylococcus aureus or complement inhibitor (SCIN) protein, or variants or derivatives or mimetics thereof (see, e.g., US Patent Publication No. 20140371133).

[0250] Various other complement inhibitors can also be used in various embodiments of the present disclosure. In some embodiments, the complement inhibitor is a naturally occurring mammalian complement regulatory protein, or a fragment or derivative thereof. For example, the complement regulatory protein can be CR1, DAF, MCP, CFH, or CFI. In some embodiments, the complement regulatory polypeptide is one that is normally membrane-bound in its naturally occurring state. In some embodiments, a fragment of such a polypeptide is used that lacks part or all of the transmembrane domain and / or the intracellular domain. For example, a soluble form of complement receptor 1 (sCR1) can also be used. For example, compounds known as TP10 or TP20 (Avant Therapeutics) can be used. C1 inhibitor (C1-INH) can also be used. In some embodiments, a soluble complement regulatory protein is used, such as CFH.

[0251] Inhibitors of C1 can also be used. For example, U.S. Patent No. 6,515,002 describes compounds that inhibit C1 (furanyl and thienyl amidines, heterocyclic amidines, and guanidines). U.S. Patent Nos. 6,515,002 and 7,138,530 describe heterocyclic amidines that inhibit C1. U.S. Patent No. 7,049,282 describes peptides that inhibit classical pathway activation. Some of the peptides include or consist of WESNGQPENN (SEQ ID NO: 73) or KTISKAKGQPREPQVYT (SEQ ID NO: 74), or peptides with significant sequence identity and / or steric structural similarity thereto. In some embodiments, these peptides are identical or substantially identical to portions of IgG or IgM molecules. U.S. Patent No. 7,041,796 discloses C3b / C4b complement receptor-like molecules and their use to inhibit complement activation. U.S. Patent No. 6,998,468 discloses anti-C2 / C2a inhibitors of complement activation.U.S. Patent No. 6,676,943 discloses human complement C3 cleavage protein from Streptococcus pneumoniae.

[0252] All publications, patent applications, patents, and other references mentioned herein, including GenBank accession numbers, are incorporated by reference in their entirety. Additionally, the materials, methods, and examples are for illustrative purposes only and not for limiting purposes. Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0253] The present disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They should not be construed as limiting the scope or content of the present disclosure in any way.

[0254] VIII. Examples Example 1: Knockdown of C3 expression in HeLa cells using siRNA cell culture HeLa cells were obtained from ATCC (ATCC in collaboration with LGC Standards, Wesel, Germany, Cat. No. ATCC-CRM-CCL-2) and cultured in HAM's F12 (#FG0815, Biochrom, Berlin, Germany) supplemented with 10% fetal bovine serum (#1248D, Biochrom GmbH, Berlin, Germany) and 100 U / ml penicillin / 100 μg / ml streptomycin (#A2213, Biochrom GmbH, Berlin, Germany) at 37°C in a humidified incubator in an atmosphere containing 5% CO2. To transfect HeLa cells with siRNA, cells were seeded in 96-well tissue culture plates (#655180, GBO, Germany) at a density of 15,000 cells / well.

[0255] siRNA 177 siRNAs were designed and synthesized to target different regions of mRNA transcript.In this experiment, the sense strand of each siRNA contained 18 nucleotides identical to the target region sequence (SEQ ID NO: 75) on C3 transcript and one additional adenine nucleotide at 3' end.Furthermore, in this experiment, the antisense strand contained 18 nucleotides identical to the target region sequence (SEQ ID NO: 75) on C3 transcript, one additional uracil nucleotide at 5' end and two additional uracil nucleotides at 3' end.

[0256] In this experiment, the siRNA contained modifications to the sense strand including the following modification patterns: xsxsXfxxxXfXfXfxxXfxxxxXfxa

[0257] The antisense strand contained the following modification pattern: usXfsxxxxxxxxxxxXfxxxxxsusu

[0258] Here, "x" represents any nucleotide, lower case letters represent nucleotides modified with a 2'-O-methyl group, and "Xf" represents a nucleotide modified with a 2'-fluoro group (where "X" can be any nucleotide). For example, "Af" represents an adenine nucleotide modified with a 2'-fluoro group. "s" represents a phosphorothioate bond.

[0259] Transfection of siRNA and C3 activity assay-Double dose experiment Transfection of siRNA was performed by reverse transfection using Lipofectamine RNAiMax (Invitrogen / Life Technologies, Karlsruhe, Germany) according to the manufacturer's instructions. In this experiment, a double dose screen was performed using quadruple siRNAs at 10 nM and 0.5 nM, respectively. siRNA targeting Aha1 was used as a nonspecific control for simultaneous C3 target mRNA expression and as a positive control to analyze transfection efficiency with respect to Aha1 mRNA levels. Firefly luciferase and Renilla luciferase were used as mock transfections.

[0260] After 24 hours of incubation with siRNA, the medium was removed and the cells were lysed in 150 μl of Medium-Lysis Mixture (1 volume of lysis mixture, 2 volumes of cell culture medium) and then incubated at 53° C. for 30 minutes. The bDNA assay (ThermoFisher QuantiGene RNA assay) was performed according to the manufacturer's instructions using a probe set for human C3 (accession number NM_000064 between bases 106 and 907 of the sequence), designed by Thermo Fisher Scientific and synthesized by Metabion International AG (Planegg, Germany). After 30 minutes of incubation at room temperature in the dark, luminescence was read using a 1420 Luminescence Counter (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Juegesheim, Germany).

[0261] Two other target-nonspecific controls (for firefly luciferase and Renilla luciferase) were used to control for Aha1 mRNA levels by hybridization with the Aha1 probe set. Transfection efficiency in each 96-well plate and at both doses in the dual dose screen was assessed by comparing Aha1 levels in each well.

[0262] The transfection efficiency with siAha1 at a dose of 10 nM was approximately 90%, and with a dose of 0.5 nM was approximately 85%, as calculated by relating Aha1-siRNA (normalized to GapDH) to the Aha1 levels obtained with the controls.

[0263] The activity of siRNA was measured by the minimum fluorescence or minimum mRNA concentration (%) of each target. For each well, the target mRNA level was normalized to the corresponding GAPDH mRNA level. The activity of a particular siRNA was expressed as the mRNA concentration (%) of each target in treated cells (normalized to GAPDH mRNA) relative to the target mRNA concentration (normalized to GAPDH mRNA) averaged across each control well.

[0264] The results of a dual dose screen of the top 24 siRNAs based on activity are shown below in Table 9. The sequences of these siRNAs are shown below in Table 10. [Table 10] [Table 11-1] [Table 11-2]

[0265] Dose-response experiments The top 12 siRNAs showing the best activity at both doses were selected to be tested in dose-response experiments (DRC). Dose-response experiments were performed with 10 concentrations of siRNAs transfected in quadruplicate, starting at 100 nM in 6-fold dilution steps down to approximately 10 fM. Mock-transfected cells were used as a control for the DRC experiments.

[0266] For each well, target mRNA levels were normalized to the corresponding GAPDH mRNA levels. The activity of a particular siRNA was expressed as the percentage of mRNA concentration of each target in treated cells (normalized to GAPDH mRNA) relative to the target mRNA concentration (normalized to GAPDH mRNA) averaged across mock-transfected wells (DRC).

[0267] The IC50 and IC80 values ​​from the DRC experiments and the maximum KD results (10 nm dose) from the dual dose experiments are shown in Table 11 below. [Table 12]

[0268] Example 2: Knockdown of C3 expression in HepG2 cells using siRNA Based on the demonstrated activity in HepG2 cells, the dual dosing experiment of Example 1 was repeated for the top 50 siRNAs (Example 1).

[0269] HePG2 cells were obtained from ATCC (a collaboration between ATCC and LGC Standards, Wesel, Germany, catalogue no. ATCC-HB-8065) ​​and cultured in MEM Eagle (#M2279, Sigma-Aldrich, Germany) supplemented with 10% fetal bovine serum (#1248D, Biochrom GmbH, Berlin, Germany), 1× non-essential amino acids (#K0293; Biochrom, Berlin, Germany), 4 mM L-glutamine (#K0283, Biochrom, Berlin, Germany), and 100 U / ml penicillin / 100 μg / ml streptomycin (#A2213, Biochrom GmbH, Berlin, Germany) at 37°C in a humidified incubator in an atmosphere containing 5% CO2.

[0270] Transfection of siRNA and C3 activity assay-Double dose experiment To transfect HePG2 cells with siRNA, cells were seeded at a density of 15,000 cells / well in collagen-coated 96-well tissue culture plates (#655150, GBO, Germany). Transfection of siRNA was performed immediately after seeding with Lipofectamine RNAiMax (Invitrogen / Life Technologies, Karlsruhe, Germany) according to the manufacturer's instructions by reverse transfection. In this experiment, a double dose screen was performed with quadruple siRNAs at 10 nM and 0.1 nM, respectively. siRNA targeting Aha1 was used as a nonspecific control for C3 target mRNA expression and as a positive control to analyze transfection efficiency with respect to Aha1 mRNA levels in parallel. Firefly luciferase and Renilla luciferase were used as mock transfections.

[0271] After 24 hours of incubation with siRNA, the medium was removed and the cells were lysed in 150 μl of Medium-Lysis Mixture (1 volume of lysis mixture, 2 volumes of cell culture medium) and then incubated at 53° C. for 30 minutes. The bDNA assay (ThermoFisher QuantiGene RNA assay) was performed according to the manufacturer's instructions using a probe set for human C3 (accession number NM_000064 between bases 106 and 907 of the sequence), designed by Thermo Fisher Scientific and synthesized by Metabion International AG (Planegg, Germany). After 30 minutes of incubation at room temperature in the dark, luminescence was read using a 1420 Luminescence Counter (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Juegesheim, Germany).

[0272] Aha1-siRNA was used as a non-specific control for C3 mRNA expression and as a positive control for analyzing transfection efficiency by measuring Aha1 mRNA levels by hybridization with the Aha1 probe set. The Aha-1 siRNA used was previously selected from a large group of candidate siRNAs and is known to have very high activity in vitro and in vivo. The transfection efficiency of each 96-well plate was calculated by analyzing Aha1 knockdown by Aha1-siRNA (normalized to GapDH) compared to the non-specific control. Aha1-siRNA (normalized to GapDH) relative to Aha1 levels obtained with the control. The transfection efficiency with siAha1 at a dose of 10 nM was approximately 90%, and at a dose of 0.5 nM, the transfection efficiency was approximately 85%.

[0273] The activity of siRNA was measured by fluorescence or mRNA concentration (%) of each target. For each well, the target mRNA level was normalized to the corresponding GAPDH mRNA level. The activity of a particular siRNA was expressed as the mRNA concentration (%) of each target in treated cells (normalized to GAPDH mRNA) relative to the target mRNA concentration (normalized to GAPDH mRNA) averaged across each control well.

[0274] The results of the dual dose screen of the top 12 siRNAs based on activity are shown below in Tables 12 and 13 for doses of 10 nm and 0.1 nm, respectively. [Table 13] [Table 14]

[0275] Example 3: C3 knockdown expression in HepG2 cells using siRNA with various modification patterns Modifications of the top six siRNAs from Examples 1 and 2 were tested.

[0276] cell culture HePG2 cells were obtained from ATCC (a collaboration between ATCC and LGC Standards, Wesel, Germany, catalogue no. ATCC-HB-8065) ​​and cultured in MEM Eagle (#M2279, Sigma-Aldrich, Germany) supplemented with 10% fetal bovine serum (#1248D, Biochrom GmbH, Berlin, Germany), 1× non-essential amino acids (#K0293; Biochrom, Berlin, Germany), 4 mM L-glutamine (#K0283, Biochrom, Berlin, Germany), and 100 U / ml penicillin / 100 μg / ml streptomycin (#A2213, Biochrom GmbH, Berlin, Germany) at 37°C in a humidified incubator in an atmosphere containing 5% CO2.

[0277] siRNA Based on the nucleotide sequences of the top siRNAs in terms of activity from Examples 1 and 2 (siRNA numbers: 1, 4, 9, 10, 16 and 22), siRNAs with different modification patterns were designed and synthesized. Five "variants" were designed and synthesized for each of the top siRNA nucleotide sequences, and each duplex is designated as "Variant 1", "Variant 2", "Variant 3", "Variant 4", "Variant 5" according to the modifications made. The siRNAs from Examples 1 and 2 (siRNA numbers: 1, 4, 9, 10, 16 and 22) are designated as "Variant 0".

[0278] The following modification patterns (5' to 3') of the sense strand of each of siRNA numbers 1, 4, 9, 10, 16, and 22 were used: xsxsXfxxxXfXfXfxxXfxxxxXfxa (pattern also used in "Variant 0" and "Variant 1" and "Variant 5") XfsxsXfxXfxXfxXfxXfxXfxXfxXfxAf (pattern used in "Variant 2", "Variant 3" and "Variant 4")

[0279] The following modification pattern (5' to 3') of the antisense strand was used: usXfsxxxxxxxxxxxXfxxxxxsusu (the pattern used in "variant 0") usXfsxxxxxxxxxxxXfxxxxxsxsx (pattern used in "Variant 1" and "Variant 3"; same pattern used in "Variant 0" except that the last two nucleotides are complementary to the C3 mRNA transcript, i.e., SEQ ID NO: 75) usXfsxXfxXfxXfxXfxXfxXfxXfxXfxXfxsxsx (the pattern used in "Variant 2") usXfsxxxXfxxxxxxxXfxXfxxxsxsx (Pattern used in "Variant 4" and "Variant 5")

[0280] Here, "x" represents any nucleotide, lower case letters represent nucleotides modified with a 2'-O-methyl group, and "Xf" represents a nucleotide modified with a 2'-fluoro group (where "X" can be any nucleotide). For example, "Af" represents an adenine nucleotide modified with a 2'-fluoro group. "s" represents a phosphorothioate bond.

[0281] Dose-response experiments Transfection of siRNA was performed by reverse transfection using Lipofectamine RNAiMax (Invitrogen / Life Technologies, Karlsruhe, Germany) according to the manufacturer's instructions.

[0282] Each siRNA was tested using a dose-response experiment (DRC) in HepG2 cells. Two additional siRNAs (siRNA numbers 26 and 27) known to knock down C3 expression were used as positive controls.

[0283] Dose-response experiments were performed using 10 concentrations of siRNA transfected in quadruplicate starting at 100 nM in 6-fold dilution steps down to approximately 10 fM. Mock-transfected cells were used as negative controls.

[0284] For each well, target mRNA levels were normalized to the corresponding GAPDH mRNA levels. The activity of a particular siRNA was expressed as the percentage of mRNA concentration of each target in treated cells (normalized to GAPDH mRNA) relative to the target mRNA concentration (normalized to GAPDH mRNA) averaged across mock-transfected wells (DRC).

[0285] The IC50, IC80 values ​​and maximum KD from the DRC experiments are shown below in Table 14. The sequences of these siRNAs are shown below in Table 15. [Table 15-1] [Table 15-2] [Table 15-3]

[0286] [Table 16-1] [Table 16-2] [Table 16-3]

[0287] The IC50 and IC80 values ​​in Table 14 show that the performance of the modification patterns differed. For example, siRNA No. 32 (using the modification pattern designated "Variant 5") had better activity (IC50 and IC80 values ​​of 0.018 nM and 0.108 nM, respectively) compared to siRNA Nos. 1 ("Variant 0"), 28 ("Variant 1"), 29 ("Variant 2"), 30 ("Variant 3"), and 31 ("Variant 4"), even though each of these siRNAs was based on the same nucleotide sequence and targeted the same region of the C3 transcript.

[0288] Furthermore, the performance of each modification pattern (i.e., different variants) appeared to vary for each siRNA nucleotide sequence (i.e., target region of the C3 transcript). For example, "variant 5" was shown to be the most effective for C3 knockdown among siRNAs based on the nucleotide sequence of siRNA1 (e.g., siRNA number 32 (variant 5) compared to siRNA number 1 (variant 0)), whereas "variant 0" was shown to be the most effective for C3 knockdown among siRNAs based on the nucleotide sequence of siRNA22 (e.g., siRNA22 (variant 0) compared to siRNA55 (variant 3)).

[0289] Example 4: In vivo evaluation of siRNA58 in non-human primates siRNA constructs siRNA53 from Example 3 was selected and further modified as described below to generate siRNA58. [Table 17]

[0290] In addition, the siRNA was linked to the GalNAc structure shown below at the 5' end of the sense strand of siRNA58 via an NHC6 linker. [ka]

[0291] The modified siRNA (hereafter referred to as "siRNA58") was then evaluated in non-human primates.

[0292] Study design Naive male cynomolgus monkeys (n=3 per group) were administered a single dose of 3 mg / kg, 10 mg / kg, or 30 mg / kg siRNA58 or vehicle (phosphate buffered saline) subcutaneously (SC) on day 1.

[0293] Serum samples were scheduled to be collected on days -5, -1, 3, 8, 15, 22, 29, 40, 57, 67, 82, 97, 112, 127, 142, 157, 172, and 184 (negative values ​​correspond to days before injection of siRNA58 or vehicle). Levels of C3 protein in serum were measured using an ELISA assay. In addition, serum samples were also analyzed for alternative complement pathway activity (AH50). Values ​​on day -1 were used as baseline.

[0294] Liver needle biopsies were performed on days 15, 46, and 79. The levels of C3 mRNA in the samples were measured using a quantitative PCR assay. In these experiments, C3 mRNA levels were normalized to the levels of ActB mRNA.

[0295] result Figure 3 depicts the time course of serum C3 protein levels after dosing for each group up to 67 days after dosing. These results show that a single subcutaneous dose of siRNA58 reduced serum C3 protein levels by 77% at the 3 mg / kg dose, 85% at the 10 mg / kg dose, and 90% at the 30 mg / kg dose compared to baseline values ​​by day 29, with reductions approaching these levels evident by day 15. Furthermore, the data in Figure 3 show that the reductions were sustained through day 67.

[0296] Figure 4 shows that a single dose of siRNA58 reduced hepatic C3 mRNA by 89% at the 3 mg / kg dose, 97% at the 10 mg / kg dose, and 99% at the 30 mg / kg dose compared to vehicle controls by day 15. The reduction continued through day 46 (Figure 5).

[0297] Figure 6 shows the time course of levels of alternative complement pathway (AH50) activity in serum collected through day 67. These results show that a single subcutaneous administration of siRNA58 reduced the levels of alternative complement pathway activity by 65% ​​at the 3 mg / kg dose, 82% at the 10 mg / kg dose, and 92% at the 30 mg / kg dose by day 29 compared to baseline values, with reductions reaching these levels by day 15. Furthermore, the reduction in activity persisted through day 67, with activity reduced by 68% at the 3 mg / kg dose, 91% at the 10 mg / kg dose, and 98% at the 30 mg / kg dose compared to baseline values.

[0298] Example 5: In vivo evaluation of siRNA60 in non-human primates siRNA constructs siRNA32 from Example 3 is selected and further modified as described below to generate siRNA58. [Table 18]

[0299] In addition, the siRNA is linked at the 5' end of the sense strand of siRNA60 via an NHC6 linker to the GalNAc structure shown below. [ka]

[0300] The modified siRNA (hereinafter referred to as "siRNA60") is then evaluated in non-human primates.

[0301] Study design Naive male cynomolgus monkeys (n=3 per group) are administered a single dose of 3 mg / kg, 10 mg / kg, or 30 mg / kg siRNA60, or vehicle (phosphate buffered saline) subcutaneously (SC) on day 1.

[0302] Serum samples are collected on days -5, -1, 3, 8, 15, 22, 29, 40, 57, 67, 82, 97, 112, 127, 142, 157, 172 and 184 (negative values ​​correspond to days before injection of siRNA60 or vehicle). Levels of C3 protein in serum are measured using an ELISA assay. In addition, serum samples are also analyzed for alternative complement pathway activity (AH50). Values ​​on day -1 are used as baseline.

[0303] Liver needle biopsies are performed on days 15, 46, and 79. The levels of C3 mRNA in the samples are measured using a quantitative PCR assay. In these experiments, C3 mRNA levels are normalized to the levels of ActB mRNA.

[0304] Example 6: Off-target analysis and safety of siRNA58 The nucleotide sequences of the sense and antisense strands of siRNA58 were analyzed for potential off-target activity (Lindow et al. 2012). The potential off-target activity of the sense and antisense strands was analyzed with mature and primary human RNA.

[0305] method The sequences analyzed are as follows: Antisense (guide) strand: 5'-uguagguauguaguggcu-3' (SEQ ID NO: 321) Sense (passenger) strand: 5'-ccaacuacaugaaccuaca-3' (SEQ ID NO: 147)

[0306] Antisense strand (mature human RNA): To identify potential off-target genes, a similarity search was performed using Smith-WaterMan gapped local alignment (sSearch) in the FASTA package (v36; Pearson 2000) with the following parameters: An E value E of less than −E 5000 corresponds to the number of search hits one can expect to find by chance when searching a database of this size, and a relatively large number ensures that all off-target sequences for hybridization are detected. -W (number of flanking positions in alignment) is set to 5 -n Nucleic acid search -f and -g Set gap creation and extension penalties to 1000 to avoid gapped alignments. ssearch36 -n -W 5 -E 5000 -f 1000 -g 1000 query refMrna.fa

[0307] Antisense strand (primary human RNA): To identify genes with potential off-target effects in the nucleus, we searched for matches between the oligonucleotide sequence and primary RNA (unspliced ​​and containing introns). Specifically, these similarity searches were performed against the human genome (version hg38) using Smith-Waterman gapped local alignments (sSearch) in the FASTA package (v36; Pearson 2000) with the following parameters: An E value E of less than −E 5000 corresponds to the number of search hits one can expect to find by chance when searching a database of this size, and a relatively large number ensures that all off-target sequences for hybridization are detected. -W (number of flanking positions in alignment) is set to 5 -n Nucleic acid search -f and -g Set gap creation and extension penalties to 1000 to avoid gapped alignments. ssearch36 -n -W 5 -E 5000 -f 1000 -g 1000 query refGene.fa

[0308] While the genome alignment allows identifying the location in the reference genome where each sequence was found, the reference genome itself does not contain the location of genes and gene bodies. Therefore, to obtain information about which genes have hybridization potential, following the alignment, the sResearch genome coordinates of each hit were converted to bed file format and annotated to intragenic hits for those genes using intersect in bedtools (v2.28.0). Gene locations were obtained from the UCSC genome browser, Table Viewer for Gencodev32 and hg38, from the "Gene and gene predictions table". For annotation using bedtools, the following command was used: bedtools cross with -a hitBedFile.bed -b geneBedFile.bed -wo -a and -b indicate the input. -wo writes out the original position of both the hit and the full annotation needed to obtain the gene name.

[0309] Sense strand (mature human RNA): To identify potential off-target genes, a similarity search was performed using Smith-WaterMan gapped local alignment (sSearch) in the FASTA package (v36; Pearson 2000) with the following parameters: An E value E of less than −E 5000 corresponds to the number of search hits one can expect to find by chance when searching a database of this size, and a relatively large number ensures that all off-target sequences for hybridization are detected. -W (number of flanking positions in alignment) is set to 5 -n Nucleic acid search -f and -g Set gap creation and extension penalties to 1000 to avoid gapped alignments.

[0310] Searches were performed to search for mature RNA sequences with (1) the longest uninterrupted complementarity greater than 11 (reduced from 13 due to the shorter sense sequence) and a number of matches (2 or 1 mismatch) greater than 16, and / or (2) 14 or more base pairs of uninterrupted complementarity with 16 matches (3 mismatches).

[0311] Sense strand (primary human RNA): To identify genes with potential off-target effects in the nucleus, we searched for matches between the oligonucleotide sequences and primary RNA (unspliced ​​and containing introns). Specifically, these similarity searches were performed against the human genome (version hg38) using Smith-Waterman gapped local alignments (sSearch) in the FASTA package (v36; Pearson 2000) with the following parameters: An E value E of less than −E 5000 corresponds to the number of search hits one can expect to find by chance when searching a database of this size, and a relatively large number ensures that all off-target sequences for hybridization are detected. -W (number of flanking positions in alignment) is set to 5 -n Nucleic acid search -f and -g Set gap creation and extension penalties to 1000 to avoid gapped alignments. ssearch36 -n -W 5 -E 5000 -f 1000 -g 1000 query refGene.fa

[0312] To obtain information about which genes have potential for off-target cleavage of their primary RNA by Argonaute, following alignment, the sResearch genomic coordinates of each hit were converted to bed file format and annotated to intragenic hits for those genes using bedtools (v2.28.0) intersect. Gene positions were obtained from the UCSC genome browser, Table Viewer for Gencodev32 and hg38, from the "Gene and gene predictions table". For annotation using bedtools, the following command was used: bedtools cross with -a hitBedFile.bed -b geneBedFile.bed -wo -a and -b indicate the input. -wo writes out the original position of both the hit and the full annotation needed to obtain the gene name.

[0313] Genetic Analysis: Genes identified based on the above search were further analyzed to understand potential safety risks. Gene expression in the major organs of oligonucleotide drug accumulation (liver and kidney) was evaluated. Using the GTEx human tissue expression atlas (GTEx Consortium 2013), log2-transformed TPM (transcript per million) expression values ​​were calculated for each gene for each subject in GTEx, and median values ​​for each gene were obtained. Values: <3 indicate very low expression, 3-4 indicate low expression, 4-6 indicate intermediate expression, and >6 indicate high expression.

[0314] Next, a search was performed for evidence of disease association with complete or partial knockdown of each target. To examine evidence of disease in the presence of complete knockdown of targets, the Online Mendelian Inheritance of Man database (OMIM; Hamosh et al. 2002) was used to look for association with rare germline mutations and human genetic diseases. It was noted that the majority of the top gene off-target hits contained multiple mismatches that significantly reduced RNAi efficiency. Although most human autosomal genes are not dosage specific (Rice & McLysaght 2017) and inactivation of one allele does not result in a phenotype, a further search for evidence of dosage-specific effects was performed using ClinGen (https: / / search.clinicalgenome.org / ), which collects information on both haploinsufficient and triploid-susceptible dosage-specific genes.

[0315] result: Antisense strand: The only perfect complementary hit to the antisense strand of siRNA58 is the target C3. The next best matches between the primary and mature RNAs all have 3 or more mismatches and are predicted to have significantly lower hybridization-dependent off-target effects. Most of the top off-target candidates showed low expression in liver and kidney and / or no association with human genetic disease. The only exception was RABL3, where a specific gain-of-function truncating mutation has been associated with a hereditary pancreatic cancer syndrome in one family. Potential knockdown of RABL3 by siRNA58 is not predicted to mimic symptoms of this novel gain-of-function mutation.

[0316] Sense strand: No perfect match was found between the primary and mature human RNAs for sequences complementary to the sense strand of siRNA58. Most of the genes with complementarity to the sense strand, including GPR173, which has only one mismatch to the sense oligonucleotide, were not significantly expressed in human liver or kidney, and they were not associated with any known genetic disease.

[0317] Example 7: In vivo evaluation of siRNA59 in rats siRNA constructs siRNA53 from Example 3 was further modified as described below to generate siRNA59. [Table 19]

[0318] In addition, the siRNA was linked to the GalNAc structure shown below at the 5' end of the sense strand of siRNA59 via an NHC6 linker. [ka]

[0319] The modified siRNA (hereafter referred to as "siRNA59") was then evaluated in Sprague-Dawley rats.

[0320] Objectives: The objective of this study was to determine the plasma pharmacokinetics (PK) and regional tissue distribution of siRNA59 at three dose levels when administered as a single subcutaneous injection to Sprague-Dawley rats. A secondary objective of this study was to compare the pharmacodynamic effects of three dose levels of siRNA59 administered as a single subcutaneous injection to equivalent doses administered over a 3-day period (once daily).

[0321] method Study design: [Table 20]

[0322] Administration: Animals in groups 1-3 and 8 received a single subcutaneous injection of PBS vehicle or 3 mg / kg, 10 mg / kg, or 30 mg / kg siRNA59 formulated in PBS at concentrations of 0.6 mg / ml, 2 mg / ml, and 6 mg / ml, respectively, in a dosing volume of 5 ml. Animals in groups 4-7 received 10 mg / ml siRNA(-) control or 1 mg / ml, 3.3 mg / ml, or 10 mg / ml siRNA59 formulated in PBS at concentrations of 0.2 mg / ml, 0.6 mg / ml, and 2 mg / ml, respectively, in a dosing volume of 5 ml, three times daily subcutaneous injections.

[0323] Sample Collection: For PD analyses, plasma and serum samples were collected at baseline and on days 3, 8, 15, 22, and 29 post-dose. Plasma samples were collected at 15 minutes and 1, 4, 8, 24, 48, and 72 hours post-dose. Liver samples were collected at necropsy on days 3 and 30 post-dose.

[0324] Bioanalytical methods: Plasma PD samples were analyzed for C3 protein concentration at Confluence Discovery Technologies (MO, USA) using a double antibody sandwich ELISA kit (Eagle Biosciences; plasma dilution 1:10,000) according to the manufacturer's instructions.

[0325] Serum PD samples were analyzed for alternative pathway (AP) complement activation at Confluence Discovery Technologies (MO, USA) using the AP Wieslab assay (Eagle Biosciences, Cat. No. COMPL AP330).

[0326] PD tissue samples were analyzed for C3 mRNA levels using a semi-validated RT-qPCR method at EpigenDx (MA, USA).

[0327] result Circulating C3: Plasma was collected at five time points to assess changes in C3 concentrations. A clear dose-dependent response to siRNA was observed across dose groups (Figure 7). Maximal levels of C3 protein reduction were achieved on study day 8 for all dose groups. Plasma C3 concentration data were comparable between single dose treatment and equivalent dose groups of QDx3. Full recovery of plasma C3 protein to baseline levels was observed only in the 3 mg / kg dose group. The 10 mg / kg and 30 mg / kg dose groups did not appear to have fully recovered to their respective baseline C3 protein levels by the final blood draw on day 29.

[0328] Alternative pathway complement activity: Serum collected from each animal treated with a single dose of siRNA59 and vehicle, as well as animals treated with multiple doses of siRNA(-) control, was used to assess complement activation using the AP Wieslab assay, an ELISA-based assay that measures the formation of C5b-9 following alternative pathway activation (Figure 8). Figure 8 shows the measurement of alternative pathway activity by ELISA detection and quantification of soluble C5b-9 complexes (optical density readings = OD) in serum from animals treated subcutaneously with vehicle (A), siRNA(-) control (B), 3 mg / kg siRNA59 (C), 10 mg / kg siRNA59 (D), and (E) 30 mg / kg siRNA59. Data represent mean ± SEM (n=3). Serum AP complement activity showed a large variability among the control groups. Serum collected from the high dose siRNA59 group showed an average reduction in alternative pathway activity of approximately 90%, which was sustained from day 8 to day 15 after treatment.

[0329] C3 Transcripts in Liver Tissue: A dose-dependent decrease in C3 mRNA in liver was observed at both endpoints of the study (Figure 9). After 3 days of treatment, liver C3 mRNA levels in single-dose PK animals were significantly reduced compared to C3 expression in vehicle controls. In animals treated with 30 mg / kg siRNA59, mean C3 expression in liver was 3% of gene expression in vehicle treated animals, but increased to 25% by day 30. C3 expression did not fully recover by the end of the study at day 30 in any of the treated animals.

[0330] Discussion: Following subcutaneous administration of siRNA59, a clear dose-dependent decrease in C3 protein in plasma and C3 mRNA in liver tissue was observed. Furthermore, the observed dose response was similar when siRNA59 was administered as a single subcutaneous bolus and as daily injections over 3 days. By days 29 and 30, respectively, C3 plasma protein concentrations and liver mRNA expression were restored only in the low-dose group.

[0331] C3 plasma protein levels and hepatic gene expression were comparable between single and multiple dose groups at all time points. Despite an approximately 90% decrease in C3 plasma protein concentrations after a dose of 10 mg / kg, no changes in AP Wieslab activity were observed in any of the dose groups, except in serum from high-dose animals. No signs of distress or behavioral changes were observed in animals of any treatment group, and no weight loss was observed after TA administration, suggesting that each dose used in this study was well tolerated.

[0332] These results indicate that C3 protein in the systemic circulation can be silenced in a dose-dependent manner following treatment with GalNAc-tagged siRNA targeting C3.

[0333] Example 8: Evaluation of C3 and C3a in blood fractions, aqueous humor (AH) and vitreous humor (VH) following administration of liver-targeted siRNA to non-human primates This example demonstrates that systemic administration of liver-targeted siRNA (targeting C3) reduces the protein concentrations, particularly of C3 and C3a, in non-human primates' plasma / serum and tissues such as AH and VH. In this example, GalNAc-conjugated siRNA ("siRNA58" in Example 4) was systemically administered to non-human primates. Specific materials and methods are described in detail below.

[0334] Study design: siRNA58 or vehicle was administered to male cynomolgus monkeys as a single subcutaneous dose once daily on days 1-4.

[0335] Serum / plasma samples were collected at weeks 2, 4 and prior to terminal necropsy (week 6). Aqueous humor (AH) and vitreous humor (VH) samples were collected from each animal prior to necropsy. Tissue samples were collected at necropsy at week 6.

[0336] The experimental design of this study is summarized in Table 20 below. [Table 21]

[0337] method C3 Analysis of Serum, AH, and VH: Each sample was analyzed for C3 protein concentration using an electrochemiluminescence detection immunoassay (Mesoscale Delivery® (MSD), Cat. No. K151XYR-2) according to the manufacturer's instructions. Prior to analysis, serum samples were diluted 1:10,000, AH samples were diluted 1:10, and VH samples were diluted 1:10. AH and VH samples were vortexed prior to analysis.

[0338] C3a analysis of plasma, AH and VH: Each sample was analyzed for C3a protein concentration using an enzyme-linked immunosorbent assay (ELISA) (Quidel®, Catalog No. A031) according to the manufacturer's instructions. Plasma samples were diluted 1:100 and AH and VH samples were diluted 1:5 prior to analysis. AH and VH samples were vortexed prior to analysis.

[0339] result Serum C3 and plasma C3a data: C3 levels in serum at different time points after administration of siRNA or vehicle are shown in Figure 10, Panels A and B. Panel A of Figure 10 shows the mean serum C3 concentration (ng / ml). Panel B of Figure 10 shows the reduction in mean serum C3 as a percentage reduction from the level before siRNA or vehicle was administered (i.e., baseline level).

[0340] The levels of C3a in plasma at different time points after administration of siRNA or vehicle are shown in panels A and B of Figure 11. Panel A of Figure 11 shows the mean plasma C3a concentration (ng / ml). Panel B of Figure 11 shows the reduction in mean serum C3a as a percentage reduction from the level before siRNA or vehicle was administered (i.e., baseline level).

[0341] These results show that C3 and C3a proteins were significantly reduced in serum / plasma (i.e., blood fraction) as a result of systemic administration of siRNA 58. The average percent reduction or knockdown (KD) of protein concentration was approximately 87-93% for both analytes C3 and C3a (see Figure 10, Panel B and Figure 11, Panel B).

[0342] Vitreous Humor C3 and C3a Data: C3 and C3a levels (ng / ml) in the vitreous humor (VH) on day 44 are shown in Figure 12, Panels A and B, respectively.

[0343] These results show that both C3 and C3a levels in the vitreous humor (VH) were significantly reduced in animals treated with siRNA58 compared to vehicle treated animals. The mean reduction in protein concentration (KD) was approximately 70% for C3 and 83% for C3a compared to baseline levels (pre-treatment). One animal in the siRNA58 treatment group was reported to have C3a levels below the limit of detection (LOD) i.e. 0.06 ng / ml.

[0344] Aqueous Humor C3 and C3a Data: C3 and C3a levels (ng / ml) in aqueous humor (AH) on day 44 are shown in Figure 13, Panels A and B, respectively.

[0345] These results show that both C3 and C3a protein levels in the AH were significantly reduced in animals treated with siRNA58 compared to vehicle treated animals. The mean reduction in protein concentration (KD) was approximately 92% for C3 and 94% for C3a compared to baseline levels (pre-treatment). One animal in the siRNA58 treatment group was reported to have C3a levels below the limit of detection (LOD) i.e. 0.06 ng / ml.

[0346] These data support the insight that systemic administration of liver-targeted siRNA reduces C3 and C3 activation products in ocular tissues and may be used to treat complement-mediated ocular diseases.

[0347] IX. Further Exemplary Sequences [Table 22]

[0348] [Table 23]

[0349] [Table 24]

[0350]

Table 25

[0351]

Table 26

[0352]

Table 27

[0353]

Table 28

[0354]

Table 29

[0355]

Table 30

[0356]

Table 31

[0357]

Table 32

[0358]

Table 33

[0359]

Table 34

[0360]

Table 35

[0361]

Table 36

[0362]

Table 37

[0363]

Table 38

[0364]

Table 39

[0365]

Table 40

[0366]

Table 41

[0367]

Table 42

[0368]

Table 43

[0369]

Table 44

[0370]

Table 45

[0371]

Table 46

[0372]

Table 47

[0373]

Table 48

[0374]

Table 49

[0375]

Table 50

[0376]

Table 51

[0377]

Table 52

[0378]

Table 53

[0379]

Table 54

[0380]

Table 55

[0381]

Table 56

[0382]

Table 57

[0383]

Table 58

[0384]

Table 59

[0385]

Table 60

[0386]

Table 61

[0387]

Table 62

[0388]

Table 63

[0389]

Table 64

[0390]

Table 65

[0391]

Table 66

[0392]

Table 67

[0393]

Table 68

[0394]

Table 69

[0395]

Table 70

[0396]

Table 71

[0397]

Table 72

[0398]

Table 73

[0399]

Table 74

[0400]

Table 75

[0401]

Table 76

[0402]

Table 77

[0403]

Table 78

[0404]

Table 79

[0405]

Table 80

[0406]

Table 81

[0407]

Table 82

[0408]

Table 83

[0409]

Table 84

[0410]

Table 85

[0411]

Table 86

[0412] [Table 87]

[0413] [Table 88]

[0414] [Table 89] [Table 90]

[0415] [Table 91]

[0416] [Table 92]

[0417] [Table 93]

[0418] [Table 94]

[0419] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the above Description, but is as set forth in the following claims.

Claims

**Claim 1**: A composition comprising siRNA targeting C3 mRNA for treating a complement-mediated eye disease of interest, wherein the composition is systemically administered to the subject. **Claim 2** The composition according to claim 1, wherein the siRNA comprises a liver targeting moiety. **Claim 3** The composition according to claim 2, wherein the liver targeting moiety is a GalNAc moiety. **Claim 4** The composition according to claim 3, wherein the siRNA comprises an antisense strand comprising a sequence set forth in Table 2A, 2B, 4, 5, 6, 10, 15, 16, 17, 18, 24 - 70, or 72 - 73, and / or a sense strand comprising a sequence set forth in Table 1, 3A, 3B, 10, 15, 16, 17, 18, or 21 - 71. **Claim 5** The composition according to claim 3, wherein the siRNA comprises an antisense strand and a sense strand, the antisense strand is complementary to a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 76 - 100, and / or the sense strand comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 76 - 100. **Claim 6** The composition according to any one of claims 1 - 5, wherein one or both of the sense strand and the antisense strand comprise at least one modified nucleotide. **Claim 7** The composition according to claim 6, wherein the at least one modified nucleotide comprises a nucleotide containing a 2'-O-methyl group, a nucleotide containing a 2'-fluoro group, and / or a phosphorothioate bond with an adjacent nucleotide. **Claim 8** The composition according to any one of claims 1 - 5, wherein the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 76 - 100, 126 - 150, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 255, 259, 264, 268, 272, 276, 325, 326, and 327. **Claim 9** The composition according to any one of claims 1 to 5, wherein the antisense strand comprises any one nucleotide sequence of SEQ ID NOs: 101 to 125, 151 to 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 257, 258, 260, 261, 262, 263, 265, 266, 267, 269, 270, 271, 273, 274, 275, 277, 278, and 300 to 324.

10. The composition according to any one of claims 2 to 5, wherein the liver targeting moiety is bound to one or more of the 5'-end of the sense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, and the 3'-end of the antisense strand.

11. The composition according to any one of claims 1 to 5, wherein the level of C3 transcript or C3 protein in the subject or a biological sample from the subject decreases relative to the level before administration of the siRNA or the composition after administration of the siRNA or the composition.

12. The composition according to any one of claims 1 to 5, wherein the siRNA or the composition is administered intravenously or subcutaneously to the subject.

13. The composition according to claim 11, wherein the siRNA or the composition is administered to the hepatocytes ex vivo.

14. The composition according to any one of claims 1 to 5, wherein the eye disease is geographic atrophy or intermediate AMD.

15. A composition comprising an siRNA targeting C3 mRNA of a subject for inhibiting or reducing the level of complement C3 in the eye of the subject relative to a control, wherein the composition is systemically administered to the subject.

16. The composition according to claim 15, wherein the siRNA comprises a liver targeting moiety.

17. The composition according to claim 16, wherein the liver targeting moiety is a GalNAc moiety.

18. The composition according to claim 17, wherein the siRNA comprises an antisense strand containing a sequence described in Table 2A, 2B, 4, 5, 6, 10, 15, 16, 17, 18, 24 to 70, or 72 to 73, and / or a sense strand containing a sequence described in Table 1, 3A, 3B, 10, 15, 16, 17, 8, or 21 to 71.

19. The composition according to claim 17, wherein the siRNA comprises an antisense strand and a sense strand, the antisense strand is complementary to a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 76 to 100, and / or the sense strand comprises a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 76 to 100.

20. The method according to any one of claims 15 to 19, wherein one or both of the sense strand and the antisense strand contain at least one modified nucleotide.

21. The composition according to claim 20, wherein the at least one modified nucleotide comprises a nucleotide containing a 2'-O-methyl group, a nucleotide containing a 2'-fluoro group, and / or a phosphorothioate bond with an adjacent nucleotide.

22. The composition according to any one of claims 15 to 19, wherein the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 76 to 100, 126 to 150, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 255, 259, 264, 268, 272, 276, 325, 326, and 327.

23. The composition according to any one of claims 15 to 19, wherein the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 101 to 125, 151 to 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 257, 258, 260, 261, 262, 263, 265, 266, 267, 269, 270, 271, 273, 274, 275, 277, 278, and 300 to 324.

24. The composition according to any one of claims 16 to 19, wherein the liver-targeted moiety is bound to one or more of the 5'-end of the sense strand, the 3'-end of the sense strand, the 5'-end of the antisense strand, and the 3'-end of the antisense strand.

25. The composition according to any one of claims 15 to 19, wherein the subject has a complement-mediated eye disease.

26. The composition according to claim 25, wherein the eye disease is geographic atrophy or intermediate AMD.

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