C3 targeted inhibitory RNA

Abstract

The present disclosure relates to C3 targeted inhibitory RNA sequences, modification patterns, and uses thereof.

Description

C3 TARGETED INHIBITORY RNACROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 556,773, filed February 22, 2024, U.S. Provisional Application No. 63 / 649,886, filed May 20, 2024, U.S. Provisional Application No. 63 / 711,985, filed October 25, 2024, and U.S. Provisional Application No. 63 / 711,856 filed, October 25, 2024, the contents of each of which are hereby incorporated by reference herein in their entirety.FIELD

[0002] The present disclosure relates to C3 targeted inhibitory RNA sequences, modification patterns, and uses thereof.BACKGROUND

[0003] Complement is a system consisting of more than 30 plasma and cell-bound proteins that plays a significant role in both innate and adaptive immunity. The proteins of the complement system act in a series of enzymatic cascades through a variety of protein interactions and cleavage events. Complement activation occurs via three main pathways: the antibody-dependent classical pathway, the alternative pathway, and the mannose-binding lectin (MBL) pathway. Inappropriate or excessive complement activation is an underlying cause or contributing factor to a number of serious diseases and conditions.SUMMARY

[0004] The present disclosure encompasses C3 targeted inhibitory RNA sequences, such as siRNAs, including compositions comprising such sequences, and methods of use thereof.

[0005] In one aspect, the present disclosure provides an isolated, modified sequence comprising any one of SEQ ID NOs: 15-28. In another aspect, the present disclosure provides an isolated, modified sequence comprising any one of SEQ ID Nos: 1-14. In some embodiments, the isolated, modified sequence further comprising at least one ligand attached to the 5’ end or the 3’ end of the sequence.

[0006] The present disclosure provides, in another aspect, an siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a nucleotide sequence comprising any one of SEQ ID NOs: 15-28 and the sense strand comprises a nucleotide sequence comprising any one of SEQ ID NOs: 1-14. In some embodiments, thesiRNA further comprises at least one ligand attached to the 5’ end or the 3’ end of either the antisense or the sense strand. In some embodiments, the ligand comprises at least one GalNAc moiety. In some embodiments, the ligand comprises an oleic moiety.

[0007] In another aspect, the present disclosure provides, an siRNA comprising an antisense strand comprising any one of SEQ ID NOs: 15-28.

[0008] In another aspect, the present disclosure provides, an siRNA comprising a sense strand comprising any one of SEQ ID NOs: 1-14.

[0009] In another aspect, the present disclosure provides, an siRNA, wherein the siRNA comprises a sense strand / antisense strand pair detailed in Table 1 A or Table A-1. In some embodiments, the siRNA further comprises at least one ligand attached to the 5’ end or the 3’ end of either the antisense or the sense strand. In some embodiments, the ligand comprises at least one GalNAc moiety. In some embodiments, the ligand comprises an oleic moiety or a C16 moiety.

[0010] In another aspect, the present disclosure provides, an siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a modification patter detailed in Table 2A or Table 2B, and the siRNA targets a C3 transcript. In some embodiments, the sense strand has a modification patte detailed in Table 3A or Table 3B. In some embodiments, the siRNA further comprises at least one ligand attached to the 5’ end of the sense strand, the 3’ end of the sense strand, the 5’ end of the antisense strand, the 3’ end of the antisense strand, or a combination thereof. In some embodiments, the ligand comprises at least one GalNAc moiety. In some embodiments, the ligand comprises three GalNAc moieties. In some embodiments, the ligand comprises an oleic moiety. In some embodiments, the siRNA has an antisense strand nudeobase sequence detailed in Table 1A or Table A-1. In some embodiments, the siRNA has a sense strand nudeobase sequence detailed in Table 1 A or Table A-1.

[0011] The present disclosure provides, in another aspect, a method of treating a subject having or at risk of a complement-mediated disorder, the method comprising administering to the subject a composition comprising an effective amount of an siRNA described herein. In some embodiments, after the administration of the composition, a level of C3 transcript or C3 protein in the subject or in a biological sample from the subject is reduced relative to a level before the administration of the composition. In some embodiments, the level of C3 transcriptor 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% or greater, relative to a level before the administration. In some embodiments, the composition is administered intrathecally, intravenously, or subcutaneously to the subject. In some embodiments, the composition is administered to a hepatocyte of the subject. In some embodiments, the composition is administered to the hepatocyte ex vivo. In some embodiments, the composition is administered to the hepatocyte in vivo.

[0012] In some embodiments, the method further comprises administering to the subject a second agent. In some embodiments, the second agent is an anti-C3 antibody or a compstatin analog. In some embodiments, the subject has a defect in complement regulation, optionally wherein the defect comprises abnormally low expression of one or more complement regulatory proteins by at least some of the subject’s cells. In some embodiments, the complement-mediated disorder is a chronic disorder.

[0013] In some embodiments, the complement-mediated disorder involves complement- mediated damage to red blood cells, optionally wherein the disorder is paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome. In some embodiments, the complement-mediated disorder is an autoimmune disease, optionally wherein the disorder is multiple sclerosis. In some embodiments, the complement-mediated disorder involves the kidney, optionally wherein the disorder is membranoproliferative glomerulonephritis, lupus nephritis, IgA nephropathy (IgAN), primary membranous nephropathy (primary MN), C3 glomerulopathy (C3G), or acute kidney injury. In some embodiments, the complement- mediated disorder involves the central or peripheral nervous system or neuromuscular junction, optionally wherein the disorder is neuromyelitis optica, Guillain-Barrd syndrome, multifocal motor neuropathy, or myasthenia gravis.

[0014] In another aspect, the present disclosure provides a composition comprising an siRNA described herein and a carrier and / or excipient.

[0015] In another aspect, the present disclosure provides a method of reducing or inhibiting complement C3 expression in a cell, the method comprising contacting the cell with an siRNA described herein. In some embodiments, after the contacting 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%, or greater, relative to a level before the contacting step. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human. In some embodiments, the subject suffers from a complement-mediated disorder.

[0016] In another aspect, the present disclosure provides a method of reducing or inhibiting expression of C3 in a subject, the method comprising contacting a cell of the subject with an siRNA described herein or a composition described herein. In some embodiments, after the contacting 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%, or greater, relative to a level before the contacting step. In some embodiments, the subject is a human. In some embodiments, the subject suffers from a complement-mediated disorder.

[0017] In another aspect, the present disclosure provides an siRNA comprising an antisense strand, wherein the antisense strand comprises any one of the siRNA antisense strand sequences detailed in Table 1 B, Table A-1 , or Table A-2. In another aspect, the present disclosure provides an siRNA comprising a sense strand, wherein the sense strand comprises any one or the siRNA sense strand sequences detailed in Table 1 B, Table A-1 , or Table A-2.

[0018] In some embodiments, an siRNA described herein comprises an antisense strand and a sense strand, wherein the antisense strand and / or the sense strand comprises a nudeobase sequence according to any one of the nudeobase sequences detailed in Table A- 1 or Table A-2; and wherein the sense strand comprises a modification patter according to any one of the modification patterns detailed in Table 3A or Table 3B and / or wherein the antisense strand comprises a modification patte according to any one of the modification patterns detailed in Table 2A or Table 2B.BRIEF DESCRIPTION OF THE DRAWING

[0019] Figure 1A and 1B: Dose response activity curves of C3-targeting siRNAs in vitro. (A) A subset of potent siRNAs from the HepG2 assay of Example 1 A were synthesized with a GalNAc conjugate and assessed for activity in a free uptake study in primary humanhepatocytes. (B) Primary human astrocytes were treated with TNF-a to stimulate C3 gene expression, then siRNAs were transfected into cells and potency was evaluated.

[0020] Figure 2: Heat map data of the immune-stimulatory potential of an siRNA from Example 1 A in a human PBMC transfection assay. The siRNA was assessed for its ability to stimulate expression of IP-10 and MIP-1a in hPBMCs from 3 donors. Positive and negative controls were used to verify functionality of the assay and ensure normal reactivity of the donor cells, respectively.

[0021] Figure 3A, 3B, 3C, 3D, and 3E: Samples from the experiment outlined in Example 2 were analyzed. C3 protein levels in (A) CSF and (B) plasma were evaluated by immunoassay. (C) C3 gene expression levels in CNS tissues and liver were analyzed by RT- qPCR and normalized to vehicle-treated animals. (D) Exposure levels of the siRNA antisense strand (AS) were quantified using an LC-MS / MS assay. (E) CSF exposure of the siRNA AS following two L-IT dose administrations.

[0022] Figure 4: Illustration of a C3 targeted siRNA with a C16 ligand on the 5’ end of the sense strand. The C16 ligand is attached via a C6 amine linker. One of skill in the art will understand that other cations in addition to Na+ are suitable in such a structure.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0023] The present disclosure encompasses C3 targeted inhibitory RNA sequences, such as siRNAs, including compositions comprising such sequences, and methods of use thereof.I. Complement System

[0024] To facilitate understanding of the disclosure, and without intending to limit the invention in any way, this section provides an overview of complement and its pathways of activation. Further details are found, e.g., in Kuby Immunology, 6th ed., 2006; Paul, W. E., Fundamental Immunology, Lippincott Williams & Wilkins; 6th ed., 2008; and Walport M J., Complement. First of two parts. N Engl J Med., 344(14): 1058-66, 2001.

[0025] Complement is an arm of the innate immune system that plays an important role in defending the body against infectious agents. The complement system comprises more than 30 serum and cellular proteins that are involved in three major pathways, known as the classical, alternative, and lectin pathways. The classical pathway is usually triggered by binding of a complex of antigen and IgM or IgG antibody to C1 (though certain other activators can also initiate the pathway). Activated C1 cleaves C4 and C2 to produce C4a and C4b, inaddition 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 produces C5 convertase, which cleaves C5 into C5a and C5b. C3a, C4a, and C5a are anaphylotoxins and mediate multiple reactions in the acute inflammatory response. C3a and C5a are also chemotactic factors that attract immune system cells such as neutrophils. It will be understood that the names “C2a” anduC2b” used initially were subsequently reversed in the scientific literature.

[0026] The alternative pathway is initiated by and amplified at, e.g., microbial surfaces and various complex polysaccharides. In this pathway, hydrolysis of C3 to C3 (H2O), which occurs spontaneously at a low level, leads to binding of factor B, which is cleaved by factor D, generating a 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 later cleaved by factor D, resulting in a C3 convertase. Surface-bound C3 convertases cleave and activate additional C3 molecules, resulting in rapid C3b deposition in dose proximity to the site of activation and leading to formation of additional C3 convertase, which in turn generates additional C3b. This process results in a cycle of C3 cleavage and C3 convertase formation that significantly amplifies the response. Cleavage of C3 and binding of another molecule of C3b to the C3 convertase gives rise to a C5 convertase. C3 and C5 convertases of this pathway are regulated by cellular molecules CR1, DAF, MCP, CD59, and fH. The mode of action of these proteins involves either decay accelerating activity (i.e., ability to dissodate convertases), ability to serve as cofadors in the degradation of C3b or C4b by factor I, or both. Normally the presence of complement regulatory proteins on cell surfaces prevents significant complement activation from occurring thereon.

[0027] The C5 convertases produced in both pathways deave C5 to produce C5a and C5b. C5b then binds to C6, C7, and C8 to form C5b-8, which catalyzes polymerization of C9 to form the C5b-9 membrane attack complex (MAC). The MAC inserts itself into target cell membranes and causes cell lysis. Small amounts of MAC on the membrane of cells may have a variety of consequences other than cell death.

[0028] The lectin complement pathway is initiated by binding of mannose-binding lectin (MBL) and MBL-assodated serine protease (MASP) to carbohydrates. The MB1-1 gene (known as LMAN-1 in humans) encodes a type I integral membrane protein localized in the intermediate region between the endoplasmic reticulum and the Golgi. The MBL-2 gene encodes the 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 a C3 convertase described above.

[0029] Complement activity is regulated by various mammalian proteins referred to as complement control proteins (CCPs) or regulators of complement activation (RCA) proteins (U.S. Pat. No. 6,897,290). These proteins differ with respect to ligand spedfidty and mechanism(s) of complement inhibition. They may accelerate the normal decay of convertases and / or function as cofactors for factor I, to enzymatically deave C3b and / or C4b into smaller fragments. CCPs are characterized by the presence of multiple (typically 4-56) homologous motifs known as short consensus repeats (SCR), complement control protein (CCP) modules, or SUSHI domains, about 50-70 amino adds in length that contain a conserved motif induding four disulfide-bonded cysteines (two disulfide bonds), proline, tryptophan, and many hydrophobic residues. The CCP family indudes complement receptor type 1 (CR1 ; C3b:C4b receptor), complement receptor type 2 (CR2), membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF), complement factor H (fH), and C4b- binding protein (C4 bp). CD59 is a membrane-bound complement regulatory protein unrelated structurally to the CCPs. Complement regulatory proteins normally serve to limit complement activation that might otherwise occur on cells and tissues of the mammalian, e.g., human host. Thus, “self cells are normally protected from the deleterious effects that would otherwise ensue were complement activation to proceed on these cells. Defidendes or defects in complement regulatory protein(s) are involved in the pathogenesis of a variety of complement- mediated disorders, e.g., as discussed herein.II. Inhibitory RNAs for C3

[0030] The disclosure includes compositions and methods related to one or more nucleotide sequences that are, comprise, or encode an inhibitory RNA that binds to and inhibits expression of messenger RNA (mRNA) produced by a target gene (e.g., C3). Inhibitory RNAs can be single stranded (e.g., an antisense oligonucleotide) or double stranded nucleic add. In some embodiments, an inhibitory RNA comprises a double stranded RNA duplex such as microRNA (miRNA) or small interfering RNA (siRNA). In some embodiments, an inhibitory RNA is an siRNA or miRNA, or a vector comprising a nudeotide sequence encoding an siRNA or miRNA.

[0031] In some embodiments, an inhibitory RNA is capable of inhibiting expression of C3 of one or more non-human spedes, e.g., a non-human primate C3, e.g., Macaca fasdcularis C3, or e.g., Chlorocebus sabaeus in addition to human C3. The Macaca fasdcularis C3 gene has been assigned NCBI Gene ID: 102131458 and the predicted amino add and nudeotide sequence of Macaca fasdcularis C3 are listed under NCBI RefSeqaccession numbers XP_005587776.1 and XM_005587719.2, respectively. In some embodiments, an inhibitory RNA comprises an antisense strand that is complementary to a target portion that is identical in the human and Macaca fasdcularis C3 transcripts. In some embodiments, an inhibitory RNA comprises an antisense strand that is complementary to a target portion of a human C3 transcript that differs by 1 , 2, or 3 nucleotides from a sequence in a Macaca fasdcularis C3 transcript. It will be appreciated that an inhibitory RNA that inhibits expression of human C3 may also inhibit expression of non-primate C3, e.g., rat or mouse C3, particularly if conserved regions of C3 transcript are targeted.

[0032] The amino add and nudeotide sequences of human C3 are known in the art and can be found in publicly available databases, for example, the National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database, where they are listed under RefSeq accession numbers NP_000055 (accession. version number NP_000055.2) and NM_000064 (accession. version number NM_000064.4), respectively (where “amino add sequence” refers to the sequence of the C3 polypeptide and “nudeotide sequence” in this context refers to the C3 mRNA sequence as represented in genomic DNA, it being understood that the actual mRNA nudeotide sequence contains U rather than T). One of ordinary skill in the art will appredate that the afore-mentioned sequences are for the complement C3 preproprotein, which indudes a signal sequence that is deaved off and is therefore not present in the mature protein. The human C3 gene has been assigned NCBI Gene ID: 718, and the genomic C3 sequence has RefSeq accession number NG_009557 (accession. version number NG_009557.1).

[0033] In some embodiments, an inhibitory RNA comprises a nudeic add strand that is complementary to a target portion of a C3 transcript, e.g., C3 mRNA. The target portion may be 15-30 nudeotides long, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nudeotides long, although shorter and longer target portions are also contemplated.

[0034] Administration of an inhibitory RNA can reduce the level of C3 transcript, C3 protein, or both C3 transcript and C3 protein, in the subject or in a biological sample compared to a level before the administration of the composition. As used herein, a biological sample may indude a biological fluid, cell, or tissue sample, induding but not limited to blood, serum or plasma sample, a sample containing hepatocytes, aqueous humor, vitreous humor, synovial fluid, cerebral spinal fluid (CSF), etc. 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%, or greater,relative to a level before the administration. Level of C3 protein can be measured, for example, in a biological sample. Non-limiting examples of biological samples in which C3 can be measured include a blood (serum or plasma) sample, a CSF sample, an aqueous / vitreous humor sample, a synovial fluid sample, etc.III. siRNAs

[0035] In some embodiments, an inhibitory RNA is a double stranded RNA (dsRNA) and inhibits C3 expression by RNA interference (RNAi). RNAi is a process of sequence-specific post-transcriptional gene silencing by which, e.g., double stranded RNA (dsRNA) 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 RNAs (siRNAs) generated from longer dsRNAs by ribonuclease III cleavage (Be stein 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 via sequence-specific RNA degradation, where sequence specificity is determined by the interaction of an siRNA with its complementary sequence within a target RNA (see, e.g., Tuschl, Chem. Biochem. 2001; 2:239-245). This disclosure includes siRNA molecules targeting C3 transcript, e.g., C3 mRNA.

[0036] Table 1A below details the nucleic add sequence (e.g. the nudeobase sequence) of certain siRNAs of the present disdosure. The sequences of Table 1A may be used with any modification pattern detailed herein, for instance, by way of non-limiting example, a modification pattern of Table 2A, Table 2B, Table 3A, or Table 3B. Table 1B and Table A-2 (in the examples) below also details nudeic add sequences (e.g., the nudeobase sequence) and the modification pattern (e.g., sugar backbone modifications, linkage modifications, 5’ terminal modifications, etc.) of certain siRNAs of the present disdosure. In certain embodiments, an siRNA of the present disdosure comprises a nudeobase sequence as detailed in Table 1A, Table 1B, or Table A-2 below. In some embodiments, an siRNA of the present disdosure comprises a nudeobase sequence as detailed in Table 1A, Table 1B, or Table A-2 with a modification pattern detailed in Table 2A or Table 2B (antisense strand modification patterns) or Table 3A or Table 3B (sense strand modification patterns) below. For instance, in some embodiments, an siRNA of the present disdosure has a nudeobase sequence as detailed in Table 1A, an antisense strand modification pattern from Table 2A or Table 2B, and a sense strand modification patter from Table 3A or Table 3B. In some embodiments, an siRNA of the present disdosure has a nudeobase sequence as detailed inTable 1B, an antisense strand modification patter from Table 2A or Table 2B, and a sense strand modification patter from Table 3A or Table 3B. In some embodiments, an siRNA of the present disclosure has a nudeobase sequence as detailed in Table A-2, an antisense strand modification patter from Table 2A or Table 2B, and a sense strand modification pattern from Table 3A or Table 3B.

[0037] With resped to Table 1A, a lower case a, c, g, or u refers to an unmodified RNA nudeotide. With resped to Table 1B, a lower-case a, c, g, or u refers to a 2’O-methyl (2’OMe) modified nudeotide. An uppercase letter followed by T refers to a 2’F modified nudeotide An 's’, when it appears between two letters, refers to a 3’ phosphorothioate bond between the nudeotide preceding the 's’ and the nudeotide following the *s.’ When a *ps’ appears at the 5’ end of a sequence, the *ps’ represents a 5’ phosphorothioate group on the first 5’ nudeotide. The abbreviation X-GNA refers to a glycerol nudeic add comprising the nudeobase “X” - by way of example, “U-GNA” refers to a glycerol nudeic add with a uradl nudeobase. The abbreviation “Xo” refers to an unlocked nudeic add comprising the nudeobase “X” - by way of example, Uo refers to an unlocked nudeic add with a uradl nudeobase. The abbreviation “(vinu)” refers to a nudeotide with a uradl nudeobase, a 2’OMe modification on the sugar, and a 5’ (E) vinylphosphonate. The abbreviation “Xm” refers to a nudeotide with an *X’ nudeobase, and a 2’ O-methoxyethyl modified sugar - for example, “Gm” refers to a nudeotide with guanine as the nudeobase and a 2’ O-methoxyethyl modified sugar. The abbreviation “isodX” refers to an isoguanine or isocytosine nudeobase. as indicated - for example, “isodG" refers to an isoguanine nudeobase.

[0038] In some embodiments, an siRNA comprises a sense strand and / or an antisense strand that comprises a nudeobase sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to the nudeobase sequences of any one of the sequences illustrated in Table 1A, Table 1B, and Table A-2. In some embodiments, an siRNA comprises a sense strand and / or an antisense strand that comprises the nudeobase sequence of any one of the sequences illustrated in Table 1A, Table 1B, and Table A-2. In some embodiments, an siRNA comprises a sense strand that comprises the nudeobase sequence of any one of the sequences illustrated in Table 1A, Table 1B, and Table A-2. In some embodiments, an siRNA comprises an anti-sense strand that comprises the nudeobase sequence of any one of the sequences illustrated in Table 1A, Table 1B, and Table A-2.

[0039] In certain instances, the sense strand nudeobase sequences detailed in Tables 1A, 1B, or A-2 may be modified such that the base at position 10 of the sense strand (counting from the 5’ end) is mismatched, such that the base at position 10 of the sensestrand does not form a canonical Watson-Crick base pair with the corresponding nudeobase in the antisense strand.

[0040] In some instances, an siRNA of the present disdosure comprises an overhang. Such an overhang may be complementary to the target sequence or may be non- complementary to the target sequence. For instance, in some embodiments, an overhang may comprise solely uradl or 2’ modified uradl. In some embodiments, an overhang may be complementary to the target sequence.Table 1A: Nudeobase sequencesTable 1B: siRNA sequences

[0041] It should be noted than an siRNA of the present disclosure (including, but not limited to, an siRNA of Table 1A, 1B, or A-2) may comprise an overhang e.g., 5" and / or 3" overhang(s) or may be blunt-ended. In this regard, the location and length of overhang(s), if present, may be varied. The term "overhang" refers to at least one unpaired nucleotide that protrudes from a duplex structure of a double-stranded nucleic add (e.g., an siRNA). For example, when a 3’-end of one strand of a double-stranded nudeic add extends beyond the 5’-end of the other complementary strand, a 3’ nudeotide overhang exists. "Blunt" or "blunt end" with resped to a double-stranded nudeic add means that there are no unpaired nudeotides at that end of the double-stranded nudeic add, i.e., there is no nudeotide overhang. A "blunt ended" inhibitory RNA (e.g., an siRNA) is thus double-stranded over its entire length, i.e., there is no nudeotide overhang at either end (though there may be one or more unpaired nudeotides within the duplex). Inhibitory RNAs (e.g., siRNAs) described herein indude inhibitory RNAs with a nudeotide overhang at one end, i.e., inhibitory RNAs with one overhang and one blunt end, inhibitory RNAs with nudeotide overhangs at both ends, and blunt-ended inhibitory (i.e., with no overhangs).

[0042] In some embodiments an overhang comprises at least 1 , 2, 3, 4, 5 or more nudeotides. An overhang may comprise or consist of nucleotide or non-nudeotide moieties or a combination thereof. An inhibitory RNA (e.g., an siRNA) of the present disclosure may contain one or more overhang regions, e.g., at the 3 -end, 5 -end, or both ends of one or both strands. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. In some embodiments, nudeotide(s) of an overhang can be present on the 5-end, the 3-end or both ends of either an antisense or sense strand of an inhibitory RNA described herein. In some embodiments, the antisense strand of a double-stranded siRNA has an overhang of 1-5 nudeotides or 5-10 nucleotides, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nudeotide overhang at the 3' end and / or the 5' end. In some embodiments, the sense strand of a double-stranded siRNA has an overhang of 1-5 nudeotides or 5-10 nudeotides, e.g., a 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nudeotide overhang at the 3' end and / or the 5' end. In some embodiments, an overhang can be 1-6 nudeotides in length, e.g., 2-6 nudeotides in length, 1-5 nudeotides in length, 2-5 nudeotides in length, 1-4 nudeotides in length, 2-4 nudeotidesin length, 1-3 nudeotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length, e.g., 2 nudeotides in length.

[0043] Overhangs can be the result of one strand being longer than the other or the result of two strands of the same length being staggered. In various embodiments, overhang may be perfectly complementary, partly complementary, or not complementary to a target RNA in a hybrid formed by the guide strand and a target RNA (i.e., perfectly complementary, partly complementary, or not complementary to its paired strand). For example, a 2 nudeotide overhang may indude nudeotides that are complementary to positions -1 and -2 relative to the target site in the mRNA. In certain embodiments, nudeotides in the overhang region of an inhibitory RNA (e.g., an siRNA) can each independently be a modified or unmodified nudeotide induding, but not limited to nudeotides that are 2 -sugar modified, such as 2'-F, 2'- 0-methyl, 2'-0-methoxyethyl-5-methyluridine (Teo), 2'-0 -methoxyethyladenosine (Aeo), 2'-0- methoxyethyl-5-methylcytidine (m5Ceo), or nudeotides that are not 2 -sugar modified, such as uradl (U), thymidine (T) or deoxythymidine (dT), and any combinations thereof. For example, an overhang sequence on the sense and / or antisense strand may comprise, UU, TT, or dTdT. In some embodiments, an overhangs may indude ribonucleotides or 2'-O-methyl modified nudeotides or a combination thereof. In some embodiments, the 5' or 3' overhang(s) of the sense strand, antisense strand or both strands of an inhibitory RNA described herein may be phosphorylated. In some embodiments, overhang region(s) contain two nudeotides having a phosphorothioate or other non-phosphodiester linkage between the two nudeotides, where the two nudeotides can be the same or different. In some embodiments, an overhang is present at the 3 'end of the sense strand, 3 ' end of the antisense strand, or 3' end of both strands. In some embodiments, a 3' overhang is present in the antisense strand. In some embodiments, a 3' overhang is present in the sense strand. In some embodiments, an inhibitory RNA may contain a single overhang. For example, an overhang may be located at the 3' end of the sense strand, with the 5' end of the sense strand (together with the 3' end of the antisense strand) forming a blunt end. Alternatively, an overhang may be located at the 3' end of the antisense strand, with the 5' end of the antisense strand (together with the 3* end of the sense strand) forming a blunt end. In some embodiments, a 3' overhang may alternatively or additionally have a moiety, e.g., a ligand, targeting moiety or lipophilic moiety, attached thereto.

[0044] In some embodiments, an inhibitory RNA of the present disclosure indudes one or more natural nudeobases and / or one or more modified nudeobases derived from a natural nudeobase. Examples indude, but are not limited to, uracil, thymine, adenine, cytosine, andguanine having their respective amino groups protected by acyl protecting groups, 2- fluorouradl, 2-fluorocytosine, 5-bromouradl, 5-iodouradl, 2,6-diaminopurine, azacytosine, pyrimidine analogs such as pseudoisocytosine and pseudouradl and other modified nudeobases such as 8-substituted purines, xanthine, or hypoxanthine (the latter two being the natural degradation products). Exemplary modified nudeobases are disdosed in Chiu and Rana, R N A, 2003, 9, 1034-1048, Limbach et al. Nudeic Adds Research, 1994, 22, 2183- 2196 and Revankar and Rao, Comprehensive Natural Products Chemistry, vol. 7, 313.

[0045] Modified nudeobases also indude expanded-size nudeobases in which one or more aryl rings, such as phenyl rings, have been added. Nudeic base replacements described in the Glen Research catalog (www.glenresearch.com); Krueger A T et al, Acc. Chem. Res., 2007, 40, 141-150; Kool, E T, Acc. Chem. Res., 2002, 35, 936-943; Benner S. A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F. E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr. Opin. Chem. Biol., 2006, 10, 622-627, are contemplated as useful for siRNA molecules described herein. Modified nudeobases also encompass structures that are not considered nudeobases but are other moieties such as, but not limited to, corrin- or porphyrin-derived rings. Porphyrin-derived base replacements have been described in Morales-Rojas, H and Kool, E T, Org. Lett., 2002, 4, 4377-4380.

[0046] In some embodiments, modified nudeobases indude any one of the following structures, optionally substituted:

[0047] In some embodiments, a modified nudeobase is fluorescent. Exemplary fluorescent modified nudeobases indude phenanthrene, pyrene, stillbene, isoxanthine, isozanthopterin, terphenyl, terthiophene, benzoterthiophene, coumarin, lumazine, tethered stillbene, benzo-uradl, and naphtho-uradl, as shown below:

[0048] In some embodiments, a modified nudeobase is unsubstituted. In some embodiments, a modified nudeobase is substituted. In some embodiments, a modified nudeobase is substituted such that it contains, e.g., heteroatoms, alkyl groups, or linking moieties connected to fluorescent moieties, biotin or avidin moieties, or other protein or peptides. In some embodiments, a modified nudeobase is a “universal base” that is not a nudeobase in the most dassical sense, but that fundions similarly to a nudeobase. One representative example of such a universal base is 3-nitropyrrole.

[0049] In some embodiments, an siRNA described herein indudes nudeosides that incorporate modified nudeobases and / or nudeobases covalently bound to modified sugars. Some examples of nudeosides that incorporate modified nudeobases indude 4- acetylcytidine; 5-(carboxyhydroxylmethyl)uridine; 2’-O-methylcytidine; 5- carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; dihydrouridine; 2-O-methylpseudouridine; beta.D-galadosylqueosine; 2’-0-methylguanosine; N6- isopentenyladenosine; 1 -methyladenosine; 1 -methylpseudouridine; 1 -methylguanosine; 1- methylinosine; 2,2-dimethylguanosine; 2-methyladenosine; 2-methylguanosine; N7- methylguanosine; 3-methyl-cytidine; 5-methylcytidine; 5-hydroxymethylcytidine; 5- formylcytosine; 5-carboxylcytosine; N6-methyladenosine; 7-methylguanosine; 5- methylaminoethyluridine; 5-methoxyaminomethyl-2-thiouridine; beta, D-man nosy Iqueosine; 5- methoxycarbonylmethyluridine; 5-methoxyuridine; 2-methylthio-N6-isopentenyladenosine; N- ((9-beta,D-ribofuranosyl-2-methylthiopurine-6-yl)carbamoyl)threonine; N-((9-beta,D- ribofuranosylpurine-6-yl)-N-methylcarbamoyl)threonine; uridine- 5-oxyacetic add methylester; uridine-5-oxyacetic add (v); pseudouridine; queosine; 2-thiocytidine; 5-methyk2-thiouridine; 2- thiouridine; 4-thiouridine; 5-methyluridine; 2’-O-methyl-5-methyluridine; and 2’-O- methyluridine.

[0050] In some embodiments, nudeosides indude 6’-modified bicydic nucleoside analogs that have either (R) or (S)-chirality at the 6 -position and indude the analogs described in U.S. Pat. No. 7,399,845. In other embodiments, nudeosides indude 5’-modified bicydic nudeoside analogs that have either (R) or (S)-chirality at the 5 -position and indude the analogs described in U.S. Publ. No. 20070287831. In some embodiments, a nudeobase or modified nudeobase is 5-bromouradl, 5-iodouradl, or 2,6-diaminopurine. In some embodiments, a nudeobase or modified nudeobase is modified by substitution with a fluorescent moiety.

[0051] Methods of preparing modified nudeobases are described in, e.g., 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.

[0052] In some embodiments, an siRNA described herein includes one or more modified nucleotides wherein a phosphate group or linkage phosphorus in the nucleotides are linked to various positions of a sugar or modified sugar. As non-limiting examples, the phosphate group or linkage phosphorus can be linked to the 2’, 3’, 4’ or 5’ hydroxyl moiety of a sugar or modified sugar. Nucleotides that incorporate modified nucleobases as described herein are also contemplated in this context.

[0053] Other modified sugars can also be incorporated within an siRNA molecule. In some embodiments, a modified sugar contains 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, wherein each R’ is independently as defined above and described herein; — O — (C1-C10 alkyl), — S— (C1-C10 alkyl), — NH— (C1-C10 alkyl), or— N(C1-C10alkyl)2; — O— (CrCio alkenyl), —S— (C2- C10 alkenyl), — NH — (C2- C10 alkenyl), or — N(C2- C10 alkenyl)2: — O — (C2- C10 alkynyl), (C2- C10 alkynyl), — NH — (C2- C10 alkynyl), or — N(C2- C10 alkynyl)2; or — O — (C1-C10 alkylene)- O— (C1-C10 alkyl), — O— (C1-C10 alkylene)-NH— (C1-C10 alkyl) or — O— (C1-C10 alkylene)-NH(CrC10 alky l)2, — NH — (C1-C10 alkylene)-O — (C1-C10 alkyl), or — N(C2- C10 alkyl)-(CrCio alkylene)- O — (C1-C10 alkyl), wherein the alkyl, alkylene, alkenyl and alkynyl may be substituted or unsubstituted. Examples of substituents include, and are not limited to, — O(CH2)nOCH3, and — O(CH2)nNH2, wherein n is from 1 to about 10, MOE, DMAOE, DMAEOE. Also contemplated herein are modified sugars described in WO 2001 / 088198; and Martin et al., Helv. Chim. Acta, 1995, 78, 486-504. See also Allerson et al (2005) J. Med. Chem. 48:901-904. In some embodiments, a 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 having similar properties. In some embodiments, modifications are made 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 sugar on the 3’- tenminal nucleotide or in the 5’ position of the 5 -terminal nucleotide.

[0054] In some embodiments, the 2’-OH of a ribose is replaced with a substituent including one of the following: — H, — F; — CF3, — CN, — N3, — NO, — NO2, — OR’, — SR’, or — N(R')2, wherein each R’ is independently as defined above and described herein; — O — (C1- C10 alkyl), — S— (C1-C10 alkyl), — NH— (C1-C10 alkyl), or— N(C1- C10alkyl)2; — O— (Cr C10 alkenyl), — S — (C2- C10 alkenyl), — NH — (C2- C10 alkenyl), or — N(C2- C10 alkenyl)2; — O — (CrC10 alkynyl), — S — (C2-C10 alkynyl), — NH — (C2-C10 alkynyl), or — N(C2-Ci0alkynyl)2; or — O — (C1-C10 alkylene)-O — (C1-C10 alkyl), — O — (C1-C10 alkylene)-NH — (C1-C10 alkyl) or — O — (Cr C10 alkylene)-NH(Ci-Cio alkyl)2, — NH— (C1-C10 alkylene)-©— (C1-C10 alkyl), or — N(Ci- C10 alkyl)-(Ci-Cio alkylene)-© — (C1-C10 alkyl), wherein the alkyl, alkylene, alkenyl and alkynyl may be substituted or unsubstituted. In some embodiments, the 2 -OH is replaced with — H (deoxyribose). In some embodiments, the 2-OH is replaced with — F. In some embodiments, the 2-OH is replaced with — OR*. In some embodiments, the 2-OH is replaced with — ©Me. In some embodiments, the 2 -OH is replaced with — OCH2CH2OMe.

[0055] Modified sugars also include locked nucleic acids (LNAs). In some embodiments, the locked nucleic add has the structure indicated below. A locked nudeic add of the structure below is indicated, wherein Ba represents a nucleobase or modified nudeobase as described herein, and wherein R28is — OCH2C4-

[0056] A modified sugar may also encompass unlocked nudeic adds (UNA)s. UNA moieties are acydic derivatives of RNA lacking the C2’-C3’-bond of the ribose ring of RNA.

[0057] In some embodiments, a modified sugar is an ENA such as those described in, e.g., Seth et al., J Am Chem Soc. 2010 Oct. 27; 132(42): 14942-14950. In some embodiments, a modified sugar is any of those found in an XNA (xenonudeic add), for instance, arabinose, anhydrohexitol, threose, 2'fluoroarabinose, or cydohexene.

[0058] Modified sugars indude sugar mimetics such as cydobutyl or cydopentyl 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 that are contemplated indude sugars in which the oxygen atom within the ribose ring is replaced by nitrogen, sulfur, selenium, or carbon. In some embodiments, a modified sugar is a modified ribose wherein the oxygen atom within the ribose ring is replaced with nitrogen, and wherein the nitrogen is optionally substituted with an alkyl group (e.g., methyl, ethyl, isopropyl, etc.).

[0059] Non-limiting examples of modified sugars include glycerol, which form glycerol nucleic acid (GNA) analogues. One example of a GNA analogue 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 C H et al., PNAS, 2007, 14598-14603. Another example of a GNA derived analogue, flexible nucleic acid (FNA) based on the mixed acetal aminal of formyl glycerol, is described in Joyce G F et al., PNAS, 1987, 84, 4398-4402 and Neuberger B D and Switzer C, J. Am. Chem. Soc., 2008, 130, 412-413. Additional non-limiting examples of modified sugars include hexopyranosyl (6* to 4), pentopyranosyl (4* to 2), pentopyranosyl (4* to 3), or tetrofuranosyl (3* to 2) sugars.

[0060] Modified sugars and sugar mimetics can be prepared by methods known in the art, including, but not limited to: 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. Sniekus, Ed., (Kluwer Academic, Netherlands, 1996), p. 293; K.-U. Schoning et al, Science (2000), 290:1347-1351; A. Eschenmoser et 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 to the 2’ modifications can be found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein. Specific modifications to the 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.

[0061] In certain embodiments, an siRNA of the present disclosure may encompass one or more glycerol nucleic add (GNA) base, one or more UNA (unlocked nudeic add) base, or a combination thereof. For instance, in certain embodiments, an siRNA disdosed herein may comprise a GNA or a UNA base at position seven of the antisense strand, wherein the numbering starts from the 5’ position.

[0062] According to certain embodiments various nudeotide modifications or nudeotide modification patterns may be used selectively in either the sense or antisense strand of an inhibitory RNA (e.g., siRNA) described herein. For example, in some embodiments one may utilize unmodified ribonudeotides in the antisense strand (at least within the duplex portion thereof) while employing modified nudeotides and / or modified or unmodified deoxyribonudeotides at some or all positions in the sense strand. In some embodiments,particular patterns of modifications are employed throughout part or all of either or both strands of an siRNA. Nucleotide modifications may occur in any of a variety of patterns. For example, an alternating patte may be used. For example, the antisense, sense strand, or both, may have 2’-O-methyl or 2 -fluoro modifications on every other nucleotide. In some embodiments, an inhibitory RNA (e.g., siRNA) comprises a sense and / or antisense strand with at least one unmodified nucleotide.

[0063] In some embodiments, every nucleotide in the sense strand and antisense strand of an inhibitory RNA (e.g., siRNA), may be modified. Each nucleotide may be modified with the same or different modification which may include, by way of non-limiting example, one or more alteration of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2’ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

[0064] In some embodiments one or more residues of the sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, GNA, HNA (1 ,5-anhydrohexrtol nucleic acid), CeNA (cydohexenyl nucleic add — a DNA mimic in which the deoxyribose is replaced by a six-membered cyclohexene ring), 2’-methoxyethyl, 2 -O-methyl, 2 -O-allyl, 2’-C- altyl, 2 -deoxy, 2 -hydroxyl, or 2’-fluoro. The strands can contain more than one modification. In some embodiments at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, e.g., 100% of the residues of the sense strand and antisense strand is independently modified with 2 -O-methyl or 2 -fluoro. In some embodiments at least two different modifications are present on the sense strand and antisense strand. Those two modifications may be the 2’-O-methyl or 2 -fluoro modifications, or others.

[0065] In certain embodiments, an siRNA of the present disclosure comprises a sense strand with a modification pattern according to any one of the modification patterns detailed in Table 3A or 3B below, and an antisense strand with a modification patter according to any one of the modification patterns detailed in Table 2A or 2B below. That is, each of the sense strand modification patterns detailed in Table 3A or 3B may be paired with any one of the antisense strand modification patterns detailed in Table 2A or 2B. Any of these sense strand / antisense strand modification pattern combinations may be used to create an siRNA duplex that targets C3. In particular, any of these sense strand / antisense strand modification pattern combinations may be used in conjunction with any one of the nucleobase sequences detailed in Table 1A, Table 1B, or Table A-2.

[0066] In some embodiments, an inhibitory RNA (e.g., an siRNA) of the present disclosure comprises an antisense strand with a modification pattern according to any one of the modification patterns depicted in Table 2A or 2B. In certain embodiments, an siRNA of the present disclosure may comprise a sense strand nucleobase sequence according to any one of the sense strands detailed in Table 1A, Table 1B, or Table A-2, and an antisense strand comprising a modification pattern according to any one of the modification patterns detailed in Table 2A or 2B. In specific embodiments, an siRNA of the present disclosure may comprise a sense strand nucleobase sequence according to any one of the sense strands detailed in Table 1A, Table 1B, or Table A-2, and an antisense nucleobase sequence according to any one of the antisense nucleobase sequences detailed in Table 1A, Table 1B, or Table A-2 with an antisense modification pattern according to any one of the modification patterns detailed in Table 2A or 2B. In certain embodiments, an siRNA of the present disclosure may comprise an antisense strand nucleobase sequence according to any one of the antisense strands detailed in Table 1A, Table 1B, or Table A-2, and a sense strand modification pattern according to any one of the modification patterns detailed in Table 3A or3B. In specific embodiments, an siRNA of the present disclosure may comprise an antisense strand nucleobase sequence according to any one of the antisense strands detailed in Table 1A, Table 1B, or Table A-2, and a sense strand nucleobase sequence according to any one of the sense strand nucleobase sequences of Table 1A, Table 1B, or Table A-2 with a sense strand modification pattern according to any one of the modification patterns detailed in Table 3A or 3B.

[0067] In some embodiments, an siRNA of the present disclosure comprises duplex number 1B, 7B, 15B, 21B, 30B, 41 B, 52B, 84B, 85B, 86B, 87B, 101B, 103B, 106B, 121B, 129B, 130B, 161B, 163B, 166B, 170B, 219B, 220B, 222B, 225B, 226B, 227B, 228B, 232 B, 238B, or 243B as detailed in Table A-2. In some embodiments, an siRNA of the present disclosure comprises duplex number 1B, 7B, 15B, 21 B, 30B, 41 B, 52B, 84B, 85B, 86B, 87B, 101B, 103B, 106B, 121B, 161 B, 163B, 166B, 170B, 219B, 220B, 225B, 227B, 228B, or243B as detailed in Table A-2. In some embodiments, an siRNA of the present disclosure comprises duplex number 129B, 130B, 222B, 226B, 232B, or 238B, as detailed in Table A-2. In some embodiments, an siRNA of the present disclosure comprises SEQ ID NO:29. In some embodiments, an siRNA of the present disclosure comprises SEQ ID NO:30. In some embodiments, an siRNA of the present disclosure comprises SEQ ID NO:31. In some embodiments, an siRNA of the present disclosure comprises SEQ ID NO:32. In some embodiments, an siRNA of the present disclosure comprises SEQ ID NO:62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, or 83. In some embodiments, an siRNA of the present disclosure comprises SEQ ID NO:72. In some embodiments, an siRNA of the present disclosure comprises SEQ ID NO:73. In some embodiments, an siRNA of the present disclosure comprises SEQ ID NO:74. In some embodiments, an siRNA of the present disclosure comprises SEQ ID NO:75. For each of these embodiments, the siRNA may comprise a ligand conjugated to a terminus (e.g., 3’ or 5’ terminus of a sense or antisense strand). In some embodiments, a ligand may be a GalNAc ligand, an oleic moiety, or a C16 moiety. For instance, an siRNA detailed herein may be conjugated at the 5’ end of the sense strand, the 3’ end of the sense strand, the 5’ end of the antisense strand, or the 3’ end of the antisense strand to a GalNAc ligand, an oleic moiety, or a C16 moiety. Methods of conjugating a ligand to an siRNA are known in the art and may include 06 amine linkers.

[0068] In some embodiments, an siRNA detailed herein may be conjugated at the 5’ end of the sense strand as illustrated in formula (I):(Ligand) - (Linker) - 5’ terminus of sense strand.

[0069] In some embodiments of formula (I), the (Ligand) is a GalNAc ligand, an oleic moiety, or a C16 moiety, and the (Linker) is any appropriate linker known in the art. In some embodiments of formula (I), the (Ligand) is a GalNAc ligand, an oleic moiety, or a C16 moiety, and the (Linker) is a C6 amine linker. For instance, the below structure illustrates one embodiment of a C16 moiety attached via a C6 amine linker to a 5’ end of a nudeobase:In some embodiments of formula (I), the (Ligand) is an oleic moiety or a C16 moiety, and the (Linker) is a 06 amine linker. In some embodiments of formula (I), the (Ligand) is a C18:1 moiety, a C16:0 moiety, or a C16:1 moiety, and the (Linker) is a 06 amine linker. In some embodiments of formula (I), the (Ligand) is a C18:1 moiety, and the (Linker) is a C6 amine linker. In some embodiments of formula (I), the (Ligand) is a C16:0 moiety and the (Linker) is a C6 amine linker. In some embodiments of formula (I), the (Ligand) is a C16:1 moiety, and the (Linker) is a C6 amine linker.

[0070] In some embodiments of formula (I), the (Linker) is a C6 amine linker, and the (Ligand) is a suitable ligand known in the art. For instance, formula (I) may be (Ligand) - NH- (CH2)6-(5’ terminus of sense strand). In some embodiments of formula (I), the (Ligand)- (Linker) is CH3(CH2)i4-CO-NH-(CH2)6-(5’ terminus of sense strand). In some embodiments of formula (I), the (Ligand)-(Linker) is CH3(CH2)i6-CO-NH-(CH2)6-(5’ terminus of sense strand). In some embodiments of formula (I), the (Ligand)-(Linker) is CH3(CH2)s-CHCH-(CH2)7-CO-NH- (CH2)6-(5’ terminus of sense strand). In some embodiments of formula (I), the (Ligand)- (Linker) is CH3(CH2)5-CHCH-(CH2)7-CO-NH-(CH2)6-(5’ terminus of sense strand).

[0071] In certain embodiments, an siRNA detailed herein may be conjugated at the 5’ end of the sense strand, via a C6 amine linker, to a C16:0 moiety. In other embodiments, an siRNA detailed herein may be conjugated at the 5’ end of the sense strand, via a C6 amine linker, to a C16:1 moiety. In still other embodiments, an siRNA detailed herein may be conjugated at the 5’ end of the sense strand, via a C6 amine linker, to a C18:1 moiety.

[0072] With respect to Table 2A, Table 2B, Table 3A, and Table 3B, “OMe” refers to a 2’O-methyl modified nucleotide. An ‘F refers to a 2’F modified nucleotide. An *s,’ when it appears between two positions, refers to a 3’ phosphorothioate between the nucleotide preceding the 's’ and the nucleotide following the *s.’ When an *ps’ appears at the 5’ end of a sequence, the *ps’ represents a 5’ phosphorothioate group on the first 5’ nucleotide. The abbreviation GNA refers to a glycerol nucleic add. The abbreviation “U” or “UNA” refers to an unlocked nudeic add. The abbreviation “5VP" refers to a 5’ (E) vinylphosphonate. The abbreviation “MOE” refers to a nudeotide with an 2’ O-methoxyethyl modified sugar. The abbreviation "isod" refers to an isoguanosine or isocytidine nudeobase. The symbol refers to an unmodified RNA base. The abbreviation “OB” refers to a nucleotide with a 2’ -Obenzyl modified sugar.Table 2A: Antisense Modification PatternsN <oTable 2B: Antisense modification patternsTable 3A: Sense strand modification patternsTable 3B: Sense strand modification patterns

[0073] In some embodiments, an siRNA of the present disclosure (for instance, an siRNA detailed in Table 1A, Table 1B, or Table A-2), may comprise a ligand (e.g., a GalNAc ligand, e.g., a GalNAc of Formula XD or XE described herein, an oleic moiety, a C16 moiety, a LAT1 binding moiety, etc.) conjugated to a terminus (e.g., 3' or 5' terminus of a sense or antisense strand). Methods of conjugating a ligand to an siRNA are known in the art and may include C6 amine linkers. In some embodiments, a ligand used herein is designed to cross the blood brain barrier.

[0074] An inhibitory RNA (e.g., siRNA) may further comprise at least one phosphorothioate and / or methylphosphonate internucleotide linkage. In some embodiments, the internucleotide linkage modification may occur on every nucleotide on the sense strand and / or antisense strand; each internucleotide linkage modification may occur 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. An alternating pattern of internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and an alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In some embodiments, an inhibitory RNA (e.g., siRNA) comprises 6-8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5'-terminus and two phosphorothioate internucleotide linkages at the 3'- terminus, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5'-terminus or the 3'-terminus. In particular embodiments, a modification pattern detailed in Table 2A, Table 2B, Table 3A, or Table 3B may comprise more or less phosphorothioate linkages than depicted in the Table. For example, a modification pattern depicted in Table 2A, Table 2B, Table 3A, or Table 3B may comprise at least one, two, three, four, five, six, seven, eight or more than eight phosphorothioate linkages. In each of these embodiments, the phosphorothioate linkage may have a specific chiral orientation, such as Rp or Sp.

[0075] In some embodiments, a ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker. In some embodiments a ligand is depicted in Formula XA, XB, or XC, or another GalNAc structure shown below. In some embodiments, an oleic moiety or a C16 moiety may be attached to an siRNA described herein by a C6-amine linker.

[0076] In some embodiments a ligand is attached to the 3' end of the sense strand. In some embodiments, an attachment is as depicted in Formula XD shown below.

[0077] In some embodiments, an inhibitory RNA (e.g., siRNA) further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

[0078] In some embodiments, a ligand targets the nucleic acid molecule to hepatocytes. For example, in some embodiments, a ligand binds to hepatocyte-specific asialoglycoprotein receptor (ASGPR), e.g., the ligand comprises a galactose derivative, e.g., GalNAc.

[0079] In some embodiments an inhibitory RNA (e.g., siRNA) is conjugated to or otherwise physically associated with one or more moieties that modulate, e.g., enhance, the activity, stability, cellular distribution, and / or cellular uptake of the inhibitory RNA (e.g., siRNA) and / or alter one or more physical properties of the inhibitory RNA (e.g., siRNA), such as charge or solubility. In some embodiments, a moiety may comprise an antibody or ligand. A ligand may be an aptamer, carbohydrate, lectin, protein, peptide, glycoprotein, lipid, cholesterol, steroid, bile acid, nucleic acid hormone, growth factor, or receptor. In some embodiments, a biologically inactive variant of a naturally occurring hormone, growth factor, or other ligand may be used. In some embodiments, a moiety comprises a targeting moiety that targets the inhibitory RNA (e.g., siRNA) to a specified cell type, e.g., a hepatocyte, a neural cell, or an ophthalmic cell type. In some embodiments, a targeting moiety binds to hepatocytespecific asialoglycoprotein receptor (ASGPR). In some embodiments, a targeting moiety binds to a receptor mediated transcytosis target such as transferrin receptor 1 (TrF1), large neutral amino acid transporter 1 (LAT1), or cluster of differentiation 98 (CD98).

[0080] In some embodiments a moiety is attached to an inhibitory RNA (e.g., siRNA) via a reversible linkage. A “reversible linkage” is a linkage that comprises a reversible bond. A “reversible bond” (also referred to as a labile bond or cleavable bond) is a covalent bond other than a covalent bond to a hydrogen atom that is capable of being selectively broken or cleaved more rapidly than other bonds in a molecule under selected conditions, the bond is capable of being selectively broken or cleaved under conditions that substantially will not break or cleave other covalent bonds in the same molecule. Cleavage or lability of a bond may be described in terms of the half-life (ti / 2) of bond cleavage (the time required for half of the bonds to cleave). Unless otherwise indicated, a reversible bond of interest herein is a “physiologically reversible bond”, by which is meant that the bond is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body. A physiologically reversible linkage is a linkage that comprises at least one physiologically reversible bond. In some embodiments, a physiologically reversible bond isreversible under mammalian intracellular conditions, which include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those found in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell, such as from proteolytic or hydrolytic enzymes. Enzymatically labile bonds are cleaved by enzymes in the body, e.g., intracellular enzymes. pH labile bonds are cleaved at a pH less than or equal to 7.0. Examples of reversible bonds and linkages and their use to conjugate moieties to an inhibitory RNA (e.g., siRNA) are described in, e.g., US Pat. App. Pub. Nos. 20130281685 and 20150273081.

[0081] In some embodiments, a moiety comprises a protein transduction domain (PTD). Protein transduction domains are polypeptides or portions thereof that facilitate uptake of heterologous molecules attached to the domain (such heterologous molecules may be referred to as “cargo”). A protein transduction domain that is a peptide may be referred to as a cell penetrating peptide (CPP). A number of protein transduction domains / peptides are known in the art. PTDs include a variety of naturally occurring or synthetic arginine-rich peptides. An arginine-rich peptide is a peptide that contains at least 30% arginine residues, e.g., at least 40%, 50%, 60%, or more. Examples of PTDs include TAT (at least amino acids 49-56), Antennopedia homeodomain, HSV VP22, RVG, angopep, and polyarginine. Such peptides may be a cationic, hydrophobic, or amphipathic peptide and may include non-standard amino acids and / or various modifications or variations such as use of circularly permuted, inverso, retro, retro-inverso, or peptidomimetic versions. The attachment of a PTD and a cargo may be covalent or noncovalent.

[0082] Exemplary PTDs that may be used are described in U.S. Pat. App. Pub. Nos. 20090093026, 20090093425, 20120142763, 20150238516, and 20160215022. A PTD may comprise two or more PTDs (e.g., between 2 and 10 PTDs), which may be the same or different. PTDs may be directly linked to one another or may be separated by a linking portion that may comprise one or more amino acids and / or one or more non-amino acid moieties, such as an alkyl chain or oligoethylene glycol moiety.

[0083] In some embodiments, an inhibitory RNA (e.g., siRNA) comprises or is physically associated with 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 a nucleic acid with which it is physically associated. One or more anionic charge neutralizing molecules or groups can be associated with a nucleic acid wherein each independently contributes to a reduction of the anionic charge and or increase in cationic charge. By chargeneutralized is meant that the anionic charge of the nucleic acid is reduced, neutralized or more cationic than the same nucleic acid in the absence of an anionic charge neutralizing molecule or group. Phosphodiester and / or phosphothioate protecting groups are examples of anionic charge neutralizing groups. In some embodiments, an inhibitory RNA (e.g., siRNA) comprises a protecting group at one or more positions that reduces the net anionic charge of a backbone that contains negatively charged groups (e.g., a phosphodiester or phosphorothioate backbone). In some embodiments, the negatively charged phosphodiester backbone is neutralized by synthesis with bioreversible phosphotriester protecting groups that are converted into charged phosphodiester bonds inside cells by the action of cytoplasmic thioesterases, resulting in an agent that is biologically active for inhibiting expression, e.g., an inhibitory RNA (e.g., siRNA) that can mediate RNAi. Such agents, which are sometimes referred to as short interfering ribonucleic neutrals (siRNNs) can therefore serve as siRNA prodrugs. It should be understood that the backbone need not be completely neutralized (i.e. , uncharged). In some embodiments, between 5% and 100% of the phosphate groups are protected, e.g., 25%-50% or 50% to 75% or 75% to 100%. In certain embodiments at least 5, 6, 7, 8, 9, or 10 of the phosphate groups on one or both strands are protected. Examples of useful phosphodiester and / or phosphothioate protecting groups, methods of making them, and their use in nucleic acids (e.g., to generate RNAi agent prodrugs) are described in the art, for instance, at US Pat. App. Pub. Nos. 20110294869, 20090093425, 20120142763, and 20150238516. In particular embodiments, an siRNA disclosed in Table 1A, Table 1B, or Table A-2 or an siRNA comprising a modification pattern disclosed in Table 2A, Table 2B, Table 3A, or Table 3B may comprise a protecting group detailed above. In various embodiments, a siRNA may comprise any of the modifications described herein. For example, in some embodiments, a modification pattern may contain 2' sugar modifications (e.g., 2'-F, 2'- O-Me). Furthermore, an siRNA may have any of the configurations or modification patterns described herein.

[0084] In some embodiments, a moiety attached to an inhibitory RNA (e.g., siRNA) comprises a carbohydrate. Representative carbohydrates include mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units. In certain embodiments, a carbohydrate comprises galactose or a galactose derivative such as galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine, and N-iso-butanoylgalactos-amine. In certain embodiments of particular interest, a galactose derivative comprises N-acetylgalactosamine (GalNAc). In certain embodiments, a moiety comprises multiple instances of the galactose or galactosederivative, e.g., multiple N-acetylgalactosamine moieties, e.g., 3 GalNAc moieties. As used herein, the term “galactose derivative” includes both galactose and derivatives of galactose having affinity for the asialoglycoprotein receptor equal to or greater than that of galactose. The term “galactose cluster” refers to a structure comprising at least 2 galactose derivatives that are physically associated with each other, typically by being covalently attached to another moiety. In some embodiments, a galactose cluster has 2-10 (e.g., 6), or 2-4 (e.g., 3) terminal galactose derivatives. A 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 serves as a branch point and that can be attached to an inhibitory RNA (e.g., siRNA). In some embodiments, a galactose derivative is linked to the moiety that serves as a branch point via a linker or spacer. In some embodiments, the moiety that serves as a branch point may be attached to an inhibitory RNA (e.g., siRNA) via a linker or spacer. For example, in some embodiments, a galactose derivative is attached to a branch point via a linker or spacer that comprises an amide, carbonyl, alkyl, oligoethylene glycol moiety, or combination thereof. In some embodiments the linkers or spacers attached to each galactose derivative are the same. In some embodiments, a galactose cluster has three terminal galactosamines or galactosamine derivatives (e.g., GalNAc) each having affinity for the asialoglycoprotein receptor. A structure in which 3 terminal GalNAc moieties are attached (e.g., through the C-1 carbons of the saccharides) to a moiety that serves as branch point may be referred to as tri-antennary N-acetylgalactosamine (GalNAc3). In some embodiments, one or more monomeric units comprising a galactose derivative may be incorporated site-specifically into an inhibitory RNA (e.g., siRNA). Such galactose derivative-containing monomeric units may comprise a galactose derivative, e.g., GalNAc, attached to a nucleoside or to a non-nucleoside moiety. In some embodiments, at least 3 nucleoside-GalNAc monomers or at least 3 non-nucleoside-GalNAc monomers are incorporated site-specifically into an inhibitory RNA (e.g., siRNA). In some embodiments, such incorporation may occur during solid-phase synthesis using phosphoramidite chemistry or via postsynthetic conjugation. In some embodiments, the galactose derivative-containing monomeric units are joined via phosphodiester bonds to each other and / or to nucleosides of the inhibitory RNA (e.g., siRNA) that do not have a galactose derivative attached. In some embodiments 2, 3, or more galactose derivative-containing monomeric units are arranged consecutively, i.e. , without any intervening units that lack a galactose derivative. In some embodiments a carbohydrate, e.g., a galactose cluster, e.g., tri-antennary N- acetylgalactosamine or two or more GalNAc-containing monomeric units, is present at the endof a strand, e.g., at the 3' end of the sense strand or at the 5' end of an antisense strand. Exemplary carbohydrates (e.g., galactose clusters), galactose derivative-containing monomeric units, carbohydrate-modified inhibitory RNAs, and methods of manufacture and use thereof are described in US Pat. App. Pub. Nos. 20090203135, 20090239814, 20110207799, 20120157509, 20150247143, US Pub. '124; Nair, J K, 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., Bioorg Med Chem Lett. 26(9):2194-7 (2016); Prakash, T P, et al., J Med Chem. 59(6):2718-33 (2016). Exemplary galactose clusters are depicted below.

[0085] Additional GalNAc structures are depicted below (and can be synthesized as described in Sharma et al., Bioconjug. Chem. 29:2478-2488 (2018)):

[0086] 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.

[0087] One of ordinary skill in the art appreciates that the structure of the linking moieties that connect each GalNAc to the branch point may vary. In some embodiments, an inhibitory RNA (e.g., siRNA) is conjugated to GalNAc as depicted below:(where said GalNAc can be conjugated at either strand (e.g., the sense strand) at the 3' or 5' end)

[0088] In some embodiments, an inhibitory RNA (e.g., siRNA) of the present disclosure is conjugated to a GalNAc ligand (e.g., a GalNAc of Formula XD or XE). For instance, an siRNA of Table 1A, Table 1B or Table A-2 may be conjugated to a GalNAc ligand (e.g., a GalNAc of Formula XD or XE).

[0089] In some embodiments, a GalNAc ligand (e.g., as shown in Formula XD or XE) is conjugated to the 3'-terminal nucleotide of the sense or antisense strand of an siRNA. In some embodiments, a GalNAc ligand (e.g., as shown in Formula XD or XE) is conjugated to the 3' position of the sugar on the 3'-terminal nucleotide of the sense or antisense strand of an siRNA. In each of these embodiments, the siRNA may be an siRNA detailed in Table 1A, Table 1B or Table A-2.

[0090] In some embodiments a GalNAc ligand (e.g., as shown in Formula XD or XE) is conjugated to the 5'-terminal nucleotide of the sense or antisense strand of an siRNA. In some embodiments a GalNAc ligand (e.g., as shown in Formula XD or XE) is conjugated to the 5' position of the 5'-terminal nucleotide of the sense or antisense strand of an siRNA. In each ofthese embodiments, the siRNA may be an siRNA detailed in Table 1 A, Table 1 B, or Table A- 2.

[0091] In some embodiments, when an inhibitory RNA is conjugated to a ligand (e.g., a GalNAc ligand), the inhibitory RNA may not include a modification (e.g., a phosphorothioate bond “PS”) to the nucleotide(s) that is / are conjugated to the ligand.

[0092] In some embodiments, an siRNA is conjugated to a GalNAc ligand (e.g., as shown in Formula XD or XE) at one terminus of either the sense or antisense strand. In some embodiments, the other three termini that are not conjugated to the GalNAc ligand contain a modification such as a phosphorothioate bond (“PS”). In some embodiments, a modification includes a PS bond between the two, three, or four 5' or 3'-most nucleotides. In some embodiments, the terminus that is conjugated to a GalNAc ligand does not contain a phosphorothioate bond between the two, three or four 5' or 3'-most nucleotides.

[0093] In some embodiments, an siRNA described herein can be conjugated to a galactose structure shown below:

[0094] In some embodiments, the linker comprises an amide, carbonyl, alkyl, oligoethylene glycol moiety, or combination thereof.

[0095] In some embodiments, an siRNA described herein can be conjugated to a galactose structure as shown below:

[0096] In some embodiments, the linker comprises an amide, carbonyl, alkyl, oligoethylene glycol moiety, or combination thereof.

[0097] Methods of synthesizing GalNAc ligands, methods of conjugating GalNAc ligands to inhibitory RNAs, and additional GalNAc ligands are known in the art and include, for example, those described in WO 2017 / 021385, WO 2017 / 178656, WO 2018 / 215391 , WO 2019 / 145543, WO 2017 / 084987, WO 2017 / 055423, and WO 2012 / 083046, which are herein incorporated by reference in their entirety.

[0098] In some embodiments an inhibitory RNA (e.g., siRNA) is conjugated to a ligand as depicted below,wherein X is O or S. In most embodiments, X is O. One of ordinary skill in the art will appreciate that the structure of the linking moiety that connects the galactose cluster to the phosphate group may vary.

[0099] In certain embodiments, a moiety comprises a lipophilic moiety. In some embodiments, the lipophilic moiety comprises a tocopherol, e.g., alpha-tocopherol. In someembodiments, 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, hexadec-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 — CH3. In some embodiments, n and m are each independently between 1 and 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 a sugar moiety as shown below.

[0100] In general, a moiety may be attached at a terminus or internal subunit of an inhibitory RNA (e.g., siRNA). In some embodiments, a moiety is attached to a modified subunit of the inhibitory RNA (e.g., siRNA). Those of ordinary skill in the art are aware of suitable methods to manufacture nucleic acids having moieties conjugated thereto. A nucleic acid strand comprising a modified nucleotide comprising a reactive functional group may be reacted with a moiety comprising a second reactive functional group, wherein the first and second reactive functional groups are capable of reacting with one another under conditions compatible with maintaining the structure of the nucleic acid strand. In some embodiments, a moiety may be attached to a sense strand or an antisense strand prior to hybridization of the strand with the complementary antisense or sense strand, respectively. In some embodiments, strands may be hybridized to form a duplex prior to incorporation of the moiety. In general, various methods of conjugation described herein may be used. See, e.g., Hermanson, G., Bioconjugate Techniques, 2nd ed., Academic Press, San Diego, 2008.

[0101] In some embodiments, an inhibitory RNA (e.g., siRNA) is a chimeric siRNA. “Chimeric” siRNAs as used herein, are siRNAs that contain two or more chemically distinct regions, each made up of at least one monomer unit, wherein the regions confer distinctproperties on the compound. In some embodiments, at least one region is modified so as to confer upon the siRNA increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity for the target nucleic acid and at least one additional region of the siRNA can serve as a substrate for enzymes (e.g., RNase H) capable of cleaving RNA:DNA or RNA:RNA hybrids. In some embodiments, at least one region of the siRNA can serve as a substrate for enzymes (e.g., RNase H) capable of cleaving RNA:DNA or RNA:RNA hybrids and at least one region can inhibit translation by steric blocking.

[0102] In some embodiments, an inhibitory RNA (e.g., siRNA) described herein can be introduced to a target cell as an annealed duplex siRNA. In some embodiments, an inhibitory RNA (e.g., siRNA) described herein is introduced to a target cell as single stranded sense and antisense nucleic acid sequences that, once within the target cell, anneal to form an inhibitory RNA (e.g., siRNA) duplex.

[0103] An inhibitory RNA described herein can be synthesized by standard methods known in the art, e.g., by use of an automated synthesizer, liquid phases synthesis, and stirbed synthesis. RNAs produced by such methodologies tend to be highly pure and to anneal efficiently to form inhibitory RNA (e.g., siRNA) duplexes. Following chemical synthesis, single stranded RNA molecules can be deprotected, annealed to form siRNAs, and purified (e.g., by gel electrophoresis or HPLC). Alternatively, standard procedures can be used for in vitro transcription of RNA from DNA templates, e.g., carrying one or more RNA polymerase promoter sequences (e.g., T7 or SP6 RNA polymerase promoter sequences). Protocols for preparation of siRNAs using T7 RNA polymerase are known in the art (see, 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 independent reactions and annealed later, or they can be synthesized simultaneously in a single reaction.IV. Compositions and Administration

[0104] Inhibitory RNAs (e.g., an siRNA described herein), or a vector comprising a nucleotide sequence encoding an siRNA or miRNA described herein, can be used to treat a complement-mediated disease or disorder, e.g., subjects suffering from or susceptible to a complement-mediated disease or disorder described herein. The route and / or mode of administration of inhibitory RNAs described herein can vary depending upon the desired results. One with skill in the art, i.e. , a physician, is aware that dosage regimens can be adjusted to provide the 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, intracerebral, intrathecal (e.g., intracisternal or via a lumbar puncture), intracerebroventricular, intravaginal, intravitreal, suprachoroidal, subretinal, transdermal, rectal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin. In some embodiments, compositions of inhibitory RNAs are delivered to the central nervous system (CNS). The mode of administration is left to the discretion of the practitioner.

[0105] One of skill in the art would understand that inhibitory RNAs (e.g., an siRNA or miRNA described herein), or a vector comprising a nucleotide sequence encoding an siRNA or miRNA described herein, may be delivered to the CNS either by local administration or systemic administration (e.g., via intrathecal, intracerebroventicular, intraparechymal, intranasal, intravenous, or subcutaneous administration) to treat a disease or disorder affecting the CNS such as multiple sclerosis, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease, other chronic demyelinating diseases (e.g., neuromyelits optica, CIDP), amyotrophic lateral sclerosis, chronic pain, stroke, allergic neuritis, progressive supranuclear palsy, Lewy body dementia (i.e. , dementia with Lewy bodies or Parkinson's disease dementia), frontotemporal dementia, corticobasal degeneration, traumatic brain injury, traumatic spinal cord injury, epilepsy, multisystem atrophy, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, gliobastoma, and leptomeningeal metastasis.

[0106] The delivery of an inhibitory RNA described herein (e.g., siRNA) to a cell can be achieved in a number of different ways. In vivo delivery may be performed by administering a composition comprising an inhibitory RNA to a subject via local delivery or systemic delivery, e.g., by parenteral administration route, e.g., subcutaneous or intravenous or intramuscular administration.

[0107] In some embodiments an inhibitory RNA is associated with a delivery agent. “Delivery agent” refers to a substance or entity that is non-covalently or covalently associated with an inhibitory RNA or is co-administered with an inhibitory RNA and serves one or more functions that increase the stability and / or efficacy of the biologically active agent beyond that which would result if the biologically active agent was delivered (e.g., administered to a subject) in the absence of the delivery agent. For example, a delivery agent may protect an inhibitory RNA from degradation (e.g., in blood), may facilitate entry of an inhibitory RNA into cells or into a cellular compartment of interest (e.g., the cytoplasm), and / or may enhance associations with particular cells containing the molecular target to be modulated. Those of ordinary skill in the art are aware of numerous delivery agents that may be used to deliver inhibitory RNA, e.g., siRNAs. See Kanasty, R., et al. Nat Mater. 12(11):967-77 (2013) forreview of some of these technologies. In some embodiments, e.g., for administering an inhibitory RNA systemically or locally, the inhibitory RNA may be associated with a delivery agent such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Without wishing to be bound by any theory, positively charged cationic delivery systems are believed to facilitate binding of a negatively charged inhibitory RNA and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an inhibitory RNA by the cell. Lipids (e.g., cationic lipids, or neutral lipids), dendrimers, or polymers may be bound to an inhibitory RNA or may form a vesicle or micelle that encapsulates an inhibitory RNA. Methods for making and administering complexes comprising a cationic agent and an inhibitory RNA are known in the art. In some embodiments it is particularly contemplated to use any of the delivery agents described in US Pub.20160298124. In some embodiments, an inhibitory RNA forms a complex with cyclodextrin for systemic administration. In some embodiments an inhibitory RNA is administered in association with a lipid or lipid-containing particle. In some embodiments an inhibitory RNA is administered in association with a cationic polymer (which may be a polypeptide or a nonpolypeptide polymer), a lipid, a peptide, PEG, cyclodextrin, or combination thereof, which may be in the form of a nanoparticle or microparticle. The lipid or peptide may be cationic. “Nanoparticle” refers to particles with lengths in two or three dimensions greater than 1 nanometer (nm) and smaller than about 150 nm e.g., 20 nm-50 nm or 50 nm-100 nm.“Microparticle” refers to particles with lengths in two or three dimensions greater than 150 nm and smaller than about 1000 nm. A nanoparticle may have a targeting moiety and / or cellpenetrating moiety or membrane active moiety covalently or noncovalently attached thereto. Nanoparticles, such as lipid nanoparticles, are described in, e.g., Tatiparti et al., Nanomaterials 7:77 (2017). Exemplary delivery agents, methods of manufacture and use in the delivery of inhibitory RNAs are described in U.S. Pat. Nos. 7,427,605; 8,158,601 ;9,012,498; 9,415,109; 9,062,021 ; 9,402,816. In some embodiments it is contemplated to use delivery technology known in the art as “Smarticles”. In some embodiments it is contemplated to use delivery technology known in the art as “stable nucleic acid lipid particles” (SNALPs), wherein the nucleic acid to be delivered is encapsulated in a lipid bilayer containing a mixture of cationic and fusogenic lipids coated with also coated with a diffusible polyethylene glycollipid (PEG-lipid) conjugate that provides a neutral, hydrophilic exterior. In some embodiments, delivery may comprise quantum dots, silica nanoparticles, nanoemulsions, micelles, or EVs.

[0108] In some embodiments a delivery agent comprises one or more amino alcohol cationic lipids, such as those described in U.S. Pat. No. 9,044,512.

[0109] In some embodiments, a delivery agent comprises one or more amino acid lipids. Amino acid lipids are molecules containing an amino acid residue (e.g., arginine, homoarginine, norarginine, nor-norarginine, ornithine, lysine, homolysine, histidine, 1- methylhistidine, pyridylalanine, asparagine, N-ethylasparagine, glutamine, 4- aminophenylalanine, the N-methylated versions thereof, and side chain modified derivatives thereof) and one or more lipophilic tails. Exemplary amino acid lipids and their use to deliver nucleic acids are described in US Pat. App. Pub. No. 20110117125 and U.S. Pat. Nos. 8,877,729, 9,139,554, and 9,339,461. In some embodiments, membrane lytic poly(amido amine) polymers and polyconjugates such as those described in U.S. Pat. App. Pub. No.20130289207 may be used. In some embodiments, a delivery agent comprises a lipopeptide compound comprising a central peptide and having lipophilic groups attached at each terminus. In some embodiments lipophilic groups can be derived from a naturally occurring lipid. In some embodiments a lipophilic group may comprise a 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 a sphinganine, or (2R,3R)-2-amino-1 ,3-octadecanediol, icosasphinganine, sphingosine, phytosphingosine, or cis-4-sphingenine. The central peptide may comprise a cationic or amphipathic amino acid sequence. Examples of such lipopeptides and their use to deliver nucleic acids are described in, e.g., U.S. Pat. No. 9,220,785.

[0110] “Masking moiety” means a molecule or group that, when physically associated with another agent (e.g., a polymer), shields, inhibits or inactivates one or more properties (biophysical or biochemical characteristics) or activities of the agent. In some embodiments, a masking moiety may be attached covalently or noncovalently to an inhibitory RNA. A masking moiety may be reversible, meaning that it is attached to the inhibitory RNA that it masks via a reversible linkage. As will be appreciated by those of ordinary skill in the art, a sufficient number of masking moieties are linked to the inhibitory RNA to be masked to achieve a desired level of inactivation.

[0111] In some embodiments an inhibitory RNA is conjugated to a delivery agent that is a polymer. Useful delivery polymers include, e.g., poly(acrylate) polymers (see, e.g., US Pat. Pub. No. 20150104408), poly(vinyl ester) polymers (see, e.g., US Pat. Pub. No.20150110732) and certain polypeptides. In some embodiments the delivery polymer is a reversibly masked membrane active polymer. In some embodiments the inhibitory RNA or polymer, or both, has a targeting moiety conjugated thereto. In some embodiments an inhibitory RNA or an inhibitory RNA-targeting moiety conjugate is co-administered with a delivery polymer but is not conjugated to the polymer. “Co-administered” in this context meansthat the inhibitory RNA and the delivery polymer are administered to the subject such that they are present in the subject during overlapping time periods. The inhibitory RNA-targeting moiety conjugate and the delivery polymer may be administered simultaneously or they may be delivered sequentially. For simultaneous administration, they may be mixed prior to administration. For 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 sufficiently close together in time such that cytoplasmic delivery of the inhibitory RNA to cells is enhanced relative to 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 has a targeting moiety attached thereto that targets the polymer to cells to which enhanced cytoplasmic delivery of the inhibitory RNA is desired. The inhibitory RNA may be targeted to the same cells, optionally using the same targeting moiety, i.e. , the inhibitory RNA may be administered as an inhibitory RNA-targeting moiety conjugate. As used herein, membrane active polymers are surface active, amphipathic polymers that are able to induce one or more of the following effects upon a biological membrane: an alteration or disruption of the membrane that allows non-membrane permeable molecules to enter a cell or cross the membrane, pore formation in the membrane, fission of membranes, or disruption or dissolving of the membrane. As used herein, a membrane, or cell membrane, comprises a lipid bilayer. The alteration or disruption of the membrane can be functionally defined by the polymer's activity in at least one the following assays: red blood cell lysis (hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis, and endosomal release. A membrane active polymer may enhance delivery of a polynucleotide to a cell by disrupting or destabilizing the plasma membrane or an internal vesicle membrane (such as an endosome or lysosome), e.g., by forming a pore in the membrane, or disrupting endosomal or lysosomal vesicles thereby permitting release of the contents of the vesicle into the cell cytoplasm. In some embodiments the targeted reversibly masked membrane active polymer is an endosomolytic polymer. Endosomolytic polymers are polymers that, in response to a change in pH, are able to cause disruption or lysis of an endosome or otherwise provide for release of a normally cell membrane impermeable compound, such as a polynucleotide or protein, from a cellular internal membrane-enclosed vesicle, such as an endosome or lysosome. In some embodiments the polymer is a reversibly modified amphipathic membrane active polyaminewherein reversible modification inhibits membrane activity, neutralizes the polyamine to reduce positive charge and form a near neutral charge polymer. The reversible modification may also provide cell-type specific targeting and / or inhibit non-specific interactions of the polymer. The polyamine may be reversibly modified through reversible modification of amines on the polyamine. The reversibly masked membrane active polymer is substantially not membrane active when masked but becomes membrane active upon unmasking. Masking moieties are generally covalently bound to the membrane active polymer through physiologically reversible linkages. By using physiologically reversible linkages, the masking moieties can be cleaved from the polymer in vivo, thereby unmasking the polymer and restoring activity of the unmasked polymer. By choosing an appropriate reversible linkage, the activity of the membrane active polymer is restored after the conjugate has been delivered or targeted to a desired cell type or cellular location. Reversibility of the linkages provides for selective activation of the membrane active polymer. The physiologically reversible bond is reversible under mammalian intracellular conditions, which include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those found in mammalian cells. In some embodiments a targeting moiety, e.g., an ASGPR targeting moiety may serve as a masking moiety. In some embodiments the ASGPR targeting moiety has a lipophilic moiety conjugated thereto. Exemplary targeting moieties (e.g., ASGPR targeting moieties), physiologically labile bonds (e.g., enzymatically labile bonds, pH labile bonds), masking moieties, membrane active polymers (e.g., endosmolytically active polymers), lipophilic moieties, RNAi agent-targeting moiety conjugates, delivery agent-targeting moiety conjugates, conjugates comprising an RNAi agent, targeting moiety, and delivery agent, and methods of delivering nucleic acids to cells (e.g., liver cells) are described in US Pat. App. Pub. Nos. 20130245091 ,20130317079, 20120157509, 20120165393, 20120172412, 20120230938, 20140135380, 20140135381 , 20150104408, and 20150110732. In some embodiments an inhibitory RNA is co-administered with a mellitin peptide, e.g., as described in US Pat. App. Pub. No. 20120165393. The inhibitory RNA, mellitin peptide, or both, may have a targeting moiety conjugated thereto, optionally via a reversible linkage. In some embodiments a masking moiety comprises a dipeptide-amidobenzyl-carbonate or disubstituted maleic anhydride masking moiety e.g., as described in US Pat. App. Pub. No. 20150110732.

[0112] In some embodiments an inhibitory RNA may be administered in “naked” form, i.e. , administered in the absence of a delivery agent. The naked inhibitory RNA may be in a suitable buffer solution. The buffer solution may, for example, comprise acetate, citrate,prolamine, carbonate, or phosphate, or any combination thereof. In some embodiments the buffer solution is phosphate buffered saline (PBS) or artificial cerebrospinal fluid. The pH and osmolarity of the buffer solution can be adjusted such that it is suitable for administering to a subject. In some embodiments an inhibitory RNA is administered not in physical association with a lipid or lipid-containing particle. In some embodiments an inhibitory RNA is administered not in physical association with a nanoparticle or microparticle. In some embodiments an inhibitory RNA is administered not in physical association with a cationic polymer. In some embodiments an inhibitory RNA is administered not in physical association with cyclodextrin. In some embodiments an inhibitory RNA administered in “naked” form comprises a targeting moiety.

[0113] Inhibitory RNAs (e.g., an siRNA or miRNA described herein), or a vector comprising a nucleotide sequence encoding an siRNA or miRNA described herein, can be incorporated into pharmaceutical compositions. Such pharmaceutical compositions are useful for, among other things, administration and delivery to a subject in vivo or ex vivo. In some embodiments, pharmaceutical compositions also contain a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent, e.g., a pharmaceutical agent that does not itself induce an immune response harmful to the individual receiving the composition, and which may be administered without undue toxicity. As used herein the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which 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, sugars and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Nonlimiting examples may include MgCI2, CaCI2, KCI, NaCI, Na3PO4. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0114] Pharmaceutical compositions may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding, free base forms. In some embodiments, a pharmaceutical composition may be a lyophilized powder.

[0115] Pharmaceutical compositions can include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, 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 pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.

[0116] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[0117] Compositions suitable for parenteral or local administration can comprise aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood or other local biological fluid of the intended recipient. Non-limiting illustrative examples include water, buffered saline, Hanks’ solution, Ringer’s solution, aCSF, dextrose, fructose, ethanol, animal, vegetable or synthetic oils. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate 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, the suspension may also contain suitable stabilizers or agents which increase the solubility to allow for the preparation of highly concentrated solutions.

[0118] Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0119] After pharmaceutical compositions have been prepared, they may be placed in an appropriate container and labeled for treatment. Such labeling can include amount, frequency, and method of administration.

[0120] Pharmaceutical compositions and delivery systems appropriate for the compositions, methods and uses of the disclosure are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy. 21st Edition. Philadelphia, Pa. Lippincott Williams & Wilkins, 2005).

[0121] The disclosure also provides methods for introducing inhibitory RNAs (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 an animal. In some embodiments, such methods include contacting a subject (e.g., a cell or tissue of a subject) with, or administering to a subject (e.g., a subject such as a mammal), an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein), such that the inhibitory RNA is expressed in the subject (e.g., in a cell or tissue of a subject). In another embodiment, a method includes providing cells of an individual (patient or subject such as a mammal) with an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein), such that the inhibitory RNA is expressed in the individual.

[0122] Compositions of an inhibitory RNA described herein (or a vector (e.g., an rAAV vector) comprising a nucleotide sequence encoding a inhibitory RNA described herein) can be administered in a sufficient or effective amount to a subject in need thereof. Doses can vary and depend upon the type, onset, progression, severity, frequency, duration, or probability of the disease to which treatment is directed, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.

[0123] The dose to achieve a therapeutic effect, e.g., the dose in vector genomes / per kilogram of body weight (vg / kg) (e.g., in the case of vector-based delivery) or mg / kg of bodyweight (mg / kg), will vary based on several factors including, but not limited to: route of administration, the level of inhibitory RNA expression required to achieve a therapeutic effect, the specific disease treated, any host immune response to the viral vector, a host immune response to the heterologous inhibitory RNA, and the stability of the inhibitory RNA expressed. One skilled in the art can determine, for vector-based deliveries of the inhibitorRNAs, a rAAV / vector genome dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors. Generally, doses will range from at least 1 *108, or more, for example, 1 x109, 1 x1010, 1 xio11, 1 xio12, 1 xio13, 1 x 1 o14, or more, vector genomes per kilogram (vg / kg) of the weight of the subject, to achieve a therapeutic effect.

[0124] In some embodiments, compositions of an inhibitory RNA are administered to a subject in an amount that is 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 0.1 mg / kg, about 0.3 mg / kg, 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 between 0.01 mg / kg and 0.1 mg / kg, between 0.01 mg / kg and 0.1 mg / kg, between 0.1 mg / kg and 1.0 mg / kg, between 1.0 mg / kg and 2.5 mg / kg, between 2.5 mg / kg and 5.0 mg / kg, between 5.0 mg / kg and 10 mg / kg, between 10 mg / kg and 20 mg / kg, between 20 mg / kg and 30 mg / kg, between 30 mg / kg and 40 mg / kg or between 40 mg / kg and 50 mg / kg. In some embodiments a fixed dose is administered. In some embodiments the dose is between 1 mg and 1.0 g, e.g., between 1 mg and 10 mg, between 10 mg and 20 mg, between 20 mg and 40 mg, between 40 mg and 80 mg, between 80 mg and 160 mg, between 160 mg and 320 mg, between 320 mg and 640 mg, between 640 mg and 1 g. In some embodiments the dose 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. In some embodiments the dose is a daily dose. In some embodiments the dose is administered according to a dosing regimen with a dosing interval of at least 2 days, e.g., at least 7 days, e.g., about 2, 3, 4, 6, or 8 weeks. For example, in some embodiments, the inhibitory RNA composition is administered according to a dosing regimen 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 longer. In some embodiments, any of the doses and / or dosing regimens described herein are administered subcutaneously. In some embodiments, the inhibitory RNA composition is administered once and levels of inhibition are subsequently measured, and once the level ofinhibition decreases to a certain level, a subsequent dose of the inhibitory composition is administered.

[0125] In some embodiments, a subject exhibits a sustained inhibition of C3, e.g., measured by C3 mRNA expression (e.g., in liver tissue, e.g., a liver biopsy) for a period of time that is at least 2 days, e.g., at least 7 days, e.g., at least about 2, 3, 4, 6, 8, 10, 12, 16, or 20 weeks post-administration. In some embodiments, a subject exhibits a reduced level of C3 protein in a biological fluid or tissue, and the reduced level of C3 protein is maintained for a period of time that is at least 2 days, e.g., at least 7 days, e.g., at least about 2, 3, 4, 6, 8, 10, 12, 16, or 20 weeks post-administration.

[0126] An effective amount or a sufficient amount can (but need not) be provided in a single administration, may require multiple administrations, and can (but need not) be, administered alone or in combination with another composition (e.g., another complement inhibitor described herein). For example, the amount may be proportionally increased as indicated by the need of the subject, type, status and severity of the disease treated or side effects (if any) of treatment. Amounts considered effective also include amounts that result in a reduction of the use of another treatment, therapeutic regimen or protocol, such as administration of another complement inhibitor described herein.

[0127] Accordingly, pharmaceutical compositions of the disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve the intended therapeutic purpose. Determining a therapeutically effective dose is well within the capability of a skilled medical practitioner using the techniques and guidance provided in the disclosure. Therapeutic doses can 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 control sequences regulating the expression levels of an inhibitory RNA described herein.Thus, a therapeutically effective amount in humans will fall in a relatively broad range that may be determined by a medical practitioner based on the response of an individual patient to vector-based treatment. Pharmaceutical compositions may be delivered to a subject, so as to allow production of an inhibitory RNA described herein in vivo by gene- and or cell-based therapies or by ex-vivo modification of the patient's or donor's cells.

[0128] Methods and uses of the disclosure include delivery and administration systemically, regionally or locally, or by any route, for example, by injection or infusion. Delivery of a pharmaceutical composition in vivo may generally be accomplished via injection using a conventional syringe, although other delivery methods such as convection-enhanced delivery can also be used (see, e.g., U.S. Pat. No. 5,720,720). For example, compositionsmay be delivered subcutaneously, epidermally, intradermally, intrathecally, intraorbitally, intramucosally, intraperitoneally, intravenously, intra-pleurally, intraarterially, orally, intrahepatically, intracerebroventricularly (e.g., via intracerebroventricular injection), via the portal vein, or intramuscularly. Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications. A clinician specializing in the treatment of patients with complement-mediated disorders may determine the optimal route for administration of inhibitory RNAs (e.g., an siRNA or miRNA described herein), or a vector comprising a nucleotide sequence encoding an siRNA or miRNA described herein.

[0129] In some embodiments, an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein) may be administered to a subject once daily, weekly, every 2, 3, or 4 weeks, or even at longer intervals. In some embodiments, an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding inhibitory RNA described herein) may be administered according to a dosing regimen that includes (i) an initial administration that is once daily, weekly, every 2, 3, or 4 weeks, or even at longer intervals; followed by (ii) a period of no administration of, e.g., 1 , 2, 3, 4, 5, 6, 8, or 10 months, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein may be administered (i) one or more times during an initial time period of up to 2, 4, or 6 weeks or less; followed by (ii) a period of no administration of, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, a subject is monitored before and / or following treatment for level of C3 expression and / or activity, e.g., as measured using an alternative pathway assay, a classical pathway assay, or both. Suitable assays are known in the art and include, e.g., a hemolysis assay. In some embodiments, a subject is treated, or is retreated, if a measured level of C3 expression and / or activity is more than 10%, 20%, 30%, 40%, 50%, 100%, 200%, or more, relative to measured level of C3 expression and / or in a control subject.V. Diseases, Disorders, and Conditions

[0130] In some embodiments, an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein) is administered to a subject suffering from or at risk of complement-mediated damage to an organ, tissue, or cells. In some embodiments, an inhibitory RNA described herein (or a vector comprising a nucleotide encoding an inhibitory RNA described herein) is administered in combination with one or more additional complement inhibitors to a subject suffering from or at risk of complement-mediated damage to an organ, tissue, or cells. In some embodiments, aninhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein) is contacted with an organ, tissue, or cells ex vivo. The organ, tissue, or cells can be introduced into a subject and can be protected from damage that would otherwise be caused by the recipient’s complement system.

[0131] Certain uses of interest include: (1) protecting red blood cells (RBCs) from complement-mediated damage in individuals with disorders such as paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome or other disorders characterized by complement-mediated RBC lysis; (2) protecting transplanted organs, tissues, and cells from complement-mediated damage; (3) reducing ischemia / reperfusion (l / R) injury (e.g., in individuals suffering from trauma, vascular obstruction, myocardial infarction, or other situations in which l / R injury may occur); and (4) protecting various body structures (e.g., the retina) or membranes (e.g., synovial membrane) that may be exposed to complement components from complement mediated damage in any of a variety of different complement- mediated disorders. The beneficial effects of inhibiting complement activation at the surface of cells or other body structures are not limited to those resulting directly from protection of the cells or structures themselves against direct complement-mediated damage (e.g., preventing cell lysis). For example, inhibiting complement activation may reduce the generation of anaphylotoxins and resulting influx / activation of neutrophils and other pro-inflammatory events and / or reduce potentially damaging release of intracellular contents, thereby potentially having beneficial effects on remote organ systems or throughout the body.A. Blood Cell Protection

[0132] In some embodiments, 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, is used to protect blood cells against complement-mediated damage. The blood cells may be any cellular component of the blood, e.g., red blood cells (RBCs), white blood cells (WBCs), and / or platelets. A variety of disorders are associated with complement-mediated damage to blood cells. Such disorders can result, for example, from deficiencies or defects in one or more of an individual’s cellular or soluble CRPs, e.g., due to (a) mutation(s) in the gene(s) encoding such proteins; (b) mutation(s) in genes required for production or proper function of one or more CRPs, and / or (c) presence of autoantibodies to one or more CRPs. Complement-mediated RBC lysis can result from the presence of autoantibodies against RBC antigens which may arise due to a diverse set of causes (often being idiopathic). Individuals having such mutation(s) in genesencoding CRPs and / or having antibodies against CRPs or against their own RBCs are at increased risk of disorders involving complement-mediated RBC damage. Individuals who have had one or more episodes characteristic of a disorder are at increased risk of a recurrence.

[0133] Paroxysmal nocturnal hemoglobinuria (PNH) is a relatively rare disorder comprising an acquired hemolytic anemia characterized by complement-mediated intravascular hemolysis, hemoglobinuria, bone marrow failure, and thrombophilia (propensity to develop blood clots). It affects an estimated 16 individuals per million worldwide, occurs in both sexes, and can arise at any age, frequently striking young adults (Bessler, M. & Hiken, J., Hematology Am Soc Hematol Educ Program, 104-110 (2008); Hillmen, P. Hematology Am Soc Hematol Educ Program, 116-123 (2008)). PNH is a chronic and debilitating disease punctuated by acute hemolytic episodes and results in significant morbidities and reduced life expectancy. In addition to anemia, many patients experience abdominal pain, dysphagia, erectile dysfunction, and pulmonary hypertension, and are at increased risk of renal failure and thromboembolic events.

[0134] PNH was first described as a distinct entity in the 1800s, but it was only in the 1950s, with discovery of the alternative pathway of complement activation, that the cause of hemolysis in PNH was firmly established (Parker C J. Paroxysmal nocturnal hemoglobinuria: an historical overview. Hematology Am Soc Hematol Educ Program. 93-103 (2008)). CD55 and CD59 are normally attached to the cell membrane via glycosyl phosphatidylinositol (GPI) anchors (glycolipid structures that anchor certain proteins to the plasma membrane). PNH arises as a consequence of nonmalignant clonal expansion of hematopoietic stem cell(s) that have acquired a somatic mutation in the PIGA gene, which encodes a protein involved in synthesis of GPI anchors (Takeda J, et al. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 73:703-711 (1993)). Progeny of such stem cells are deficient in GPI-anchored proteins, including CD55 and CD59. This defect renders these cells susceptible to complement-mediated RBC lysis. Flow cytometric analysis using antibodies to GPI-anchored proteins is often used for diagnosis. It detects deficiency of GPI-anchored proteins at the cell surface and allows determination of the degree of deficiency and the proportion of affected cells (Brodsky R A. Advances in the diagnosis and therapy of paroxysmal nocturnal hemoglobinuria. Blood Rev. 22(2):65-74 (2008). PNH type III RBCs are completely deficient in GPI-linked proteins and are highly sensitive to complement whereas PNH type II RBCs have a partial deficiency and are less sensitive. FI.AER is a fluorescently labeled inactive variant of proaerolysin (a bacterialtoxin that binds GPI anchors) and is increasingly used together with flow cytometry for diagnosis of PNH. Lack of binding of FLAER to granulocytes is sufficient for diagnosis of PNH. In some embodiments, an inhibitory RNA described herein (or a vector encoding an inhibitory RNA described herein), alone or in combination with one or more additional complement inhibitors described herein, protects PNH RBCs, from deposition of C3b. In some embodiments an inhibitory RNA described herein (or a vector encoding an inhibitory RNA described herein), alone or in combination with one or more additional complement inhibitors described herein, inhibits intravascular and extravascular hemolysis in a subject suffering from PNH.

[0135] In some embodiments, 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, is administered to a subject suffering from atypical hemolytic syndrome (aHUS). aHUS is a chronic disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure and is caused by inappropriate complement activation, often due to mutations in genes encoding complement regulatory proteins (Warwicker, P., et al. Kidney Int 53, 836-844 (1998); Kavanagh, D. & Goodship, T. Pediatr Nephrol 25, 2431-2442 (2010). Mutations in the complement factor H (CFH) gene are the most common genetic abnormality in patients with aHUS, and 60-70% of these patients die or reach end stage renal failure within one year after disease onset (Kavanagh & Goodship, supra.) Mutations in factor I, factor B, C3, factor H- related proteins 1-5, and thrombomodulin have also been described. Other causes of aHUS include autoantibodies against complement regulatory proteins such as CFH. In some embodiments, 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, is administered to a subject that has been identified as having a mutation in factor I, factor B, C3, factor H-related proteins 1-5, or thrombomodulin or has been identified as having antibodies against a complement regulatory protein, e.g., CFH.

[0136] Complement-mediated hemolysis occurs in a diverse group of other conditions including autoimmune hemolytic anemias that involve antibodies that bind to RBCs and lead to complement-mediated hemolysis. For example, such hemolysis can occur in primary chronic cold agglutinin disease and certain reactions to drugs and other foreign substances (Berentsen, S., et al., Hematology 12, 361-370 (2007); Rosse, W. F., Hillmen, P. & Schreiber, A. D. Hematology Am Soc Hematol Educ Program, 48-62 (2004)). In some embodiments, aninhibitory 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, is administered to a subject suffering from or at risk of chronic cold agglutinin disease. In another embodiment, 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, is used to treat a subject suffering from or at risk of the HELLP syndrome, which is defined by the existence of hemolysis, elevated liver enzymes, and low platelet count and is associated with mutations in complement regulatory protein(s) in at least some subjects (Fakhouri, F., et al., 112: 4542-4545 (2008)).

[0137] In some embodiments, 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, is administered to a subject suffering from or at risk of warm autoimmune hemolytic anemia.

[0138] In other embodiments, 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, is used to protect RBCs or other cellular components of blood to be transfused into a subject. Certain examples of such uses are discussed further in below.B. Transplantation

[0139] Transplantation is a therapeutic approach of increasing importance, providing a means to replace organs and tissues that have been damaged through trauma, disease, or other conditions. Kidneys, liver, lungs, pancreas, and heart are among the organs that can be successfully transplanted. Tissues that are frequently transplanted include bones, cartilage, tendons, cornea, skin, heart valves, and blood vessels. Pancreatic islet or islet cell transplantation is a promising approach for treatment of diabetes, e.g., type I diabetes. For purposes of the present disclosure, an organ, tissue, or cell (or population of cells) that is be transplanted, is being transplanted, or has been transplanted may be referred to as a “graft”. For purposes hereof, a blood transfusion is considered a “graft”.

[0140] Transplantation subjects the graft to a variety of damaging events and stimuli that can contribute to graft dysfunction and, potentially, failure. For example, ischemia-reperfusion (l / R) injury is a common and significant cause of morbidity and mortality in the case of many grafts (particularly solid organs) and can be a major determinant of likelihood of graft survival.Transplant rejection is one of the major risks associated with transplants between genetically different individuals and can lead to graft failure and a need to remove the graft from the recipient.

[0141] In some embodiments, 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, is used to protect a graft from complement-mediated damage. For example, a cell-reactive compstatin analog reacts with cells of the graft, becomes covalently attached thereto, and inhibits complement activation. A cell-targeted compstatin analog binds to a target molecule in the graft (e.g., expressed by endothelial cells or other cells in the graft) and inhibits complement activation. A target molecule may be, e.g., is a molecule whose expression is induced or stimulated by a stimulus such as injury or inflammation, molecule that would be recognized as “non-self’ by the recipient, a carbohydrate xenoantigen to which antibodies are commonly found in human beings such as a blood group antigen or a xenoantigen, e.g., a molecule comprising an alphagal epitope. In some embodiments, a reduction in complement activation can be demonstrated by a reduction in average C4d deposition in blood vessels of grafts that have been contacted 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, as compared with the average level of C4d deposition in grafts that have not been contacted 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 (e.g., in subjects who are matched with respect to the grafts and other therapy that they receive).

[0142] A graft can be contacted 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, that inhibits C3 expression prior to, during, and / or after being transplanted, in various embodiments of the disclosure. For example, prior to transplantation a graft removed from a donor can be contacted with a liquid comprising a cell-reactive, long-acting, or targeted compstatin analog. For example, the graft can be bathed in and / or perfused with the solution. In another embodiment, 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, is administered to a donor prior toremoval of the graft. In some embodiments, 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, is administered to a recipient during and / or after the introduction of the graft. In some embodiments, 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, is delivered locally to the transplanted graft. In some embodiments a cell-reactive, long-acting, or targeted compstatin analog is administered systemically, e.g., intravenously or subcutaneously. In some embodiments, 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, is administered to a recipient prior to the introduction of the graft. In some embodiments the subject receives one or more additional doses of the inhibitory RNA, vector encoding the inhibitory RNA, and / or one or more additional complement inhibitors after receiving the graft.

[0143] The disclosure provides a composition comprising: (a) an isolated graft; and (b) an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein) that inhibits C3 expression. The disclosure also provides a composition comprising: (a) an isolated graft; (b) a cell-reactive, long-acting, or targeted compstatin analog and (c) an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein) that inhibits C3 expression. In some embodiments the composition further comprises a liquid solution suitable for contacting (e.g., suitable for rinsing, washing, bathing, perfusing, maintaining, or storing) a graft (e.g., an organ) such as an isolated graft that has been removed from a donor and is awaiting transplantation to a recipient. In some embodiments the disclosure provides a composition comprising: (a) a liquid solution suitable for contacting a graft (e.g., an organ); and (b) an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein) that inhibits C3 expression. In some embodiments the composition further comprises a cell-reactive, long-acting, or targeted compstatin analog. The liquid solution can be any liquid solution that is physiologically acceptable to the graft (e.g., appropriate osmotic composition, non-cytotoxic) and medically acceptable in view of the subsequent introduction of the graft into the recipient (e.g., preferably sterile or at least reasonably free from microorganisms or other contaminants) and compatible with the cell-reactive compstatin analog (i.e., will not destroy the reactivity of thecompstatin analog) or compatible with the long-acting or targeted compstatin analog. In some embodiments, a solution is any solution known in the art for any such purposes. In some embodiments, a liquid solution is Marshall’s or Hyperosmolar Citrate (Soltran®, Baxter Healthcare), University of Wisconsin (UW) solution (ViaSpan™, Bristol Myers Squibb), Histidine Tryptophan Ketoglutarate (HTK) solution (Custodial®, Kohler Medical Limited), EuroCollins (Fresenius), and Celsior® (Sangstat Medical), Polysol, IGL-1 , or AQIX® RS-1. Of course other solutions, e.g., containing equivalent or similar ingredients in the same or different concentrations could be used within the scope of physiologically acceptable compositions. In some embodiments a solution does not contain ingredient(s) with which the cell-reactive compstatin analog would be expected to significantly react, and any solution may be modified or designed to lack such ingredients. In some embodiments, the cell-reactive compstatin analog is present in the graft-compatible solution at a concentration of, e.g., between 0.01 mg / ml and 100 mg / ml or may be added to the solution to achieve such concentration.

[0144] In some embodiments, a graft is or comprises a solid organ such as a kidney, liver, lung, pancreas, or heart. In some embodiments, a graft is or comprises bone, cartilage, fascia, tendon, ligament, cornea, sclera, pericardium, skin, heart valve, blood vessel, amniotic membrane, or dura mater. In some embodiments, a graft comprises multiple organs such as a heart-lung or pancreas-kidney graft. In some embodiments, a graft comprises less than a complete organ or tissue. For example, a graft may contain a portion of an organ or tissue, e.g., a liver lobe, section of blood vessel, skin flap, or heart valve. In some embodiments, a graft comprises a preparation comprising isolated cells or tissue fragments that have been isolated from their tissue of origin but retain at least some tissue architecture, e.g., pancreatic islets. In some embodiments, a preparation comprises isolated cells that are not attached to each other via connective tissue, e.g., hematopoietic stem cells or progenitor cells derived from peripheral and / or cord blood, or whole blood or any cell-containing blood product such as red blood cells (RBCs) or platelets. In some embodiments a graft is obtained from a deceased donor (e.g., a “donation after brain death” (DBD) donor or “donation after cardiac death” donor). In some embodiments, depending on the particular type of graft, a graft is obtained from a living donor. For example, kidneys, liver sections, blood cells, are among the types of grafts that can often be obtained from a living donor without undue risk to the donor and consistent with sound medical practice.

[0145] In some embodiments, a graft is a xenograft (i.e. , the donor and recipient are of different species). In some embodiments a graft is an autograft (i.e., a graft from one part ofthe body to another part of the body in the same individual). In some embodiments, a graft is an isograft (i.e. , the donor and recipient are genetically identical). In most embodiments, the graft is an allograft (i.e., the donor and recipient are genetically non-identical members of the same species). In the case of an allograft, the donor and recipient may or may not be genetically related (e.g., family members). Typically, the donor and recipient have compatible blood groups (at least ABO compatibility and optionally Rh, Kell and / or other blood cell antigen compatibility). The recipient’s blood may have been screened for alloantibodies to the graft and / or the recipient and donor since the presence of such antibodies can lead to hyperacute rejection (i.e., rejection beginning almost immediately, e.g., within several minutes after the graft comes into contact with the recipient’s blood). A complement-dependent cytotoxicity (CDC) assay can be used to screen a subject’s serum for anti-HLA antibodies. The serum is incubated with a panel of lymphocytes of known HLA phenotype. If the serum contains antibodies against HLA molecules on the target cells, cell death due to complement- mediated lysis occurs. Using a selected panel of target cells allows one to assign specificity to the detected antibody. Other techniques useful for determining the presence or absence anti- HLA antibodies and, optionally, determining their HLA specificity, include ELISA assays, flow cytometry assays, microbead array technology (e.g., Luminex technology). The methodology for performing these assays is well known, and a variety of kits for performing them are commercially available.

[0146] In some embodiments 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, inhibits complement- mediated rejection. For example, in some embodiments, 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, inhibits hyperacute rejection. Hyperacute rejection is caused at least in part by antibody- mediated activation of the recipient’s complement system via the classical pathway and resulting MAC deposition on the graft. It typically results from the presence in the recipient of pre-existing antibodies that react with the graft. While it is desirable to attempt to avoid hyperacute rejection by appropriate matching prior to transplantation, it may not always possible to do so due, e.g., to time and / or resource constraints. Furthermore, some recipients (e.g., multiply transfused individuals, individuals who have previously received transplants, women who have had multiple pregnancies) may already have so many pre-formed antibodies, potentially including antibodies to antigens that are not typically tested for, that itcan be difficult or perhaps almost impossible to obtain with confidence a compatible graft in a timely manner. Such individuals are at increased risk of hyperacute rejection.

[0147] In some embodiments, 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, inhibits acute rejection or graft failure. As used herein, “acute rejection” refers to rejection occurring between at least 24 hours, typically at least several days to a week, after a transplant, up to 6 months after the transplant. Acute antibody-mediated rejection (AMR) often involves an acute rise in donorspecific alloantibody (DSA) in the first few weeks after transplantation. Without wishing to be bound by any theory, it is possible that pre-existing plasma cells and / or the conversion of memory B cells to new plasma cells play a role in the increased DSA production. Such antibodies can result in complement-mediated damage to the graft, which can be inhibited by contacting the graft with a cell-reactive compstatin analog. Without wishing to be bound by any theory, inhibiting complement activation at the graft may reduce leukocyte (e.g., neutrophil) infiltration, another contributor to acute graft failure.

[0148] In some embodiments, 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, inhibits complement- mediated l / R injury to a graft. As discussed further below, l / R injury can occur upon reperfusion of tissue whose blood supply has been temporarily disrupted, as occurs in transplanted organs. Reducing l / R injury would reduce the likelihood of acute graft dysfunction or reduce its severity, and reduce the likelihood of acute graft failure.

[0149] In some embodiments, 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, inhibits chronic rejection and / or chronic graft failure. As used herein, “chronic rejection or graft failure” refers to rejection or failure occurring at least 6 months post-transplant, e.g., between 6 months and 1 , 2, 3, 4, 5 years, or more post-transplant, often after months to years of good graft function. It is caused by a chronic inflammatory and immune response against the graft. For purposes hereof, chronic rejection can include chronic allograft vasculopathy, a term used to refer to fibrosis of the internal blood vessels of the transplanted tissue. As immunosuppressive regimens have reduced the incidence of acute rejection, chronic rejection is becoming more prominent as a cause of graft dysfunction and failure. There is increasing evidence that B-cell production of alloantibody is an important element in the genesis of chronic rejection and graftfailure (Kwun J. and Knechtle S J, Transplantation, 88(8):955-61 (2009). Earlier damage to the graft may be a contributing factor leading to chronic processes such as fibrosis that can ultimately lead to chronic rejection. Thus, inhibiting such earlier damage using a cell-reactive compstatin analog may delay and / or reduce the likelihood or severity of chronic graft rejection.

[0150] In some embodiments, 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, is administered to a graft recipient to inhibit graft rejection and / or graft failure.C. Ischemia / Reperfusion Injury

[0151] Ischemia-reperfusion (l / R) injury is an important cause of tissue damage following trauma and in other conditions associated with temporary disruption of blood flow such as myocardial infarction, stroke, severe infection, vascular disease, aneurysm repair, cardiopulmonary bypass, and transplantation.

[0152] In the setting of trauma, systemic hypoxemia, hypotension, and local interruption of the blood supply resulting from contusions, compartment syndrome, and vascular injuries cause ischemia that damages metabolically active tissues. Restoration of the blood supply triggers an intense systemic inflammatory reaction that is often more harmful than the ischemia itself. Once the ischemic region is reperfused, factors that are produced and released locally enter the circulatory system and reach remote locations, sometimes causing significant damage to organs not affected by the original ischemic insult, such as the lungs and intestine, leading to single and multiple organ dysfunction. Complement activation occurs soon after reperfusion and is a key mediator of post-ischemic damage, both directly and through its chemoattractive and stimulatory effects on neutrophils. All three major complement pathways are activated and, acting cooperatively or independently, are involved in l / R related adverse events affecting numerous organ systems. In some embodiments of the disclosure, 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, is administered to a subject who has recently (e.g., within the preceding 2, 4, 8, 12, 24, or 48 hours) experienced trauma, e.g., trauma that puts the subject at risk of l / R injury, e.g., due to systemic hypoxemia, hypotension, and / or local interruption of the blood supply. In some embodiments the cell-reactive compstatin analog may be administered intravascularly, optionally into a blood vessel that supplies an injuredbody part or directly to the body part. In some embodiments, the subject suffers from spinal cord injury, traumatic brain injury, burn, and / or hemorrhagic shock.

[0153] In some embodiments, 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, is administered to a subject prior to, during, or after a surgical procedure, e.g., a surgical procedure that is expected to temporarily disrupt blood flow to a tissue, organ, or portion of the body. Examples of such procedures include cardiopulmonary bypass, angioplasty, heart valve repair / replacement, aneurysm repair, or other vascular surgeries. 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, may be administered prior to, after, and / or during an overlapping time period with the surgical procedure.

[0154] In some embodiments, 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, is administered to a subject who has suffered an Ml, thromboembolic stroke, deep vein thrombosis, or pulmonary embolism. 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, may be administered in combination with a thrombolytic agent such as tissue plasminogen activator (tPA) (e.g., alteplase (Activase), reteplase (Retavase), tenecteplase (TNKase)), anistreplase (Eminase), streptokinase (Kabikinase, Streptase), or urokinase (Abbokinase). 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, may be administered prior to, after, and / or during an overlapping time period with the thrombolytic agent.

[0155] In some embodiments, 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, is administered to a subject to treat l / R injury.D. Other Complement-Mediated Disorders

[0156] In some embodiments, 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, may be administered systemically or locally introduced into the eye for treatment of an eye disorder such as macular degeneration (e.g., age-related macular degeneration (AMD) and Stargardt macular dystrophy), diabetic retinopathy, glaucoma, or uveitis. For example, 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, may be administered systemically, or introduced into the vitreous cavity (e.g., by intravitreal injection) or introduced into the subretinal space (e.g., by subretinal injection), for treatment of a subject suffering from or at risk of AMD. In some embodiments the AMD is neovascular (wet) AMD. In some embodiments the AMD is dry AMD. As will be appreciated by those of ordinary skill in the art, dry AMD encompasses geographic atrophy (GA), intermediate AMD, and early AMD. In some embodiments, a subject with GA is treated in order to slow or halt progression of the disease. For example, in some embodiments, treatment of a subject with GA reduces the rate of retinal cell death. A reduction in the rate of retinal cell death may be evidenced by a reduction in the rate of GA lesion growth 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, as compared with control (e.g., patients given a sham administration). In some embodiments, a subject has intermediate AMD. In some embodiments, a subject has early AMD. In some embodiments, a subject with intermediate or early AMD is treated in order to slow or halt progression of the disease. For example, in some embodiments, treatment of a subject with intermediate AMD may slow or prevent progression to an advanced form of AMD (neovascular AMD or GA). In some embodiments, treatment of a subject with early AMD may slow or prevent progression to intermediate AMD. In some embodiments an eye has both GA and neovascular AMD. In some embodiments an eye has GA but not wet AMD. In some embodiments, 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, is administered to the suprachoroidal space, e.g., by suprachoroidal injection, for treatment of an eye disorder such as macular degeneration (e.g., age-related macular degeneration (AMD) and Stargardt macular dystrophy), diabeticretinopathy, glaucoma, or uveitis. In some embodiments 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, is administered, e.g., by intravitreal injection or subretinal injection to treat glaucoma, uveitis (e.g., posterior uveitis), or diabetic retinopathy. In some embodiments 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, is introduced into the anterior chamber, e.g., to treat anterior uveitis.

[0157] In some embodiments 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, is used to treat a subject suffering from or at risk of an autoimmune disease, e.g., an autoimmune disease mediated at least in part by antibodies against one or more self antigens.

[0158] 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 may be introduced into the synovial cavity, e.g., in a subject suffering from arthritis (e.g., rheumatoid arthritis).

[0159] In some embodiments, 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, is used to treat a subject suffering from or at risk of an intracerebral hemorrhage.

[0160] In some embodiments 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, is used to treat a subject suffering from or at risk of myasthenia gravis (e.g., generalized myasthenia gravis), chronic inflammatory demyelinating polyneuropathy (CIDP), or multifocal motor neuropathy (MMN).

[0161] In some embodiments 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, is used to treat a subject suffering from or at risk of hidratenitis suppurativa.

[0162] In some embodiments 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, is used to treat a subject suffering from or at risk of immune-mediated necrotizing myopathy.

[0163] In some embodiments 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, is used to treat a subject suffering from or at risk of neuromyelitis optica (NMO).

[0164] In some embodiments 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, is used to treat a subject suffering from or at risk of a disorder affecting the kidney, e.g., the glomeruli of the kidney. In some embodiments the disorder is membranoproliferative glomerulonephritis (MPGN), e.g., MPGN type I, MPGN type II, or MPGN type III. In some embodiments the disorder is IgA nephropathy (IgAN). In some embodiments the disorder is primary membranous nephropathy. In some embodiments the disorder is C3 glomerulopathy. In some embodiments the disorder is characterized by glomerular deposits containing one or more complement activation products, e.g., C3b, in the kidney. In some embodiments treatment as described herein reduces the level of such deposits. In some embodiments a subject suffering from a complement-mediated kidney disorder suffers from proteinuria (an abnormally high level of protein in the urine) and / or an abnormally low glomerular filtration rate (GFR). In some embodiments treatment as described herein results in decreased proteinuria and / or an increased or stabilized GFR.

[0165] In some embodiments, 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, is used to treat a subject suffering from or at risk of a neurodegenerative disease. In some embodiments, 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, is used to treat a subject suffering from neuropathic pain or at risk of developing neuropathic pain. In some embodiments, 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, is used to treat a subject suffering from or at risk of rhinosinusitis or nasal polyposis. In some embodiments, 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, is used to treat a subject suffering from or at risk of cancer, including neurologic cancers such as glioma, glioblastoma andleptomeningeal disease. In some embodiments, 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, is used to treat a subject suffering from or at risk of sepsis. In some embodiments, 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, is used to treat a subject suffering from or at risk of adult respiratory distress syndrome.

[0166] In some embodiments, 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, is used to treat a subject suffering from or at risk of anaphylaxis or infusion reaction. For example, in some embodiments, a subject may be treated prior to, during, or after receiving a drug or a vehicle that may cause anaphylaxis or infusion reaction. In some embodiments, a subject at risk of or suffering from anaphylaxis from a food (e.g., peanut, shellfish, or other food allergens), insect sting (e.g., bee, wasp), is 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.

[0167] 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, may be administered locally or systemically, in various embodiments of the disclosure.

[0168] In some embodiments, 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, is used to treat a respiratory disease.

[0169] In some aspects, methods of treating a complement-mediated disorder, e.g., a chronic complement-mediated disorder, are provided, the methods comprising administering 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, to a subject in need of treatment for the disorder. In some aspects, methods of treating a Th17-associated disorder are provided, the methods comprising administering an inhibitory RNA described herein (or a vector comprising a nucleotide sequence encoding an inhibitory RNA described herein), alone or in combinationwith one or more additional complement inhibitors described herein, to a subject in need of treatment for the disorder.

[0170] In some aspects, a “chronic disorder” is a disorder that persists for at least 3 months and / or is accepted in the art as being a chronic disorder. In many embodiments, a chronic disorder persists for at least 6 months, e.g., at least 1 year, or more, e.g., indefinitely. One of ordinary skill in the art will appreciate that at least some manifestations of various chronic disorders may be intermittent and / or may wax and wane in severity over time. A chronic disorder may be progressive, e.g., having a tendency to become more severe or affect larger areas over time. A number of chronic complement-mediated disorders are discussed herein. A chronic complement-mediated disorder may be any chronic disorder in which complement activation (e.g., excessive or inappropriate complement activation) is involved, e.g., as a contributing and / or at least partially causative factor. For convenience, disorders are sometimes grouped by reference to an organ or system that is often particularly affected in subjects suffering from the disorder. It will be appreciated that a number of disorders can affect multiple organs or systems, and such classification(s) are in no way limiting. Furthermore, a number of manifestations (e.g., symptoms) may occur in subjects suffering from any of a number of different disorders. Non-limiting information regarding disorders of interest herein may be found, e.g., in standard textbooks of internal medicine such as Cecil Textbook of Medicine (e.g., 23rd edition), Harrison's Principles of Internal Medicine (e.g., 17th edition), and / or standard textbooks focusing on particular areas of medicine, particular body systems or organs, and / or particular disorders.

[0171] In some embodiments, a chronic complement-mediated disorder is a Th2- associated disorder. As used herein, a Th2-associated disorder is a disorder characterized by an excessive number and / or excessive or inappropriate activity of CD4+ helper T cells of the Th2 subtype (“Th2 cells”) in the body or a portion thereof, e.g., in at least one tissue, organ, or structure. For example, there may be a predominance of Th2 cells relative to CD4+ helper T cells of the Th1 subtype (“Th1 cells”) e.g., in at least one tissue, organ, or structure affected by a disorder. As known in the art, Th2 cells typically secrete characteristic cytokines such as interleukin-4 (IL-4), interleukin-5 (IL- 5), and interleukin-13 (IL-13), while Th1 cells typically secrete interferon-7 (IFN-y) and tumor necrosis factor p (TNF 0). In some embodiments, a Th2-associated disorder is characterized by excessive production and / or amount of IL-4, IL-5, and / or IL-13, e.g., relative to IFN-y and / or TNF p e.g., in at least some at least one tissue, organ, or structure

[0172] In some embodiments, a chronic complement-mediated disorder is a Th17- associated disorder. In some aspects, as described in further detail in PCT / US2012 / 043845, filed Jun. 22, 2012, entitled “Methods of Treating Chronic Disorders with Complement Inhibitors,” complement activation and Th17 cells participate in a cycle that involves dendritic cells and antibodies and that contributes to maintenance of a pathologic immunologic microenvironment underlying a range of disorders. Without wishing to be bound by any theory, the pathologic immunologic microenvironment, once established, is self-sustaining and contributes to cell and tissue injury. In some aspects, long-acting compstatin analogs are of use to treat Th17-associated disorders.

[0173] As used herein, a Th17-associated disorder is a disorder characterized by an excessive number and / or excessive or inappropriate activity of CD4+ helper T cells of the Th17 subtype (“Th17 cells”) in the body or a portion thereof, e.g., in at least one tissue, organ, or structure. For example, there may be a predominance of Th17 cells relative to Th1 and / or Th2 cells, e.g., in at least one tissue, organ, or structure affected by a disorder. In some embodiments a predominance of Th17 cells is a relative predominance, e.g., the ratio of Th17 cells to Th1 cells and / or the ratio of Th17 cells to Th2 cells, is increased relative to normal values. In some embodiments the ratio of Th17 cells to T regulatory cells(CD4+CD25+regulatory T cells, also termed “Treg cells”), is increased relative to normal values. Formation of Th17 cells and / or activation of Th 17 cells is promoted by various cytokines, e.g., interleukin 6 (IL-6), interleukin 21 (IL-21), interleukin 23 (IL-23), and / or interleukin 1 p (IL-1 P). Formation of Th17 cells encompasses differentiation of precursor T cells, e.g., naive CD4+ T cells, towards a Th17 phenotype and their maturation into functional Th17 cells. In some embodiments, formation of Th17 cells encompasses any aspect of development, proliferation (expansion), survival, and / or maturation of Th17 cells. In some embodiments, a Th17-associated disorder is characterized by excessive production and / or amount of IL-6, IL-21 , IL-23, and / or IL-1 p. Th 17 cells typically secrete characteristic cytokines such as interleukin-17A (IL-17A), interleukin-17F (IL-17F), interleukin-21 (IL-21), and interleukin-22 (IL-22). In some embodiments, a Th17-associated disorder is characterized by excessive production and / or amount of a Th17 effector cytokine, e.g., IL-17A, IL-17F, IL-21 , and / or IL-22. In some embodiments excessive production or amount of a cytokine is detectable in the blood. In some embodiments excessive production or amount of a cytokine is detectable locally, e.g., in at least one tissue, organ or structure. In some embodiments a Th17-associated disorder is associated with a decreased number of Tregs and / or decreased amount of a Treg-associated cytokine. In some embodiments a Th17 disorder is any chronicinflammatory disease, which term encompasses a range of ailments characterized by self- perpetuating immune insults to a variety of tissues and that seem to be dissociated from the initial insult that caused the ailment (which may be unknown). In some embodiments a Th17- associated disorder is any autoimmune disease. Many if not most “chronic inflammatory diseases” may in fact be autoimmune diseases. Examples of Th17-associated disorders include inflammatory skin diseases such as psoriasis and atopic dermatitis; systemic scleroderma and sclerosis; inflammatory bowel disease (IBD) (such as Crohn's disease and ulcerative colitis); Behcet's Disease; dermatomyositis; polymyositis; multiple sclerosis (MS); dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; lupus nephritis; rheumatoid arthritis (RA), Sjogren's syndrome, multiple sclerosis, vasculitis; central nervous system (CNS) inflammatory disorders, chronic hepatitis; chronic pancreatitis, glomerulonephritis; sarcoidosis; thyroiditis, pathologic immune responses to tissue / organ transplantation (e.g., transplant rejection); COPD, asthma, bronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), periodontitis, and gingivitis. In some embodiments a Th17 disease is a classically known autoimmune disease such as Type I diabetes or psoriasis. In some embodiments a Th17-associated disorder is age-related macular degeneration.

[0174] In some embodiments, a chronic complement-mediated disorder is an IgE- associated disorder. As used herein, an “IgE-associated disorder” is a disorder characterized by excessive and / or inappropriate production and / or amount of IgE, excessive or inappropriate activity of IgE producing cells (e.g., IgE producing B cells or plasma cells), and / or excessive and / or inappropriate activity of IgE responsive cells such as eosinophils or mast cells. In some embodiments, an IgE-associated disorder is characterized by elevated levels of total IgE and / or in some embodiments, allergen-specific IgE, in the plasma of a subject and / or locally.

[0175] In some embodiments, a chronic complement-mediated disorder is characterized by the presence of autoantibodies and / or immune complexes in the body, which may activate complement via, e.g., the classical pathway. Autoantibodies may, for example, bind to selfantigens, e.g., on cells or tissues in the body. In some embodiments, autoantibodies bind to antigens in blood vessels, skin, nerves, muscle, connective tissue, heart, kidney, thyroid, etc. In some embodiments, a subject has neuromyelitis optica and produces an autoantibody (e.g., an IgG autoantibody) to aquaporin 4. In some embodiments, a subject has pemphigoid and produces an autoantibody (e.g., an IgG or IgE autoantibody) to a structural component of the hemidesmosome (e.g., transmembrane collagen XVII (BP180 or BPAG2) and / or plakin familyprotein BP230 (BPAG1). In some embodiments, a chronic complement-mediated disorder is not characterized by autoantibodies and / or immune complexes.

[0176] In some embodiments, a chronic complement-mediated disorder is a respiratory disorder. In some embodiments, a chronic respiratory disorder is asthma or chronic obstructive pulmonary disease (COPD). In some embodiments, a chronic respiratory disorder is pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), radiation-induced lung injury, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis (also known as allergic alveolitis), eosinophilic pneumonia, interstitial pneumonia, sarcoid, Wegener's granulomatosis, or bronchiolitis obliterans. In some embodiments, the disclosure provides a method of treating a subject in need of treatment for a chronic respiratory disorder, e.g., asthma, COPD, pulmonary fibrosis, radiation-induced lung injury, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis (also known as allergic alveolitis), eosinophilic pneumonia, interstitial pneumonia, sarcoid, Wegener's granulomatosis, or bronchiolitis obliterans, the method comprising administering 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, to a subject in need of treatment for the disorder.

[0177] In some embodiments, a chronic complement-mediated disorder is allergic rhinitis, rhinosinusitis, or nasal polyposis. In some embodiments, the disclosure provides a method of treating a subject in need of treatment for allergic rhinitis, rhinosinusitis, or nasal polyposis, the method comprising administering 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, to a subject in need of treatment for the disorder.

[0178] In some embodiments, a chronic complement-mediated disorder is a disorder that affects the musculoskeletal system. Examples of such disorders include inflammatory joint conditions (e.g., arthritis such as rheumatoid arthritis or psoriatic arthritis, juvenile chronic arthritis, spondyloarthropathies Reiter's syndrome, gout). In some embodiments, a musculoskeletal system disorder results in symptoms such as pain, stiffness and / or limitation of motion of the affected body part(s). Inflammatory myopathies include dermatomyositis, polymyositis, and various others are disorders of chronic muscle inflammation of unknown etiology that result in muscle weakness. In some embodiments, a chronic complement- mediated disorder is myasthenia gravis. In some embodiments, the disclosure provides a method of treating any of the foregoing disorders affecting the musculoskeletal system, themethod comprising administering 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, to a subject in need of treatment for the disorder.

[0179] In some embodiments, a chronic complement-mediated disorder is a disorder that affects the integumentary system. Examples of such disorders include, e.g., atopic dermatitis, psoriasis, pemphigoid, pemphigus, systemic lupus erythematosus, dermatomyositis, scleroderma, sclerodermatomyositis, Sjogren syndrome, and chronic urticaria. In some aspects, the disclosure provides a method of treating any of the foregoing disorders affecting the integumentary system, the method comprising administering 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, to a subject in need of treatment for the disorder.

[0180] In some embodiments, a chronic complement-mediated disorder affects the nervous system, e.g., the central nervous system (CNS) and / or peripheral nervous system (PNS). Examples of such disorders include, e.g., multiple sclerosis, other chronic demyelinating diseases (e.g., neuromyelits optica, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy), amyotrophic lateral sclerosis, chronic pain, stroke, allergic neuritis, Huntington's disease, Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy, Lewy body dementia (i.e. , dementia with Lewy bodies or Parkinson's disease dementia), frontotemporal dementia, traumatic brain injury, traumatic spinal cord injury, multisystem atrophy, corticobasal degeneration, epileptic disorders, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, glioma, glioblastoma, and leptomeningeal metastasis. In some embodiments, the disclosure provides a method of treating any of the foregoing disorders affecting the nervous system, the method comprising administering 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, to a subject in need of treatment for the disorder.

[0181] In some embodiments, a chronic complement-mediated disorder affects the circulatory system. For example, in some embodiments the disorder is a vasculitis or other disorder associated with vessel inflammation, e.g., blood vessel and / or lymph vessel inflammation. In some embodiments, a vasculitis is polyarteritis nodosa, Wegener's granulomatosis, giant cell arteritis, Churg-Strauss syndrome, microscopic polyangiitis,Henoch-Schonlein purpura, Takayasu's arteritis, Kawasaki disease, or Behcet's disease. In some embodiments, a subject, e.g., a subject in need of treatment for vasculitis, is positive for antineutrophil cytoplasmic antibody (ANCA).

[0182] In some embodiments, a chronic complement-mediated disorder affects the gastrointestinal system. For example, the disorder may be inflammatory bowel disease, e.g., Crohn's disease or ulcerative colitis. In some embodiments, the disclosure provides a method of treating a chronic complement-mediated disorder that affects the gastrointestinal system, the method comprising administering 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, to a subject in need of treatment for the disorder.

[0183] In some embodiments, a chronic complement-mediated disorder is a thyroiditis (e.g., Hashimoto's thyroiditis, Graves' disease, post-partum thyroiditis), myocarditis, hepatitis (e.g., hepatitis C), pancreatitis, glomerulonephritis (e.g., membranoproliferative glomerulonephritis or membranous glomerulonephritis), or panniculitis.

[0184] In some embodiments, the disclosure provides methods of treating a subject suffering from chronic pain, the methods comprising administering 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, to a subject in need thereof. In some embodiments, a subject suffers from neuropathic pain. Neuropathic pain has been defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system, in particular, pain arising as a direct consequence of a lesion or disease affecting the somatosensory system. For example, neuropathic pain may arise from lesions that involve the somatosensory pathways with damage to small fibers in peripheral nerves and / or to the spino-thalamocortical system in the CNS. In some embodiments, neuropathic pain arises from autoimmune disease (e.g., multiple sclerosis), metabolic disease (e.g., diabetes), infection (e.g., viral disease such as shingles or HIV), vascular disease (e.g., stroke), trauma (e.g., injury, surgery), or cancer. For example, neuropathic pain can be pain that persists after healing of an injury or after cessation of a stimulus of peripheral nerve endings or pain that arises due to damage to nerves. Exemplary conditions of or associated with neuropathic pain include painful diabetic neuropathy, postherpetic neuralgia (e.g., pain persisting or recurring at the site of acute herpes zoster 3 or more months after the acute episode), trigeminal neuralgia, cancer related neuropathic pain, chemotherapy-associated neuropathic pain, HIV-related neuropathic pain (e.g., from HIVneuropathy), central / post-stroke neuropathic pain, neuropathy associated with back pain, e.g., low back pain (e.g., from radiculopathy such as spinal root compression, e.g., lumbar root compression, which compression may arise due to disc herniation), spinal stenosis, peripheral nerve injury pain, phantom limb pain, polyneuropathy, spinal cord injury related pain, myelopathy, and multiple sclerosis. In certain embodiments of the disclosure, 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, is administered according to a dosing schedule to treat neuropathic pain in a subject with one or more of the afore-mentioned conditions.

[0185] In some embodiments, a chronic complement-mediated disorder is a chronic eye disorder. In some embodiments, the chronic eye disorder is characterized by macular degeneration, choroidal neovascularization (CNV), retinal neovascularization (RNV), ocular inflammation, or any combination of the foregoing. Macular degeneration, CNV, RNV, and / or ocular inflammation may be a defining and / or diagnostic feature of the disorder. Exemplary disorders that are 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. Macular degeneration related conditions include, e.g., age-related macular degeneration (AMD) and Stargardt macular dystrophy. In some embodiments, a subject is in need of treatment for wet AMD. In some embodiments, a subject is in need of treatment for dry AMD. In some embodiments, a subject is in need of treatment for geographic atrophy (GA). In some embodiments, a subject is in need of treatment for ocular inflammation. Ocular inflammation can affect a large number of eye structures such as the conjunctiva (conjunctivitis), cornea (keratitis), episclera, sclera (scleritis), uveal tract, retina, vasculature, and / or optic nerve. Evidence of ocular inflammation can include the presence of inflammation-associated cells such as white blood cells (e.g., neutrophils, macrophages) in the eye, the presence of endogenous inflammatory mediator(s), one or more symptoms such as eye pain, redness, light sensitivity, blurred vision and floaters, etc. Uveitis is a general term that refers to inflammation in the uvea of the eye, e.g., in any of the structures of the uvea, including the iris, ciliary body or choroid. Specific types of uveitis include iritis, iridocyclitis, cyclitis, pars planitis and choroiditis. In some embodiments, the chronic eye disorder is an eye disorder characterized by optic nerve damage (e.g., optic nerve degeneration), such as glaucoma.

[0186] In some embodiments methods disclosed herein inhibit (interfere with, disrupt) the DC-Th17-B-Ab-C-DC cycle discussed above. For example, administration of an inhibitoryRNA 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, may break the cycle by which complement stimulates DC cells to promote the Th17 phenotype. As a result, the number and / or activity of Th17 cells diminishes, which in turn reduces the amount of Th17-mediated stimulation of B cells and polyclonal antibody production. In some embodiments, these effects result in “resetting” the immunological microenvironment to a more normal, less pathological state. As described in Example 1 of PCT / US2012 / 043845 (WO / 2012 / 178083) and US Publ. No. 20140371133 evidence supporting the capacity of complement inhibition to have a prolonged inhibitory effect on Th17-associated cytokine production has been obtained in an animal model of asthma.

[0187] In some embodiments, inhibiting the DC-Th17-B-Ab-C-DC cycle has a diseasemodifying effect. Without wishing to be bound by any theory, rather than merely treating symptoms of a disorder, inhibiting the DC-Th17-B-Ab-C-DC cycle may interfere with fundamental pathologic mechanisms that may contribute to ongoing tissue damage even when symptoms are well controlled and / or that may contribute to exacerbations of the disease. In some embodiments, inhibiting the DC-Th17-B-Ab-C-DC cycle causes a chronic disorder to go into remission. In some embodiments, remission refers to a state of absence or substantial absence of disease activity in a subject with a chronic disorder, with the possibility of return of disease. In some embodiments remission may be sustained for a prolonged period of time (e.g., at least 6 months, e.g., 6-12 months, 12-24 months, or more) in the absence of continued therapy or with a reduced dose or increased dosing interval. In some aspects, inhibition of complement may change the immunological micro-environment of a tissue that is rich in Th17 cells and modify it into a micro-environment that is rich in regulatory T cells (Tregs). Doing so could allow the immune system to “reset” itself and go into a state of remission. In some embodiments, for example, remission may be sustained until occurrence of a triggering event. A triggering event may be, for example, an infection (which may result in production of polyclonal antibodies that react both with an infectious agent and a self protein), exposure to particular environmental conditions (e.g., high levels of air pollutants such as ozone or particulate matter or components of smoke such as cigarette smoke, allergens), etc. Genetic factors may play a role. For example, individuals having particular alleles of genes encoding complement components may have a higher baseline level of complement activity, a more reactive complement system and / or a lower baseline level of endogenous complement regulatory protein activity. In some embodiments an individual has a genotype associated withincreased risk of AMD. For example, the subject may have a polymorphism in a gene encoding a complement protein or complement regulatory protein, e.g., CFH, C3, factor B, wherein the polymorphism is associated with an increased risk of AMD.

[0188] In some embodiments an immunologic microenvironment may become progressively more polarized towards a pathological state over time, e.g., in a subject who has not yet developed symptoms of a chronic disorder or in a subject who has developed the disorder and has been treated as described herein. Such a transition may occur stochastically (e.g., due at least in part to apparently random fluctuations in antibody levels and / or affinity) and / or as a result of accumulated “sub-threshold” trigger events that are not of sufficient intensity to trigger a symptomatic outbreak of a disorder.

[0189] In some embodiments it is contemplated that a relatively short course of 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, e.g., between 1 week and 6 weeks, e.g., about 2-4 week, may provide a long-lasting benefit. In some embodiments, a remission is achieved for a prolonged period of time, e.g., 1-3 months, 3-6 months, 6-12 months, 12-24 months, or more. In some embodiments a subject may be monitored and / or treated prophylactically before recurrence of symptoms. For example, a subject may be treated prior to or upon exposure to a triggering event. In some embodiments a subject may be monitored, e.g., for an increase in a biomarker, e.g., a biomarker comprising an indicator of Th 17 cells or Th17 cell activity, or complement activation, and may be treated upon increase in the level of such biomarker. See, e.g., PCT / US2012 / 043845 for further discussion.VI. Combination Therapy

[0190] In some aspects, methods of the present disclosure involve administering an inhibitory RNA described herein, alone or in combination with one or more additional complement inhibitors. In some embodiments, an inhibitory RNA is administered to a subject already receiving therapy with another complement inhibitor; in some embodiments, another complement inhibitor is administered to a subject receiving an inhibitory RNA. In some embodiments, both an inhibitory RNA and another complement inhibitor are administered to the subject.

[0191] In some embodiments administration of an inhibitory RNA may allow for administering a reduced dosing regimen of (e.g., involving a smaller amount in an individual dose, reduced frequency of dosing, reduced number of doses, and / or reduced overallexposure to) a second complement inhibitor, as compared to administration of a second complement inhibitor as single therapy. Without wishing to be bound by any theory, in some embodiments a reduced dosing regimen of a second complement inhibitor may avoid one or more undesired adverse effects that could otherwise result.

[0192] In some aspects, administration of an inhibitory RNA in combination with a second complement inhibitor can reduce the amount of C3 in the subject's blood sufficiently such that a reduced dosing regimen of an inhibitory RNA and / or the second complement inhibitor is required to achieve a desired degree of complement inhibition.

[0193] In some aspects, administration of an inhibitory RNA in combination with a second complement inhibitor can reduce the amount of C3 in the subject's blood sufficiently such that a reduced dosing regimen of an inhibitory RNA and / or the second complement inhibitor is required to achieve a desired level of, or a desired amount of improvement in, one or more signs, symptoms, biomarkers, or outcome measures, of a complement-mediated disorder. Any complement inhibitor, e.g., a complement inhibitor known in the art, can be administered in combination with an inhibitory RNA described herein. In some embodiments, a complement inhibitor is compstatin or a compstatin analog.

[0194] Compstatin is a cyclic peptide that binds to C3 and inhibits complement activation. As used herein, the term “compstatin analog” includes compstatin and any complement inhibiting analog thereof. The term “compstatin analog” encompasses compstatin and other compounds designed or identified based on compstatin and whose complement inhibiting activity is at least 50% as great as that of compstatin as measured, e.g., using any complement activation assay accepted in the art or substantially similar or equivalent assays. Certain suitable assays are described in U.S. Pat. No. 6,319,897, W02004 / 026328, Morikis, supra, Mallik, supra, Katragadda 2006, supra, WC2007062249 (PCT / US2006 / 045539); WC2007044668 (PCT / US2006 / 039397), WC / 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 be an ELISA assay. In some embodiments, an assay described in WO / 2010 / 135717 (PCT / US2010 / 035871) is used.

[0195] Compstatin analogs may be prepared by various synthetic methods of peptide synthesis known in the art via condensation of amino acid residues, e.g., in accordance with conventional peptide synthesis methods, may be prepared by expression in vitro or in living cells from appropriate nucleic acid sequences encoding them using methods known in the art. For example, peptides may be synthesized using standard solid-phase methodologies as described in Malik, supra, Katragadda, supra, W02004026328, and / or W02007062249.Potentially reactive moieties such as amino and carboxyl groups, reactive functional groups, etc., may be protected and subsequently deprotected using various protecting groups and methodologies known in the art. See, e.g., “Protective Groups in Organic Synthesis”, 3rded. Greene, T. W. and Wuts, P. G., Eds., John Wiley & Sons, New York: 1999. Peptides may be purified using standard approaches such as reversed-phase HPLC. Separation of diasteriomeric peptides, if desired, may be performed using known methods such as reversed-phase HPLC. Preparations may be lyophilized, if desired, and subsequently dissolved in a suitable solvent, e.g., water. The pH of the resulting solution may be adjusted, e.g. to physiological pH, using a base such as NaOH. Peptide preparations may be characterized by mass spectrometry if desired, e.g., to confirm mass and / or disulfide bond formation. See, e.g., Mallik, 2005, and Katragadda, 2006.

[0196] A compstatin analog can be modified by addition of a molecule such as polyethylene glycol (PEG) to stabilize the compound, reduce its immunogenicity, increase its lifetime in the body, increase or decrease its solubility, and / or increase its resistance to degradation. Methods for pegylation are well known in the art (Veronese, F. M. & Harris, Adv. Drug Deliv. Rev. 54, 453-456, 2002; Davis, F. F., Adv. Drug Deliv. Rev. 54, 457-458, 2002); Hinds, K. D. & Kim, S. W. Adv. Drug Deliv. Rev. 54, 505-530 (2002; Roberts, M. J., Bentley, M. D. & Harris, J. M. Adv. Drug Deliv. Rev. 54, 459-476; 2002); Wang, Y. S. et al. Adv. Drug Deliv. Rev. 54, 547-570, 2002). A wide variety of polymers such as PEGs and modified PEGs, including derivatized PEGs to which polypeptides can conveniently be attached are described in Nektar Advanced Pegylation 2005-2006 Product Catalog, Nektar Therapeutics, San Carlos, Calif., which also provides details of appropriate conjugation procedures.

[0197] An exemplary PEGylated compstatin analog is pegcetacoplan. Pegcetacoplan is also referred to as Poly(oxy-1 ,2-ethanediyl), a-hydro-co-hydroxy-, 15,15 -diester with N-acetyl- L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophyl-L-glutaminyl-L-a-aspartyl-L- tryptophylglycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-L-threonyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-N6-carboxy-L-lysinamide cyclic (2->12)-(disulfide); or 0,0 - bis[(S2,S12-cyclo{N-acetyl-L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophyl-L-glutaminyl-L- a-aspartyl-L-tryptophylglycyl-L-alanyl-L-histidyl-L-arginyl-L- cysteinyl-L-threonyl-2-[2-(2- aminoethoxy)ethoxy]acetyl-L-lysinamide})-N6 15-carbonyl]polyethylene glycol (n=800-1100). Additional compstatin analogs are described in, e.g., WO 2012 / 155107 and WO 2014 / 078731.

[0198] In some embodiments, a composition comprising one or more inhibitory RNAs (e.g., an siRNA or miRNA described herein), or comprising a vector comprising a nucleotide sequence encoding an siRNA or miRNA described herein, is administered to a subject incombination with a compstatin analog, such that the compstatin analog and / or the inhibitory RNA composition is administered less frequently and / or at a lower dosage.

[0199] In some embodiments, a complement inhibitor is an antibody, e.g., an anti-C3 and / or anti-C5 antibody, or a fragment thereof. In some embodiments, an antibody fragment may be used to inhibit C3 or C5 activation. The fragmented anti-C3 or anti-C5 antibody may be 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 de-immunized. In some embodiments 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, a complement inhibitor is an antibody, e.g., an anti-C3 and / or anti-C5 antibody, or a fragment thereof.

[0200] In some embodiments, a complement inhibitor is a polypeptide inhibitor and / or a nucleic acid aptamer (see, e.g., U.S. Publ. No. 20030191084). Exemplary polypeptide inhibitors include an enzyme that degrades C3 or C3b (see, e.g., U.S. Pat. No. 6,676,943). Additional polypeptide inhibitors include mini-factor H (see, e.g., U.S. Publ. No. 20150110766), Efb protein or complement inhibitor (SCIN) protein from Staphylococcus aureus, or a variant or derivative or mimetic thereof (see, e.g., U.S. Publ. 20140371133).

[0201] A variety of other complement inhibitors can also be used in various embodiments of the 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 may be CR1 , DAF, MCP, CFH, or CFI. In some embodiments, the complement regulatory polypeptide is one that is normally membranebound in its naturally occurring state. In some embodiments, a fragment of such polypeptide that lacks some or all of a transmembrane and / or intracellular domain is used. Soluble forms of complement receptor 1 (sCR1), for example, can also be used. For example the 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 control protein, e.g., CFH, is used.

[0202] Inhibitors of C1s can also be used. For example, U.S. Pat. No. 6,515,002 describes compounds (furanyl and thienyl amidines, heterocyclic amidines, and guanidines) that inhibit Cis. U.S. Pat. Nos. 6,515,002 and 7,138,530 describe heterocyclic amidines that inhibit C1s. U.S. Pat. No. 7,049,282 describes peptides that inhibit classical pathway activation. In some embodiments these peptides are identical or substantially identical to a portion of an IgG or IgM molecule. U.S. Pat. No. 7,041 ,796 discloses C3b / C4b ComplementReceptor-like molecules and uses thereof to inhibit complement activation. U.S. Pat. No.6,998,468 discloses anti-C2 / C2a inhibitors of complement activation. U.S. Pat. No. 6,676,943 discloses human complement C3-degrading protein from Streptococcus pneumoniae.DEFINITIONS

[0203] Antibody: As used herein, the term “antibody” refers to an immunoglobulin or a derivative thereof containing an immunoglobulin domain capable of binding to an antigen. The antibody can be of any species, e.g., human, rodent, rabbit, goat, chicken, etc. The antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE, or subclasses thereof such as lgG1 , lgG2, etc. In various embodiments of the present disclosure, the antibody is a fragment such as an Fab', F(ab')2, scFv (single-chain variable) or other fragment that retains an antigen binding site, or a recombinantly produced scFv fragment, including recombinantly produced fragments. See, e.g., Allen, T., Nature Reviews Cancer, Vol. 2, 750-765, 2002, and references therein. The antibody can be monovalent, bivalent or multivalent. The antibody may be a chimeric or “humanized” antibody in which, for example, a variable domain of rodent origin is fused to a constant domain of human origin, thus retaining the specificity of the rodent antibody. The domain of human origin need not originate directly from a human in the sense that it is first synthesized in a human being. Instead, “human” domains may be generated in rodents whose genome incorporates human immunoglobulin genes. See, e.g., Vaughan, et al., (1998), Nature Biotechnology, 16: 535-539. The antibody may be partially or completely humanized. An antibody may be polyclonal or monoclonal, though for purposes of the present disclosure, 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 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.

[0204] Approximately: As used herein, the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0205] Complementary: As used herein, in accordance with its art-accepted meaning, “complementary” refers to the capacity for precise pairing between particular bases, nucleosides, nucleotides or nucleic acids. For example, adenine (A) and uridine (U) are complementary; adenine (A) and thymidine (T) are complementary; and guanine (G) and cytosine (C), are complementary and are referred to in the art as Watson-Crick base pairings. If a nucleotide at a certain position of a first nucleic acid sequence is complementary to a nucleotide located opposite in a second nucleic acid sequence when the strands are aligned in anti-parallel orientation, 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 may be evaluated by aligning them in antiparallel orientation for maximum complementarity over a window of evaluation, determining the total number of nt in both strands that form complementary base pairs within the window, dividing by the total number of nt within the window, and multiplying by 100. For example, AAAAAAAA and TTTGTTAT are 75% complementary since there are 12 nt in complementary base pairs out of a total of 16 nt. When computing the number of complementary nt needed to achieve a particular % complementarity, fractions are rounded to the nearest whole number. A position occupied by non-complementary nucleotides constitutes a mismatch, i.e., the position is occupied by a non-complementary base pair. In certain embodiments a window of evaluation has the length described herein for duplex portions or target portions. Complementary sequences include base-pairing of a polynucleotide comprising a first nucleotide sequence to a polynucleotide comprising a second nucleotide sequence over the entire length of both nucleotide sequences (if the same length) or over the entire length of the shorter sequence (if different lengths). Such sequences can be referred to as “perfectly complementary” (100% complementarity) with respect to each other herein. Nucleic acids that are at least 70% complementary over a window of evaluation are considered “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 window of evaluation. Where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences may be perfectly complementary or they may comprise one or more unmatched bases upon hybridization, e.g., up to about 5%, 10%, 15%, 20%, or 25% unmatched bases upon hybridization, e.g., 1 , 2, 3, 4, 5, or 6 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their intended use. It should be understood that where two oligonucleotides are designed to form, upon hybridization, one ormore single stranded overhangs, such overhangs are not regarded as mismatches or unpaired nucleotides with regard to the determination of percent complementarity. For example, the two strands of a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is perfectly complementary to the shorter oligonucleotide and a 2 nucleotide overhang, may be referred to as “perfectly complementary” herein. “Complementary” sequences, as used herein may include one or more non-Watson- Crick base pairs and / or base pairs formed from non-natural and other modified nucleotides, in so far as the requirements with respect to their ability to hybridize are fulfilled. Such non- Watson-Crick base pairs include, but are not limited to, G:ll Wobble or Hoogsteen base pairing. Those of ordinary skill in the art are aware that guanine, cytosine, adenine, and uracil can be replaced by other bases without substantially altering the base pairing properties of a polynucleotide comprising a nucleotide bearing such bases, according to the so-called “wobble” rules (see, e.g., Murphy, F V IV & V Ramakrishnan, V., Nature Structural and Molecular Biology 11 : 1251-1252 (2004)). For example, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Thus, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of an Inhibitory RNA described herein by a nucleotide containing, for example, inosine. It will be understood that the terms “complementary”, “perfectly complementary”, and “substantially complementary” can be used with respect to the base matching between any two nucleic acids, e.g., the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a ds inhibitory RNA (e.g., an siRNA) and a target sequence, or between an antisense oligonucleotide and a target sequence, as will be evident from the context. “Hybridize”, as used herein, refers to the interaction between two nucleic acid sequences comprising or consisting of complementary portions such that a duplex structure is formed that is stable under the particular conditions of interest, as will be understood by the ordinary skilled artisan.

[0206] Complement component: As used herein, the terms “complement component” or “complement protein” is a molecule that is involved in activation of the complement system or participates in one or more complement-mediated activities. Components of the classical complement pathway include, e.g., Clq, C1 r, C1s, C2, C3, C4, C5, C6, C7, C8, C9, and the C5b-9 complex, also referred to as the membrane attack complex (MAC) and active fragments or enzymatic cleavage products of any of the foregoing (e.g., C3a, C3b, C4a, C4b, C5a, etc.). Components of the alternative pathway include, e.g., factors B, D, H, and I, andproperdin, with factor H being a negative regulator of the pathway. Components of the lectin pathway include, e.g., MBL2, MASP-1 , and MASP-2. Complement components also include cell-bound receptors for soluble complement components. Such receptors include, e.g., C5a receptor (C5aR), C3a receptor (C3aR), Complement Receptor 1 (CR1), Complement Receptor 2 (CR2), Complement Receptor 3 (CR3), etc. It will be appreciated that the term “complement component” is not intended to include those molecules and molecular structures that serve as “triggers” for complement activation, e.g., antigen-antibody complexes, foreign structures found on microbial or artificial surfaces, etc.

[0207] Host cell: As used herein, the term “host cell” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Persons of skill upon reading this disclosure will understand 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 succeeding generations due to either mutation 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 that are suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence). Exemplary cells include those of prokaryotes and eukaryotes (single-cell or multiple-cell), bacterial cells (e.g., strains of E. coli, Bacillus spp., Streptomyces spp., etc.), mycobacteria 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 such as, for example, hybridomas or quadromas. In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is eukaryotic and is selected from the following cells: CHO (e.g., CHO K1 , DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1 , kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, W138, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1 , U937, 3T3, L cell, C127 cell, SP2 / 0, NS-0, MMT 060562, Sertoli cell, BRL 3 A cell, HT1080 cell, myeloma cell, tumor cell, and a cell line derived from an aforementioned cell. In some embodiments, the cell comprises one or more viral genes.

[0208] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if theirsequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned 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 length of a reference sequence. The nucleotides at corresponding positions are then compared. When 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, then the molecules are identical at that position. The percent identity between the 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, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use 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 an NWSgapdna.CMP matrix.

[0209] Linked: As used herein, the term “linked”, when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another to form a molecular structure that is sufficiently stable so that the moieties remain associated under the conditions in which the linkage is formed and, preferably, under the conditions in which the new molecular structure is used, e.g., physiological conditions. In certain preferred embodiments of the present disclosure, the linkage is a covalent linkage. In other embodiments the linkage is noncovalent. Moieties may be linked either directly or indirectly. When two moieties are directly linked, they are either covalently bonded to one another or are in sufficiently close proximity such that intermolecular forces between the two moieties maintain their association. When two moieties are indirectly linked, they are each linked either covalently or noncovalently to a third moiety, which maintains the association between the twomoieties. In general, when two moieties are referred to as being linked by a “linker” or “linking moiety” or “linking portion”, the linkage between the two linked moieties is indirect, and typically each of the linked moieties is covalently bonded to the linker. The linker can be any suitable moiety that reacts with the two moieties to be linked within a reasonable period of time, under conditions consistent with stability of the moieties (which may be protected as appropriate, depending upon the conditions), and in sufficient amount, to produce a reasonable yield.

[0210] MicroRNA (miRNA): As used herein, the term “microRNA” or “miRNA” refers to a small non-coding RNA molecule that can function in transcriptional and / or post-transcriptional regulation of target gene expression. The terms encompass a mature miRNA sequence or a precursor miRNA sequence, including a primary transcript (pri-miRNA) and a stem-loop precursor (pre-miRNA). The biogenesis of a naturally occurring miRNA initiates in the nucleus by RNA polymerase II transcription, generating a primary transcript (pri-miRNA). The primary transcript is cleaved by Drosha ribonuclease III enzyme to produce an approximately 70 nt stem-loop precursor miRNA (pre-miRNA). The pre-miRNA is then actively exported to the cytoplasm where it is cleaved by Dicer ribonuclease to form the mature miRNA, which includes an “antisense strand” or “guide strand” (that includes a region that is substantially complementary to a target sequence) and a “sense strand” or “passenger strand” (that includes a region that is substantially complementary to a region of the antisense strand). Those of ordinary skill in the art will appreciate that a guide strand may be perfectly complementary to a target region of a target RNA or may have less than perfect complementarity to a target region of a target RNA. The guide strand of this miRNA is incorporated into an RNA-induced silencing complex (RISC) that recognizes target mRNAs through base pairing with the miRNA, and commonly results in translational inhibition or destabilization of the target mRNA. As is understood in the field, for naturally occurring miRNAs, target mRNA recognition occurs through imperfect base pairing with the mRNA. In some embodiments, an miRNA is synthetic or engineered, and target mRNA recognition occurs through perfect base pairing with the mRNA. Typically, the target mRNA contains a sequence complementary to a “seed” sequence of the miRNA, which usually corresponds to nucleotides 2-8 of the miRNA. Information concerning miRNAs and associated pri-miRNA and pre-miRNA sequences is available in miRNA databases such as miRBase (Griffiths-Jones et al. 2008 Nucl Acids Res 36, (Database Issue: D154-D158) and the NCBI human genome database.

[0211] Operably linked: As used herein, the term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element “operably linked” to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, “operably linked” control elements are contiguous (e.g., covalently linked) with the coding elements of interest; in some embodiments, control elements act in trans to or otherwise at a from the functional element of interest.

[0212] Recombinant: As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and / or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and / or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs 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 such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc.).

[0213] RNA interference: As used herein, the term “RNA interference” or “RNAi” refers generally to a process in which a double-stranded RNA molecule or a short hairpin RNA molecule reduces or inhibits expression of a nucleic acid sequence with which the doublestranded or short hairpin RNA molecule shares substantial or total homology. Without wishing to be bound by any theory, it is believed that, in nature, the RNAi pathway is initiated by a Type III endonuclease known as Dicer, which cleaves long double-stranded RNA (dsRNA) into double-stranded fragments typically of 21-23 base pairs with 2-base 3' overhangs (although variations in length and overhangs (e.g. overhangs of 1 , 2, 3, 4, 5, and more than 5)are also contemplated), referred to as “short interfering RNAs” (“siRNAs”). Such siRNAs comprise two single-stranded RNAs (ssRNAs), with an “antisense strand” or “guide strand” that includes a region that is substantially complementary to a target sequence, and a “sense strand” or “passenger strand” that includes a region that is substantially complementary to a region of the antisense strand. Those of ordinary skill in the art will appreciate that a guide strand may be perfectly complementary to a target region of a target RNA or may have less than perfect complementarity to a target region of a target RNA.

[0214] Subject: As used herein, the term “subject” or “test subject” refers to any organism to which a provided compound or composition is administered in accordance with the present disclosure e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans; insects; worms; etc.) and plants. In some embodiments, a subject may be suffering from, and / or susceptible to a disease, disorder, and / or condition.

[0215] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and / or chemical phenomena.

[0216] 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 a disease, disorder, and / or condition.

[0217] Target gene: A “target gene”, as used herein, refers to a gene whose expression is to be modulated, e.g., inhibited. As used herein, the term “target RNA” refers to an RNA to be degraded or translationally repressed or otherwise inhibited using one or more miRNAs. A target RNA may also be referred to as a target sequence or target transcript. The RNA may be a primary RNA transcript transcribed from the target gene (e.g., a pre-mRNA) or a processed transcript, e.g., mRNA encoding a polypeptide. As used herein, the term “target portion” or “target region” refers to a contiguous portion of the nucleotide sequence of a target RNA. In some embodiments, a target portion 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 a suitable inhibitory RNA. A target portion may be from about 8-36 nucleotides in length, e.g., about 10-20 or about 15-30 nucleotides in length. A target portion length may have specific value or subrange within the afore-mentioned ranges. For example, in certainembodiments a target portion may be between 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.

[0218] Therapeutic agent: As used herein, the phrase “therapeutic agent” refers to any agent that, when administered to a subject, has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0219] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces 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.

[0220] Treating: As used herein, the term “treating” refers to providing treatment, i.e., providing any type of medical or surgical management of a subject. The treatment can be provided in order to reverse, alleviate, inhibit the progression of, prevent or reduce the likelihood of a disease, disorder, or condition, or in order to reverse, alleviate, inhibit or prevent the progression of, prevent or reduce the likelihood of one or more symptoms or manifestations of a disease, disorder or condition. “Prevent” refers to causing a disease, disorder, condition, or symptom or manifestation of such not to occur for at least a period of time in at least some individuals. Treating can include administering an agent to the subjectfollowing the development of one or more symptoms or manifestations indicative of a complement-mediated condition, e.g., in order to reverse, alleviate, reduce the severity of, and / or inhibit or prevent the progression of the condition and / or to reverse, alleviate, reduce the severity of, and / or inhibit or one or more symptoms or manifestations of the condition. A composition of the disclosure can be administered to a subject who has developed a complement-mediated disorder or is at increased risk of developing such a disorder relative to a member of the general population. A composition of the disclosure can be administered prophylactically, i.e. , before development of any symptom or manifestation of the condition. Typically in this case the subject will be at risk of developing the condition.

[0221] Nucleic acid: The term “nucleic acid” includes any nucleotides, analogs thereof, and polymers thereof. The term “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecules and, thus, include double- and singlestranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. In some embodiments, an siRNA of the present invention may comprise a 5’ cap, such as vinyl phosphonate, on the antisense strand. The terms encompass poly- or oligoribonucleotides (RNA) and poly- or oligo-deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides 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 encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, and are not limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. In some embodiments, the prefix poly- refers to a nucleic acid containing 2 to about 10,000, 2 to about 50,000, or 2 to about 100,000 nucleotide monomer units. In some embodiments, the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units.

[0222] Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNAsegments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may 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) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0223] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

[0224] All publications, patent applications, patents, and other references mentioned herein, including GenBank Accession Numbers, are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein 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.ADDITIONAL EXEMPLARY EMBODIMENTS

[0225] One embodiment is an isolated, modified sequence comprising any one of SEQ ID NOs: 50-153 or 209-327 (i.e., any one of the antisense strand sequences in Table 1 B or Table A-2). Such isolated, modified sequence may further comprise at least one ligand attached to the 5’ end or the 3’ end, wherein each ligand is individually selected from the group consisting of a GalNAc moiety, a C16 lipid, and a C18 lipid. Such ligands may be attached to the sequence via a linker.

[0226] One embodiment is an isolated, modified sequence comprising any one of SEQ ID NOs: 29-49, or 154-208 (i.e., any one of the sense strand sequences in Table 1 B or Table A- 2). Such isolated, modified sequence may further comprise at least one ligand attached to the 5’ end or the 3’ end, wherein each ligand is individually selected from the group consisting of a GalNac moiety, a C16 lipid, and a C18 lipid. Such ligands may be attached to the sequence via a linker.

[0227] One embodiment is an siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a nucleobase sequence comprising any one of SEQ ID NOs: 50-153 or 209-327 (i.e., any one of the antisense strand sequences in Table 1 B or Table A-2) and the sense strand comprises a nucleobase sequence comprising any one of SEQ ID NOs: 29-49, or 154-208 (i.e., any one of the sense strand sequences in Table 1 B or Table A-2). Such siRNA may further comprise at least one ligand attached to the 5’ end or the 3’ end of either the antisense or the sense strand, or any combination thereof, wherein each ligand is individually selected from the group consisting of a GalNAc moiety, a C16 lipid, and a C18 lipid.

[0228] One embodiment is an siRNA comprising an antisense strand comprising any one of SEQ ID NOs: 50-153 or 209-327.

[0229] One embodiment is an siRNA comprising a sense strand comprising any one of SEQ ID NOs: 29-49, or 154-208.

[0230] One embodiment is an siRNA, wherein the siRNA comprises a sense strand / antisense strand pair detailed in Table 1 B or Table A-2. Such siRNA may further comprise at least one ligand attached to the 5’ end or the 3’ end of either the antisense or the sense strand, or any combination thereof, wherein each ligand is individually selected from the group consisting of a GalNac moiety, a C16 lipid, and a C18 lipid.

[0231] One embodiment is a duplex of Table 1 B or A-2, wherein the sense strand of the duplex comprises a ligand at the 5’ end, wherein the ligand is selected from the group consisting of a GalNac moiety, a C16 lipid, and a 018 lipid.

[0232] One embodiment is an siRNA comprising a sense strand sequence of SEQ ID NO:32, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32.

[0233] One embodiment is an siRNA comprising an antisense strand sequence of SEQ ID NO:75, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 75.

[0234] One embodiment is an siRNA comprising a sense strand sequence of SEQ ID NO:32, or a sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 32 and an antisense strand sequence of SEQ ID NO:75, or a sequence that is at least 80%, 85%,90%, or 95% identical to SEQ ID NO: 75. Such an siRNA may comprise a ligand attached to the 5’ end of the sense strand, wherein the ligand is selected from the group consisting of a GalNac moiety, a C16 lipid, and a C18 lipid. In some embodiments, such a ligand may be attached via a C6 amine linker.

[0235] One embodiment is an siRNA as depicted in Figure 4.

[0236] One embodiment is an siRNA comprising an antisense strand, wherein the antisense strand comprises a modification pattern detailed in Table 2A or 2B, and the siRNA targets a C3 transcript. Such siRNAs may further comprise a sense strand, wherein the sense strand has a modification pattern detailed in Table 3A or 3B. Such siRNA may further comprise at least one ligand attached to the 5’ end or the 3’ end of either the antisense or the sense strand, or any combination thereof, wherein each ligand is individually selected from the group consisting of a GalNac moiety, a C16 lipid, and a 018 lipid. Such siRNA may comprise an antisense strand nucleobase sequence detailed in Table 1A, 1 B, A-1 or A-2. Such siRNA may also comprise a sense strand nucleobase sequence detailed in Table 1A, 1 B, A-1 or A-2.

[0237] One embodiment is a method of treating a subject having or at risk of a complement-mediated disorder, the method comprising administering to the subject a composition comprising an effective amount of an siRNA detailed herein. Such a method comprises a reduction in 03 transcript or 03 protein in the subject or in a biological sample from the subject relative to a level before the administration of the siRNA. For instance, the level of 03 transcript or 03 protein may be 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% or greater, relative to a level before the administration of the siRNA. The siRNA may be administered intravenously or subcutaneously to the subject. In some embodiments, the siRNA is administered to a hepatocyte of the subject (e.g., ex vivo or in vivo). The subject may be administered a second agent, such as an anti-C3 antibody or a compstatin analog.

[0238] One embodiment is a composition comprising an siRNA detailed herein and a carrier and / or excipient.

[0239] One embodiment is a method of reducing or inhibiting complement 03 expression in a cell, the method comprising contacting the cell with the siRNA detailed herein. Such a method comprises a reduction in 03 transcript or 03 protein in the cell relative to a level before the administration of the siRNA. For instance, the level of 03 transcript or 03 protein may be reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 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% or greater, relative to a level before the administration of the siRNA. In some embodiments, the cell is in a subject (e.g., a human subject). In some embodiments, a subject suffers from a complement- mediated disorder.EXAMPLES

[0240] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.Example 1 : Knockdown of C3 Expression in HepG2 Cells Using siRNAs

[0241] Duplex pairs from Table 1B were tested.

[0242] Cell Culture

[0243] HepG2 cells were obtained from ATCC (ATCC in partnership with LGC Standards, Wesel, Germany, cat. #ATCC-HB-8065) and cultured in MEM Eagle (#M2279, Sigma- Aldrich, Germany), supplemented to contain 10% fetal calf 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 pg / ml Streptomycin (#A2213, Biochrom GmbH, Berlin, Germany) at 37° C. in an atmosphere with 5% CO2in a humidified incubator.

[0244] siRNAs

[0245] siRNAs were designed and synthesized as detailed in Table 1B.

[0246] Dose Response Experiments

[0247] T ransfection of siRNA was carried out with Lipofectamine RNAiMax (Invitrogen / Life Technologies, Karlsruhe, Germany) according to manufacturer's instructions for reverse transfection.

[0248] Each siRNA was tested using dose response experiments (DRC) in HepG2 cells. Dose-response experiments were done with siRNA in 10 concentrationstransfected in quadruplicates, starting at 100 nM in 6-fold dilutions steps down to ~10 fM. Mock transfected cells served as negative controls.

[0249] For each well, the target mRNA level was normalized to the respective GAPDH mRNA level. The activity of a given siRNA was expressed as percent mRNA concentration of the respective target (normalized to GAPDH mRNA) in treated cells, relative to the target mRNA concentration (normalized to GAPDH mRNA) averaged across mock transfected wells (DRCs). IC20, IC50, IC80 values, and Max KD from the DRC experiments are shown in Table A-1 below. The sequences for these siRNAs are shown in Table 1B above.

[0250] IC50 and IC80 values in Table A-1 indicate that the modification patterns varied in performance. (N / M in Table A stands for “not measured.”)Table A-1Example 2: Knockdown of C3 Expression in HepG2 Cells Using siRNAs

[0251] Duplex pairs were tested.Cell Culture

[0252] HepG2 cells were obtained from ATCC (ATCC in partnership with LGC Standards, Wesel, Germany, cat. #ATCC-HB-8065) and cultured in MEM Eagle (#M2279, Sigma-Aldrich, Germany), supplemented to contain 10% fetal calf 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 pg / mlStreptomycin (#A2213, Biochrom GmbH, Berlin, Germany) at 37° C. in an atmosphere with 5% CO2in a humidified incubator. siRNAs

[0253] siRNAs were designed and synthesized with sequences and modification patterns detailed herein.Experiments

[0254] Transfection of siRNA was carried out with Lipofectamine RNAiMax (Invitrogen / Life Technologies, Karlsruhe, Germany) according to manufacturer's instructions for reverse transfection.

[0255] Each siRNA detailed in Table A-2 below was tested in HepG2 cells. using siRNA at 2 concentrations: 10nM and 0.1 nM.

[0256] For each well, the target mRNA level was normalized to the respective GAPDH mRNA level. The activity of a given siRNA was expressed as percent mRNA concentration of the respective target (normalized to GAPDH mRNA) in treated cells, relative to the target mRNA concentration (normalized to GAPDH mRNA) averaged across mock transfected wells.

[0248] In a further experiment, a subset of potent siRNAs from the HepG2 assay were synthesized with a GalNAc conjugate and assessed for activity in a free uptake study in primary human hepatocytes. A dose response activity curve of an exemplary conjugated siRNA is shown in Figure 1A.

[0249] In a further experiment, primary human astrocytes were treated with TNF-a to stimulate C3 gene expression, then exemplary siRNAs were transfected into cells and potency was evaluated. Figure 1B shows a dose response activity curve an exemplary conjugated siRNA in primary human astrocytes.

[0250] In a further in vitro experiment, an exemplary siRNA from this Example was assessed for its ability to stimulate expression of IP-10 and MIP-1a in hPBMCs from 3 donors. Figure 2 shows heat map data of the immune-stimulatory potential of the exemplary siRNA in a human PBMC transfection assay. Positive and negative controls were used to verify functionality of the assay and ensure normal reactivity of the donor cells, respectively.Table A-2

[0257] With respect to Table A-2, a lower-case a, c, g, or u refers to a 2’O-methyl (2’OMe) modified nucleotide. An uppercase letter followed by ‘f refers to a 2’F modified nucleotide. A uppercase letter A, C. G. or U, not followed by another character, refers to an unmodified ribonucleotide. An ‘s’, when it appears between two letters, refers to a 3’ phosphorothioate bond between the nucleotide preceding the ‘s’ and the nucleotide following the ‘s.’ When a ‘ps’ appears at the 5’ end of a sequence, the ‘ps’ represents a 5’ phosphorothioate group on the first 5’ nucleotide. The abbreviation X-GNA refers to a glycerol nucleic acid comprising the nucleobase “X” - by way of example, “U-GNA” refers to a glycerol nucleic acid with a uracil nucleobase. The abbreviation “Xo” refers to an unlocked nucleic acid comprising the nucleobase “X” - by way of example, Uo refers to an unlocked nucleic acid with a uracil nucleobase. The abbreviation “(vinu)” refers to a nucleotide with a uracil nucleobase, a 2’OMe modification on the sugar, and a 5’ (E) vinylphosphonate. The abbreviation “Xm” refers to a nucleotide with an ‘X’ nucleobase, and a 2’ O-methoxyethyl modified sugar - for example, “Gm” refers to a nucleotide with guanine as the nucleobase and a 2’ O-methoxyethy! modified sugar. The abbreviation “Bn-X” refers to a nucleotide with an ‘X’ nucleobase, and a 2’ OBenzyl modified sugar - for example, “Bn-G” refers to a nucleotide with guanine as the nucleobase and a 2* OBenzyl modified sugar. The abbreviation “isodX” refers to an isoguanine or isocytosine nucleobase, as indicated ~ for example, “IsodG” refers to an isoguanine nucleobase.Example 3: Evaluation of intrathecally dosed siRNA in NHPs.

[0258] Non-naTve cynomolgus monkeys were implanted with intrathecal catheters as part of a previous study. Animals were dosed via lumbar puncture on Day 1 and Day 43 of the study with either artificial cerebrospinal fluid (aCSF) or an siRNA selected from Example 1 or 2 conjugated to a C16 fatty acid. CSF was collected via the catheters throughout the study inlife and animals were sacrificed 6 weeks post-final dose (Day 85). The data in Figure 3 is derived from siRNA duplex 28A. C3 protein levels in (Figure 3A) CSF and (Figure 3B) plasma were evaluated by immunoassay. C3 gene expression levels in CNS tissues and liver were analyzed by RT-qPCR and normalized to vehicle-treated animals (Figure 3C). Exposure levels of the C16 conjugated siRNA antisense strand (AS) were quantified using an LC- MS / MS assay (Figure 3D). CSF exposure of the C16 conjugated siRNA AS following two L-IT dose administrations (Figure 3E). These studies highlight the discovery of a potent C3- targeting siRNA, with silencing activity in Alzheimer’s relevant brain regions in NHP. Thestudies further suggest a C16 conjugated siRNA is non-immunostimulatory and is tolerated in NHPs.EQUIVALENTS

[0259] 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 present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMSWhat is claimed is:

1. An isolated, modified sequence comprising any one of SEQ ID NOs: 15-28.

2. An isolated, modified sequence comprising any one of SEQ ID Nos: 1-14.

3. The isolated, modified sequence of claim 1 or claim 2, further comprising at least one ligand attached to the 5’ end or the 3’ end of the sequence.

4. An siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a nucleotide sequence comprising any one of SEQ ID NOs: 15-28 and the sense strand comprises a nucleotide sequence comprising any one of SEQ ID NOs: 1-14.

5. The siRNA of claim 4, further comprising at least one ligand attached to the 5’ end or the3’ end of either the antisense or the sense strand.

6. The siRNA of claim 5, wherein the ligand comprises at least one GalNAc moiety.

7. The siRNA of claim 5, wherein the ligand comprises an oleic moiety.

8. An siRNA comprising an antisense strand comprising any one of SEQ ID NOs: 15-28 and50-153.

9. An siRNA comprising a sense strand comprising any one of SEQ ID NOs: 1-14 and 29-47.

10. An siRNA, wherein the siRNA comprises a sense strand / antisense strand pair detailed in Table 1 A, Table 1 B, Table A-1 , or Table A-2.11 . The siRNA of claim 10, further comprising at least one ligand attached to the 5’ end or the 3’ end of either the antisense or the sense strand.

12. The siRNA of claim 11 , wherein the ligand comprises at least one GalNAc moiety.

13. The siRNA of claim 11 , wherein the ligand comprises an oleic moiety or a C16 moiety.

14. An siRNA comprising an antisense strand and a sense strand, wherein the antisense strand comprises a modification pattern detailed in Table 2A or Table 2B, and the siRNA targets a 03 transcript.

15. The siRNA of claim 14, wherein the sense strand has a modification pattern detailed in Table 3A or Table 3B.

16. The siRNA of claim 14 or 15, further comprising at least one ligand attached to the 5’ end of the sense strand, the 3’ end of the sense strand, the 5’ end of the antisense strand, the 3’ end of the antisense strand, or a combination thereof.

17. The siRNA of claim 16, wherein the ligand comprises at least one GalNAc moiety.

18. The siRNA of claim 16, wherein the ligand comprises three GalNAc moieties.

19. The siRNA of claim 16, wherein the ligand comprises an oleic moiety.

20. The siRNA of any one of claims 14-19, wherein the siRNA has an antisense strand nucleobase sequence detailed in Table 1 A or Table A-1 .21 . The siRNA of any one of claims 14-20, wherein the siRNA has a sense strand nucleobase sequence detailed in Table 1 A or Table A-1 .

22. A method of treating a subject having or at risk of a complement-mediated disorder, the method comprising administering to the subject a composition comprising an effective amount of the siRNA of any one of claims 4-21 , 47, or 48.

23. The method of claim 22, wherein after the administration of the composition, a level of C3 transcript or C3 protein in the subject or in a biological sample from the subject is reduced relative to a level before the administration of the composition.

24. The method of claim 23, wherein 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% or greater, relative to a level before the administration.

25. The method of any one of claims 22-24, wherein the composition is administered intrathecally, intravenously, or subcutaneously to the subject.

26. The method of any one of claims 22-24, wherein the composition is administered to a hepatocyte of the subject.

27. The method of claim 26, wherein the composition is administered to the hepatocyte ex vivo.

28. The method of claim 26, wherein the composition is administered to the hepatocyte in vivo.

29. The method of any one of claims 22-28, further comprising administering to the subject a second agent.

30. The method of claim 29, wherein the second agent is an anti-C3 antibody or a compstatin analog.31 . The method of any one of claims 22-30, wherein the subject has a defect in complement regulation, optionally wherein the defect comprises abnormally low expression of one or more complement regulatory proteins by at least some of the subject’s cells.

32. The method of any one of claims 22-31 , wherein the complement-mediated disorder is a chronic disorder.

33. The method of any one of claims 22-32, wherein the complement-mediated disorder involves complement-mediated damage to red blood cells, optionally wherein the disorder is paroxysmal nocturnal hemoglobinuria or atypical hemolytic uremic syndrome.

34. The method of any one of claims 22-32, wherein the complement-mediated disorder is an autoimmune disease, optionally wherein the disorder is multiple sclerosis.

35. The method of any one of claims 22-32, wherein the complement-mediated disorder involves the kidney, optionally wherein the disorder is membranoproliferative glomerulonephritis, lupus nephritis, IgA nephropathy (IgAN), primary membranous nephropathy (primary MN), C3 glomerulopathy (C3G), or acute kidney injury.

36. The method of any one of claims 22-32, wherein the complement-mediated disorder involves the central or peripheral nervous system or neuromuscular junction, optionally wherein the disorder is neuromyelitis optica, Guillain-Barre syndrome, multifocal motor neuropathy, or myasthenia gravis.

37. A composition comprising the siRNA of any one of claims 4-21 , 47, or 48 and a carrier and / or excipient.

38. A method of reducing or inhibiting complement C3 expression in a cell, the method comprising contacting the cell with the siRNA of any one of claims 4-21.

39. The method of claim 38, wherein after the contacting 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%, or greater, relative to a level before the contacting step.

40. The method of claim 38 or 39, wherein the cell is in a subject.41 . The method of any one of claims 22-36 or 40, wherein the subject is a human.

42. The method of claim 41 , wherein the subject suffers from a complement-mediated disorder.

43. A method of reducing or inhibiting expression of C3 in a subject, the method comprising contacting a cell of the subject with the siRNA of any one of claims 4-21 or the composition of claim 37.

44. The method of claim 43, wherein after the contacting 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%, or greater, relative to a level before the contacting step.

45. The method of claim 43 or 44, wherein the subject is a human.

46. The method of claim 45, wherein the subject suffers from a complement-mediated disorder.

47. An siRNA comprising an antisense strand, wherein the antisense strand comprises any one of the siRNA antisense strand sequences detailed in Table 1 B, Table A-1 or Table A-2.

48. An siRNA comprising a sense strand, wherein the sense strand comprises any one or the siRNA sense strand sequences detailed in Table 1 B, Table A-1 or Table A-2.

49. The siRNA of claim 10, wherein the sense strand has the sequence of SEQ ID NO:32.

50. The siRNA of claim 10, wherein the antisense strand has the sequence of SEQ ID NO:75.51 . The siRNA of claim 49, wherein the siRNA has the sequence of duplex 14A-36A in Table 1 B.

52. The siRNA of any of claims 49 50, wherein the siRNA further comprises a GalNac moiety.

53. The siRNA of any of claims 49-50, wherein the siRNA further comprises a C16 moiety.

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