Nucleic acids for inhibiting expression of MASP-2 in cells

Abstract

The present invention relates to double-stranded nucleic acid molecules that disrupt or inhibit mannan-binding lectin serine protease 2 (MASP-2) gene expression, which further relates to therapeutic uses of such inhibition, such as for the treatment of diseases and disorders associated with deregulation of the complement pathway, in particular the lectin pathway, and / or associated with overactivation or ectopic expression or localization or accumulation of MASP-2 in the body.

Description

[Technical field]

[0001] The present invention relates to double-stranded nucleic acid molecules that disrupt or inhibit mannan-binding lectin serine protease 2 (MASP-2) gene expression, which further relates to therapeutic uses of such inhibition, such as for the treatment of diseases and disorders associated with deregulation of the complement pathway, in particular the lectin pathway, and / or associated with overactivation or ectopic expression or localization or accumulation of MASP-2 in the body. [Background technology]

[0002] Double-stranded RNA (dsRNA), capable of binding to expressed mRNA through complementary base pairing, has been shown to block gene expression by a mechanism termed "RNA interference (RNAi)" (Fire et al., 1998, Nature. 1998 Feb 19;391 (6669):806-11 and Elbashir et al., 2001, Nature. 2001 May 24;411 (6836):494-8). Short dsRNA induces gene-specific posttranscriptional silencing in many organisms, including vertebrates, making it a useful tool for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a sequence-specific multicomponent nuclease that degrades messenger RNAs with sufficient complementarity or homology to the silencing trigger loaded into the RISC complex. Interfering RNA, such as siRNA, antisense RNA and microRNA, is an oligonucleotide that prevents the formation of protein by inhibiting gene translation of protein through degradation of mRNA molecule.Gene silencing agents are becoming more and more important for therapeutic application in the medical field.

[0003] According to Watts and Corey in the Journal of Pathology (2012; Vol 226, pp. 365-379), there are algorithms that can be used to design nucleic acid silencing triggers, but all of these have significant limitations. Because the algorithms do not take into account factors such as the tertiary structure of the target mRNA or the involvement of RNA-binding proteins, various experimental methods may be required to identify potent siRNAs. Therefore, the discovery of potent nucleic acid silencing triggers with minimal off-target effects is a complex process. For the pharmaceutical development of these highly charged molecules, it is necessary that they can be synthesized economically, distributed to target tissues, and can enter cells and function within acceptable toxicity limits.

[0004] The complement system or pathway is part of the innate immune system of host defense against invading pathogens. It consists of several proteins that circulate in the bloodstream mainly in precursor form. Most of the proteins that form the complement system, including the complement component protein C3 (also referred to simply as C3 herein), are synthesized and secreted into the bloodstream, mostly by hepatocytes in the liver. Activation of this system triggers an inflammatory response, leading to the attraction and opsonization of phagocytes and the resulting clearance of pathogens, immune complexes, and cellular debris (Janeway's Immunobiology 9th Edition). The complement system consists of three pathways (classical, lectin, and alternative), all of which converge on the formation of the so-called complement component 3 convertase complex. These enzyme complexes cleave the complement component C3 protein into C3a and C3b. Once cleaved, C3b forms part of a complex that cleaves C5 into C5a and C5b. After cleavage, C5b is one of the key components of the major complement pathway effector, the membrane attack complex. C3 is therefore a key component of the complement system activation pathway.

[0005] Complement factor B (CFB or "factor B") is involved in activation of the alternative pathway. Binding of CFB to C3b (e.g., on the cell surface) renders it susceptible to cleavage by factor D to form the serine protease C3Bb, which is itself a C3 convertase, resulting in an amplification loop of C3 activation. CFB is primarily synthesized in the liver, and at lower levels in several extrahepatic sites as well.

[0006] The mannan-binding lectin serine protease 2 (also known as mannose-binding protein-associated serine protease 2, or mannan-binding lectin serine peptidase 2, or MBL-associated serine protease 2) (MASP-2) gene, which encodes the protein MASP-2, is the major enzyme involved in the activation of the lectin pathway of the complement system. MASP-2 can also activate the alternative pathway of the complement system through the C4 / C2 bypass pathway, leading to MASP-2-dependent C3 activation by an unknown mechanism. The MASP-2 gene encodes a member of the peptidase S1 family of serine proteases. The encoded preproprotein is proteolytically processed to generate A and B chains, which heterodimerize to form the mature protease. This protease cleaves complement components C2 and C4 in the lectin pathway of the complement system to generate C3 convertase. The encoded protease also plays a role in the coagulation cascade to form thrombin through cleavage of prothrombin. Alternative splicing results in multiple transcript variants, at least one of which encodes an isoform that is proteolytically processed.

[0007] The MASP-2 gene encodes the 76 kDa serine protease MASP-2 (long isoform) that is highly expressed in the liver and the 19 kDa alternative splicing product Map19 (short isoform) present in plasma.

[0008] Several diseases are associated with abnormal acquisition of the complement pathway or its genetic activation, as well as abnormal or excessive expression of MASP-2.

[0009] Currently, there are only a few treatments for diseases, disorders, and syndromes mediated by the complement system. The monoclonal humanized antibody eculizumab is one of them. It is known to bind to complement protein C5, thereby blocking the membrane attack complex at the end of the complement cascade (Hillmen et al., 2006 NEJM). However, only a portion of patients suffering from diseases mediated by the complement system respond to eculizumab therapy. Thus, there is a high unmet need for medical treatment of diseases mediated by or related to complement. Inhibiting the expression of MASP-2 represents a promising therapeutic strategy for many complement-mediated diseases. For example, targeting MASP-2 expression or activity via antisense oligonucleotides or inhibitors has been presented as a promising treatment strategy for a variety of complement-mediated conditions, including rheumatoid arthritis (Holers et al., Front Immunol. 2020 Feb. 21; 11:201), IgA nephropathy, lupus nephritis (Lafayette et al., Kidney Int Rep. 2020 Aug. 13; 5(11):2032-2041), thrombotic microangiopathy, and atypical hemolytic uremic syndrome (Elhadad et al., Clin Exp Immunol. 2021 Jan. 203(1):96-104; Young et al., Bone Marrow Transplant. 2021 Aug. 56(8):1805-1817). It has been proposed to investigate MASP-2 inhibition (via antibodies) for treating pneumococcal meningitis (Kasanmoentalib et al., J Neuroinflammation. 2017 Apr 6;14(1):77) and coronavirus infection (Flude et al., Viruses, 2021 Feb 17;13(2):312).

[0010] WO2012139081A2, Lafayette et al. (Kidney Int Rep. 2020 Aug. 13;5(11):2032-2041), Young et al. (Bone Marrow Transplant. 2021 Aug;56(8):1805-1817, and Elhadad et al. (Clin Exp Immunol. 2021 Jan;203(1):96-104) describe MASP-2 antibodies (e.g., narsoplimab) for treating conditions having MASP-2-dependent complement activation. US20200032270A1 and WO2021168148A1 describe double-stranded RNA agents targeting the MASP-2 gene. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2012139081A2 [Patent Document 2] US20200032270A1 [Patent Document 3] WO2021168148A1 [Patent Document 4] U.S. Patent No. 5,885,968 [Patent Document 5] European Patent No. 0758904 B1 [Patent Document 6] WO2017 / 174657 [Non-patent literature]

[0012] [Non-Patent Document 1] Fire et al., 1998, Nature. 1998 Feb 19;391 (6669):806-11 [Non-Patent Document 2] Elbashir et al., 2001, Nature. 2001 May 24;411 (6836):494-8 [Non-Patent Document 3] Watts and Corey in the Journal of Pathology (2012; Vol 226, pp. 365-379) [Non-Patent Document 4] Janeway's Immunobiology 9th Edition [Non-Patent Document 5] Hillmen et al., 2006 NEJM [Non-Patent Document 6] Holers et al., Front Immunol. 2020 Feb 21; 11:201 [Non-Patent Document 7] Lafayette et al., Kidney Int Rep. 2020 Aug 13;5(11):2032-2041 [Non-Patent Document 8] Elhadad et al., Clin Exp Immunol. 2021 Jan;203(1):96-104 [Non-Patent Document 9] Young et al., Bone Marrow Transplant. 2021 Aug;56(8):1805-1817 [Non-Patent Document 10] Kasanmoentalib et al. J Neuroinflammation. 2017 Apr 6;14(1):77 [Non-Patent Document 11] Flude et al., Viruses, 2021 Feb 17;13(2):312 [Non-Patent Document 12] Dubber et al. (Bioconjug. Chem. 2003 Jan-Feb;14(1):239-46.) [Non-Patent Document 13] Weigel, PH et al., Biochim. Biophys. Acta. 2002 Sep 19;1572(2-3):341-63 [Non-Patent Document 14] Lshibashi, S. et al., J Biol. Chem. 1994 Nov 11;269(45):27803-6 [Non-Patent Document 15] Akinc et al., Mol Ther. 2010 Jul; 18(7): 1357-64

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[0013] One aspect of the present invention is a double-stranded nucleic acid for inhibiting expression of mannan-binding lectin serine protease 2 (MASP-2), comprising a first strand and a second strand, wherein an unmodified equivalent of the sequence of the first strand comprises a sequence of at least 15 nucleotides that differs by no more than three nucleotides from any one of the sequences of the first strand shown in Table 5a (Table 8).

[0014] An unmodified equivalent of a first strand sequence can include, for example, a sequence of at least 15 nucleotides that differs by no more than 3 nucleotides from any one of the first strand sequences listed in Table 1.

[0015] The nucleic acids described herein are therefore preferably double-stranded nucleic acids capable of inhibiting expression of MASP-2 in a cell and may find use as therapeutic or diagnostic agents, for example in related diagnostic or therapeutic methods. The nucleic acids comprise or consist of a first strand and a second strand, the first strand typically comprising a sequence sufficiently complementary to MASP-2 mRNA so as to mediate RNA interference.

[0016] One aspect relates to a composition comprising a nucleic acid as disclosed herein and a solvent (preferably water) and / or a delivery vehicle and / or a physiologically acceptable excipient and / or a carrier and / or a salt and / or a diluent and / or a buffer and / or a preservative.

[0017] One aspect relates to a composition comprising a nucleic acid disclosed herein and an additional therapeutic agent selected from, for example, an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody, and a peptide.

[0018] One aspect relates to the nucleic acids disclosed herein, or compositions comprising same, for use as therapeutic or diagnostic agents, e.g., in related methods.

[0019] One aspect relates to a nucleic acid disclosed herein, or a composition comprising same, for use in the prevention or treatment of a disease, disorder, or syndrome.

[0020] One aspect relates to the use of the nucleic acids disclosed herein, or compositions comprising same, in the prevention or treatment of a disease, disorder, or syndrome.

[0021] One aspect relates to the use of a nucleic acid disclosed herein, or a composition comprising same, in the preparation of a medicament for the prevention or treatment of a disease, disorder, or syndrome.

[0022] One aspect relates to a method of prevention or treatment of a disease, disorder, or syndrome, comprising administering to an individual in need of treatment a pharma- ceutical effective dose or amount of a nucleic acid disclosed herein or a composition comprising same, preferably, the nucleic acid or composition is administered to the subject subcutaneously, intravenously, or by oral, rectal, intrapulmonary, intramuscular, or intraperitoneal administration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention relates to nucleic acids and compositions thereof that are double-stranded and contain a sequence substantially homologous to the expressed RNA transcript of MASP-2. These nucleic acids, their conjugates, and compositions containing them may be used in the prevention and treatment of various diseases, disorders, and syndromes in which it is desirable to reduce the expression of the MASP-2 gene product.

[0024] The first aspect of the present invention is a double-stranded nucleic acid for inhibiting the expression of MASP-2, preferably in a cell, comprising a first strand and a second strand, wherein the unmodified equivalent of the sequence of the first strand comprises a sequence of at least 15 nucleotides that differs by no more than three nucleotides from any one of the sequences of the first strand shown in Table 5a (Table 8). These nucleic acids have the advantage, inter alia, of being active in a variety of species relevant for preclinical and clinical development and / or having few associated off-target effects. Having few associated off-target effects means that the nucleic acid specifically inhibits the intended target and does not significantly inhibit other genes or inhibits only one or a few other genes at a therapeutically acceptable level.

[0025] For example, an unmodified equivalent of a first strand sequence can include a sequence of at least 15 nucleotides that differs by no more than 3 nucleotides from any one of the first strand sequences listed in Table 1.

[0026] [Table 1]

[0027] Preferably, the unmodified equivalent of the first strand sequence comprises a sequence of at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all 19 nucleotides that differ by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotides, from any one of the first strand sequences shown in Table 1 or Table 5a.

[0028] For example, an unmodified equivalent of a first strand sequence can include a sequence that differs by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotides, at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all 19 nucleotides, from any one of the first strand sequences listed in Table 1 or Table 5a.

[0029] Preferably, the unmodified equivalent of the first strand sequence of the nucleic acid consists of one of the first strand sequences shown in Table 1 or Table 5a. The sequence may, however, be modified by some nucleic acid modifications that do not change the identity of the nucleotides. For example, modifications of the backbone or sugar residues of the nucleic acid do not change the identity of the nucleotides, since the bases themselves remain the same as in the reference sequence.

[0030] For example, an unmodified equivalent of a first strand sequence of a nucleic acid may consist of one of the first strand sequences shown in Table 1 or Table 5a, optionally modified by one or more of the nucleic acid modifications described above.

[0031] A nucleic acid comprising a sequence according to a reference sequence herein means that the nucleic acid comprises a sequence of contiguous nucleotides in the order defined in the reference sequence.

[0032] In this specification, when a reference sequence that comprises, consists essentially of, or consists of nucleotides is made, this reference is not limited to sequences with unmodified nucleotides.The same reference also includes the same nucleotide sequence in which one, several, for example, two, three, four, five, six, seven, or more, for example, all nucleotides are modified, for example, by 2'-OMe, 2'-F, ligand, linker, 3'-end or 5'-end modification, or any other modification.It also relates to sequences in which two or more nucleotides are linked to each other by natural phosphodiester linkages or any other linkages, for example, phosphorothioate or phosphorodithioate linkages.

[0033] A double-stranded nucleic acid is a nucleic acid in which a first strand and a second strand can hybridize to each other over at least a portion of their length and thus form a duplex region under physiological conditions, e.g., at a concentration of 1 μM of each strand in PBS at 37° C. The first and second strands can preferably hybridize to each other and thus form a duplex region over a region of at least 15 nucleotides, preferably 16, 17, 18, or 19 nucleotides. This duplex region preferably includes nucleotide base pairing between the two strands based on Watson-Crick base pairing and / or wobble base pairing (e.g., GU base pairing). Not all nucleotides of the two strands in the duplex region necessarily need to base pair with each other to form a duplex region. A certain number of mismatches, deletions, or insertions between the nucleotide sequences of the two strands are allowed. Overhangs at either end of the first or second strand, or unpaired nucleotides at one end of the double-stranded nucleic acid are also possible. The double-stranded nucleic acid is preferably a double-stranded nucleic acid under physiological conditions, and preferably has a melting temperature (Tm) of 45° C. or higher, preferably 50° C. or higher, more preferably 55° C. or higher, for example in PBS, at a concentration of 1 μM of each strand.

[0034] A double-stranded nucleic acid that is stable under physiological conditions is, for example, a double-stranded nucleic acid that has a Tm of 45° C. or higher, preferably 50° C. or higher, more preferably 55° C. or higher, in PBS at a concentration of 1 μM for each strand.

[0035] The first and second strands are preferably capable of forming a double-stranded region (i.e., complementary to each other) i) over at least a portion of their length, preferably over at least 15 nucleotides of both their lengths, ii) over the entire length of the first strand, iii) over the entire length of the second strand, or iv) over the entire length of both the first and second strands. Strands that are complementary to each other over a certain length means that the strands can base-pair with each other over that length, either by Watson-Crick or wobble base pairing. It is not necessary that each nucleotide of that length can base-pair with its counterpart in the other strand over the entire length, as long as a stable double-stranded nucleotide can be formed under physiological conditions. However, in certain embodiments, it is preferred if each nucleotide of that length can base-pair with its counterpart in the other strand over the entire length.

[0036] A certain number of mismatches, deletions, or insertions between the first strand and the target sequence, or between the first strand and the second strand, may be tolerated in the context of the nucleic acids according to the invention, and may even, in certain cases, increase RNA interference (e.g., inhibitory) activity.

[0037] The inhibitory activity of the nucleic acid according to the present invention depends on the formation of a duplex region between all or a portion of the first strand and a portion of the target nucleic acid. The portion of the target nucleic acid that forms a duplex region with the first strand, defined as starting with the first base pair formed between the first strand and the target sequence and ending with the last base pair formed between the first strand and the target sequence (inclusive), is the target nucleic acid sequence, or simply the target sequence. The duplex region formed between the first strand and the second strand does not have to be the same as the duplex region formed between the first strand and the target sequence. That is, the second strand may have a different sequence from the target sequence, but the first strand must be able to form a duplex structure with both the second strand and the target sequence, at least under physiological conditions.

[0038] The complementarity between the first strand and the target sequence can be perfect (i.e., 100% identity with no nucleotide mismatches, insertions or deletions in the first strand compared to the target sequence).

[0039] The complementarity between the first strand and the target sequence does not have to be perfect. The complementarity can be from about 70% to about 100%. More specifically, the complementarity can be at least 70%, 80%, 85%, 90%, or 95%, as well as intermediate values.

[0040] The identity between the first strand and the complementary sequence of the target sequence can range from about 75% to about 100%. More specifically, the complementarity can be at least 75%, 80%, 85%, 90%, or 95%, as well as intermediate values, provided that the nucleic acid is capable of reducing or inhibiting the expression of MASP-2.

[0041] A nucleic acid having less than 100% complementarity between the first strand and the target sequence may be capable of reducing expression of MASP-2 to the same level as a nucleic acid having complete complementarity between the first strand and the target sequence. Alternatively, it may be capable of reducing expression of MASP-2 to a level that is 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the level of reduction achieved by a nucleic acid having complete complementarity.

[0042] The nucleic acids of the present disclosure can be isolated nucleic acids.

[0043] The nucleic acid of the present disclosure is (a) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than three nucleotides from any one of the first strand sequences in Table 5a (Table 8), and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than three nucleotides from the corresponding second strand sequence in Table 5a (Table 8); (b) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than two nucleotides from any one of the first strand sequences in Table 5a (Table 8), and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than two nucleotides from the corresponding second strand sequence in Table 5a (Table 8); (c) the unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than one nucleotide from any one of the first strand sequences in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than one nucleotide from the corresponding second strand sequence in Table 5a (Table 8); (d) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5a (Table 8); (e) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5a (Table 8); (f) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5a (Table 8); (g) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 1-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5a (Table 8); (h) the unmodified equivalent of the first strand sequence comprises any one of the sequences of the first strand sequences having a given SEQ ID NO shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5a (Table 8); (i) the unmodified equivalent of the first strand sequence consists essentially of any one of the first strand sequences having a given SEQ ID NO shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence consists essentially of the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5a (Table 8); (j) the unmodified equivalent of the first strand sequence consists of any one of the first strand sequences having a given SEQ ID NO shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5a (Table 8); (k) the unmodified equivalent of the first strand sequence consists of a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8); the unmodified equivalent of the first strand sequence further consists of one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8); Optionally, the unmodified equivalent of the second strand sequence comprises, or consists essentially of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5a (Table 8); (l) the unmodified equivalent of the first strand sequence consists of a sequence corresponding to nucleotides 1-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8); the unmodified equivalent of the first strand sequence further consists of one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8); The unmodified equivalent of the first strand sequence consists of a continuous region of 17 to 25 nucleotides in length, preferably 18 to 24 nucleotides in length, that is complementary to the MASP-2 transcript of SEQ ID NO: 821; Optionally, the unmodified equivalent of the second strand sequence comprises, or consists essentially of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5a (Table 8); (m) the unmodified equivalent of the first strand and the unmodified equivalent of the second strand of any one of the nucleic acid molecules of subparagraphs (a)-(l) above are present on a single strand, and the unmodified equivalent of the first strand and the unmodified equivalent of the second strand are capable of hybridizing to one another, thereby forming a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; or (n) the unmodified equivalent of the first strand and the unmodified equivalent of the second strand of any one of the nucleic acid molecules of subparagraphs (a)-(l) above are on two separate strands that are capable of hybridizing to each other to thereby form a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; It may be a nucleic acid.

[0044] For example, the nucleic acid of the disclosure may be (a) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than three nucleotides from any one of the first strand sequences in Table 1, and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than three nucleotides from the corresponding second strand sequence in Table 1; (b) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than two nucleotides from any one of the first strand sequences in Table 1, and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than two nucleotides from the corresponding second strand sequence in Table 1; (c) the unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than one nucleotide from any one of the first strand sequences in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than one nucleotide from the corresponding second strand sequence in Table 1; (d) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 1; (e) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 1; (f) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 1; (g) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 1-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 1; (h) the unmodified equivalent of the first strand sequence comprises any one of the sequences of the first strand sequences having a given SEQ ID NO shown in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 1; (i) the unmodified equivalent of the first strand sequence consists of any one of the first strand sequences having a given SEQ ID NO shown in Table 1, and optionally the unmodified equivalent of the second strand sequence consists of the corresponding second strand sequence having a given SEQ ID NO shown in Table 1; (j) the unmodified equivalent of the first strand sequence consists essentially of any one of the first strand sequences having a given SEQ ID NO shown in Table 1, and optionally the unmodified equivalent of the second strand sequence consists essentially of the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 1; (k) the unmodified equivalent of the first strand sequence consists of a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1; the unmodified equivalent of the first strand sequence further comprises one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1; Optionally, the unmodified equivalent of the second strand sequence comprises, or essentially consists of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 1. (l) the unmodified equivalent of the first strand sequence consists of a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1; the unmodified equivalent of the first strand sequence further comprises one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1; The unmodified equivalent of the first strand sequence consists of a continuous region of 17 to 25 nucleotides in length, preferably 18 to 24 nucleotides in length, that is complementary to the MASP-2 transcript of SEQ ID NO: 821; Optionally, the unmodified equivalent of the second strand sequence comprises, or consists essentially of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 1. (m) the unmodified equivalent of the first strand and the unmodified equivalent of the second strand of any one of the nucleic acid molecules of subparagraphs (a)-(l) above are present on a single strand, and the unmodified equivalent of the first strand and the unmodified equivalent of the second strand are capable of hybridizing to one another, thereby forming a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; or (n) the unmodified equivalent of the first strand and the unmodified equivalent of the second strand of any one of the nucleic acid molecules of subparagraphs (a)-(l) above are on two separate strands that are capable of hybridizing to each other to thereby form a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; It may be a nucleic acid.

[0045] By "corresponding to" a second strand is meant a second strand that is present in the same duplex as a given first strand in Table 5a, 5b, or 5c (Tables 8, 9, or 10), or is listed as the corresponding second strand sequence in Table 1 or Table 2, as the context requires, i.e., a first strand and its corresponding second strand are designated as the "A" and "B" strands, respectively, of a duplex having a given duplex identifier in Table 5a, 5b, or 5c (Tables 8, 9, or 10), or are so listed in Tables 1 and 2.

[0046] In one embodiment, if the 5'-most nucleotide of the first strand is a nucleotide other than A or U, then it is replaced by A or U. Preferably, if the 5'-most nucleotide of the first strand is a nucleotide other than U, then it is replaced by U, more preferably by U with a 5' vinyl phosphonate.

[0047] When a nucleic acid of the present invention does not contain the entire sequence of a reference first strand and / or second strand sequence (e.g., provided in Tables 1, 2, 5a, 5b or 5c (Tables 1, 2, 8, 9 or 10)), or one or both strands differ by one, two or three nucleotides from the corresponding reference sequence, this nucleic acid preferably retains at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably at least 100% MASP-2 inhibitory activity compared to the inhibitory activity of a corresponding nucleic acid comprising the entire reference sequence of the first strand and the second strand in comparable experiments.

[0048] A nucleic acid that can hybridize under physiological conditions is a nucleic acid that can form base pairs, preferably Watson-Crick or wobble base pairs, between at least a portion of the opposing nucleotides in the strand, so as to form at least a double-stranded region.Such a double-stranded nucleic acid is preferably a stable double-stranded nucleic acid under physiological conditions (e.g., at 37°C in PBS, at a concentration of 1 μM of each strand), which means that under such conditions, the two strands remain hybridized to each other.The Tm of the double-stranded nucleotide is preferably 45°C or higher, preferably 50°C or higher, more preferably 55°C or higher.

[0049] One aspect of the present invention relates to a nucleic acid for inhibiting expression of MASP-2, comprising a first sequence of at least 15, preferably at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all nucleotides that differ from any of the sequences of the unmodified equivalents of the first strand of Table 5a (Table 8) or Table 1 (Table 1) by no more than three nucleotides, preferably no more than two nucleotides, more preferably no more than one nucleotide, and most preferably no nucleotides, and wherein the first sequence is capable of hybridizing to a target gene transcription product (e.g., mRNA) under physiological conditions. Preferably, the nucleic acid further comprises a second sequence of at least 15, preferably at least 16, more preferably at least 17, even more preferably at least 18, and most preferably all nucleotides that differ from any of the sequences of the unmodified equivalents of the corresponding second strand in Table 5a (Table 8) or Table 1 (Table 1) by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide, and most preferably no nucleotides, and the second sequence is capable of hybridizing to the first sequence under physiological conditions, and preferably the nucleic acid is an siRNA capable of inhibiting MASP-2 expression via the RNAi pathway.

[0050] One aspect relates to any double-stranded nucleic acid disclosed in Tables 1, 2, 5a, 5b or 5c (Tables 1, 2, 8, 9 or 10), each of which may be referenced by a given double-stranded identifier, preferably for inhibiting expression of MASP-2, provided that the double-stranded nucleic acid is capable of inhibiting expression of MASP-2. All of these nucleic acids are siRNAs.

[0051] One aspect relates to a double-stranded nucleic acid, preferably capable of inhibiting expression of MASP-2 in a cell, for use as a therapeutic or diagnostic agent, e.g. in a related therapeutic or diagnostic method, preferably comprising or consisting of a first strand and a second strand, preferably wherein the first strand comprises a sequence sufficiently complementary to MASP-2 mRNA so as to mediate RNA interference.

[0052] The nucleic acids described herein may be capable of inhibiting the expression of MASP-2. The inhibition may be complete, i.e., 0% expression remains. The inhibition of MASP-2 expression may be partial, i.e., it may be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more of the MASP-2 expression level in the absence of the nucleic acid of the invention, or an intermediate inhibition. The level of inhibition may be measured by comparing the treated sample to an untreated sample, or to a control, such as a sample treated with a siRNA that does not target MASP-2. The inhibition may be measured by measuring MASP-2 mRNA and / or protein levels, or the level of a biomarker or indicator that correlates with the presence or activity of MASP-2. It may be measured in cells that may have been treated in vitro with the nucleic acids described herein. Alternatively or additionally, inhibition may be measured in cells, e.g., liver cells, or tissues, e.g., liver tissue, or organs, e.g., liver, or body fluids, e.g., blood, serum, lymph, or any other body part or fluid, taken from a subject previously treated with a nucleic acid disclosed herein. Preferably, inhibition of MASP-2 expression is determined by comparing MASP-2 mRNA levels measured in cells expressing MASP-2 after 24 or 48 hours of in vitro treatment with the double-stranded RNA disclosed herein under ideal conditions (see Examples for suitable concentrations and conditions) with MASP-2 mRNA levels measured in control cells that are untreated, mock-treated, or treated with a control double-stranded RNA under the same conditions.

[0053] One aspect of the invention relates to a nucleic acid, wherein a first strand and a second strand are present on a single strand of the looped nucleic acid, such that the first strand and the second strand can hybridize to each other, thereby forming a double stranded nucleic acid having a duplex region.

[0054] Preferably, the first and second strands of the nucleic acid are separate strands. The two separate strands are preferably each 17-25 nucleotides long, more preferably 18-25 nucleotides long. The two strands may be of the same length or of different lengths. The first strand may be 17-25 nucleotides long, preferably it may be 18-24 nucleotides long, it may be 18, 19, 20, 21, 22, 23, or 24 nucleotides long. Most preferably, the first strand is 19 nucleotides long. The second strand may independently be 17-25 nucleotides long, preferably it may be 18-24 nucleotides long, it may be 18, 19, 20, 21, 22, 23, or 24 nucleotides long. More preferably, the second strand is 18 or 19 or 20 nucleotides long, most preferably it is 19 nucleotides long.

[0055] Preferably, the first and second strands of the nucleic acid form a duplex region 17-25 nucleotides in length. More preferably, the duplex region is 18-24 nucleotides in length. The duplex region may be 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In the most preferred embodiment, the duplex region is 18 or 19 nucleotides in length. The duplex region is defined herein as the region between the 5'-most nucleotide of the first strand that is base-paired to a nucleotide of the second strand and the 3'-most nucleotide of the first strand that is base-paired to a nucleotide of the second strand, inclusive. The duplex region may contain nucleotides in either or both strands that are not base-paired to a nucleotide in the other strand. It may contain 1, 2, 3, or 4 such nucleotides on the first strand and / or the second strand. However, preferably, the duplex region consists of 17-25 consecutive nucleotide base pairs. In other words, it preferably contains 17-25 contiguous nucleotides on both strands that all base pair with nucleotides on the other strand.More preferably, the duplex region consists of 18 or 19, most preferably 18 contiguous nucleotide base pairs.

[0056] In each of the embodiments disclosed herein, the nucleic acid may be blunt ended on both ends, may have an overhang on one end and a blunt end on the other end, or may have an overhang on both ends.

[0057] The nucleic acid may have an overhang on one end and a blunt end on the other end. The nucleic acid may have an overhang on both ends. The nucleic acid may be blunt ended on both ends. The nucleic acid may be blunt ended at the 5' end of the first strand and the 3' end of the second strand, or at the 3' end of the first strand and the 5' end of the second strand.

[0058] The nucleic acid may include an overhang at the 3' or 5' end. The nucleic acid may have a 3' overhang on the first strand. The nucleic acid may have a 3' overhang on the second strand. The nucleic acid may have a 5' overhang on the first strand. The nucleic acid may have a 5' overhang on the second strand. The nucleic acid may have an overhang on both the 5' and 3' ends of the first strand. The nucleic acid may have an overhang on both the 5' and 3' ends of the second strand. The nucleic acid may have a 5' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may have a 3' overhang on the first strand and a 5' overhang on the second strand. The nucleic acid may have a 3' overhang on the first strand and a 3' overhang on the second strand. The nucleic acid may have a 5' overhang on the first strand and a 5' overhang on the second strand.

[0059] The overhang at the 3' or 5' end of the second or first strand may be 1, 2, 3, 4, or 5 nucleotides in length. Optionally, the overhang may be 1 or 2 nucleotides, which may or may not be modified.

[0060] In one embodiment, the 5' end of the first strand is a single-stranded overhang of 1, 2, or 3 nucleotides, preferably 1 nucleotide.

[0061] Preferably, the nucleic acid is an siRNA. siRNA is a small interfering or silencing RNA that can inhibit the expression of a target gene through the RNA interference (RNAi) pathway. Inhibition occurs post-transcriptionally through targeted degradation of the mRNA transcript of the target gene. siRNA forms part of the RISC complex. RISC complex specifically targets the target RNA by the sequence complementarity of the first (antisense) strand with the target sequence.

[0062] Preferably, the nucleic acid mediates RNA interference (RNAi). Preferably, the nucleic acid mediates RNA interference with an inhibition efficiency of at least 50%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%. Inhibition efficiency is preferably measured by comparing MASP-2 mRNA levels in cells, e.g., hepatocytes, treated with MASP-2-specific siRNA with MASP-2 mRNA levels in cells treated with a control in comparable experiments. The control can be treatment with non-MASP-2 targeting siRNA or without siRNA. The nucleic acid, or at least the first strand of the nucleic acid, is thus preferably capable of being incorporated into the RISC complex. As a result, the nucleic acid, or at least the first strand of the nucleic acid, can thus guide the RISC complex to a specific target RNA to which the nucleic acid, or at least the first strand of the nucleic acid, is at least partially complementary. The RISC complex then specifically cleaves this target RNA, resulting in inhibition of expression of the gene from which the RNA was derived.

[0063] Nucleic acid modification The nucleic acids discussed herein include unmodified RNA as well as RNA that has been modified, for example, to improve efficacy or stability. Unmodified RNA refers to a molecule in which the components of the nucleic acid, i.e., sugar, base, and phosphate moieties, are the same or essentially the same as those that occur naturally, e.g., in the human body. The term "modified nucleotide" as used herein refers to a nucleotide in which one or more of the components of the nucleotide, i.e., sugar, base, and phosphate moieties, are different from those that occur naturally. The term "modified nucleotide" also refers to a molecule that, in certain cases, is not a nucleotide in the strict sense of the term because it lacks or has a substitute for an essential component of a nucleotide, e.g., sugar, base, or phosphate moiety. A nucleic acid that contains such modified nucleotides is still understood to be a nucleic acid even if one or more of the nucleotides of the nucleic acid are replaced by modified nucleotides that lack or have a substitute for an essential component of a nucleotide.

[0064] Modification of the nucleic acid of the present invention generally provides a powerful tool in overcoming potential limitations inherent to native RNA molecules, including but not limited to in vitro and in vivo stability and bioavailability. The nucleic acid according to the present invention may be modified by chemical modification. Modified nucleic acid may also minimize the possibility of inducing interferon activity in humans. Modification may further enhance functional delivery of the nucleic acid to target cells. Modified nucleic acid of the present invention may include one or more chemically modified ribonucleotides in either or both of the first or second strand. Ribonucleotides may include chemical modifications of base, sugar, or phosphate moieties. Ribonucleic acid may be modified by substitution with or insertion of nucleic acid or base analogs.

[0065] Throughout the present description, "the same or common modification" refers to the same modification for any nucleotide, be it A, G, C, or U modified with a group, e.g., methyl (2'-OMe) or fluoro (2'-F) group. For example, 2'-F-dU, 2'-F-dA, 2'-F-dC, 2'-F-dG are all considered to be the same or common modification, as are 2'-OMe-rU, 2'-OMe-rA, 2'-OMe-rC, 2'-OMe-rG. In contrast, the 2'-F modification is a different modification compared to the 2'-OMe modification.

[0066] Preferably, at least one nucleotide of the first and / or second strand of the nucleic acid is a modified nucleotide, preferably a non-naturally occurring nucleotide, such as preferably a 2'-F modified nucleotide.

[0067] Modified nucleotides can be nucleotides that have modified sugar groups. The 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.

[0068] Examples of "oxy"-2' hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl (e.g., methyl), cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG), O(CH2CHO) n CH2CH2OR; "locked" nucleic acids (LNA) in which the 2' hydroxyl is connected, for example, by a methylene bridge, to the 4' carbon of the same ribose sugar; O-amine (amine = NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino), and aminoalkoxy, O(CH2) n Amines, (eg, amine = NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).

[0069] "Deoxy" modifications include hydrogen, halogen, amino (e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH) n Included are CH2CH2-amine (amine = NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino), -NHC(O)R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl, and alkynyl, which may be optionally substituted, for example, with amino functionalities. Other substituents of certain embodiments include 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro.

[0070] The sugar group can also contain one or more carbons that have the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified nucleotides can contain sugars such as arabinose.

[0071] Modified nucleotides can also include "abasic" sugars that lack a nucleobase at C-1'. These abasic sugars can further contain modifications to one or more of the constituent sugar atoms.

[0072] The 2' modifications may be used in combination with one or more phosphate internucleoside linker modifications (eg, phosphorothioate or phosphorodithioate).

[0073] One or more nucleotides of the nucleic acid of the invention may be modified. The nucleic acid may comprise at least one modified nucleotide. The modified nucleotide may be in the first strand. The modified nucleotide may be in the second strand. The modified nucleotide may be in the duplex region. The modified nucleotide may be outside the duplex region, i.e. in the single strand region. The modified nucleotide may be on the first strand and outside the duplex region. The modified nucleotide may be on the second strand and outside the duplex region. The 3' terminal nucleotide of the first strand may be a modified nucleotide. The 3' terminal nucleotide of the second strand may be a modified nucleotide. The 5' terminal nucleotide of the first strand may be a modified nucleotide. The 5' terminal nucleotide of the second strand may be a modified nucleotide.

[0074] The nucleic acids of the invention can have one modified nucleotide, or the nucleic acids of the invention can have about 2-4 modified nucleotides, or the nucleic acids can have about 4-6 modified nucleotides, about 6-8 modified nucleotides, about 8-10 modified nucleotides, about 10-12 modified nucleotides, about 12-14 modified nucleotides, about 14-16 modified nucleotides, about 16-18 modified nucleotides, about 18-20 modified nucleotides, about 20-22 modified nucleotides, about 22-24 modified nucleotides, about 24-26 modified nucleotides, or about 26-28 modified nucleotides. In each case, the nucleic acid containing the modified nucleotides retains at least 50% of its activity compared to the same nucleic acid without the modified nucleotides, or vice versa. The nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of its activity compared to the same nucleic acid without the modified nucleotide, as well as any intermediate values, or may have greater than 100% of the activity of the same nucleic acid without the modified nucleotide.

[0075] The modified nucleotide may be a purine or a pyrimidine. At least half of the purines may be modified. At least half of the pyrimidines may be modified. All of the purines may be modified. All of the pyrimidines may be modified. The modified nucleotide may be selected from the group consisting of 3' terminal deoxythymine (dT) nucleotides, 2'-O-methyl (2'-OMe) modified nucleotides, 2' modified nucleotides, 2' deoxy modified nucleotides, locked nucleotides, abasic nucleotides, 2' amino modified nucleotides, 2' alkyl modified nucleotides, 2'-deoxy-2'-fluoro (2'-F) modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, nucleotides containing 5'-phosphorothioate groups, nucleotides containing 5' phosphates or 5' phosphate mimetics, and terminal nucleotides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

[0076] Nucleic acids may include nucleotides containing modified bases, where the base is 2-aminoadenosine, 2,6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridines (e.g., 5-bromouridine), 6-azapyrimidines, 6-alkylpyrimidines (e.g., 6-methyluridine), propyne, queosine, 2-thiouridine, 4-thiouridine, wybutoxine, wybutoxine, 4-acetylcytidine, 5-(carboxymethyl)pyr ... 5-methyl-2-thiouridine, 2-methyl-N6-isopentenyladenosine, beta-D-mannosylqueuosine, uridine-5-oxyacetic acid, and 2-thiocytidine.

[0077] Many of the modifications that occur in nucleic acids described herein, such as modifications of bases, or phosphate moieties, or non-linked O of phosphate moieties, will be repeated in a polynucleotide molecule. In some cases, modifications occur at all possible positions / nucleotides in a polynucleotide, but in many cases, they do not. Modifications can occur only at the 3' or 5' terminal positions, and can occur only in terminal regions, such as positions on terminal nucleotides or the last 2, 3, 4, 5, or 10 nucleotides of a strand. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in double-stranded regions of the nucleic acids of the invention, or only in single-stranded regions of the nucleic acids of the invention. Phosphorothioate or phosphorodithioate modifications at non-linked O positions can occur only at one or both ends, or can occur only in terminal regions, such as positions on terminal nucleotides or the last 2, 3, 4, or 5 nucleotides of a strand, or can occur in double-stranded and / or single-stranded regions, especially at the ends. The 5' and / or 3' ends may be phosphorylated.

[0078] The stability of the double-stranded nucleic acid of the present invention can be increased by including specific bases in the overhang or by including modified nucleotides in the single-stranded overhang, for example, in the 5' or 3' overhang or both. Purine nucleotides may be included in the overhang. All or some of the bases in the 3' or 5' overhang can be modified. Modifications can include the use of modifications at the 2' OH group of the ribose sugar, the use of deoxyribonucleotides instead of ribonucleotides, and modifications at the phosphate group, for example, phosphorothioate or phosphorodithioate modifications. The overhang does not necessarily have to be homologous to the target sequence.

[0079] Nucleases can hydrolyze nucleic acid phosphodiester bonds, however, chemical modifications to nucleic acids can confer improved properties and make oligoribonucleotides more stable to nucleases.

[0080] Modified nucleic acid, as used herein, means (i) the alteration, e.g., replacement, of one or both of the non-linked phosphate oxygens and / or one or more of the linking phosphate oxygens (referred to as linkages even when at the 5' and 3' ends of the nucleic acids of the invention); (ii) modifying, e.g., replacing, a component of the ribose sugar, e.g., the 2' hydroxyl of the ribose sugar; (iii) replacement of the phosphate moiety with a “dephospho” linker; (iv) modification or replacement of naturally occurring bases; (v) replacement or modification of the ribose-phosphate backbone, and (vi) modification of the 3' or 5' termini of the first and / or second strand, e.g., removal, modification, or replacement of a terminal phosphate group, or conjugation of a moiety, e.g., a fluorescent labeling moiety, to the 3' or 5' termini of one or both strands; may include one or more of:

[0081] The terms "replacement," "modification," and "alteration" refer to differences from naturally occurring molecules.

[0082] Specific modifications are discussed in more detail below.

[0083] The nucleic acid may comprise one or more nucleotides of the second and / or first strand that are modified. Alternating nucleotides may be modified to form modified nucleotides.

[0084] "Alternating" as described herein means occurring one by one in a regular manner. In other words, alternating means occurring in a repetitive order. For example, one nucleotide is modified, the next consecutive nucleotide is not modified, the next consecutive nucleotide is modified, etc. One nucleotide can be modified with a first modification, the next consecutive nucleotide can be modified with a second modification, the next consecutive nucleotide is modified with the first modification, etc., where the first and second modifications are different.

[0085] Some representative modified nucleic acid sequences of the invention are presented in the Examples, which are meant to be representative and not limiting.

[0086] In one embodiment of the nucleic acid, at least nucleotides 2 and 14 of the first strand are preferably modified with a first common modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand. The first modification is preferably a 2'-F.

[0087] In one embodiment, at least one, some, or preferably all even-numbered nucleotides of the first strand are preferably modified with a first common modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand. The first modification is preferably a 2'-F.

[0088] In one embodiment, at least one, some, or preferably all odd-numbered nucleotides of the first strand are modified, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5'-end of the first strand. Preferably, they are modified by a second modification. This second modification is preferably different from the first modification, if the nucleic acid also contains the first modification, for example, those of nucleotides 2 and 14 of the first strand, or those of all even-numbered nucleotides. The first modification is preferably any 2'-ribose modification that is the same size or smaller in volume than a 2'-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2'-fluoroarabinonucleic acid (FANA) modification. The 2'-ribose modification that is the same size or smaller in volume than a 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halo, or 2'-NH2. The second modification is preferably any 2'-ribose modification that is bulkier than the 2'-OH group. The 2'-ribose modification that is bulkier than the 2'-OH group can be, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl, or 2'-O-alkyl, provided that the nucleus is capable of reducing the expression of the target gene under comparable conditions at least to the same extent as the same nucleic acid without the modification. The first modification is preferably 2'-F, and / or the second modification is preferably 2'-OMe.

[0089] In the context of the present disclosure, the size or volume of a substituent, such as a 2' ribose modification, is preferably measured as a van der Waals volume.

[0090] In one embodiment, at least one, some, or preferably all nucleotides of the second strand at positions corresponding to even-numbered nucleotides of the first strand are preferably modified by a third modification. Preferably, in the same nucleic acid, nucleotides 2 and 14 of the first strand, or all even-numbered nucleotides, are modified with a first modification. Additionally or alternatively, odd-numbered nucleotides of the first strand are modified with a second modification. Preferably, the third modification is different from the first modification and / or the third modification is the same as the second modification. The first modification is preferably any 2' ribose modification that is the same size or smaller in volume than the 2'-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2'-fluoroarabinonucleic acid (FANA) modification. A 2'-ribose modification that is the same size or smaller in volume than a 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halo, or 2'-NH2. The second and / or third modification is preferably any 2'-ribose modification that is larger in volume than a 2'-OH group. A 2'-ribose modification that is larger in volume than a 2'-OH group can be, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl, or 2'-O-alkyl, provided that the nucleic acid is capable of reducing the expression of a target gene to at least the same extent as the same nucleic acid without the modification under comparable conditions. The first modification is preferably 2'-F, and / or the second and / or third modification is preferably 2'-OMe. The nucleotides of the first strand are numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand.

[0091] For example, a nucleotide of the second strand that is at a position corresponding to an even-numbered nucleotide of the first strand is the nucleotide of the second strand that is base paired with the even-numbered nucleotide of the first strand.

[0092] In one embodiment, at least one, some, or preferably all nucleotides of the second strand at positions corresponding to odd-numbered nucleotides of the first strand are preferably modified with a fourth modification. Preferably, in the same nucleic acid, nucleotides 2 and 14 of the first strand, or all even-numbered nucleotides, are modified with a first modification. Additionally or alternatively, the odd-numbered nucleotides of the first strand are modified with a second modification. Additionally or alternatively, all nucleotides of the second strand at positions corresponding to even-numbered nucleotides of the first strand are modified with a third modification. The fourth modification is preferably different from the second modification, preferably different from the third modification, and the fourth modification is preferably the same as the first modification. The first and / or fourth modification is preferably any 2' ribose modification that is the same size or smaller in volume than a 2'-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2'-fluoroarabinonucleic acid (FANA) modification. The 2'-ribose modification that is the same size or smaller in volume than the 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halo, or 2'-NH2. The second and / or third modification is preferably any 2'-ribose modification that is larger in volume than the 2'-OH group. The 2'-ribose modification that is larger in volume than the 2'-OH group can be, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl, or 2'-O-alkyl, provided that the nucleic acid is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification under comparable conditions. The first and / or fourth modification is preferably a 2'-OMe modification, and / or the second and / or third modification is preferably a 2'-F modification. The nucleotides of the first strand are numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand.

[0093] In one embodiment of the nucleic acid, the nucleotide(s) of the second strand at a position corresponding to nucleotide 11, or nucleotide 13, or nucleotides 11 and 13, or nucleotides 11 to 13 of the first strand are modified with a fourth modification. Preferably, all nucleotides of the second strand other than the nucleotide(s) at a position corresponding to nucleotide 11, or nucleotide 13, or nucleotides 11 and 13, or nucleotides 11 to 13 of the first strand are modified with a third modification. Preferably, in the same nucleic acid, nucleotides 2 and 14, or all even-numbered nucleotides of the first strand are modified with a first modification. Additionally or alternatively, the odd-numbered nucleotides of the first strand are modified with a second modification. The fourth modification is preferably different from the second modification, preferably different from the third modification, and the fourth modification is preferably the same as the first modification. The first and / or fourth modification is preferably any 2' ribose modification that is the same size as or smaller in volume than a 2'-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2'-fluoroarabinonucleic acid (FANA) modification. The 2' ribose modification that is the same size as or smaller in volume than a 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halo, or 2'-NH2. The second and / or third modification is preferably any 2' ribose modification that is larger in volume than a 2'-OH group. The 2' ribose modification that is larger in volume than a 2'-OH group can be, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl, or 2'-O-alkyl, provided that the nucleic acid is capable of reducing the expression of a target gene to at least the same extent as the same nucleic acid without the modification under comparable conditions. The first and / or fourth modification is preferably a 2'-OMe modification and / or the second and / or third modification is preferably a 2'-F modification. The nucleotides of the first strand are numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand.

[0094] In one embodiment of the nucleic acid, all even numbered nucleotides of the first strand are modified by a first modification, all odd numbered nucleotides of the first strand are modified by a second modification, all nucleotides of the second strand at positions corresponding to even numbered nucleotides of the first strand are modified by a third modification, and all nucleotides of the second strand at positions corresponding to odd numbered nucleotides of the first strand are modified by a fourth modification, wherein the first and / or fourth modification is 2'-F and / or the second and / or third modification is 2'-OMe.

[0095] In one aspect of the nucleic acid, all even-numbered nucleotides of the first strand are modified by a first modification, all odd-numbered nucleotides of the first strand are modified by a second modification, all nucleotides of the second strand at positions corresponding to nucleotides 11-13 of the first strand are modified by a fourth modification, and all nucleotides of the second strand other than the nucleotides corresponding to nucleotides 11-13 of the first strand are modified by a third modification, the first and fourth modifications being 2'-F and the second and third modifications being 2'-OMe. In one embodiment within this aspect, the 3'-terminal nucleotide of the second strand is an inverted RNA nucleotide (i.e., the nucleotide is linked to the 3'-end of the strand through its 3' carbon, rather than through its 5' carbon as is customary). When the 3'-terminal nucleotide of the second strand is an inverted RNA nucleotide, the inverted RNA nucleotide is preferably an unmodified nucleotide, in the sense that it does not contain any modifications compared to its natural nucleotide counterpart. Specifically, the inverted RNA nucleotide is preferably a 2'-OH nucleotide. Preferably, in this embodiment, when the 3'-terminal nucleotide of the second strand is an inverted RNA nucleotide, the nucleic acid is blunt-ended at least at the end including the 5'-end of the first strand.

[0096] One aspect of the present invention is a nucleic acid disclosed herein for inhibiting expression of the MASP-2 gene, preferably in a cell, wherein the first strand is a double-stranded nucleic acid comprising modified or unmodified nucleotides at multiple positions to facilitate processing of the nucleic acid by RISC.

[0097] In one embodiment, "promotes processing by RISC" means that the nucleic acid can be processed by RISC, e.g., any modifications present allow the nucleic acid to be processed by RISC, and preferably are beneficial to processing by RISC, and may suitably result in siRNA activity.

[0098] The nucleic acids disclosed herein are such that the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'-OMe modification, and the nucleotide(s) on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11, 12, and 13 of the first strand are not modified with a 2'-OMe modification (in other words, they are unmodified or modified with a modification other than 2'-OMe).

[0099] In one embodiment, the nucleotide on the second strand that corresponds to position 13 of the first strand is the nucleotide that base pairs with position 13 (from the 5' end) of the first strand.

[0100] In one embodiment, the nucleotide on the second strand that corresponds to position 11 of the first strand is the nucleotide that base pairs with position 11 (from the 5' end) of the first strand.

[0101] In one embodiment, the nucleotide on the second strand that corresponds to position 12 of the first strand is the nucleotide that base pairs with position 12 (from the 5' end) of the first strand.

[0102] For example, in a double-stranded, blunt-ended 19-mer nucleic acid, position 13 (from the 5' end) of the first strand will pair with position 7 (from the 5' end) of the second strand. Position 11 (from the 5' end) of the first strand will pair with position 9 (from the 5' end) of the second strand. This nomenclature can be applied to other positions on the second strand.

[0103] In one embodiment, in the case of partially complementary first and second strands, the nucleotides on the second strand that "correspond" to a position on the first strand do not necessarily form base pairs if that position is one where there is a mismatch, but the principles of nomenclature still apply.

[0104] One embodiment is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2'-OMe modification, and the nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2'-F modification.

[0105] One embodiment is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2'-F modification, and the nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand are not modified with a 2'-OMe modification.

[0106] One embodiment is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are modified with a 2'-F modification, and the nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2'-F modification.

[0107] One aspect is a nucleic acid disclosed herein wherein greater than 50% of the nucleotides of the first and / or second strand comprise a 2'-OMe modification, for example, preferably greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, or greater than 85%, or more, of the first and / or second strand comprise a 2'-OMe modification, measured as a percentage of the total nucleotides of both the first and second strand.

[0108] One embodiment is a nucleic acid disclosed herein in which more than 50% of the nucleotides of the first and / or second strand comprise a naturally occurring RNA modification, for example, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or more than 85% or more of the first and / or second strand, measured as a percentage of the total nucleotides of both the first and second strand, comprise such a modification. Suitable naturally occurring modifications include 2'-OMe as well as other 2' sugar modifications, particularly 2'-H modifications that result in DNA nucleotides.

[0109] One aspect is a nucleic acid as disclosed herein comprising, on the first and / or second strand, preferably as a percentage of the total nucleotides of both the first and second strand, 20% or less, e.g., 15% or less, e.g., 10% or less, of nucleotides having a 2' modification that is not a 2'-OMe modification.

[0110] One embodiment is a nucleic acid disclosed herein, wherein the number of nucleotides in the first and / or second strand that have a 2'-modification that is not a 2'-OMe modification is 7 or less, more preferably 5 or less, and most preferably 3 or less.

[0111] One aspect is a nucleic acid disclosed herein that comprises 20% or less (e.g., 15% or less, or 10% or less) 2'-F modifications on the first strand and / or the second strand, preferably as a percentage of total nucleotides of both strands.

[0112] One embodiment is a nucleic acid as disclosed herein, wherein the number of nucleotides in the first and / or second strand having a 2'-F modification is 7 or less, more preferably 5 or less, most preferably 3 or less.

[0113] One embodiment is a nucleic acid as disclosed herein, in which all nucleotides except for positions 2 and 14 from the 5' end of the first strand and nucleotides on the second strand corresponding to positions 11, or 13, or 11 and 13, or positions 11-13 of the first strand are modified with a 2'-OMe modification. Preferably, the nucleotides that are not modified with 2'-OMe are modified with a fluoro at the 2' position (2'-F modification).

[0114] In certain embodiments, a preferred aspect is a nucleic acid disclosed herein, in which all nucleotides of the nucleic acid are modified at the 2' position of the sugar. Preferably, these nucleotides are modified with a 2'-F modification, if the modification is not a 2'-OMe modification.

[0115] In one embodiment, the nucleic acid is modified with alternating 2'-OMe and 2-F modifications in the first strand, with positions 2 and 14 (starting from the 5' end) being modified with 2'-F. Preferably, the second strand is modified with 2'-F modifications at nucleotides on the second strand corresponding to positions 11, or 13, or positions 11 and 13, or positions 11-13 of the first strand. Preferably, the second strand is modified with 2'-F modifications at positions 11-13, counting from the 3' end starting at the first position of the complementary (double-stranded) region, with the remaining modifications being naturally occurring modifications, preferably 2'-OMe. The complementary region in at least this case starts at the first position of the second strand with the corresponding nucleotide in the first strand, regardless of whether the two nucleotides can base pair with each other.

[0116] In one embodiment of the nucleic acid, each of the nucleotides of the first strand and the second strand is a modified nucleotide.

[0117] The term "odd-numbered" as used herein means numbers that are not divisible by 2. Examples of odd numbers are 1, 3, 5, 7, 9, 11, etc. The term "even-numbered" as used herein means numbers that are divisible by 2. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14, etc.

[0118] Unless specifically indicated otherwise, herein, the nucleotides of a first strand are numbered consecutively starting with nucleotide number 1 at the 5'-end of the first strand. The nucleotides of a second strand are numbered consecutively starting with nucleotide number 1 at the 3'-end of the second strand.

[0119] One or more nucleotides of the first and / or second strand may be modified to form modified nucleotides. One or more of the odd-numbered nucleotides of the first strand may be modified. One or more of the even-numbered nucleotides of the first strand may be modified by at least a second modification, where the at least a second modification is different from the modification in the one or more odd-numbered nucleotides. At least one of the one or more modified even-numbered nucleotides may be adjacent to at least one of the one or more modified odd-numbered nucleotides.

[0120] A plurality of odd-numbered nucleotides in the first strand may be modified in the nucleic acid of the present invention. A plurality of even-numbered nucleotides in the first strand may be modified by a second modification. The first strand may comprise adjacent nucleotides modified by a common modification. The first strand may also comprise adjacent nucleotides modified by a second different modification (i.e., the first strand may comprise adjacent nucleotides modified by a first modification, as well as adjacent nucleotides modified by a second modification different from the first modification).

[0121] One or more of the odd-numbered nucleotides of the second strand (the nucleotides are numbered consecutively starting with nucleotide number 1 at the 3' end of the second strand) may be modified by a modification different from the modification of the odd-numbered nucleotides of the first strand (the nucleotides are numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand) and / or one or more of the even-numbered nucleotides of the second strand may be modified by the same modification as the odd-numbered nucleotides of the first strand. At least one of the one or more modified even-numbered nucleotides of the second strand may be adjacent to one or more modified odd-numbered nucleotides. A plurality of odd-numbered nucleotides of the second strand may be modified by a common modification and / or a plurality of even-numbered nucleotides may be modified by the same modification present in the odd-numbered nucleotides of the first strand. A plurality of odd-numbered nucleotides of the second strand may be modified by a modification different from the modification of the odd-numbered nucleotides of the first strand.

[0122] The second strand can include adjacent nucleotides modified with a common modification, which can be a different modification than the modification of the odd-numbered nucleotides of the first strand.

[0123] In some embodiments of the nucleic acids of the invention, each odd-numbered nucleotide of the first strand and each even-numbered nucleotide of the second strand may be modified with a common modification, each even-numbered nucleotide may be modified with a different modification in the first strand, and each odd-numbered nucleotide may be modified with a different modification in the second strand.

[0124] The nucleic acids of the invention may have a modified nucleotide in a first strand that is shifted by at least one nucleotide compared to an unmodified or differently modified nucleotide in a second strand.

[0125] In certain embodiments, one or more, or each, of the odd-numbered nucleotides may be modified in the first strand and one or more, or each, of the even-numbered nucleotides may be modified in the second strand. One or more, or each, of the alternating nucleotides of either or both strands may be modified by a second modification. One or more, or each, of the even-numbered nucleotides may be modified in the first strand and one or more, or each, of the even-numbered nucleotides may be modified in the second strand. One or more, or each, of the alternating nucleotides of either or both strands may be modified by a second modification. One or more, or each, of the odd-numbered nucleotides may be modified in the first strand and one or more, or each, of the odd-numbered nucleotides may be modified in the second strand by a common modification. One or more, or each, of the alternating nucleotides of either or both strands may be modified by a second modification. One or more, or each, of the even-numbered nucleotides may be modified in the first strand and one or more, or each, of the odd-numbered nucleotides may be modified with a common modification in the second strand. One or more, or each, of alternating nucleotides in either or both strands may be modified with a second modification.

[0126] The nucleic acids of the invention may comprise single-stranded or double-stranded constructs that comprise at least two regions of alternating modifications in one or both of the strands. These alternating regions may comprise up to about 12 nucleotides, but preferably comprise from about 3 to about 10 nucleotides. The regions of alternating nucleotides may be located at the termini of one or both strands of the nucleic acids of the invention. The nucleic acids may comprise from 4 to about 10 nucleotides of alternating nucleotides at each of the termini (3' and 5'), and these regions may be separated by about 5 to about 12 consecutive nucleotides that are unmodified or that are differently or commonly modified.

[0127] The odd-numbered nucleotides of the first strand may be modified and the even-numbered nucleotides may be modified with a second modification. The second strand may include adjacent nucleotides modified with a common modification, which may be the same as the modification of the odd-numbered nucleotides of the first strand. One or more nucleotides of the second strand may also be modified with a second modification. The one or more nucleotides with the second modification may be adjacent to each other and to a nucleotide with a modification that is the same as the modification of the odd-numbered nucleotides of the first strand. The first strand may also include a phosphorothioate linkage between the two nucleotides at the 3' and 5' ends, or a phosphorodithioate linkage between the two nucleotides at the 3' end. The second strand may include a phosphorothioate or phosphorodithioate linkage between the two nucleotides at the 5' end. The second strand may also be conjugated to a ligand at the 5' end.

[0128] The nucleic acid of the present invention may comprise a first strand comprising adjacent nucleotides modified with a common modification. One or more such nucleotides may be adjacent to one or more nucleotides that may be modified with a second modification. The one or more nucleotides with the second modification may be adjacent. The second strand may comprise adjacent nucleotides modified with a common modification, which may be the same as one of the modifications of one or more nucleotides of the first strand. One or more nucleotides of the second strand may also be modified with a second modification. The one or more nucleotides with the second modification may be adjacent. The first strand may also comprise a phosphorothioate linkage between the two nucleotides at the 3'-end and the 5'-end, or a phosphorodithioate linkage between the two nucleotides at the 3'-end. The second strand may comprise a phosphorothioate or phosphorodithioate linkage between the two nucleotides at the 3'-end. The second strand may also be conjugated to a ligand at the 5'-end. Nucleotides 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25, numbered 5' to 3' in the first strand and 3' to 5' in the second strand, can be modified by a modification in the first strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 can be modified by a second modification in the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 can be modified by a modification in the second strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 can be modified by a second modification in the second strand. The nucleotides are numbered, for the purposes of the nucleic acids of the present invention, from 5' to 3' in the first strand and 3' to 5' in the second strand.

[0129] Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 may be modified by a modification in the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a second modification in the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a modification in the second strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 may be modified by a second modification in the second strand.

[0130] Specifically, if the first and / or second strand is shorter than 25 nucleotides in length, e.g., 19 nucleotides in length, there are no modified nucleotides numbered 20, 21, 22, 23, 24, and 25. The skilled artisan will understand that the above description applies to shorter strands as appropriate.

[0131] One or more modified nucleotides of the first strand may be paired with modified nucleotides of the second strand with a common modification. One or more modified nucleotides of the first strand may be paired with modified nucleotides of the second strand with a different modification. One or more modified nucleotides of the first strand may be paired with modified nucleotides of the second strand with a different modification. One or more modified nucleotides of the first strand may be paired with unmodified nucleotides of the second strand. One or more modified nucleotides of the second strand may be paired with unmodified nucleotides of the first strand. In other words, alternating nucleotides may be aligned on the two strands, such that, for example, all modifications in the alternating region of the second strand are paired with the same modification in the first strand, or alternatively, the modifications may be offset by one nucleotide with a common modification in the alternating region of one strand being paired with a different modification (i.e., a second or further modification) in the other strand. Another option is to have different modifications in each of the strands.

[0132] The modifications in the first strand may be shifted by one nucleotide relative to the modified nucleotides in the second strand such that the common modified nucleotides do not pair with each other.

[0133] The modification(s) may each and individually be selected from the group consisting of 3'-terminal deoxythymine, 2'-OMe, 2'-deoxy modification, 2'-amino modification, 2'-alkyl modification, morpholino modification, phosphoramidate modification, 5'-phosphorothioate group modification, 5'-phosphate or 5'-phosphate mimetic modification, and cholesteryl derivative or dodecanoic acid bisdecylamide group modification, and / or the modified nucleotide may be any one of a locked nucleotide, an abasic nucleotide, or a nucleotide containing a non-natural base. At least one modification may be 2'-OMe, and / or at least one modification may be 2'-F. Further modifications as described herein may be present in the first and / or second strand.

[0134] The nucleic acid of the present invention may comprise an inverted RNA nucleotide at one or more of the ends of the strands. Such an inverted nucleotide provides stability to the nucleic acid. Preferably, the nucleic acid comprises at least one inverted nucleotide at the 3' end of the first and / or second strand and / or at the 5' end of the second strand. More preferably, the nucleic acid comprises an inverted nucleotide at the 3' end of the second strand. Most preferably, the nucleic acid comprises an inverted RNA nucleotide at the 3' end of the second strand, which nucleotide is preferably an inverted A. An inverted nucleotide is a nucleotide that is linked to the 3' end of the nucleic acid through its 3' carbon instead of its 5' carbon as usual, or that is linked to the 5' end of the nucleic acid through its 5' carbon instead of its 3' carbon as usual. The inverted nucleotide is preferably present at the end of a strand, opposite the corresponding nucleotide of the other strand, rather than as an overhang. Thus, the nucleic acid is preferably blunt-ended at the end that comprises the inverted RNA nucleotide. The inverted RNA nucleotide is present at the end of the chain, which means that the last nucleotide at this end of the chain is preferably an inverted RNA nucleotide.The nucleic acid having such a nucleotide is stable and easy to synthesize.The inverted RNA nucleotide is preferably an unmodified nucleotide, meaning that it does not contain any modification compared to the natural nucleotide counterpart.Specifically, the inverted RNA nucleotide is preferably a 2'-OH nucleotide.

[0135] The nucleic acids of the invention may comprise one or more nucleotides modified with a 2'-H at the 2' position, and thus have a DNA nucleotide within the nucleic acid. The nucleic acids of the invention may comprise a DNA nucleotide at position 2 and / or 14 of the first strand, counting from the 5' end of the first strand. The nucleic acids may comprise a DNA nucleotide on the second strand corresponding to position 11, or 13, or positions 11 and 13, or positions 11-13 of the first strand.

[0136] In one embodiment, there is no more than one DNA nucleotide per nucleic acid of the invention. The nucleic acids of the invention may comprise one or more LNA nucleotides. The nucleic acids of the invention may comprise an LNA nucleotide at position 2 and / or 14 of the first strand, counting from the 5' end of the first strand. The nucleic acids may comprise an LNA on the second strand corresponding to position 11, or 13, or positions 11 and 13, or positions 11-13 of the first strand.

[0137] Some representative modified nucleic acid sequences of the invention are presented in the Examples, which are meant to be representative and not limiting.

[0138] In certain preferred embodiments, the nucleic acid may comprise a first modification and a second or further modification, each and individually selected from the group comprising a 2'-OMe modification and a 2'-F modification. The nucleic acid may comprise a modification that is 2'-OMe, which may be the first modification, and a second modification that is 2'-F. The nucleic acid of the invention may also comprise phosphorothioate or phosphorodithioate modifications, and / or deoxy modifications, which may be present at or between two or three nucleotides at each or either end of each strand or both strands when the nucleic acid has two strands.

[0139] In one embodiment of the nucleic acid, at least one nucleotide of the first and / or second strand is a modified nucleotide, and when the first strand comprises at least one modified nucleotide: (i) at least one or both of nucleotides 2 and 14 of the first strand are modified by a first modification; and / or (ii) at least one, some, or all of the even-numbered nucleotides of the first strand are modified by a first modification; and / or (iii) at least one, some, or all of the odd-numbered nucleotides of the first strand are modified by a second modification; and / or If the second strand contains at least one modified nucleotide, (iv) at least one, some, or all of the nucleotides of the second strand that are located at positions corresponding to even-numbered nucleotides of the first strand are modified by a third modification; and / or (v) at least one, some, or all of the nucleotides of the second strand that are located at positions corresponding to odd-numbered nucleotides of the first strand are modified by a fourth modification; and / or (vi) at least one, some, or all of the nucleotides of the second strand that are located at positions corresponding to nucleotide 11, or nucleotide 13, or nucleotides 11 and 13, or nucleotides 11 to 13 of the first strand are modified by a fourth modification; and / or (vii) at least one, some, or all of the nucleotides of the second strand at positions other than nucleotide 11, or nucleotide 13, or nucleotides 11 and 13, or nucleotides 11-13 of the first strand are modified by a third modification; wherein the nucleotides of the first strand are numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand; The modifications are preferably as follows: (a) the first modification is preferably different from the second and third modifications; (b) the first modification is preferably the same as the fourth modification; (c) the second and third modifications are preferably the same modifications; (d) the first modification is preferably a 2'-F modification; (e) the second modification is preferably a 2'-OMe modification; (f) the third modification is preferably a 2'-OMe modification, and / or (g) the fourth modification is preferably a 2'-F modification; At least one of Optionally, the nucleic acid is conjugated to a ligand.

[0140] One embodiment is a double-stranded nucleic acid for inhibiting expression of MASP-2, preferably in a cell, comprising a first strand and a second strand, wherein an unmodified equivalent of the sequence of the first strand comprises a sequence of at least 15 nucleotides that differs by no more than three nucleotides from any one of the sequences of the first strand shown in Table 5a (Table 8) or Table 1 (Table 1), wherein all even-numbered nucleotides of the first strand are modified by a first modification, all odd-numbered nucleotides of the first strand are modified by a second modification, all nucleotides of the second strand that are located at positions corresponding to the even-numbered nucleotides of the first strand are modified by a third modification, and all nucleotides of the second strand that are located at positions corresponding to the odd-numbered nucleotides of the first strand are modified by a fourth modification, wherein the first and fourth modifications are 2'-F and the second and third modifications are 2'-OMe.

[0141] One embodiment is a double-stranded nucleic acid that inhibits expression of MASP-2, preferably in a cell, comprising a first strand and a second strand, wherein an unmodified equivalent of the sequence of the first strand comprises a sequence of at least 15 nucleotides that differs by no more than three nucleotides from any one of the sequences of the first strand shown in Table 5a (Table 8) or Table 1 (Table 1), wherein all even-numbered nucleotides of the first strand are modified by a first modification, all odd-numbered nucleotides of the first strand are modified by a second modification, all nucleotides of the second strand at positions corresponding to nucleotides 11 to 13 of the first strand are modified by a fourth modification, and all nucleotides of the second strand other than the nucleotides corresponding to nucleotides 11 to 13 of the first strand are modified by a third modification, wherein the first and fourth modifications are 2'-F and the second and third modifications are 2'-OMe.

[0142] The 3' and 5' ends of the oligonucleotide may be modified. Such modifications may be at the 3' or 5' end or at both ends of the molecule. They may include modification or replacement of the entire terminal phosphate or one or more of the atoms of the phosphate group. For example, the 3' and 5' ends of the oligonucleotide may be conjugated to other functional molecular entities, such as labeling moieties, such as fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3, or Cy5 dyes), or protecting groups (e.g., sulfur, silicon, boron, or ester-based). The functional molecular entities may be attached to the sugar through the phosphate group and / or a linker. The terminal atom of the linker may connect to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S, or C group of the sugar. Alternatively, the linker may connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNA). These spacers or linkers include, for example, -(CH2) n -, -(CH2) n N-, -(CH2) n O-, -(CH2) n S-, -(CH2CH2O) n Examples include CH2CH2O- (e.g., n=3 or 6), abasic sugar, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. The 3' terminus can be an -OH group.

[0143] Other examples of terminal modifications include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, EDTA, lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid (cholenic acid), acid), dimethoxytrityl, or phenoxazine), and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

[0144] Terminal modifications may also be useful for monitoring distribution, in which case the group to be added may include a fluorophore, such as fluorescein or an Alexa dye.Terminal modifications may also be useful for enhancing uptake, and useful modifications for this include cholesterol.Terminal modifications may also be useful for crosslinking an RNA agent to another moiety.

[0145] Terminal modifications may be added for several reasons, including modulating activity or modulating resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5' end with a phosphate or phosphate analog. The nucleic acids of the invention may be 5' phosphorylated or contain a phosphoryl analog at the 5' prime end, on either the first or second strand. 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate ((HO)2(O)PO-5'); 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'); '; 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'; 5'-monothiophosphate (phosphorothioate; (HO)2(S)PO-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5'), 5'-phosphorothioate any additional combination of oxygen / sulfur substituted monophosphates, diphosphates, and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'- (where R is alkyl), (OH)2(O)P-5'-CH2-), 5' vinylphosphonates, 5'-alkyl ether phosphonates (alkyl ether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'- (where R is alkyl ether)).

[0146] Certain moieties can be linked to the 5' end of the first or second strand. These include abasic ribose moieties, abasic deoxyribose moieties, modified abasic ribose and abasic deoxyribose moieties, such as 2'-O alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and their modified forms, C6-imino-Pi; mirror nucleotides, such as L-DNA and L-RNA; 5'OMe nucleotides; and nucleotide analogs, such as 4',5'-methylene nucleotides; 1-(β-D-erythrofuranosyl) nucleotides; 4'-thionucleotides, carbocyclic nucleotides; 5'-amino-alkyl phosphates; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotides; alpha-nucleotides; threo-pentofuranosyl nucleotides; acyclic 3',4'-seconucleotides; 3,4-dihydroxybutyl nucleotides; 3,5-dihydroxypentyl nucleotides, 5'-5'-inverted abasic moieties; 1,4-butanediol phosphate; 5'-amino; and bridged or unbridged methylphosphonate and 5'-mercapto moieties.

[0147] In each sequence described herein, a C-terminal "-OH" moiety may be replaced with a C-terminal "-NH2" moiety, and vice versa.

[0148] The invention also provides a nucleic acid according to any aspect of the invention described herein, wherein the first strand has a terminal 5'(E)-vinylphosphonate nucleotide at its 5' end. The terminal 5'(E)-vinylphosphonate nucleotide is preferably linked to the second nucleotide of the first strand by a phosphodiester linkage. Preferably, the terminal 5'(E)-vinylphosphonate ("vp") nucleotide is a uridine ("vp-U").

[0149] The first strand of the nucleic acid has formula (I): (vp)-N (po) [N (po) ]n- (I) It may include, where "(vp)-" is 5' (E)-vinyl phosphonate, "N" is a nucleotide, "po" is a phosphodiester linkage, and n is 1 to (total number of nucleotides in the first strand minus 2), preferably, n is 1 to (total number of nucleotides in the first strand minus 3), and more preferably, n is 1 to (total number of nucleotides in the first strand minus 4).

[0150] Preferably, the terminal 5' (E)-vinylphosphonate nucleotide is an RNA nucleotide, preferably (vp)-U.

[0151] A terminal 5' (E)-vinylphosphonate nucleotide is a nucleotide in which the phosphate group at the 5' end of the ribose is replaced with an E-vinylphosphonate:

[0152] [ka]

[0153] In one embodiment, the first strand has a terminal 5' (E)-vinyl phosphonate nucleotide at its 5' end, the terminal 5' (E)-vinyl phosphonate nucleotide is linked to a second nucleotide of the first strand by a phosphodiester linkage, and the first strand includes a) more than one phosphodiester linkage, b) phosphodiester linkages between at least the terminal three 5' nucleotides, and / or c) phosphodiester linkages between at least the terminal four 5' nucleotides.

[0154] In one embodiment, the first strand and / or the second strand of the nucleic acid comprises at least one phosphorothioate (ps) and / or at least one phosphorodithioate (ps2) linkage between two nucleotides.

[0155] In one embodiment, the first strand and / or the second strand of the nucleic acid comprises more than one phosphorothioate and / or more than one phosphorodithioate linkages.

[0156] In one embodiment, the first and / or second strand of the nucleic acid comprises phosphorothioate or phosphorodithioate linkages between the terminal two 3' nucleotides or phosphorothioate or phosphorodithioate linkages between the terminal three 3' nucleotides. Preferably, the linkages between the other nucleotides in the first and / or second strand are phosphodiester linkages.

[0157] In one embodiment, the first strand and / or the second strand of the nucleic acid comprises phosphorothioate linkages between the terminal two 5' nucleotides or comprises phosphorothioate linkages between the terminal three 5' nucleotides.

[0158] In one embodiment, the nucleic acid of the present invention comprises one or more phosphorothioate or phosphorodithioate modifications at one or more of the ends of the first and / or second strand. Optionally, each or either end of the first strand may comprise one, two, or three phosphorothioate or phosphorodithioate modified nucleotides (internucleoside linkages). Optionally, each or either end of the second strand may comprise one, two, or three phosphorothioate or phosphorodithioate modified nucleotides (internucleoside linkages).

[0159] In one embodiment, the nucleic acid comprises phosphorothioate linkages between the two or three 3' and / or 5' nucleotides at the ends of the first and / or second strand. Preferably, the nucleic acid comprises phosphorothioate linkages between each of the three 3' and three 5' nucleotides at the ends of the first and second strand. Preferably, all remaining linkages between nucleotides of the first and / or second strand are phosphodiester linkages.

[0160] In one embodiment, the nucleic acid comprises a phosphorodithioate linkage between each of the two, three, or four terminal nucleotides at the 3' end of a first strand, and / or a phosphorodithioate linkage between each of the two, three, or four terminal nucleotides at the 3' end of a second strand and / or a phosphorodithioate linkage between each of the two, three, or four terminal nucleotides at the 5' end of a second strand, and comprises linkages other than phosphorodithioate linkages between the two, three, or four terminal nucleotides at the 5' end of the first strand.

[0161] In one embodiment, the nucleic acid comprises phosphorothioate linkages between the terminal three 3' nucleotides and the terminal three 5' nucleotides of the first and second strands. Preferably, all remaining linkages between nucleotides of the first and / or second strands are phosphodiester linkages.

[0162] In one embodiment, the nucleic acid is (i) having phosphorothioate linkages between the terminal three 3' nucleotides and the terminal three 5' nucleotides of the first strand; (ii) is conjugated to a triantennary ligand at either the 3'-terminal nucleotide or the 5'-terminal nucleotide of the second strand; (iii) at the end of the second strand opposite that which is conjugated to the triantennary ligand, have phosphorothioate linkages between the terminal three nucleotides; (iv) Optionally, all remaining linkages between nucleotides of the first and / or second strand are phosphodiester linkages.

[0163] In one embodiment, the nucleic acid is (i) having a terminal 5' (E)-vinylphosphonate nucleotide at the 5' end of the first strand; (ii) having phosphorothioate linkages between the three 3' nucleotides at the ends of the first and second strands and between the three 5' nucleotides at the end of the second strand, or having phosphorodithioate linkages between the two 3' nucleotides at the ends of the first and second strands and between the two 5' nucleotides at the end of the second strand; (iii) Optionally, all remaining linkages between nucleotides of the first and / or second strand are phosphodiester linkages.

[0164] In one embodiment, the nucleic acid has a terminal 5' (E)-vinylphosphonate nucleotide at the 5' end of the first strand, phosphorothioate linkages between the terminal three 3' nucleotides of the first strand and between the terminal three 3' nucleotides of the second strand, and optionally all remaining linkages between nucleotides of the first strand and / or second strand are phosphodiester linkages.

[0165] The use of phosphorodithioate linkages in the nucleic acid of the present invention reduces the variability in the stereochemical structure of a population of nucleic acid molecules compared to molecules that contain phosphorothioate at the same position.Phosphorothioate linkages introduce chiral centers, and it is difficult to control which non-linked oxygen is replaced with sulfur.The use of phosphorodithioate ensures that there are no chiral centers in the linkage, thus reducing or eliminating any variability in a population of nucleic acid molecules, depending on the number of phosphorodithioate and phosphorothioate linkages used in the nucleic acid molecule.

[0166] In one embodiment, the nucleic acid comprises phosphorodithioate linkages between the two terminal nucleotides at the 3' end of the first strand, phosphorodithioate linkages between the two terminal nucleotides at the 3' end of the second strand, and phosphorodithioate linkages between the two terminal nucleotides at the 5' end of the second strand, and linkages other than phosphorodithioate linkages between the two, three, or four terminal nucleotides at the 5' end of the first strand. Preferably, the first strand has a terminal 5' (E)-vinyl phosphonate nucleotide at its 5' end. This terminal 5' (E)-vinyl phosphonate nucleotide is preferably linked to the second nucleotide of the first strand by a phosphodiester linkage. Preferably, all linkages between the nucleotides of both strands, other than the linkages between the two terminal nucleotides at the 3' end of the first strand and the linkages between the two terminal nucleotides at the 3' end and 5' end of the second strand, are phosphodiester linkages.

[0167] In one embodiment, the nucleic acid comprises phosphorothioate linkages between each of the three terminal 3' nucleotides and / or each of the three terminal 5' nucleotides of the first strand and / or between each of the three terminal 3' nucleotides and / or each of the three terminal 5' nucleotides of the second strand, if there are no phosphorodithioate linkages at the ends. Absence of phosphorodithioate linkages at the ends means that the linkages between the two, or preferably the three, terminal nucleotides of the nucleic acid ends in question are other than phosphorodithioate linkages.

[0168] In one embodiment, all linkages of the nucleic acid between nucleotides of both strands other than the linkages between the two terminal nucleotides at the 3' end of the first strand and the linkages between the two terminal nucleotides at the 3' and 5' ends of the second strand are phosphodiester linkages.

[0169] Other phosphate-linked modifications are possible.

[0170] Phosphate linkers can also be modified by replacement of the linking oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Replacement can occur at the terminal oxygen. Replacement of a non-linking oxygen with a nitrogen is possible.

[0171] The phosphate groups may also be individually replaced with non-phosphorus containing connecting agents.

[0172] Examples of moieties that can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino. In certain embodiments, replacements can include methylenecarbonylamino and methylenemethylimino groups.

[0173] The phosphate linker and ribose sugar may be replaced by nuclease-resistant nucleotides. Examples include morpholino, cyclobutyl, pyrrolidine, and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

[0174] In one embodiment, the nucleic acid, preferably an siRNA, which inhibits the expression of MASP-2 via RNAi, preferably in a cell, is (i) modified nucleotides, (ii) modified nucleotides other than 2'-OMe modified nucleotides at positions 2 and 14 from the 5' end of the first strand having a given SEQ ID NO, preferably 2'-F modified nucleotides; (iii) each of the odd-numbered nucleotides of the first strand, numbered starting from 1 at the 5' end of the first strand having a given SEQ ID NO, is a 2'-OMe modified nucleotide; (iv) each even-numbered nucleotide of the first strand, numbered starting from 1 at the 5' end of the first strand having a given SEQ ID NO, is a 2'-F modified nucleotide; (v) the nucleotides of the second strand corresponding to positions 11 and / or 13 or positions 11-13 of the first strand having a given SEQ ID NO are modified by a modification other than a 2'-OMe modification, preferably one or both or all of these positions contain a 2'-F modification; (vi) an inverted nucleotide, preferably a 3'-3' linkage at the 3' end of the second strand having the given SEQ ID NO; (vii) one or more phosphorothioate linkages; (viii) one or more phosphorodithioate linkages, and / or (ix) the first strand having a given SEQ ID NO has a terminal 5'(E)-vinylphosphonate nucleotide at its 5' end, wherein the terminal 5'(E)-vinylphosphonate nucleotide is preferably a uridine and is preferably linked to a second nucleotide of the first strand by a phosphodiester linkage; Includes one or more or all of the following:

[0175] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 15 nucleotides that differs by no more than 3 nucleotides from any one of the first strand sequences having a given SEQ ID NO: shown in Table 5b.

[0176] The nucleic acid of the present disclosure is (a) the first strand sequence comprises a sequence that differs by no more than three nucleotides from any one of the first strand sequences in Table 5b (Table 9), and optionally the second strand sequence comprises a sequence that differs by no more than three nucleotides from the corresponding second strand sequence in Table 5b (Table 9); (b) the first strand sequence comprises a sequence that differs by no more than two nucleotides from any one of the first strand sequences in Table 5b (Table 9), and optionally the second strand sequence comprises a sequence that differs by no more than two nucleotides from the corresponding second strand sequence in Table 5b (Table 9); (c) the first strand sequence comprises a sequence that differs by no more than one nucleotide from any one of the first strand sequences in Table 5b (Table 9), and optionally the second strand sequence comprises a sequence that differs by no more than one nucleotide from the corresponding second strand sequence in Table 5b (Table 9); (d) the first strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of any one of the first strand sequences having a given SEQ ID NO shown in Table 5b (Table 9), and optionally the second strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5b (Table 9); (e) the first strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of any one of the first strand sequences having a given SEQ ID NO shown in Table 5b (Table 9), and optionally the second strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5b (Table 9); (f) the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO shown in Table 5b (Table 9), and optionally the second strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5b (Table 9); (g) the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO shown in Table 5b (Table 9), and optionally the second strand sequence comprises a sequence corresponding to nucleotides 1-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5b (Table 9); (h) the first strand sequence comprises any one of the first strand sequences having a given SEQ ID NO shown in Table 5b (Table 9), and optionally the second strand sequence comprises the corresponding second strand sequence having a given SEQ ID NO shown in Table 5b (Table 9); (i) the first strand sequence consists essentially of any one of the first strand sequences having a given SEQ ID NO shown in Table 5b (Table 9), and optionally the second strand sequence consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5b (Table 9); (j) the first strand sequence consists of any one of the first strand sequences having a given SEQ ID NO shown in Table 5b (Table 9), and optionally the second strand sequence consists of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5b (Table 9); (k) the sequence of the first strand consists of a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5b (Table 9); the first strand sequence further comprises one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5b (Table 9); Optionally, the second strand sequence comprises, consists essentially of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5b (Table 9); (l) the sequence of the first strand consists of a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5b (Table 9); the first strand sequence further comprises one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5b (Table 9); The sequence of the first strand consists of a continuous region of 17 to 25 nucleotides, preferably 18 to 24 nucleotides, that is complementary to the MASP-2 transcript of SEQ ID NO: 821; Optionally, the second strand sequence comprises, consists essentially of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5b (Table 9); (m) the first strand and the second strand of any one of the nucleic acid molecules of subparagraphs (a)-(l) above are present on a single strand, and the first strand and the second strand are capable of hybridizing to each other, thereby forming a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; or (n) the first strand and the second strand of any one of the nucleic acid molecules of subparagraphs (a) through (l) above are on two separate strands that are capable of hybridizing to one another to thereby form a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; It may be a nucleic acid.

[0177] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 15 nucleotides that differs by no more than 3 nucleotides from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0178] [Table 2]

[0179] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 17 nucleotides that differs by no more than 3 nucleotides from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0180] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 17 nucleotides that differs by no more than two nucleotides from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0181] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 17 nucleotides that differs by no more than one nucleotide from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0182] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 18 nucleotides that differs by no more than 3 nucleotides from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0183] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 18 nucleotides that differs by no more than two nucleotides from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0184] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 18 nucleotides that differs by no more than one nucleotide from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0185] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 19 nucleotides that differs by no more than 3 nucleotides from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0186] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 19 nucleotides that differs by no more than two nucleotides from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0187] A nucleic acid of the disclosure can include a first strand and a second strand, where the sequence of the first strand includes a sequence of at least 19 nucleotides that differs by no more than one nucleotide from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0188] The nucleic acids of the present disclosure can include a first strand and a second strand, where the sequence of the first strand consists essentially of or consists of a sequence derived from any one of the first strand sequences having a given SEQ ID NO: shown in Table 2.

[0189] For example, the nucleic acid of the disclosure may be (a) the first strand sequence comprises a sequence that differs by no more than three nucleotides from any one of the first strand sequences in Table 2, and optionally the second strand sequence comprises a sequence that differs by no more than three nucleotides from the corresponding second strand sequence in Table 2; (b) the first strand sequence comprises a sequence that differs by no more than two nucleotides from any one of the first strand sequences in Table 2, and optionally the second strand sequence comprises a sequence that differs by no more than two nucleotides from the corresponding second strand sequence in Table 2; (c) the first strand sequence comprises a sequence that differs by no more than one nucleotide from any one of the first strand sequences in Table 2, and optionally the second strand sequence comprises a sequence that differs by no more than one nucleotide from the corresponding second strand sequence in Table 2; (d) the first strand sequence comprises a sequence corresponding to nucleotides 2 to 17 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2, and optionally the second strand sequence comprises a sequence corresponding to nucleotides 2 to 17 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (e) the first strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2, and optionally the second strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (f) the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO shown in Table 2, and optionally the second strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO shown in Table 2; (g) the first strand sequence comprises a sequence corresponding to nucleotides 2 to 19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2, and optionally the second strand sequence comprises a sequence corresponding to nucleotides 1 to 18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (h) the first strand sequence comprises any one of the first strand sequences in Table 2, and optionally the second strand sequence comprises the corresponding second strand sequence in Table 2; (i) the first strand sequence consists essentially of any one of the first strand sequences in Table 2, and optionally the second strand sequence consists essentially of the sequence of the corresponding second strand sequence in Table 2; (j) the first strand sequence consists of any one of the first strand sequences in Table 2, and optionally the second strand sequence consists of the corresponding second strand sequence in Table 2; (k) the sequence of the first strand consists of a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the sequences of the first strand having a given SEQ ID NO: shown in Table 2; the first strand sequence further comprises one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2; Optionally, the second strand sequence comprises, consists essentially of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (l) the sequence of the first strand consists of a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the sequences of the first strand having a given SEQ ID NO: shown in Table 2; the first strand sequence further comprises one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2; The sequence of the first strand consists of a continuous region of 17 to 25 nucleotides, preferably 18 to 24 nucleotides, that is complementary to the MASP-2 transcript of SEQ ID NO: 821; Optionally, the second strand sequence comprises, consists essentially of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (m) the first strand and the second strand of any one of the nucleic acid molecules of subparagraphs (a)-(l) above are present on a single strand, and the first strand and the second strand are capable of hybridizing to each other, thereby forming a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; or (n) the first strand and the second strand of any one of the nucleic acid molecules of subparagraphs (a) through (l) above are on two separate strands that are capable of hybridizing to one another to thereby form a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; It may be a nucleic acid.

[0190] All of the features of the nucleic acids can be combined with all other aspects of the invention disclosed herein.

[0191] different parts The nucleic acid of the present invention may be conjugated to a heterologous moiety. A heterologous moiety is any moiety that is not a nucleic acid molecule capable of inhibiting the expression of MASP-2. A heterologous moiety may be or may include a peptide (or polypeptide), a sugar (or polysaccharide), a lipid, a different nucleic acid, or any other suitable molecule.

[0192] Any given nucleic acid may be conjugated to multiple heterologous moieties, which may be the same or different.

[0193] An individual heterologous moiety may itself comprise one or more functional moieties (e.g., a targeting agent, described in more detail below), each covalently associated to a nucleic acid, optionally via a linker.

[0194] Heterologous moiety or its functional component can function, for example, to modulate bioavailability or pharmacokinetics. For example, it can increase half-life in vivo. Alternatively, heterologous moiety (or its functional component) can include a targeting agent. Efficient delivery of oligonucleotide, especially the double-stranded nucleic acid of the present invention, to cells in vivo is important and requires specific targeting and substantial protection from extracellular environment, especially serum proteins. One way to achieve specific targeting is to conjugate a targeting agent to nucleic acid, where the targeting agent serves to target nucleic acid to target cells that have cell surface receptors that bind to the targeting agent.

[0195] In this context, the term "receptor" is used to include any molecule on the surface of a target cell that can bind to a targeting agent, and is not intended to imply any particular function of the cell surface receptor. A targeting agent may be considered as the "ligand" of a cell surface receptor. The terms "targeting agent" and "ligand" may be used interchangeably. Again, this term is not intended to imply any particular function of the targeting agent or cell surface receptor, or any particular relationship between two molecules other than the ability of one to bind to the other.

[0196] Thus, the targeting agent can be any moiety that has affinity for a selected receptor. It can be, for example, an affinity protein (e.g., an antibody or fragment thereof that has affinity for a selected receptor), an aptamer, or any other suitable moiety. In some embodiments, the targeting agent can be a physiological ligand of the receptor.

[0197] The binding between the targeting agent and the receptor can promote the uptake of the conjugated nucleic acid by the target cell, for example, via the internalization of the receptor or any other suitable mechanism. Thus, in order for the conjugated nucleic acid to be taken up by the target cell by a mechanism, for example, a different receptor-mediated endocytosis pathway or a functionally similar process, a suitable ligand for the desired receptor molecule can be used as a targeting agent. In other embodiments, a ligand that can mediate the internalization of the nucleic acid into the target cell by a mechanism other than receptor-mediated endocytosis can alternatively be conjugated to the nucleic acid of the present invention for cell- or tissue-specific targeting.

[0198] One example of a ligand that mediates receptor-mediated endocytosis is the GalNAc moiety described herein, which has high affinity for the asialoglycoprotein receptor complex (ASGP-R). The ASGP-R complex is composed of membrane ASGR1 and ASGR2 receptor multimers in various ratios that are highly abundant in hepatocytes. One of the first disclosures of the use of triantennary cluster glycosides as conjugated ligands was in U.S. Pat. No. 5,885,968. Conjugates with three GalNAc ligands and containing phosphate groups are known and described in Dubber et al. (Bioconjug. Chem. 2003 Jan-Feb;14(1):239-46.). The ASGP-R complex exhibits a 50-fold higher affinity for N-acetyl-D-galactosamine (GalNAc) than D-Gal.

[0199] The ASGP-R complex specifically recognizes the terminal β-galactosyl subunits of glycosylated proteins or other oligosaccharides (Weigel, PH et al., Biochim. Biophys. Acta. 2002 Sep 19;1572(2-3):341-63) and can be used to deliver drugs to hepatocytes in the liver that express the receptor complex by covalently coupling galactose or galactosamine to the drug substance (Ishibashi, S. et al., J Biol. Chem. 1994 Nov 11;269(45):27803-6). Furthermore, the binding affinity can be greatly increased by the multivalent effect achieved by the repetition of the targeting moiety (Biessen EA et al., J Med Chem. 1995 Apr 28;38(9):1538-46).

[0200] ASGP-R complex is a mediator of the active uptake of glycoproteins containing terminal β-galactosyl into cellular endosomes.Therefore, ASGPR is highly suitable for the targeted delivery of drug candidates, such as nucleic acids, conjugated to such ligands to cells expressing the receptor (Akinc et al., Mol Ther. 2010 July; 18(7): 1357-64).

[0201] More generally, the ligand may comprise a sugar that is selected to have affinity for at least one type of receptor on the target cell, in particular the receptor being on the surface of mammalian liver cells, such as the aforementioned hepatic asialoglycoprotein receptor complex (ASGP-R).

[0202] The sugar may be selected from N-acetylgalactosamine, mannose, galactose, glucose, glucosamine, and fucose. The sugar may be N-acetylgalactosamine (GalNAc). The heterologous moiety may contain multiple such sugars, for example, two or particularly three such sugars, for example, three GalNAc groups.

[0203] A heterologous moiety may thus comprise (i) one or more functional moieties, and (ii) a linker, where the linker conjugates the functional moiety to a nucleic acid as defined in any preceding aspect. The linker may be a monovalent structure or a bivalent, trivalent or tetravalent branched structure. The nucleotides may be modified as defined herein.

[0204] The functional moieties may therefore be ligands (or targeting agents). When multiple functional moieties are present, they may be the same or different. When the functional moieties are ligands, they may be sugars and thus may be (or may include) GalNAc.

[0205] In one embodiment, the nucleic acid has the formula (II): [SX 1 -PX 2 ]3-AX 3 - (II) is conjugated to a heterologous moiety comprising a compound of During the ceremony, S represents a functional moiety, e.g., a ligand, e.g., a sugar, preferably the sugar is N-acetylgalactosamine; X 1 is C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2, or 3; P is a phosphate or a modified phosphate, preferably a thiophosphate; X 2 is an alkylene or a group of the formula (-CH2) n -O-CH2- alkylene ether, where n=1 to 6; A is a branching unit, X 3 represents a cross-linking unit, The nucleic acids according to the invention are linked to X via a phosphate or modified phosphate, preferably a thiophosphate. 3 is conjugated to

[0206] In formula (II), the branching unit "A" is preferably three-branched to accommodate three sugar ligands. The branching unit is preferably covalently attached to the ligand and the remaining tether portion of the nucleic acid. The branching unit may include a branched aliphatic group, including a group selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether, and hydroxyamino groups. The branching unit may include a group selected from alkyl and ether groups.

[0207] The branching unit A is

[0208] [ka]

[0209] may have a structure selected from Each A1 independently represents O, S, C=O, or NH, and each n independently represents an integer of 1 to 20.

[0210] The branching units are

[0211] [ka]

[0212] may have a structure selected from Each A1 independently represents O, S, C=O, or NH, and each n independently represents an integer of 1 to 20.

[0213] The branching units are

[0214] [ka]

[0215] may have a structure selected from A1 is O, S, C═O, or NH, and each n independently represents an integer of 1 to 20.

[0216] The branching unit has the structure:

[0217] [ka]

[0218] may have:

[0219] The branching unit has the structure:

[0220] [ka]

[0221] may have:

[0222] The branching unit has the structure:

[0223] [ka]

[0224] may have:

[0225] Alternatively, the branching unit A is

[0226] [ka]

[0227] may have a structure selected from During the ceremony, R1 is hydrogen or C1-C10 alkylene; R2 is a C1 to C10 alkylene.

[0228] Optionally, the branching units consist only of carbon atoms.

[0229] "X 3 The " portion is a bridging unit. The bridging unit is linear and covalently binds to the branching unit and the nucleic acid.

[0230] X3 is -C1~C 20 Alkylene-, -C2~C 20 Alkenylene, formula -(C1-C 20 Alkylene)-O-(C1-C 20 Alkylene ether of -C(O)-C1-C 20 may be selected from alkylene-, -C0-C4 alkylene(Cy)C0-C4 alkylene- (wherein Cy represents a substituted or unsubstituted 5- or 6-membered cycloalkylene, arylene, heterocyclylene, or heteroarylene ring), -C1-C4 alkylene-NHC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)NH-C1-C4 alkylene-, -C1-C4 alkylene-SC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)S-C1-C4 alkylene-, -C1-C4 alkylene-OC(O)-C1-C4 alkylene-, -C1-C4 alkylene-C(O)O-C1-C4 alkylene-, and -C1-C6 alkylene-SS-C1-C6 alkylene-.

[0231] X 3 is the formula -(C1~C 20 Alkylene)-O-(C1-C 20 It may be an alkylene ether of X 3 is the formula -(C1~C 20 Alkylene)-O-(C4~C 20 alkylene)-, wherein the (C4 to C 20 alkylene) is linked to Z. 3 -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C6H 12 - and -CH2-O-CH 16 -, especially -CH2-O-C4H8-, -CH2-O-C6H 12 - and -CH2-O-CH 16 -, in each case, the -CH2- group is linked to A.

[0232] In one embodiment, the nucleic acid has the formula (III): [SX1 -PX 2 ]3-AX 3 - (III) is conjugated to a heterologous moiety of During the ceremony, S represents a functional moiety, e.g., a ligand, e.g., a sugar, preferably GalNAc; X 1 is C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2, or 3; P is a phosphate or a modified phosphate, preferably a thiophosphate; X 2 is C1-C8 alkylene, A is,

[0233] [ka]

[0234] is a branching unit selected from X 3 is a cross-linking unit, The nucleic acids according to the invention are linked to X via a phosphate or modified phosphate, preferably a thiophosphate. 3 is conjugated to

[0235] The branching unit A has the structure:

[0236] [ka]

[0237] may have:

[0238] The branching unit A has the structure:

[0239] [ka]

[0240] It may have X 3 is attached to a nitrogen atom.

[0241] X 3 is C1~C 20 It may be alkylene. Preferably, X 3 -C3H6-, -C4H8-, -C6H 12 - and -C8H 16 -, especially -C4H8-, -C6H 12 - and -C8H 16 - is selected from the group consisting of

[0242] In one embodiment, the nucleic acid has the formula (IV): [SX 1 -PX 2 ]3-AX 3 - (IV) is conjugated to a ligand comprising a compound of During the ceremony, S represents a functional moiety, e.g., a ligand, e.g., a sugar, preferably GalNAc; X 1 is C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2, or 3; P is a phosphate or a modified phosphate, preferably a thiophosphate; X 2 is an alkylene ether of formula -C3H6-O-CH2-, A is a branching unit, X 3 -CH2-O-CH2-, -CH2-O-C2H4-, -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C5H 10 -,-CH2-O-C6H 12 -, -CH2-O-CH 14 - and -CH2-O-CH 16 -, in each occurrence, the -CH2- group is linked to A, X 3is conjugated to the nucleic acid according to the invention by a phosphate or a modified phosphate, preferably a thiophosphate.

[0243] The branching units may include carbon. Preferably, the branching units are carbon.

[0244] X 3 -CH2-O-C4H8-, -CH2-O-C5H 10 -,-CH2-O-C6H 12 -,-CH2-O-C7H 14 - and -CH2-O-CH 16 -. Preferably, X 3 -CH2-O-C4H8-, -CH2-O-C6H 12 - and -CH2-O-CH 16 is selected from the group consisting of:

[0245] X 1 can be (-CH-CH-O)(-CH)-. X 1 can be (-CH-CH-O)(-CH)-. 1 can be (-CH-CH-O)(-CH)-. Preferably, X 1 can be (-CH-CH-O)(-CH)-. Alternatively, X 1 represents C3-C6 alkylene. X 1 X may be propylene. 1 may be butylene. X 1 can be pentylene. X 1 can be hexylene. Preferably, the alkyl is a straight chain alkylene. In particular, X 1 can be butylene.

[0246] X 2 represents an alkylene ether of the formula -C3H6-O-CH2-, i.e., C3 alkoxymethylene, or -CH2CH2CH2OCH2-.

[0247] For any of the above embodiments, when P represents a modified phosphate group, P is

[0248] [ka]

[0249] It can be expressed by In the formula, Y 1 and Y 2 are independently =O, =S, -O - , -OH, -SH, -BH3, -OCH2CO2, -OCH2CO2R x , -OCH2C(S)OR x , and -OR x R x represents C1-C6 alkyl,

[0250] [ka]

[0251] indicates the bond to the remainder of the compound.

[0252] Modified phosphate refers to a phosphate group in which one or more of the non-linking oxygens are replaced. Examples of modified phosphate groups include phosphorothioates, phosphorodithioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates are those in which both non-linking oxygens are replaced with sulfur. One, each, or both non-linking oxygens of the phosphate group can be independently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0253] The phosphate can also be modified by replacement of the linking oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Replacement can occur at the terminal oxygen. Replacement of a non-linking oxygen with a nitrogen is possible.

[0254] For example, Y 1 may represent -OH, Y 2 may represent =O or =S, or Y 1 -O - can be represented, Y 2 may represent =O or =S, Y 1 can represent =O, and Y 2 -CH3, -SH, -OR x or -BH3, Y 1 can represent =S, and Y 2 -CH3, OR x , or -SH.

[0255] A person skilled in the art would recognize that in a particular case, Y 1 and Y 2 It will be appreciated that there will be delocalization between

[0256] Preferably, the modified phosphate group is a thiophosphate group. The thiophosphate group may be a bithiophosphate (i.e., Y 1 represents =S, and Y 2 Ga-S - (i.e., Y 1 -O - represents Y 2 represents =S or Y 1 represents =O, and Y 2 Ga-S - Preferably, P is a monothiophosphate. The inventors have found that conjugates having a thiophosphate group instead of a phosphate group have improved potency and duration of action in vivo.

[0257] P also represents ethyl phosphate (i.e., Y 1 represents =0, and Y 2 represents OCH2CH3).

[0258] The ligand, e.g., sugar, can be selected to have affinity for at least one type of receptor on the target cell. In particular, the receptor is on the surface of mammalian liver cells, e.g., the hepatic asialoglycoprotein receptor complex (ASGP-R).

[0259] For both the above and the below embodiments, the sugar can be selected from N-acetyl with one or more of galactosamine, mannose, galactose, glucose, glucosamine, and fructose.Typically, the ligand to be used in the present invention can include N-acetylgalactosamine (GalNAc).Preferably, the compound of the present invention can have three ligands, each of which preferably includes N-acetylgalactosamine.

[0260] "GalNAc" refers to 2-(acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetylgalactosamine. Reference to "GalNAc" or "N-acetylgalactosamine" includes both the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose. In certain embodiments, both the β form: 2-(acetylamino)-2-deoxy-β-D-galactopyranose and the α form: 2-(acetylamino)-2-deoxy-α-D-galactopyranose may be used interchangeably. Preferably, the compounds of the present invention include the β form of 2-(acetylamino)-2-deoxy-β-D-galactopyranose.

[0261] [ka]

[0262] 2-(Acetylamino)-2-deoxy-D-galactopyranose

[0263] [ka]

[0264] 2-(Acetylamino)-2-deoxy-β-D-galactopyranose

[0265] [ka]

[0266] 2-(Acetylamino)-2-deoxy-α-D-galactopyranose

[0267] In one embodiment, the nucleic acid is a conjugated nucleic acid, where the nucleic acid has the following structure, which may be referred to as a "triantennary ligand" for ease of reference:

[0268] [ka]

[0269] [ka]

[0270] [ka]

[0271] [ka]

[0272] [ka]

[0273] and conjugated to a heterologous moiety having one of the following: Z is any nucleic acid as defined herein.

[0274] In certain embodiments, nucleic acid Z is conjugated to a triantennary ligand via a phosphate or thiophosphate group linking the triantennary ligand to the 3' or 5' position of the sugar of the terminal nucleotide of nucleic acid Z, specifically the 3' or 5' position of the ribose.

[0275] In certain embodiments, the heterologous moiety ("triantennary ligand") is conjugated to the 3' position of the ribose of the terminal nucleotide of the second (sense) strand of Z (also referred to as strand "B" in Tables 5a, 5b, 5c (Tables 8, 9, 10)).

[0276] In other embodiments, the heterologous moiety ("triantennary ligand") is conjugated to the 5' position of the ribose of the terminal nucleotide of the second (sense) strand of Z (also referred to as strand "B" in Tables 5a, 5b, 5c (Tables 8, 9, 10)).

[0277] In other embodiments, the heterologous moiety ("triantennary ligand") is conjugated to the 3' position of the ribose of the terminal nucleotide of the first (antisense) strand of Z (also referred to as strand "A" in Tables 5a, 5b, 5c (Tables 8, 9, 10)).

[0278] Preferably, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid has the following structure:

[0279] [ka]

[0280] and is conjugated to a triantennary ligand having one of the following structures: Z is any nucleic acid as defined herein.

[0281] In a preferred embodiment, nucleic acid Z is conjugated to a triantennary ligand via a phosphate or thiophosphate group linking the triantennary ligand to the 3' or 5' position of the ribose of the terminal nucleotide of said nucleic acid Z.

[0282] Preferably, the triantennary ligand is conjugated to the 5' position of the ribose of the terminal nucleotide of the second (sense) strand of Z (also referred to as strand "B" in Tables 5a, 5b, 5c (Tables 8, 9, 10)).

[0283] A heterologous moiety of formula (II), (III), or (IV), or any one of the tri-antennary ligands disclosed herein, may be attached to either the 3'-end of the first (antisense) strand and / or the 3'- and / or 5'-end of the second (sense) strand. A nucleic acid may contain more than one heterologous moiety of formula (II), (III), or (IV), or any one of the tri-antennary ligands disclosed herein. However, a single heterologous moiety of formula (II), (III), or (IV), or any one of the tri-antennary ligands disclosed herein, is preferred, since a single such moiety is sufficient to efficiently target a nucleic acid to a target cell. Preferably, in that case, at least the last two, preferably at least the last three, more preferably at least the last four nucleotides of the end of the nucleic acid to which the ligand is attached are linked by phosphodiester linkages.

[0284] Preferably, the 5' end of the first (antisense) strand is not conjugated to a heterologous moiety, as conjugation at this position could potentially interfere with the biological activity of the nucleic acid.

[0285] A nucleic acid having a single heterologous moiety (e.g., of formula (II), (III), or (IV), or any one of the triantennary ligands disclosed herein) at the 5'-end of the strand is easier and therefore cheaper to synthesize than the same nucleic acid having the same group at the 3'-end. Preferably, therefore, a single heterologous moiety (e.g., of formula (II), (III), or (IV), or any one of the triantennary ligands disclosed herein) is covalently attached (conjugated) to the 5'-end of the second strand of the nucleic acid.

[0286] In one embodiment, the first strand of the nucleic acid is a compound of formula (V):

[0287] [ka]

[0288] In the formula, b is preferably 0 or 1, The second strand is a compound of formula (VI):

[0289] [ka]

[0290] During the ceremony, c and d are independently preferably 0 or 1; Z1 and Z2 are the first and second strands of a nucleic acid, respectively; Y is independently O or S; n is independently 0, 1, 2, or 3; L1 is a linker to which the ligand is attached, L1 is the same or different in formulas (V) and (VI), and when L1 occurs more than once in the same formula, it is the same or different in formulas (V) and (VI), L1 is preferably formula (VII), and b+c+d is preferably 2 or 3.

[0291] Preferably, L1 in formula (V) and (VI) is of formula (VII):

[0292] [ka]

[0293] During the ceremony, L is -(CH2) r C(O)-, where r=2 to 12; -(CH2-CH2-O) s -CH2-C(O)- (wherein s=1 to 5), -(CH2) t -CO-NH-(CH2) t -NH-C(O)- (wherein t is independently 1 to 5); -(CH2) u -CO-NH-(CH2) u -C(O)-, where u is independently 1 to 5, and -(CH2) v -NH-C(O)- (wherein v is 2 to 12) and preferably selected from the group consisting of: the terminal C(O) is attached to X of formula (VII) if present, or to W1 of formula (VII) if X is absent, or to V of formula (VII) if W1 is absent; W1, W3, and W5 are individually absent, or -(CH2) r (Wherein, r=1 to 7), -(CH2) s -O-(CH2) s - (wherein s is independently 0 to 5); -(CH2) t -S-(CH2)t (wherein t is independently 0 to 5) and preferably selected from the group comprising: X is absent or is selected from the group comprising or preferably consisting of NH, NCH3, or NC2H5; V is CH, N,

[0294] [ka]

[0295] and preferably selected from the group consisting of: B, if present, is a modified or natural nucleobase.

[0296] In one embodiment, the first chain is a compound of formula (VIII):

[0297] [ka]

[0298] In the formula, b is preferably 0 or 1, The second chain is a compound of formula (IX):

[0299] [ka]

[0300] wherein c and d are independently preferably 0 or 1; During the ceremony, Z1 and Z2 are the first and second strands of a nucleic acid, respectively; Y is independently O or S; R1 is H or methyl; n is independently preferably 0, 1, 2 or 3; L is the same or different in formulae (VIII) and (IX), and if L occurs more than once in the same formula, it is the same or different in formulae (VIII) and (IX), -(CH2) r C(O)-, where r=2 to 12; -(CH2-CH2-O) s -CH2-C(O)- (wherein s=1 to 5), -(CH2) t -CO-NH-(CH2) t-NH-C(O)- (wherein t is independently 1 to 5); -(CH2) u -CO-NH-(CH2) u -C(O)- (wherein u is independently 1 to 5); -(CH2) v -NH-C(O)- (wherein v is 2 to 12) and preferably selected from the group consisting of: The terminal C(O), if present, is attached to an NH group (of the linker, not the targeting ligand); b+c+d is preferably 2 or 3.

[0301] In one embodiment, the first strand of the nucleic acid is a compound of formula (X):

[0302] [ka]

[0303] In the formula, b is preferably 0 or 1, The second chain is a compound of formula (XI):

[0304] [ka]

[0305] During the ceremony, c and d are independently preferably 0 or 1; Z1 and Z2 are the first and second RNA strands of the nucleic acid, respectively; Y is independently O or S; n is independently preferably 0, 1, 2 or 3; L2 is the same or different in formulas (X) and (XI) and is the same or different in the portions bracketed by b, c, and d;

[0306] [ka]

[0307] or preferably selected from the group consisting of: or n is 0 and L2 is

[0308] [ka]

[0309] and the terminal OH group is absent such that the moiety

[0310] [ka]

[0311] During the ceremony, F is a saturated branched or unbranched (e.g., unbranched) C 1~8 Alkyl (e.g., C 1~6 alkyl) chain, one of the carbon atoms is optionally replaced with an oxygen atom, provided that said oxygen atom is separated from another heteroatom (e.g., an O or N atom) by at least two carbon atoms; L is the same or different in formulae (X) and (XI), -(CH2) r C(O)-, where r=2 to 12; -(CH2-CH2-O) s -CH2-C(O)- (wherein s=1 to 5), -(CH2) t -CO-NH-(CH2) t -NH-C(O)- (wherein t is independently 1 to 5); -(CH2) u -CO-NH-(CH2) u -C(O)-, where u is independently 1 to 5, and -(CH2) v -NH-C(O)- (wherein v is 2 to 12) and preferably selected from the group comprising or consisting of: The terminal C(O), if present, is attached to an NH group (of the linker, not the targeting ligand); b+c+d is preferably 2 or 3.

[0312] In one embodiment, in any of the nucleic acids of formula (V) and (VI), or (VIII) and (IX), or (X) and (XI), b is 0, c is 1, and d is 1, or b is 1, c is 0, d is 1, or b is 1, c is 1, and d is 0, or b is 1, c is 1, and d is 1. Preferably, b is 0, c is 1, and d is 1, or b is 1, c is 0, d is 1, or b is 1, c is 1, and d is 1. Most preferably, b is 0, c is 1, and d is 1.

[0313] In one embodiment, in any of the nucleic acids of formula (V) and (VI), or (VIII) and (IX), or (X) and (XI), Y is O. In another embodiment, Y is S. In preferred embodiments, Y is independently selected from O or S at different positions in the formula.

[0314] In one embodiment, in any of the nucleic acids of formula (VIII) and (IX), R1 is H or methyl. In one embodiment, R1 is H. In another embodiment, R1 is methyl.

[0315] In one embodiment, in any of the nucleic acids of formula (V) and (VI), or (VIII) and (IX), or (X) and (XI), n is 0, 1, 2, or 3. Preferably, n is 0.

[0316] Examples of F moieties in any of the nucleic acids of formula (X) and (XI) include (CH2) 1~6 , for example, (CH2) 1~4 , for example, CH2, (CH2)4, (CH2)5, or (CH2)6, or CH2O(CH2) 2~3 For example, CH2O(CH2)CH3.

[0317] In one embodiment, L2 in formulae (X) and (XI) is

[0318] [ka]

[0319] It is.

[0320] In one embodiment, L2 is

[0321] [ka]

[0322] It is.

[0323] In one embodiment, L2 is

[0324] [ka]

[0325] It is.

[0326] In one embodiment, L2 is

[0327] [ka]

[0328] It is.

[0329] In one embodiment, n is 0 and L2 is

[0330] [ka]

[0331] and the terminal OH group is absent such that the moiety

[0332] [ka]

[0333] Y is O or S.

[0334] In one embodiment, L in the nucleic acid of formula (V) and (VI), or (VIII) and (IX), or (X) and (XI) is -(CH2) r C(O)-, where r=2 to 12; -(CH2-CH2-O) s -CH2-C(O)- (wherein s=1 to 5), -(CH2) t -CO-NH-(CH2) t -NH-C(O)- (wherein t is independently 1 to 5); -(CH2) u -CO-NH-(CH2) u -C(O)-, where u is independently 1 to 5, and -(CH2) v -NH-C(O)- (wherein v is 2 to 12) and preferably selected from the group comprising or consisting of: The terminal C(O) is bonded to an NH group.

[0335] Preferably, L is -(CH2) r -C(O)-, in which r=2 to 12, more preferably r=2 to 6, and even more preferably r=4 or 6, for example 4.

[0336] Preferably, L is

[0337] [ka]

[0338] It is.

[0339] Within the portion bracketed by b, c, and d, L2 in the nucleic acids of formula (X) and (XI) is typically the same. Between the portions bracketed by b, c, and d, L2 may be the same or different. In some embodiments, L2 in the portion bracketed by c is the same as L2 in the portion bracketed by d. In some embodiments, L2 in the portion bracketed by c is not the same as L2 in the portion bracketed by d. In some embodiments, L2 in the portions bracketed by b, c, and d are the same, for example, when the linker moiety is a serinol-derived linker moiety.

[0340] The serinol-derived linker moiety may be based on serinol in any stereochemical configuration, i.e., derived from L-serine isomers, D-serine isomers, racemic serine, or other combinations of isomers. In a preferred embodiment of the invention, the serinol-GalNAc moiety (SerGN) has the following stereochemical configuration:

[0341] [ka]

[0342] i.e. based on (S)-serinol-amidite or (S)-serinol succinate solid supported building blocks derived from L-serine isomers.

[0343] In a preferred embodiment, the first strand of the nucleic acid is a compound of formula (VIII) and the second strand of the nucleic acid is a compound of formula (IX), wherein b is 0; c and d are 1; n is 0, Z1 and Z2 are the first and second strands of a nucleic acid, respectively; Y is S, R1 is H, L is -(CH2)4-C(O)-, where the terminal C(O) of L is attached to an N atom of the linker (i.e., not to an available N atom of the targeting ligand).

[0344] In another preferred embodiment, the first strand of the nucleic acid is a compound of formula (V) and the second strand of the nucleic acid is a compound of formula (VI), wherein: b is 0; c and d are 1; n is 0, Z1 and Z2 are the first and second strands of a nucleic acid, respectively; Y is S, L1 is of formula (VII), W1 is -CH2-O-(CH2)3-, W3 is -CH2-, W5 is non-existent, V is CH; X is NH; L is -(CH2)4-C(O)-, and the terminal C(O) of L is bonded to the N atom of X in formula (VII).

[0345] In another preferred embodiment, the first strand of the nucleic acid is a compound of formula (V) and the second strand of the nucleic acid is a compound of formula (VI), wherein: b is 0; c and d are 1; n is 0, Z1 and Z2 are the first and second strands of a nucleic acid, respectively; Y is S, L1 is of formula (VII), W1, W3, and W5 are absent; V is

[0346] [ka]

[0347] and X is non-existence, L is -(CH2)4-C(O)-NH-(CH2)5-C(O)-, and the terminal C(O) of L is bonded to the N atom of V in formula (VII).

[0348] In one embodiment, the nucleic acid is conjugated to a triantennary ligand having the structure:

[0349] [ka]

[0350] Nucleic acids are a) at the 3' position of the ribose of the terminal nucleotide of the second (sense) strand of Z (also referred to as strand "B" in Tables 5a, 5b, 5c (Tables 8, 9, 10)), b) at the 5' position of the ribose of the terminal nucleotide of the second (sense) strand of Z (also referred to as strand "B" in Tables 5a, 5b, 5c (Tables 8, 9, 10)); or c) at the 3' position of the ribose of the terminal nucleotide of the first (antisense) strand of Z (also referred to as strand "A" in Tables 5a, 5b (Tables 8, 9) The triantennary ligand is conjugated via the phosphate group of the ligand.

[0351] In one embodiment of the nucleic acid, the cell targeted by the nucleic acid bearing the ligand is a hepatocyte.

[0352] In any one of the above-mentioned ligands in which GalNAc is present, GalNAc may be replaced with any other selected targeting ligand, such as those mentioned herein, in particular mannose, galactose, glucose, glucosamine, and fucose.

[0353] In one embodiment, the nucleic acid is conjugated to a heterologous moiety comprising a lipid, more preferably cholesterol.

[0354] In one embodiment, the double-stranded nucleic acid for inhibiting expression of MASP-2 is one of the duplexes shown in Table 5c (Table 10), which may be referenced by their duplex identifier numbers.

[0355] In one preferred embodiment, the double-stranded nucleic acid for inhibiting expression of MASP-2 is a double-stranded nucleic acid having the duplex identification number EM1203, EM1204, EM1205, EM1206, EM1207, EM1208, EM1209, EM1210, EM1211, EM1212, or EM1213.

[0356] In one preferred embodiment, the double-stranded nucleic acid for inhibiting expression of MASP-2 is a nucleic acid in which the sequence of the first strand comprises (vp)-mU fC mG fG mA fG mC fG mG fA mA fG mG fU mA fA mU (ps) fG (ps) mU (sequence number 804), and optionally the sequence of the second strand comprises [ST23 (ps)]3ST41 (ps) mA mC mA mU mU mA fC fC fU mU mC mC mG mC mU mC mC (ps) mG (ps) mA (sequence number 805).

[0357] In one preferred embodiment, the double-stranded nucleic acid for inhibiting expression of MASP-2 is a nucleic acid in which the sequence of the first strand consists of (vp)-mU fC mG fG mA fG mC fG mG fA mA fG mG fU mA fA mU (ps) fG (ps) mU (sequence number 804), and optionally the sequence of the second strand consists of [ST23 (ps)]3ST41 (ps) mA mC mA mU mU mA fC fC fU mU mC mC mG mC mU mC mC (ps) mG (ps) mA (sequence number 805).

[0358] In one preferred embodiment, the double-stranded nucleic acid for inhibiting expression of MASP-2 is a nucleic acid in which the sequence of the first strand includes (vp)-mU fA mC fG mA fA mG fU mC fG mU fA mC fU mC fG mC (ps) fA (ps) mG (sequence number 806), and optionally the sequence of the second strand includes [ST23 (ps)]3ST41 (ps) mC mU mG mC mG mA fG fU fA mC mG mA mC mU mU mC mG (ps) mU (ps) mC (sequence number 807).

[0359] In one preferred embodiment, the double-stranded nucleic acid for inhibiting expression of MASP-2 is a nucleic acid in which the sequence of the first strand consists of (vp)-mU fA mC fG mA fA mG fU mC fG mU fA mC fU mC fG mC (ps) fA (ps) mG (sequence number 806), and optionally the sequence of the second strand consists of [ST23 (ps)]3ST41 (ps) mC mU mG mC mG mA fG fU fA mC mG mA mC mU mU mC mG (ps) mU (ps) mC (sequence number 807).

[0360] In one preferred embodiment, the double-stranded nucleic acid for inhibiting expression of MASP-2 is a nucleic acid in which the sequence of the first strand includes (vp)-mU fA mA fG mG fU mA fA mU fG mU fC mC fA mG fG mC (ps) fU (ps) mG (sequence number 810), and optionally the sequence of the second strand includes [ST23 (ps)]3ST41 (ps) mC mA mG mC mC mU fG fG fA mC mA mU mU mA mC mC mU (ps) mU (ps) mC (sequence number 811).

[0361] In one preferred embodiment, the double-stranded nucleic acid for inhibiting expression of MASP-2 is a nucleic acid in which the sequence of the first strand consists of (vp)-mU fA mA fG mG fU mA fA mU fG mU fC mC fA mG fG mC (ps) fU (ps) mG (sequence number 810), and optionally the sequence of the second strand consists of [ST23 (ps)]3ST41 (ps) mC mA mG mC mC mU fG fG fA mC mA mU mU mA mC mC mU (ps) mU (ps) mC (sequence number 811).

[0362] Compositions, Uses, and Methods The present invention also provides compositions comprising the nucleic acids of the present invention. The nucleic acids and compositions can be used as therapeutic or diagnostic agents, alone or in combination with other agents. For example, one or more nucleic acids of the present invention can be combined with a delivery vehicle (e.g., liposome) and / or excipient, such as a carrier, a diluent. Other agents, such as preservatives and stabilizers, can also be added. Any pharmaceutically acceptable salt or solvate of the nucleic acids of the present invention is also within the scope of the present invention. Methods for delivery of nucleic acids are known in the art and within the knowledge of the skilled artisan.

[0363] The compositions disclosed herein are, in particular, pharmaceutical compositions. Such compositions are suitable for administration to a subject.

[0364] In one aspect, the composition comprises a nucleic acid disclosed herein, or a pharma- ceutically acceptable salt or solvate thereof, and a solvent (preferably water), and / or a delivery vehicle, and / or a physiologically acceptable excipient, and / or a carrier, and / or a salt, and / or a diluent, and / or a buffer, and / or a preservative.

[0365] Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, intranasal, or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration.The formulations can conveniently be presented in unit dosage form and can be prepared by any of the methods well known in the field of pharmacy.Subcutaneous or transdermal modes of administration can be particularly suitable for the compounds described herein.

[0366] The prophylactic or therapeutically effective amount of the nucleic acid of the present invention depends on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal being considered. These factors and their relationships for determining this amount are well known to those skilled in the medical field. This amount and method of administration can be adjusted to achieve optimal effectiveness and can depend on factors such as body weight, diet, concomitant medications, and other factors well known to those skilled in the medical field. The dosage size and dosage regimen most suitable for human use can be guided by the results obtained by the present invention and can be confirmed in appropriately designed clinical trials.

[0367] Effective dosages and treatment protocols can be determined by conventional means, starting with low doses in laboratory animals, then increasing the dosage while monitoring the effects, as well as systematically varying the dosage regimen. Numerous factors can be considered by the clinician when determining the optimal dosage for a given subject. Such considerations are known to those skilled in the art.

[0368] The nucleic acids of the invention, or salts thereof, can be formulated as pharmaceutical compositions prepared for storage or administration, which typically contain a prophylactically or therapeutically effective amount of the nucleic acids of the invention, or salts thereof, in a pharma- ceutically acceptable carrier.

[0369] The nucleic acids of the invention, or conjugated nucleic acids, may also be administered in combination with other therapeutic compounds, either separately or simultaneously, e.g., administered as a combined unit dose. The invention also includes compositions comprising one or more nucleic acids according to the invention in a physiological / pharmaceutical acceptable excipient, e.g., stabilizers, preservatives, diluents, buffers, etc.

[0370] In one embodiment, the composition comprises a nucleic acid as disclosed herein and an additional therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody, and a peptide. Preferably, the additional therapeutic agent is an agent that targets, preferably inhibits the expression or activity of, MASP-2 or another element of the immune system or more specifically the complement pathway, such as a protein. Preferably, the additional therapeutic agent is one of the following: a) a peptide that inhibits the expression or activity of a component of the complement pathway, preferably one of MASP-2, C4, C2, C3, C5, or one of their subunits or protein cleavage products, b) an antibody that specifically binds to a component of the complement pathway, preferably one of MASP-2, C4, C2, C3, C5, or one of their subunits or protein cleavage products under physiological conditions, c) eculizumab or an antigen-binding derivative thereof, d) narsoplimab (OMS721) or an antigen-binding derivative thereof.

[0371] Eculizumab is a humanized monoclonal antibody that specifically binds complement component C5 and is commercialized under the trade name SOLIRIS®. It specifically binds complement component C5 with high affinity and inhibits cleavage of C5 into C5a and C5b. This antibody is described, for example, in EP 0758904 B1 and its family members.

[0372] In certain embodiments, two or more nucleic acids of the invention having different sequences may be administered simultaneously or sequentially.

[0373] In another aspect, the present invention provides compositions, eg, pharmaceutical compositions, comprising one or a combination of the different nucleic acids of the present invention and at least one pharma- ceutically acceptable carrier.

[0374] Dosage levels of the therapeutic agents and compositions of the present invention can be determined by one of skill in the art through experimentation. In one embodiment, a unit dose can contain between about 0.01 mg / kg and about 100 mg / kg of nucleic acid or conjugated nucleic acid. Alternatively, the dose can be 10 mg / kg to 25 mg / kg of body weight, or 1 mg / kg to 10 mg / kg of body weight, or 0.05 mg / kg to 5 mg / kg of body weight, or 0.1 mg / kg to 5 mg / kg of body weight, or 0.1 mg / kg to 1 mg / kg of body weight, or 0.1 mg / kg to 0.5 mg / kg of body weight, or 0.5 mg / kg to 1 mg / kg of body weight. Alternatively, the dose may be about 0.5 mg / kg to about 10 mg / kg body weight, or about 0.6 mg / kg to about 8 mg / kg body weight, or about 0.7 mg / kg to about 7 mg / kg body weight, or about 0.8 mg / kg to about 6 mg / kg body weight, or about 0.9 mg / kg to about 5.5 mg / kg body weight, or about 1 mg / kg to about 5 mg / kg body weight, or about 1 mg / kg body weight, or about 3 mg / kg body weight, or about 5 mg / kg body weight, where "about" refers to a deviation of up to 30%, preferably up to 20%, more preferably up to 10%, even more preferably up to 5%, and most preferably 0% from the indicated value. Dosage levels may also be calculated according to other parameters, such as body surface area, etc.

[0375] Dose units of these nucleic acids preferably contain from about 1 mg / kg to about 5 mg / kg body weight, or from about 1 mg / kg to about 3 mg / kg body weight, or about 1 mg / kg body weight, or about 3 mg / kg body weight, or about 5 mg / kg body weight. The MASP-2 mRNA levels in the liver and / or MASP-2 protein levels in the plasma or blood of subjects treated with a dose unit of nucleic acid are preferably reduced by at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% at the time of maximum effect compared to controls not treated with the nucleic acid or treated with a control nucleic acid under comparable conditions.

[0376] Dosage and frequency of administration may vary depending on whether the treatment is therapeutic or prophylactic (e.g., preventative), and may be adjusted during the course of treatment. In certain preventative applications, relatively low dosages are administered at relatively infrequent intervals for relatively long periods of time. Some subjects may continue to receive treatment throughout their lifespan. In certain therapeutic applications, relatively high dosages are often required at relatively short intervals until the progression of the disease is reduced or the patient shows partial or complete recovery of the symptoms of the disease. The patient may then be switched to a suitable preventive dosing regimen.

[0377] The actual dosage level of the nucleic acid of the present invention, alone or in combination with one or more other active ingredients in the pharmaceutical composition of the present invention, can be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without causing adverse side effects to the subject or patient. The selected dosage level depends on various factors, such as pharmacokinetic factors, such as the activity of the particular nucleic acid or composition used, the route of administration, the time of administration, the excretion rate of the particular nucleic acid used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the subject or patient being treated, and similar factors well known in the medical field.

[0378] The pharmaceutical compositions may be in sterile injectable aqueous suspensions or solutions, or may be in lyophilized form.

[0379] The pharmaceutical composition may be in unit dosage form. In such form, the composition is divided into unit doses containing an appropriate amount of active ingredient. The unit dosage form may be a packaged preparation, the package containing discrete amounts of the preparation, for example, individually packaged tablets, capsules, and powders in vials or ampoules. The unit dosage form may also be a capsule, cachet, or tablet itself, or the appropriate number of any of these packaged forms. The unit dosage form may also be provided in a single-dose injectable form, for example, in the form of a pen. The composition may be formulated for any suitable route and means of administration.

[0380] The therapeutic agents and pharmaceutical compositions of the present invention can be administered to a mammalian subject in a pharmacologic effective dose. The mammal can be selected from humans, non-human primates, monkeys or prosimians, dogs, cats, horses, cows, pigs, goats, sheep, mice, rats, hamsters, hedgehogs, guinea pigs, or other related species. On this basis, "MASP-2" as used herein refers to a nucleic acid or protein when naturally or artificially expressed in any of the above-mentioned species, although preferably the term refers to a human nucleic acid or protein.

[0381] The pharmaceutical composition of the present invention may be administered alone or in combination with one or more other therapeutic or diagnostic agents. Combination therapy may include the nucleic acid of the present invention in combination with at least one other therapeutic agent selected based on the specific patient, disease, or condition to be treated. Examples of such other agents include, among others, therapeutically active small molecules or polypeptides, single-chain antibodies, classical antibodies or fragments thereof, or nucleic acid molecules that modulate the gene expression of one or more additional genes, and similar modulating therapeutic agents that may complement or otherwise be beneficial to therapeutic or prophylactic treatment regimes.

[0382] The pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier may be, for example, a solvent or dispersion medium containing water, alcohol, such as ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixture. Proper fluidity can be maintained, for example, by the use of coating agents, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants according to formulation chemistry well known in the art. In certain embodiments, isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, may be desirable in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as monostearate salts and gelatin.

[0383] One aspect of the invention is a nucleic acid or composition disclosed herein for use as a therapeutic agent. The nucleic acid or composition is preferably for use in the prevention or treatment of a disease, disorder, or syndrome.

[0384] The present invention provides nucleic acids for use alone or in combination with one or more additional therapeutic agents in pharmaceutical compositions for the treatment or prevention of conditions, diseases and disorders responsive to inhibition of MASP-2 expression.

[0385] One aspect of the invention is the use of the nucleic acids or compositions disclosed herein in the prevention or treatment of a disease, disorder, or syndrome.

[0386] The nucleic acids and pharmaceutical compositions of the present invention can be used in the treatment of various conditions, disorders, or diseases. Treatment with the nucleic acids of the present invention preferably results in MASP-2 depletion in vivo, preferably in the liver and / or blood. Therefore, the nucleic acids of the present invention and compositions comprising them will be useful in methods for treating various pathological disorders in which it may be beneficial to inhibit the expression of MASP-2. The present invention provides a method for treating a disease, disorder, or syndrome, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the nucleic acid of the present invention.

[0387] The present invention therefore provides a method for the prevention or treatment of a disease, disorder, or syndrome, comprising administering to a subject (e.g., a patient) in need thereof a therapeutically effective amount of a nucleic acid or a pharmaceutical composition comprising a nucleic acid of the invention.

[0388] The most desirable therapeutically effective amount is the amount that will result in the desired efficacy of the particular treatment selected by the skilled artisan for a given subject in need thereof. This amount will vary according to various factors understood by the skilled artisan, including, but not limited to, the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological state of the subject (including age, sex, type and stage of disease, general physical condition, responsiveness to a given dosage, and type of drug), the nature of the pharmacologic carrier(s) in the formulation, and the route of administration. A skilled artisan in the clinical and pharmacological fields will be able to determine the therapeutically effective amount through experimentation, i.e., by monitoring the subject's response to administration of the compound and adjusting the dosage accordingly. See, for example, Remington: The Science and Practice of Pharmacy 21st Edition, Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

[0389] In certain embodiments, the nucleic acids and pharmaceutical compositions of the invention can be used to treat or prevent a disease, disorder, or syndrome.

[0390] In certain embodiments, the invention provides a method for the prevention or treatment of a disease, disorder, or syndrome in a mammalian subject, e.g., a human, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a nucleic acid disclosed herein.

[0391] Administration of a "therapeutically effective dose" of the nucleic acids of the invention may result in a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of impairment or disability due to disease affliction.

[0392] The nucleic acids of the invention may be useful for treating or diagnosing a disease, disorder, or syndrome that can be diagnosed or treated using the methods described herein. Treatment and diagnosis of other diseases, disorders, or syndromes are also contemplated to fall within the scope of the invention.

[0393] One aspect of the invention is a method of prevention or treatment of a disease, disorder, or syndrome, comprising administering to an individual in need of treatment a pharma- ceutical effective dose or amount of a nucleic acid or composition disclosed herein, preferably, the nucleic acid or composition is administered to the subject subcutaneously, intravenously, or by oral, rectal, intrapulmonary, intramuscular, or intraperitoneal administration. Preferably, it is administered subcutaneously.

[0394] The associated disease, disorder or syndrome is preferably a complement-mediated disease, disorder or syndrome or a disease, disorder or syndrome associated with the complement pathway, in particular the alternative complement pathway.

[0395] The disease, disorder, or syndrome is typically associated with abnormal activation and / or excessive activation (hyperactivation) of the complement pathway (particularly the lectin pathway) and / or overexpression or ectopic expression or localization or accumulation of MASP-2. One example of a disease with accumulation of MASP-2 is IgA nephropathy, in which the lectin pathway contributes to the activation and deposition of C3 and C4 and the induction of renal damage. The abnormal or excessive activation of the complement pathway may have a genetic cause or may be acquired.

[0396] The disease, disorder, or syndrome is a) C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), lupus nephritis, IgA nephropathy (IgA N), cold agglutinin disease (CAD), myasthenia gravis (MG), primary membranous nephropathy, immune complex-mediated glomerulonephritis (IC-mediated GN), post-infectious glomerulonephritis (PIGN), systemic lupus erythematosus (SLE), ischemia / reperfusion injury, age-related macular degeneration (AMD), rheumatoid arthritis (RA), antineutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), dysbiotic periodontitis, malarial anemia, neuromyelitis optica, post-HCT / solid organ transplant (TMA), Guillain-Barre syndrome, membranous glomerulonephritis, thrombotic thrombocytopenic purpura, and sepsis; or b) C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uraemic syndrome (aHUS), lupus nephritis, IgA nephropathy (IgA or c) may be selected from the group including or preferably consisting of C3 glomerulopathy (C3G), antineutrophil cytoplasmic autoantibody associated vasculitis (ANCA-AV), atypical hemolytic uremic syndrome (aHUS), cold agglutinin disease (CAD), myasthenia gravis (MG), IgA nephropathy (IgA N), paroxysmal nocturnal hemoglobinuria (PNH); or d) may be selected from the group including or preferably consisting of C3 glomerulopathy (C3G), antineutrophil cytoplasmic autoantibody associated vasculitis (ANCA-AV), atypical hemolytic uremic syndrome (aHUS), antineutrophil cytoplasmic autoantibody associated vasculitis (ANCA-AV), IgA nephropathy (IgA N), paroxysmal nocturnal hemoglobinuria (PNH); N), post-HCT / solid organ transplant (TMA), Guillain-Barre syndrome, paroxysmal nocturnal hemoglobinuria (PNH), membranous glomerulonephritis, lupus nephritis, and thrombotic thrombocytopenic purpura, e) it may be C3 glomerulopathy (C3G), cold agglutinin disease (CAD), and IgA nephropathy (IgA N), or f) it is C3 glomerulopathy (C3G). The subject to be treated with the nucleic acid or composition according to the invention is preferably a subject suffering from or at risk of suffering from one of these diseases, disorders, or syndromes.

[0397] The nucleic acid or composition disclosed herein may be for use in a regimen that includes treatment once or twice a week, every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every 9 weeks, every 10 weeks, every 11 weeks, every 12 weeks, every 3 months, every 4 months, every 5 months, every 6 months, or in a regimen that uses a variable dosing frequency, for example, a combination of the aforementioned intervals. The nucleic acid or composition may be for use subcutaneously, intravenously, or using any other application route, for example, oral, rectal, intrapulmonary, or intraperitoneal. Preferably, it is for use subcutaneously.

[0398] In cells and / or subjects treated or receiving the nucleic acids or compositions disclosed herein, expression of MASP-2 may be inhibited by 15% up to 100%, but at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%, or in the range of intermediate values, compared to untreated cells and / or subjects. The level of inhibition may allow for the treatment of diseases associated with MASP-2 expression or overexpression or complement hyperactivation, or may serve to further investigate the function and physiological role of the MASP-2 gene product. The level of inhibition is preferably measured in the liver or blood or kidneys of the subject treated with the nucleic acid or composition, preferably in the blood.

[0399] One aspect is the use of the nucleic acids or compositions disclosed herein in the manufacture of a medicament for treating a disease, disorder or syndrome, such as those listed above, or preferably a further pathology associated with elevated levels of MASP-2 in the blood or kidneys or overactivation of the complement pathway, or in a further therapeutic approach in which inhibition of MASP-2 expression is desired. The medicament is a pharmaceutical composition.

[0400] Each of the nucleic acids of the invention, and their pharma- ceutically acceptable salts and solvates, constitutes an individual embodiment of the invention.

[0401] Also included in the present invention are methods of prevention or treatment of diseases, disorders, or syndromes, such as those listed above, comprising administration of a composition comprising a nucleic acid or composition described herein to an individual in need thereof. The nucleic acid or composition may be administered, for example, twice a week, once a week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, or every 8-12 weeks or more, in a regimen that includes weekly treatment, or in a regimen with varying dosing frequencies, for example, a combination of the aforementioned intervals. The nucleic acid, or conjugated nucleic acid, may be for use subcutaneously or intravenously, or by other application routes, such as oral, rectal, or intraperitoneal.

[0402] The nucleic acids of the invention may be administered by any suitable route of administration known in the art, including, but not limited to, aerosol, enteral, intranasal, intraocular, oral, parenteral, rectal, intravaginal, or transdermal (e.g., topical administration using a cream, gel, or ointment, or a transdermal patch). "Parenteral administration" typically refers to injection or communication with an intended site of action, and includes suborbital, infusion, intraarterial, intravesical, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration.

[0403] The use of chemical modification patterns of nucleic acids confers nuclease stability in serum, making, for example, subcutaneous application routes feasible.

[0404] Solutions or suspensions used for intradermal or subcutaneous application typically contain one or more of the following: sterile diluents, such as water for injection, saline, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and / or tonicity adjusters, such as sodium chloride or dextrose. pH can be adjusted using acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers containing citrates, phosphates, acetates, and the like. Such preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0405] Sterile injectable solutions can be prepared by incorporating nucleic acid in the required amount in a suitable solvent that contains one or a combination of the above-mentioned components, and then optionally performing sterilization filtration.Dispersion can be prepared by incorporating active compound in a sterile vehicle that contains dispersion medium and optionally other components, such as those mentioned above.In the case of sterile powder for preparing sterile injectable solutions, the preparation method is vacuum drying and freeze-drying (lyophilization), which obtains powder of active ingredient in addition to any additional desired ingredients from their sterile filtered solutions.

[0406] When a prophylactically or therapeutically effective amount of the nucleic acid of the present invention is administered, for example, by intravenous, cutaneous, or subcutaneous injection, the nucleic acid will be in the form of a pyrogen-free parenterally acceptable aqueous solution. Methods for preparing parenterally acceptable solutions, taking into account appropriate pH, isotonicity, stability, etc., are within the skill of those of ordinary skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to the nucleic acid, an isotonic vehicle, such as sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection, or other vehicles known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those of ordinary skill in the art.

[0407] The amount of nucleic acid that can be combined with carrier materials to produce a single dosage form will vary depending on a variety of factors, including the subject being treated and the particular mode of administration. Generally, it will be the amount of composition that will produce an appropriate therapeutic effect under the particular circumstances. Generally, out of 100 percent, this amount will range from about 0.01% to about 99% nucleic acid, about 0.1% to about 70%, or about 1% to about 30% nucleic acid in combination with a pharma- ceutically acceptable carrier.

[0408] Nucleic acid can be prepared with carriers that protect compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems.Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Many methods for preparing such formulations are patented or generally known to those skilled in the art.See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0409] The dosage regimen can be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a dose can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased in each case as indicated by the specific circumstances of the therapeutic situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in dosage unit form when administered to a subject or patient. As used herein, dosage unit form refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated, each unit containing a predetermined amount of active compound calculated to provide a desired therapeutic effect. The specifications of the dosage unit form of the present invention depend on the specific characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, as well as the treatment and sensitivity of any individual patient.

[0410] A nucleic acid, or composition of the invention, can be produced using methods conventional in the art, including chemical synthesis, eg, solid phase chemical synthesis.

[0411] The nucleic acid or composition of the present invention can be administered using one or more of various medical devices known in the art.For example, in one embodiment, the nucleic acid of the present invention can be administered using a needleless subcutaneous injection device.Examples of well-known implants and modules useful in the present invention are in the art, including, for example, implantable microinfusion pumps for controlled rate delivery, devices for administration through the skin, infusion pumps for delivery at precise infusion rates, variable flow implantable infusion devices for continuous drug delivery, and osmotic drug delivery systems.These and other such implants, delivery systems, and modules are known to those skilled in the art.

[0412] In certain embodiments, the nucleic acids or compositions of the present invention can be formulated to ensure a desired distribution in vivo. To target the therapeutic compounds or compositions of the present invention to a specific in vivo location, they can be formulated, for example, in liposomes, which can contain one or more moieties that are selectively transported to specific cells or organs, thereby enhancing targeted drug delivery.

[0413] The present invention is characterized by high specificity at the molecular and tissue-directed delivery level. The sequences of the nucleic acids of the present invention are highly specific to their targets, which means that they do not inhibit the expression of genes that are not designed to target, or only inhibit the expression of genes that are not designed to target, and / or inhibit the expression of only a small number of genes that are not designed to target. An additional level of specificity is achieved when the nucleic acid is linked to a ligand that is specifically recognized and internalized by a particular cell type. This is the case, for example, when the nucleic acid is linked to a ligand that contains a GalNAc moiety that is specifically recognized and internalized by hepatocytes. This results in the nucleic acid inhibiting the expression of its target only in the cells that are targeted by the linked ligand. These two levels of specificity are likely to confer a better safety profile than currently available treatments. In certain embodiments, the present invention thus provides the nucleic acid of the present invention linked to a ligand that contains one or more GalNAc moieties, or one or more other moieties that confer cell type or tissue specific internalization of the nucleic acid, thereby conferring additional specificity of target gene knockdown by RNA interference.

[0414] The nucleic acid described herein can be formulated in the form of liposomes with lipids. Such formulations can be described in the art as lipoplexes. The composition with lipids / liposomes can be used to assist in the delivery of the nucleic acid of the present invention to target cells. The lipid delivery system described herein can be used as an alternative to conjugated ligands. The modifications described herein can be present when the nucleic acid of the present invention is used with lipid delivery systems or ligand conjugated delivery systems.

[0415] Such lipoplexes are i) a cationic lipid, or a pharma- ceutically acceptable salt thereof; ii) steroids, iii) phosphatidylethanolamine phospholipids, and / or iv) PEGylated lipids The lipid composition may include a lipid composition comprising:

[0416] The cationic lipid can be an amino cationic lipid.

[0417] The cationic lipid has the formula (XII):

[0418] [ka]

[0419] or a pharma- ceutically acceptable salt thereof, wherein: X represents O, S, or NH; R 1 and R 2 are independently C4 to C 22 Straight or branched alkyl chain, or C4-C with one or more double bonds 22 represents a straight or branched alkenyl chain, the alkyl or alkenyl chain optionally containing an intervening ester, amide, or disulfide; When X represents S or NH, R 3 and R 4each independently represents hydrogen, methyl, ethyl, a mono- or polyamine moiety, or R 3 and R 4 taken together form a heterocyclyl ring, When X represents O, R3 and R4 each independently represent hydrogen, methyl, ethyl, a mono- or polyamine moiety, or R 3 and R 4 taken together form a heterocyclyl ring, or R 3 represents hydrogen, and R 4 represents C(NH)(NH2).

[0420] The cationic lipid has the formula (XIII):

[0421] [ka]

[0422] or a pharma- ceutically acceptable salt thereof.

[0423] The cationic lipid has the formula (XIV):

[0424] [ka]

[0425] or a pharma- ceutically acceptable salt thereof.

[0426] The content of the cationic lipid component may be from about 55 mol% to about 65 mol% of the total lipid content of the composition. In particular, the cationic lipid component is about 59 mol% of the total lipid content of the composition.

[0427] The composition may further comprise a steroid. The steroid may be cholesterol. The steroid content may be about 26 mol% to about 35 mol% of the total lipid content of the lipid composition. More specifically, the steroid content may be about 30 mol% of the total lipid content of the lipid composition.

[0428] Phosphatidylethanolamine phospholipids are 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimethano ... The phospholipid may be selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2-disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE), and 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (SLPE). The phospholipid content may be about 10 mol% of the total lipid content of the composition.

[0429] The PEGylated lipid may be selected from the group consisting of 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG), and C16-ceramide-PEG. The content of the PEGylated lipid may be about 1-5 mol% of the total lipid content of the composition.

[0430] The content of the cationic lipid component in the composition can be about 55 mol % to about 65 mol % of the total lipid content of the lipid composition, preferably about 59 mol % of the total lipid content of the lipid composition.

[0431] The composition may have a molar ratio of components i):ii):iii):iv) selected from 55:34:10:1, 56:33:10:1, 57:32:10:1, 58:31:10:1, 59:30:10:1, 60:29:10:1, 61:28:10:1, 62:27:10:1, 63:26:10:1, 64:25:10:1, and 65:24:10:1.

[0432] The composition has the structure

[0433] [ka]

[0434] A cationic lipid having the formula: structure

[0435] [ka]

[0436] steroids having the formula structure

[0437] [ka]

[0438] phosphatidylethanolamine phospholipid having and structure

[0439] [ka]

[0440] PEGylated lipids having may include.

[0441] Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions can be formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes can be formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition can be formed from phosphatidylcholine (PC), such as soybean PC and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0442] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of the plasma membrane of tissue culture cells. DOTMA analogs can also be used to form liposomes.

[0443] Derivatives and analogs of the lipids described herein can also be used to form liposomes.

[0444] Liposomes containing nucleic acid can be prepared by various methods. In one example, the lipid components of the liposome are dissolved in a detergent, so that micelles with the lipid components are formed. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholic acid, CHAPS, octylglucoside, deoxycholic acid, and lauroyl sarcosine. The nucleic acid preparation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the nucleic acid and aggregate around the nucleic acid to form liposomes. After aggregation, the detergent is removed, for example, by dialysis, to obtain a liposome preparation of nucleic acid.

[0445] If necessary, a carrier compound that aids in aggregation may be added during the aggregation reaction, for example, by controlled addition. For example, the carrier compound may be a polymer other than a nucleic acid, such as spermine or spermidine. The pH may also be adjusted to aid in aggregation.

[0446] The nucleic acid formulation of the present invention may include a surfactant. In one embodiment, the nucleic acid is formulated as an emulsion that includes a surfactant.

[0447] Surfactants that are not ionized are nonionic surfactants. Examples include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, etc., nonionic alkanolamides, and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block copolymers.

[0448] Surfactants that have a negative charge when dissolved or dispersed in water are anionic surfactants. Examples include carboxylates, such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid, such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates, such as alkyl benzene sulfonates, acyl isethionates, acyltaurates, and sulfosuccinates, and phosphates.

[0449] Surfactants that have a positive charge when dissolved or dispersed in water are cationic surfactants. Examples include quaternary ammonium salts and ethoxylated amines.

[0450] Surfactants capable of carrying either a positive or negative charge are amphoteric surfactants. Examples include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0451] A "micelle" is defined herein as a specific type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all the hydrophobic parts of the molecule face inward and the hydrophilic parts remain in contact with the surrounding aqueous phase. The reverse orientation exists when the environment is hydrophobic. Micelles can be formed by mixing an aqueous solution of nucleic acid with an alkali metal alkyl sulfate and at least one micelle-forming compound.

[0452] Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharma- ceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleic acid, monolauric acid, borage oil, evening primrose oil, menthol, trihydroxyoxocholanylglycine and pharma-ceutically acceptable salts thereof, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ethers and analogs thereof, polidocanol alkyl ethers and analogs thereof, chenodeoxycholic acid, deoxycholic acid, and mixtures thereof.

[0453] Phenol and / or m-cresol may be added to the mixed micellar composition to act as a stabilizer and preservative. An isotonicity agent, such as glycerin, may be added as well.

[0454] The nucleic acid preparation may be incorporated into particles, such as microparticles, which may be produced by spray drying, freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these methods.

[0455] definition As used herein, the terms "inhibit", "downregulate" or "reduce" in reference to gene expression means that the expression of a gene or the level of an RNA molecule or equivalent RNA molecule encoding one or more proteins or protein subunits (e.g., mRNA), or the activity of one or more proteins or protein subunits is reduced below that observed in the absence of the nucleic acid or conjugated nucleic acid of the present invention, or compared to that obtained with an siRNA molecule that has no known homology to the human transcript (referred to herein as a non-silencing control). Such controls can be conjugated and modified in a similar manner to the molecules of the present invention and delivered to target cells by the same route. Expression after treatment with the nucleic acid of the present invention can be reduced to 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or 0% of that observed in the absence of the nucleic acid or conjugated nucleic acid, or to intermediate values ​​therebetween or less. Expression can be measured in cells to which the nucleic acid is applied. Alternatively, especially when the nucleic acid is administered to a subject, the levels can be measured in different cell populations, or tissues or organs, or body fluids, such as blood or plasma. The level of inhibition is preferably measured under selected conditions, since they show the highest effect of the nucleic acid on target mRNA levels in cells treated with the nucleic acid in vitro. The level of inhibition can be measured, for example, 24 hours or 48 hours after treatment with the nucleic acid at a concentration between 0.038 nM and 10 mM, preferably 0.5 nM, 1 nM, 10 nM, or 100 nM. These conditions can be different for different nucleic acid sequences or different types of nucleic acid, for example unmodified or modified, or conjugated or not to a ligand. Examples of suitable conditions for determining the level of inhibition are described in the examples.

[0456] Nucleic acid refers to a nucleic acid that comprises two strands that contain nucleotides and can interfere with gene expression. Inhibition can be complete or partial, and can result in downregulation of gene expression in a targeted manner. Nucleic acid comprises two separate polynucleotide strands, a first strand that can be a guide strand and a second strand that can be a passenger strand. The first strand and the second strand can be part of the same polynucleotide molecule, which is self-complementary and "folds" to form a double-stranded molecule. Nucleic acid can be an siRNA molecule.

[0457] A nucleic acid may include ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogs, non-nucleotides that can mimic nucleotides so that they can "pair" with corresponding bases in a target sequence or complementary strand. A nucleic acid may also include a double-stranded nucleic acid portion or duplex region formed by all or a portion of a first strand (also known in the art as a guide strand) and all or a portion of a second strand (also known in the art as a passenger strand). A duplex region is defined as beginning at the first base pair formed between the first strand and the second strand and ending at the last base pair formed between the first strand and the second strand, including the endpoints.

[0458] Duplex region refers to the region in two complementary or substantially complementary oligonucleotides that base pair with each other by either Watson-Crick base pairing or any other manner that allows duplexing between complementary or substantially complementary oligonucleotide strands. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, but only 19 nucleotides in each strand are complementary or substantially complementary, so the "duplex region" consists of 19 base pairs. The remaining base pairs can be present as 5' and 3' overhangs or as single-stranded regions. Furthermore, 100% complementarity is not required within the duplex region, and substantial complementarity is allowed within the duplex region. Substantial complementarity refers to the complementarity between strands such that they can anneal under biological conditions. Techniques for experimentally determining whether two strands can anneal under biological conditions are well known in the art. Alternatively, the two strands may be synthesized and added together under biological conditions to determine whether they anneal to each other. The portions of the first strand and the second strand that form at least one double-stranded region may be fully complementary or at least partially complementary to each other. Depending on the length of the nucleic acid, a perfect match in terms of base complementarity between the first strand and the second strand is not necessarily required. However, the first and second strands must be able to hybridize under physiological conditions.

[0459] As used herein, the term "non-base paired nucleotide analog" refers to a nucleotide analog that contains a non-base paired portion, including, but not limited to, 6-desaminoadenosine (nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me ribo T, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, and N3-Me dC. In some embodiments, the non-base paired nucleotide analog is a ribonucleotide. In other embodiments, it is a deoxyribonucleotide.

[0460] As used herein, the term "terminal functional group" includes, without limitation, halogen, alcohol, amine, carboxylic acid, ester, amide, aldehyde, ketone, and ether groups.

[0461] "Overhang" as used herein has its usual meaning in the art, i.e., a single-stranded portion of a nucleic acid that extends beyond the terminal nucleotide of the complementary strand in a double-stranded nucleic acid. The term "blunt end" includes double-stranded nucleic acids whereby both strands terminate at the same position, regardless of whether the terminal nucleotides are base-paired or not. The terminal nucleotides of the first and second strands at the blunt end may be base-paired. The terminal nucleotides of the first and second strands at the blunt end may be unpaired. The two terminal nucleotides of the first and second strands at the blunt end may be base-paired. The two terminal nucleotides of the first and second strands at the blunt end may be unpaired.

[0462] The term "serinol derived linker moiety" means that the linker moiety comprises the following structure:

[0463] [ka]

[0464] The O atom of the structure typically links to the RNA strand, and the N atom typically links to the targeting ligand.

[0465] The terms "patient," "subject," and "individual" may be used interchangeably and refer to either a human or non-human animal. These terms include mammals, such as humans, primates, livestock animals (e.g., cows, pigs), pet animals (e.g., dogs, cats), and rodents (e.g., mice and rats).

[0466] As used herein, the term "treating" or "treatment" and grammatical variations thereof refer to an approach to obtain beneficial or desired clinical results. The term may refer to slowing the onset or development of a condition, disorder, or disease, reducing or alleviating symptoms associated therewith, causing complete or partial regression of the condition, or any combination of any of the above. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, whether detectable or undetectable, attenuation of the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or relief of the disease state, and remission (whether partial or total). "Treatment" may also mean prolonging survival compared to the expected survival in the absence of treatment. A subject (e.g., a human) in need of treatment may therefore be a subject already suffering from the disease or disorder in question. The term "treatment" includes inhibition or reduction of an increase in the severity of a pathological state or symptom compared to the absence of treatment, and is not necessarily meant to indicate a complete cessation of the associated disease, disorder, or condition.

[0467] As used herein, the term "prevention" and its grammatical variants refer to an approach to inhibit or prevent the onset, progression, or time or rate of onset of a condition, disease, or disorder, which may be associated with a pathology and / or symptoms. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, prevention, inhibition, or slowing of disease symptoms, progression, or onset, whether detectable or undetectable. A subject (e.g., a human) in need of prevention may therefore be a subject who is not yet afflicted with the disease or disorder in question. The term "prevention" includes slowing the onset of a disease compared to the absence of treatment, and is not necessarily meant to indicate a permanent prevention of the associated disease, disorder, or condition. Thus, "prevention" of a condition refers in certain contexts to reducing the risk of the onset of the condition, or preventing, inhibiting, or delaying the onset of symptoms associated with the condition. It will be understood that prevention may be considered as treatment or therapy.

[0468] As used herein, an "effective amount," "prophylactically effective amount," "therapeutically effective amount," or "effective dose" is an amount of a composition (e.g., a therapeutic composition or therapeutic agent) that provides at least one desired therapeutic effect in a subject, e.g., preventing or treating a targeted condition, or beneficially alleviating a symptom associated with a condition.

[0469] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is not harmful to the patient or subject to whom the salt in question is administered. It may be, for example, a salt selected from acid addition salts and base salts. Examples of acid addition salts include chloride salts, citrate salts, and acetate salts. Examples of base salts include salts in which the cation is an alkali metal cation, such as sodium or potassium ion, an alkaline earth metal cation, such as calcium or magnesium ion, and a substituted ammonium ion, such as N(R 1 )(R 2 )(R 3 )(R 4 ) + Ions of the type (wherein R 1, R 2 , R 3 , and R 4 are independently typically selected from hydrogen, an optionally substituted C1-6-alkyl group, or an optionally substituted C2-6-alkenyl group. Examples of relevant C1-6-alkyl groups include methyl, ethyl, 1-propyl, and 2-propyl groups. Examples of potentially relevant C2-6-alkenyl groups include ethenyl, 1-propenyl, and 2-propenyl. Other examples of pharma- ceutically acceptable salts are described in "Remington's Pharmaceutical Sciences", 17th ed., Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and newer editions thereof), "Encyclopaedia of Pharmaceutical Technology", 3rd ed., James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and J. Pharm. Sci. 66: 2 (1977). A "pharmaceutically acceptable salt" qualitatively retains the desired biological activity of the parent compound without imparting any undesirable effects to the compound. Examples of pharmaceutically acceptable salts include acid addition salts and base addition salts. Acid addition salts include salts derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphorous acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, etc., or from non-toxic organic acids, such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, etc. Base addition salts include salts derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium, etc., and from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, etc.

[0470] The term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985). For example, sterile saline and phosphate buffered saline at slightly acidic or physiological pH can be used. Exemplary pH buffering agents include phosphoric acid, citric acid, acetate, tris / hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, the preferred buffers histidine, arginine, lysine, or acetate, or mixtures thereof. The term further encompasses any agent listed in the United States Pharmacopoeia for use in animals, including humans. "Pharmaceutical acceptable carrier" includes any and all physiologically acceptable, i.e., compatible, solvents, dispersion media, coatings, antimicrobial agents, isotonic agents, and absorption delaying agents, and the like. In certain embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration selected, the nucleic acid may be coated with a material or materials intended to protect the compound from the action of acids and other natural inactivating conditions to which the nucleic acid may be exposed when administered to a subject by a particular route of administration.

[0471] The term "solvate" in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (in this case, a nucleic acid compound according to the present invention or its pharma- ceutically acceptable salt) and a solvent. In this context, the solvent can be, for example, water or another pharma- ceutically acceptable, typically small molecule organic species, for example, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such a solvate is usually referred to as a hydrate.

[0472] The invention will now be described with reference to the following non-limiting figures and examples. [Brief description of the drawings]

[0473] [Figure 1] Figure 1 shows the relative MASP-2 mRNA expression in primary cynomolgus hepatocytes after treatment with GalNAc-siRNA conjugates targeting MASP-2 at five different concentrations, 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM, normalized to PPIB mRNA. Mean values ​​compared to untreated (ut). Cells treated with MASP-2 non-specific siRNA were used as control (Ctr). [Diagram 2] Figure 1 shows the relative MASP-2 mRNA expression in primary cynomolgus hepatocytes after treatment with GalNAc-siRNA conjugates targeting MASP-2 at five different concentrations, 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM, normalized to PPIB mRNA. Mean values ​​compared to untreated (ut). Cells treated with MASP-2 non-specific siRNA were used as control (Ctr). [Diagram 3] Figure 1 shows the relative MASP-2 mRNA expression in primary human hepatocytes after treatment with GalNAc-siRNA conjugates targeting MASP-2 at five different concentrations, 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM, normalized to PPIB mRNA. Mean values ​​compared to untreated (ut). Cells treated with MASP-2 non-specific siRNA were used as control (Ctr). [Figure 4] Figure 1 shows the relative MASP-2 mRNA expression in primary human hepatocytes after treatment with GalNAc-siRNA conjugates targeting MASP-2 at five different concentrations, 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM, normalized to PPIB mRNA. Mean values ​​compared to untreated (ut). Cells treated with MASP-2 non-specific siRNA were used as control (Ctr). [Diagram 5]Figure 1 shows the relative MASP-2 mRNA expression in primary monkey hepatocytes after treatment with GalNAc-siRNA conjugates targeting MASP-2 at five different concentrations, 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM, normalized to PPIB mRNA. Mean values ​​compared to untreated (ut). Cells treated with MASP-2 non-specific siRNA were used as control (Ctr). [Figure 6] This figure shows the relative MASP-2 mRNA expression in liver samples isolated on day 15 after subcutaneous injection of GalNAc-siRNA conjugates targeting MASP-2 at two concentrations, 1 or 5 mg / kg, normalized to APOB mRNA. Mean values ​​compared to PBS-treated controls. [Figure 7] Figure 1 shows the relative MASP-2 mRNA expression in primary cynomolgus hepatocytes after treatment with GalNAc-siRNA conjugates targeting MASP-2 at five different concentrations, 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM, normalized to PPIB mRNA. Mean values ​​compared to untreated (ut). Cells treated with MASP-2 non-specific siRNA were used as control (Ctr). [Figure 8] Figure 1 shows the relative MASP-2 mRNA expression in primary human hepatocytes after treatment with GalNAc-siRNA conjugates targeting MASP-2 at five different concentrations, 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM, normalized to PPIB mRNA. Mean values ​​compared to untreated (ut). Cells treated with MASP-2 non-specific siRNA were used as control (Ctr). [Figure 9]Figure 1 shows relative MASP-2 mRNA expression in primary monkey hepatocytes after treatment with GalNAc-siRNA conjugates targeting Masp-2 at five different concentrations, 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM, normalized to PPIB mRNA. Mean values ​​compared to untreated (ut). Cells treated with MASP-2 non-specific siRNA were used as control (Ctr). [Figure 10] This figure shows the change in MASP-2 protein levels in cynomolgus monkey serum isolated on days 2, 3, 8, 15, 22, 29, and 57 after a single subcutaneous injection of vehicle or 3 mg / kg GalNAc-conjugated siRNA EM1208, EM1209, or EM1211, normalized to pre-dosing values. Mean and SEM are shown. [Figure 11] This figure shows the change in MASP-2 protein levels in cynomolgus monkey serum isolated on days 3, 8, 15, 22, 29, 43, 57, 71, and 85 after a single subcutaneous injection of vehicle or 0.3, 1, 3, or 10 mg / kg GalNAc-conjugated siRNA EM1208, normalized to pre-dosing values. Mean and SEM are shown. EXAMPLES

[0474] Example 1 In vitro studies in HepG2 cells showing the MASP-2 knockdown efficacy of the tested siRNAs following transfection of 0.5 nM or 10 nM siRNA. The MASP-2 knockdown efficacy of siRNAs EM1001-EM1192 (Table A (Table 3) and Table 5b (Table 9)) was determined after transfection of 0.5 or 10 nM of siRNA into HepG2 cells. The results are shown in Table A (Table 3) below. At 10 nM, residual MASP-2 levels after knockdown reached a minimum of 42%, and at 0.5 nM, residual MASP-2 levels reached a minimum of 63%. At 10 nM, the most potent siRNAs were EM1172, EM1128, EM1137, EM1189, and EM1192.

[0475] For transfection of siRNA into HepG2 cells, cells were seeded in 96-well tissue culture plates (TPP, Cat. 92096, Switzerland) at a density of 15,000 cells / well. Transfection of siRNA was performed immediately before seeding using Lipofectamine RNAiMax (Invitrogen / Life Technologies, Cat. 13778-500, Germany) according to the manufacturer's instructions. A double dose screen was performed in triplicate with 10 nM and 0.5 nM MASP-2 siRNA, respectively, with scrambled siRNA and luciferase-targeting siRNA as non-specific controls. After 24 h incubation with siRNA, the medium was removed and the cells were lysed in 250 μl lysis buffer (InviTrap RNA Cell HTS96 Kit / C (Stratec, Cat. 7061300400, Germany)) and then frozen at -80°C. RNA was isolated using the InviTrap RNA Cell HTS96 Kit / C (Stratec, Cat. No. 7061300400, Germany). RT-qPCR was performed in a singleplex 384-well format using MASP-2 and HPRT specific primer probe sets and Takyon™ One-Step Low Rox Probe 5X MasterMix dTTP in a QuantStudio6 device from Applied Biosystems. Expression differences were calculated using the delta delta Ct method, and the relative expression of MASP-2 normalized to the housekeeping gene HPRT was determined. The results are presented in Table A as % remaining MASP-2 mRNA after siRNA transfection.

[0476] [Table 3A]

[0477] [Table 3B]

[0478] [Table 3C]

[0479] [Table 3D]

[0480] [Table 3E]

[0481] [Table 3F]

[0482] [Table 3G]

[0483] Example 2 In vitro studies in HepG2 cells showing the MASP-2 knockdown efficacy of the tested siRNAs following transfection of 5 nM or 10 nM siRNA. The MASP-2 knockdown efficacy of siRNAs EM1001 to EM1192 (Table B (Table 4) and Table 5b (Table 9)) was determined after transfection of 5 or 10 nM of siRNA into HepG2 cells. The results are shown in Table B (Table 4) below. At 10 nM, residual MASP-2 levels after knockdown reached a minimum of 42%, and at 5 nM, residual MASP-2 levels reached a minimum of 45%. At 10 nM, the most potent siRNAs were EM1172, EM1128, EM1137, EM1089, and EM1192.

[0484] For transfection of siRNA into HepG2 cells, cells were seeded in 96-well tissue culture plates (TPP, Cat. 92096, Switzerland) at a density of 40,000 cells / well. Transfection of siRNA was performed immediately before seeding using Lipofectamine RNAiMax (Invitrogen / Life Technologies, Cat. 13778-500, Germany) according to the manufacturer's instructions. A dual dose screen was performed in triplicate with MASP-2-siRNA at 10 nM and 5 nM, respectively, with scrambled siRNA and luciferase-targeting siRNA as non-specific controls. After 24 h of incubation with siRNA, the medium was removed and the cells were lysed in 250 μl of lysis buffer (InviTrap RNA Cell HTS96 Kit / C (Stratec, Cat. 7061300400, Germany)) and then frozen at -80°C. RNA was isolated using the InviTrap RNA Cell HTS96 Kit / C (Stratec, Cat. No. 7061300400, Germany). RT-qPCR was performed in a singleplex 384-well format using MASP-2 and HPRT specific primer probe sets and Takyon™ One-Step Low Rox Probe 5X MasterMix dTTP in a QuantStudio6 device from Applied Biosystems. Expression differences were calculated using the delta delta Ct method, and the relative expression of MASP-2 normalized to the housekeeping gene HPRT was determined. The results are presented in Table B as % remaining MASP-2 mRNA after siRNA transfection.

[0485] [Table 4A]

[0486] [Table 4B]

[0487] [Table 4C]

[0488] [Table 4D]

[0489] [Table 4E]

[0490] [Table 4F]

[0491] [Table 4G]

[0492] Example 3 In vitro studies in HepG2 cells showing MASP-2 knockdown efficacy of the tested siRNAs following transfection of 20, 4, 0.8, or 0.16 nM siRNA. The MASP-2 knockdown efficacy of selected siRNAs (Table 3 and Table 5b) was determined after transfection of 20, 4, 0.8, or 0.16 nM of siRNA into HepG2 cells. The results are shown in Table 3 below. At 20 nM, the remaining MASP-2 levels after knockdown reached a minimum of 22% and at 4 nM a minimum of 32%. At 20 nM, the most potent siRNAs were EM1192, EM1074, EM1119, EM1089, and EM1155.

[0493] For siRNA transfection into HepG2 cells, cells were seeded in 96-well tissue culture plates (TPP, Catalog 92096, Switzerland) at a density of 40,000 cells / well. siRNA transfection was performed immediately before seeding using Lipofectamine RNAiMax (Invitrogen / Life Technologies, Catalog 13778-500, Germany) according to the manufacturer's instructions. Dose-response screening was performed in triplicate with 20, 4, 0.8, or 0.16 nM MASP-2 siRNA, respectively, with scrambled siRNA and luciferase-targeting siRNA as nonspecific controls. After 24 hours of incubation with siRNA, the medium was removed and the cells were lysed in 250 μl of lysis buffer (InviTrap RNA Cell HTS96 Kit / C (Stratec, Cat. No. 7061300400, Germany)) and then frozen at -80°C. RNA was isolated using InviTrap RNA Cell HTS96 Kit / C (Stratec, Cat. No. 7061300400, Germany). RT-qPCR was performed in singleplex 384-well format using MASP-2 and HPRT specific primer probe sets and Takyon™ One-Step Low Rox Probe 5X MasterMix dTTP on a QuantStudio6 device from Applied Biosystems. Expression differences were calculated using the delta delta Ct method and the relative expression of MASP-2 normalized to the housekeeping gene HPRT was determined. The results are presented in Table 3 as % remaining MASP-2 mRNA after siRNA transfection.

[0494] [Table 5A]

[0495] [Table 5B]

[0496] [Table 5C]

[0497] [Table 5D]

[0498] Example 4 In vitro study in primary cynomolgus monkey hepatocytes showing the MASP-2 knockdown efficacy of the GalNAc-siRNA conjugates tested. MASP-2 mRNA expression was assessed after incubation with 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM GalNAc-siRNA conjugates EM1193, EM1194, EM1195, EM1196, EM1197, EM1198, EM1199, EM1200, EM1201, EM1202 (further described in Table 5c). The mRNA levels of the housekeeping gene PPIB served as a control.

[0499] To test the knockdown efficacy of GalNAc-conjugated siRNA targeting MASP-2 in primary cynomolgus hepatocytes, 45,000 cells per well (supplier: Life Technologies) were added to siRNA in seeding medium (Life Technologies) at final concentrations of 100 nM to 0.16 nM in collagen-coated 96-well plates (Life Technologies). 24 hours after treatment, cells were lysed using an InviTrap RNA Cell HTS96 Kit / C (Stratec). RT-qPCR was performed using mRNA-specific primers and probes for MASP-2 and PPIB. Differences in expression were calculated using the delta-delta Ct method, and the relative expression of MASP-2 normalized to the housekeeping gene PPIB was determined. Results are expressed as the ratio of MASP-2 to PPIB mRNA compared to untreated levels and can be seen in Figure 1.

[0500] Dose-dependent knockdown of MASP-2 mRNA was observed for the eight GalNAc conjugates tested. At 100 nM, remaining MASP-2 levels after knockdown ranged from 11% to 86%. The most potent knockdown was observed with 100 nM EM1201, EM1202, EM1200, and EM1198, with remaining MASP-2 levels of 11%, 12%, 17%, and 18%, respectively.

[0501] Example 5 In vitro study in primary cynomolgus monkey hepatocytes showing the MASP-2 knockdown efficacy of the GalNAc-siRNA conjugates tested. MASP-2 mRNA expression was assessed after incubation with 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM GalNAc-siRNA conjugates EM1193, EM1194, EM1195, EM1196, EM1197, EM1198, EM1199, EM1200, EM1201, EM1202 (further described in Table 5c). The mRNA levels of the housekeeping gene PPIB served as a control.

[0502] To test the knockdown efficacy of GalNAc-conjugated siRNA targeting MASP-2 in primary cynomolgus hepatocytes, 45,000 cells per well (supplier: Life Technologies) were added to siRNA in seeding medium (Life Technologies) at final concentrations of 100 nM to 0.16 nM in collagen-coated 96-well plates (Life Technologies). 24 hours after treatment, cells were lysed using an InviTrap RNA Cell HTS96 Kit / C (Stratec). RT-qPCR was performed using mRNA-specific primers and probes for MASP-2 and PPIB. Differences in expression were calculated using the delta-delta Ct method, and the relative expression of MASP-2 normalized to the housekeeping gene PPIB was determined. Results are expressed as the ratio of MASP-2 to PPIB mRNA compared to untreated levels and can be seen in Figure 2.

[0503] Dose-dependent knockdown of MASP-2 mRNA was observed for the eight GalNAc conjugates tested. At 100 nM, remaining MASP-2 levels after knockdown ranged from 17% to 80%. The most potent knockdown was observed with 100 nM EM1201, EM1202, EM1200, and EM1198, with remaining MASP-2 levels of 17%, 21%, 21%, and 24%, respectively.

[0504] Example 6 In vitro study in primary human hepatocytes showing the MASP-2 knockdown efficacy of the GalNAc-siRNA conjugates tested. MASP-2 mRNA expression was assessed after incubation with 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM GalNAc-siRNA conjugates EM1193, EM1194, EM1195, EM1196, EM1197, EM1198, EM1199, EM1200, EM1201, EM1202 (further described in Table 5c). The mRNA levels of the housekeeping gene PPIB served as a control.

[0505] To test the knockdown efficacy of GalNAc-conjugated siRNA targeting MASP-2 in primary human hepatocytes, 35,000 cells per well (supplier: Life technologies) were added to siRNA in seeding medium (Life Technologies) at final concentrations of 100 nM to 0.16 nM in collagen-coated 96-well plates (Life technologies). 24 hours after treatment, cells were lysed using the InviTrap RNA Cell HTS96 Kit / C (Stratec). RT-qPCR was performed using mRNA-specific primers and probes for MASP-2 and PPIB. Differences in expression were calculated using the delta-delta Ct method, and the relative expression of MASP-2 was normalized to the expression of the housekeeping gene PPIB. Results are expressed as the ratio of MASP-2 to PPIB mRNA compared to untreated levels and can be seen in Figure 3.

[0506] Dose-dependent knockdown of MASP-2 mRNA was observed for the five GalNAc conjugates tested. At 100 nM, remaining MASP-2 levels after knockdown ranged from 34% to 99%. The most potent knockdown was observed with 100 nM EM1202, EM1201, EM1198, and EM1200, with remaining MASP-2 levels of 40%, 42%, 45%, and 47%, respectively.

[0507] Example 7 In vitro study in primary human hepatocytes showing the MASP-2 knockdown efficacy of the GalNAc-siRNA conjugates tested. MASP-2 mRNA expression was assessed after incubation with 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM GalNAc-siRNA conjugates EM1193, EM1194, EM1195, EM1196, EM1197, EM1198, EM1199, EM1200, EM1201, EM1202 (further described in Table 5c). The mRNA levels of the housekeeping gene PPIB served as a control.

[0508] To test the knockdown efficacy of GalNAc-conjugated siRNA targeting MASP-2 in primary human hepatocytes, 35,000 cells per well (supplier: Life technologies) were added to siRNA in seeding medium (Life Technologies) at final concentrations of 100 nM to 0.16 nM in collagen-coated 96-well plates (Life technologies). 24 hours after treatment, cells were lysed using the InviTrap RNA Cell HTS96 Kit / C (Stratec). RT-qPCR was performed using mRNA-specific primers and probes for MASP-2 and PPIB. Differences in expression were calculated using the delta-delta Ct method, and the relative expression of MASP-2 was normalized to the expression of the housekeeping gene PPIB. Results are expressed as the ratio of MASP-2 to PPIB mRNA compared to untreated levels and can be seen in Figure 4.

[0509] Dose-dependent knockdown of MASP-2 mRNA was observed for the five GalNAc conjugates tested. At 100 nM, remaining MASP-2 levels after knockdown ranged from 37% to 88%. The most potent knockdown was observed with 100 nM EM1201, EM1195, and EM1198, with remaining MASP-2 levels of 37%, 37%, and 48%, respectively.

[0510] Example 8 In vitro study in primary mouse hepatocytes showing the MASP-2 knockdown efficacy of the GalNAc-siRNA conjugates tested. MASP-2 mRNA expression was assessed after incubation with 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM of GalNAc-siRNA conjugates EM1193, EM1194, EM1195 (further described in Table 5c). The mRNA levels of the housekeeping gene PPIB served as a control.

[0511] To test the knockdown efficacy of GalNAc-conjugated siRNA against MASP-2 in primary mouse hepatocytes, 25,000 cells per well (supplier: Life Technologies) were added to siRNA in seeding medium (Life Technologies) at final concentrations of 100 nM to 0.01 nM in collagen-coated 96-well plates (Life technologies). 24 hours after treatment, cells were lysed using the InviTrap RNA Cell HTS96 Kit / C (Stratec). qPCR was performed using mRNA-specific primers and probes for MASP-2 and PPIB. Differences in expression were calculated using the delta-delta Ct method, and the relative expression of MASP-2 was normalized to the expression of the housekeeping gene PPIB. Results are expressed as the ratio of MASP-2 to PPIB mRNA compared to untreated levels and can be seen in Figure 5.

[0512] Dose-dependent knockdown of MASP-2 mRNA was observed for all GalNAc conjugates tested. At 100 nM, remaining MASP-2 levels after knockdown ranged from 23% to 39%. The most potent knockdown was observed with 100 nM EM1195 and EM1194, with remaining MASP-2 levels of 23% and 25%, respectively.

[0513] Example 9 In vivo study showing knockdown of MASP-2 mRNA in mouse liver tissue following a single subcutaneous dose of 1 or 5 mg / kg GalNAc-conjugated siRNA at 2 weeks post-dosing. GalNAc-siRNA conjugates EM1193, EM1194, EM1195 (further described in Table 5c) were tested. The mRNA levels of the housekeeping gene APOB served as a housekeeping control.

[0514] Eight-week-old male C57BL / 6 mice were obtained from Janvier, France. Animal experiments were performed in accordance with the ethical guidelines of the July 2013 version of the German Protection of Animals Act. Mice were randomized into groups of 4-5 according to body weight. On day 1 of the study, animals received a single subcutaneous dose of 1 or 5 mg / kg siRNA dissolved in phosphate-buffered saline (PBS) or PBS alone as a control. Mice viability, body weight, and behavior were monitored throughout the study and there were no pathological findings.

[0515] On day 15, the study was terminated, animals were euthanized, and liver samples were flash frozen and stored at -80°C until further analysis. Briefly, for analysis, total RNA was prepared using the RNeasy Fibrous Tissue Mini kit (QIAGEN, Venlo, Netherlands) according to the manufacturer's instructions. To assess the integrity of the isolated RNA, automated electrophoresis was performed using a 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, USA). 100 ng of total RNA per reaction was used for RT-qPCR using amplicon sets specific for mouse MASP-2 and APOB.

[0516] Differences in expression were calculated using the delta-delta Ct method, and the relative expression of MASP-2 compared to the housekeeping gene APOB normalized to PBS was used to compare the different siRNAs. All GalNAc conjugates tested induced a dose-dependent knockdown of MASP-2 mRNA in the liver. At 5 mg / kg, the remaining MASP-2 levels after knockdown ranged from 28% to 61%. The maximal knockdown achieved of 72% was observed using EM1195 with 5 mg / kg siRNA (Figure 6).

[0517] Example 10 In vitro study in primary cynomolgus monkey hepatocytes showing the MASP-2 knockdown efficacy of the GalNAc-siRNA conjugates tested. MASP-2 mRNA expression was assessed after incubation with 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM of siRNA conjugates EM1203, EM1204, EM1205, EM1206, EM1207, EM1208, EM1209, EM1210, EM1211, EM1212, EM1213 (further described in Table 5c). The mRNA levels of the housekeeping gene PPIB served as a control.

[0518] To test the knockdown efficacy of GalNac-conjugated siRNA targeting MASP-2 in primary cynomolgus hepatocytes, 45,000 cells per well (suppliers: Life Technologies and Primacyt) were seeded in collagen-coated 96-well plates (Life Technologies). siRNA at concentrations between 100 nM and 0.16 nM was added immediately after seeding. 24 hours after treatment, cells were lysed using the InviTrap RNA Cell HTS96 Kit / C (Stratec). RT-qPCR was performed using mRNA-specific primers and probes for MASP-2 and PPIB. Differences in expression were calculated using the delta-delta Ct method, and the relative expression of MASP-2 normalized to the housekeeping gene PPIB was determined. Results are expressed as the ratio of MASP-2 to PPIB mRNA compared to untreated levels and can be seen in Figure 7.

[0519] Dose-dependent knockdown of MASP-2 mRNA was observed for the eight GalNAc conjugates tested. At 100 nM, remaining MASP-2 levels after knockdown ranged from 8% to 96%. The most potent knockdown was observed with 100 nM EM1211, EM1208, EM1209, and EM1212, with remaining MASP-2 levels of 8%, 11%, 12%, and 16%, respectively.

[0520] Example 11 In vitro study in primary human hepatocytes showing the MASP-2 knockdown efficacy of the GalNAc-siRNA conjugates tested. MASP-2 mRNA expression was assessed following incubation with 100 nM, 20 nM, 4 nM, 0.8 nM, and 0.16 nM GalNAc siRNA conjugates EM1203, EM1204, EM1205, EM1206, EM1207, EM1208, EM1209, EM1210, EM1211, EM1212, EM1213 (further described in Table 5c). mRNA levels of the housekeeping gene PPIB served as a control.

[0521] To test the knockdown efficacy of GalNAc-conjugated siRNA targeting MASP-2 in primary human hepatocytes, 35,000 cells per well (supplier: Life technologies) were seeded in collagen-coated 96-well plates (Life technologies). siRNA at concentrations between 100 nM and 0.16 nM was added immediately after seeding. 24 hours after treatment, cells were lysed using the InviTrap RNA Cell HTS96 Kit / C (Stratec). RT-qPCR was performed using mRNA-specific primers and probes for MASP-2 and PPIB. Differences in expression were calculated using the delta-delta Ct method, and the relative expression of MASP-2 was normalized to the expression of the housekeeping gene PPIB. Results are expressed as the ratio of MASP-2 to PPIB mRNA compared to untreated levels and can be seen in Figure 8.

[0522] Dose-dependent knockdown of MASP-2 mRNA was observed for all GalNAc conjugates tested. At 100 nM, remaining MASP-2 levels after knockdown ranged from 10% to 86%. The most potent knockdown was observed with 100 nM EM1211, EM1208, and EM1209, with remaining MASP-2 levels of 10%, 22%, and 31%, respectively.

[0523] Example 12 In vitro study in primary mouse hepatocytes showing the MASP-2 knockdown efficacy of the GalNAc-siRNA conjugates tested. MASP-2 mRNA expression was assessed following incubation with 100 nM, 10 nM, 1 nM, 0.1 nM, and 0.01 nM of GalNAc-siRNA conjugates EM1203, EM1204, and EM1205 (further described in Table 5c). The mRNA levels of the housekeeping gene PPIB served as a control.

[0524] To test the knockdown efficacy of GalNAc-conjugated siRNA targeting MASP-2 in primary mouse hepatocytes, 25,000 cells per well (suppliers: Life Technologies and Primacyt) were seeded in collagen-coated 96-well plates (Life Technologies). siRNA at concentrations between 100 nM and 0.01 nM was added immediately after seeding. 24 hours after treatment, cells were lysed using the InviTrap RNA Cell HTS96 Kit / C (Stratec). qPCR was performed using mRNA-specific primers and probes for MASP-2 and PPIB. Differences in expression were calculated using the delta-delta Ct method, and the relative expression of MASP-2 was normalized to the expression of the housekeeping gene Ppib. Results are expressed as the ratio of MASP-2 to PPIB mRNA compared to untreated levels and can be seen in Figure 9.

[0525] Dose-dependent knockdown of MASP-2 mRNA was observed for all GalNAc conjugates tested. At 100 nM, remaining MASP-2 levels after knockdown ranged from 19% to 38%. The most potent knockdown was observed with 100 nM EM1204 and EM1205, with remaining MASP-2 levels of 19% and 22%, respectively.

[0526] Example 13 The synthesis of (vp)-mU-phos (Table 4) was carried out as described in Prakash, Nucleic Acids Res. 2015, 43(6), 2993-3011 and Haraszti, Nucleic Acids Res. 2017, 45(13), 7581-7592. The synthesis of phosphoramidite derivatives of ST41 (ST41-phos) (Table 4) and ST23 (ST23-phos) (Table 4) can be carried out as described in WO2017 / 174657. The synthesis of phosphorothioamidites was carried out as described in Caruthers, J. Org. Chem. 1996, 61, 4272-4281.

[0527] Example 14 Exemplary compounds were synthesized according to the methods described below and known to those skilled in the art: The assembly of the oligonucleotide chains and linker building blocks was carried out by solid phase synthesis applying the phosphoramidite technique.

[0528] Downstream cleavage, deprotection, and purification were performed according to standard procedures well known in the art.

[0529] Oligonucleotide synthesis was carried out on an AKTA oligopilot 10 using commercially available 2'O-methyl RNA and 2'fluoro-2'deoxy RNA base loaded CPG (= controlled pore glass) solid support and phosphoramidites (all standard protection, ChemGenes, LinkTech).

[0530] Ancillary reagents were purchased from EMP Biotech and Biosolve. Synthesis was performed using 0.1 M solutions of phosphoramidites in dry acetonitrile (<20 ppm H2O) and benzylthiotetrazole (BTT) was used as activating agent (0.3 M in acetonitrile). Coupling time was 10 min. When phosphorothioamidites were used to introduce phosphorodithioate linkages (PS2), repeated coupling wash cycles were performed for 60 min. Cap / OX / Cap or Cap / Thio / Cap cycles were applied (Cap: Ac2O (= acetic anhydride) / NMI (= N-methylimidazole) / lutidine / acetonitrile, OX (= oxidizing agent): 0.05 M I2 / H2O in pyridine, Thio = thiolation reagent, 50 mM EDITH (= 3-ethoxy-1,2,4-dithiazolin-5-one)). Phosphorothioates and phosphorodithioates were introduced using the commercially available thiolation reagent EDITH (Link technologies) 50 mM in acetonitrile. DMT (=dimethoxytrityl) cleavage was achieved by treatment with 3% dichloroacetic acid in toluene. Diethylamine (DEA) washes were performed after completion of the programmed synthesis cycle. All oligonucleotides were synthesized in DMT-off mode.

[0531] To synthesize the triantennary GalNAc clusters ([ST23(ps)]3ST41(ps) and [ST23]3ST41, Table 4), ST23 (Table 4) and ST41 (Table 4) were introduced by successive coupling of a branched treble amidite derivative (C4XLT-phos) followed by a GalNAc amidite (ST23-phos, Table 4). The attachment of the (vp)-mU moiety (Table 4) was achieved by the use of (vp)-mU-phos (Table 4)) in the last synthesis cycle. (vp)-mU-phos does not provide a hydroxy group suitable for further synthesis extension and therefore does not bear a DMT group. Thus, the coupling of (vp)-mU-phos results in the termination of the synthesis.

[0532] For removal of the methyl ester masking the vinyl phosphonate (vp), the CPG with the fully assembled oligonucleotide was dried under reduced pressure and transferred to a 20 mL PP (=polypropylene) syringe reactor for solid-phase peptide synthesis equipped with a fritted disk (Carl Roth GmbH). The CPG was then contacted with a solution of 250 μL TMSBr (=bromotrimethylsilane) and 177 μL pyridine (0.5 ml / μmol of solid support-bound oligonucleotide) in CHCl at room temperature, and the reactor was sealed with a Luer cap. The reaction vessel was slightly agitated for a period of 2×15 min, the excess reagent was discarded, and the remaining CPG was washed twice with 10 ml of acetonitrile. Further downstream processing was unchanged from any other exemplary compound.

[0533] The single strands were cleaved from the CPG by treatment with 40% aqueous methylamine (in the presence of 20 mM DTT if phosphorodithioate linkages were present) for 90 min at room temperature. The resulting crude oligonucleotides were purified by ion exchange chromatography (Resource Q, 6 ml, GE Healthcare) on an AKTA Pure HPLC system using a sodium chloride gradient. Fractions containing the product were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech), and lyophilized until further use.

[0534] All final single-stranded products were analyzed by AEX-HPLC to verify their purity. The identity of each single-stranded product was verified by LC-MS analysis.

[0535] Example 15 Each single strand was dissolved in H2O to a concentration of 60 OD / ml. Both individual oligonucleotide solutions were added together to a reaction vessel. To make the reaction easier to monitor, a titration was performed. The first strand was added in 25% excess over the second strand, as judged by UV absorption at 260 nm. The reaction mixture was heated to 80°C for 5 minutes and then cooled slowly to room temperature. The formation of the double strand was monitored by ion-pairing reversed-phase HPLC. From the UV area of ​​the remaining single strand, the required amount of the second strand was calculated and added to the reaction mixture. The reaction was heated again to 80°C and cooled slowly to room temperature. This procedure was repeated until less than 10% of the remaining single strand was detected.

[0536] (Example 16) In vitro studies in HepG2 cells showing MASP-2 knockdown efficacy of the tested siRNAs following transfection of 20, 4, 0.8, 0.16, or 0.032 nM siRNA. The MASP-2 knockdown efficacy of selected siRNAs (Table 5b) was determined after transfection of 20, 4, 0.8, or 0.16 nM of siRNA into HepG2 cells. The results are shown in Table 6 below. At 20 nM, the remaining MASP-2 levels after knockdown reached a minimum of 22%, and at 4 nM, a minimum of 20%. At 20 nM, the most potent siRNAs were EM1223 and EM1217.

[0537] For siRNA transfection into HepG2 cells, cells were seeded in 96-well tissue culture plates (TPP, Catalog 92096, Switzerland) at a density of 40,000 cells / well. siRNA transfection was performed immediately before seeding using Lipofectamine RNAiMax (Invitrogen / Life Technologies, Catalog 13778-500, Germany) according to the manufacturer's instructions. Dose-response screening was performed in triplicate with 20, 4, 0.8, 0.16, or 0.032 nM MASP-2 siRNA, respectively, with scrambled siRNA and luciferase-targeting siRNA as nonspecific controls. After 24 hours of incubation with siRNA, the medium was removed and the cells were lysed in 250 μl of lysis buffer (InviTrap RNA Cell HTS96 Kit / C (Stratec, Cat. No. 7061300400, Germany)) and then frozen at -80°C. RNA was isolated using InviTrap RNA Cell HTS96 Kit / C (Stratec, Cat. No. 7061300400, Germany). RT-qPCR was performed in singleplex 384-well format using MASP-2 and PPIB specific primer probe sets and Takyon™ One-Step Low Rox Probe 5X MasterMix dTTP on a QuantStudio6 device from Applied Biosystems. Expression differences were calculated using the delta delta Ct method and the relative expression of MASP-2 normalized to the housekeeping gene PPIB was determined. The results are presented in Table 6 as % remaining MASP-2 mRNA after siRNA transfection.

[0538] [Table 6]

[0539] (Example 17) In vivo study showing knockdown of MASP-2 mRNA in non-human primates (NHPs). The objective of this experiment was to determine the protein knockdown efficacy of siRNA GalNAc conjugates targeting MASP-2 in vivo in non-human primates (NHPs).

[0540] Experimentally bred cynomolgus monkeys (8-15 years old, female) were assigned to different treatment groups (4 animals per group). On day 1, each group was treated with a single dose of GalNAc siRNA at 3 mg / kg body weight by subcutaneous injection, while control animals received the vehicle 0.9% saline by subcutaneous injection. Serial blood sampling was performed before administration (days -7 and 1), 2, 3, 8, 15, 22, 29, and 57. MASP-2 protein expression was measured in serum using a specific ELISA assay (Abcam ab278121).

[0541] The results of MASP-2 protein level reduction over 8 weeks after a single subcutaneous administration of GalNAc-conjugated siRNA EM1208, EM1209, and EM1211 are shown in Figure 10. MASP-2 protein levels in cynomolgus serum collected 3 weeks after a single treatment with EM1208 were reduced by an average of 89%, and 91% after 8 weeks. MASP-2 protein levels in cynomolgus serum collected 3 weeks after a single treatment with EM1209 were reduced by an average of 74%, and 55% after 8 weeks. MASP-2 protein levels in cynomolgus serum collected 3 weeks after a single treatment with EM1211 were reduced by an average of 55%, and 20% after 8 weeks.

[0542] (Example 18) The aim of the second experiment in NHPs was to determine the duration of action of a single siRNA GalNAc conjugate targeting MASP-2 by analyzing protein knockdown efficacy.

[0543] Experimentally bred cynomolgus monkeys (8-15 years old, male and female) were assigned to different treatment groups (4 animals per group). On day 1, each group was treated with a single dose of either 0.3, 1, 3, or 10 mg / kg body weight of GalNAc siRNA by subcutaneous injection, while control animals received the vehicle 0.9% saline by subcutaneous injection. Serial blood sampling was performed before administration (day -7 and day 1), on days 3, 8, 15, 22, 29, 43, 57, 71, and 85. MASP-2 protein expression was measured in serum using a specific ELISA assay (Abcam ab278121).

[0544] The results of MASP-2 protein level reduction over 12 weeks after a single subcutaneous administration of GalNAc-conjugated siRNA EM1208 are shown in Figure 11. Dose-dependent MASP-2 protein knockdown in cynomolgus monkey serum was observed after a single administration of EM1208. After 4 weeks of treatment with 10 mg / kg EM1208, MASP-2 protein levels were reduced by an average of 98%, after 8 weeks by 99%, and after 12 weeks by 95%. When EM1208 was administered at 3 mg / kg, MASP-2 protein levels in serum collected after 4 weeks were reduced by an average of 91%, after 8 weeks by 84%, and after 12 weeks by 62%.

[0545] [Table 7A]

[0546] [Table 7B]

[0547] The abbreviations set forth in Table 4 may be used herein. The list of abbreviations may not be exhaustive and additional abbreviations and their meanings may be found throughout this document.

[0548] [Table 8A]

[0549] [Table 8B]

[0550] [Table 8C]

[0551] [Table 8D]

[0552] [Table 8E]

[0553] [Table 8F]

[0554] [Table 8G]

[0555] [Table 8H]

[0556] [Table 8I]

[0557] The duplexes listed in Table 5b (Table 9) have the various modifications indicated, see Table 4 (Table 7) for explanations of the abbreviations used. Where appropriate, the sequences of the equivalent unmodified strands from Table 5a (Table 8) are also shown.

[0558]

Table 9A

[0559]

Table 9B

[0560]

Table 9C

[0561]

Table 9D

[0562]

Table 9E

[0563]

Table 9F

[0564]

Table 9G

[0565]

Table 9H

[0566]

Table 9I

[0567]

Table 9J

[0568]

Table 9K

[0569] [Table 9L]

[0570] [Table 9M]

[0571] [Table 9N]

[0572] Table 9O

[0573] [Table 9P]

[0574] [Table 9Q]

[0575] [Table 10A]

[0576] [Table 10B]

[0577] [Table 10C]

[0578] SEQ ID NO:821 >NM_006610.4 Homo sapiens MBL-associated serine protease 2 (MASP-2), transcript variant 1, mRNA

[0579] [ka]

[0580] statement The following statements represent aspects of the present invention. 1. A double-stranded nucleic acid for inhibiting expression of MASP-2, comprising a first strand and a second strand, wherein the unmodified equivalent of the sequence of the first strand comprises a sequence of at least 15 nucleotides that differs by three or fewer nucleotides from any one of the first strand sequences shown in Table 5a (Table 8) or Table 1 (Table 1).

[0581] 2. The nucleic acid of statement 1, wherein the first strand and the second strand are separate strands, each between 18 and 25 nucleotides in length.

[0582] 3. The nucleic acid of statement 1 or statement 2, wherein the first strand and the second strand form a duplex region of 17 to 25 nucleotides in length.

[0583] 4. A nucleic acid according to any one of statements 1 to 3, wherein the double-stranded region consists of 17 to 25 consecutive nucleotide base pairs.

[0584] 5. a) Both ends are blunt; b) has an overhang at one end and a blunt end at the other end, or c) having overhangs on both ends; A nucleic acid according to any one of statements 1 to 4.

[0585] 6. A nucleic acid according to any one of statements 1 to 5 which is a siRNA.

[0586] 7. A nucleic acid according to any one of statements 1 to 6, which mediates RNA interference.

[0587] 8. (a) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than three nucleotides from any one of the first strand sequences in Table 5a (Table 8), and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than three nucleotides from the corresponding second strand sequence in Table 5a (Table 8); (b) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than two nucleotides from any one of the first strand sequences in Table 5a (Table 8), and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than two nucleotides from the corresponding second strand sequence in Table 5a (Table 8); (c) the unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than one nucleotide from any one of the first strand sequences in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than one nucleotide from the corresponding second strand sequence in Table 5a (Table 8); (d) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5a (Table 8); (e) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5a (Table 8); (f) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5a (Table 8); (g) an unmodified equivalent of a first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5a (Table 8), and optionally an unmodified equivalent of a second strand sequence having a given SEQ ID NO: shown in Table 5a (Table 8) comprises a sequence corresponding to nucleotides 1-18 from the 5' end of the corresponding second strand sequence; (h) the unmodified equivalent of the first strand sequence comprises any one of the sequences of the first strand sequences having a given SEQ ID NO shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence comprises the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5a (Table 8); or (i) the unmodified equivalent of the first strand sequence consists essentially of any one of the first strand sequences having a given SEQ ID NO shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence consists essentially of the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5a (Table 8); (j) the unmodified equivalent of the first strand sequence consists of any one of the first strand sequences having a given SEQ ID NO shown in Table 5a (Table 8), and optionally the unmodified equivalent of the second strand sequence consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5a (Table 8); A nucleic acid according to any one of statements 1 to 7.

[0588] 9. (a) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than three nucleotides from any one of the first strand sequences in Table 1, and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than three nucleotides from the corresponding second strand sequence in Table 1; (b) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than two nucleotides from any one of the first strand sequences in Table 1, and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than two nucleotides from the corresponding second strand sequence in Table 1; (c) the unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than one nucleotide from any one of the first strand sequences in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than one nucleotide from the corresponding second strand sequence in Table 1; (d) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of any one of the first strand sequences in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of the corresponding second strand sequence in Table 1; (e) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of any one of the first strand sequences in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of the corresponding second strand sequence in Table 1; (f) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of the corresponding second strand sequence in Table 1; (g) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 1-18 from the 5' end of the corresponding second strand sequence in Table 1; (h) the unmodified equivalent of the first strand sequence comprises any one of the first strand sequences in Table 1, and optionally the unmodified equivalent of the second strand sequence comprises the corresponding second strand sequence in Table 1; or (i) the unmodified equivalent of the first strand sequence consists essentially of any one of the first strand sequences in Table 1, and optionally the unmodified equivalent of the second strand sequence consists essentially of the sequence of the corresponding second strand sequence in Table 1; (j) the unmodified equivalent of the first strand sequence consists of any one of the first strand sequences in Table 1, and optionally the unmodified equivalent of the second strand sequence consists of the sequence of the corresponding second strand sequence in Table 1; (k) an unmodified equivalent of a first strand sequence consists of a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1, said unmodified equivalent of a first strand sequence further consisting of one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1, and optionally an unmodified equivalent of a second strand sequence comprises, consists essentially of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 1; (l) an unmodified equivalent of a first strand sequence comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1, and said unmodified equivalent of a first strand sequence further comprises one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 21, 22, and 23) or five (nucleotides 21, 22, and 23) at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 1; and wherein the unmodified equivalent of the first strand sequence comprises a contiguous region of 17 to 25 nucleotides in length, preferably 18 to 24 nucleotides in length, that is complementary to the MASP-2 transcript of SEQ ID NO: 821, and optionally the unmodified equivalent of the second strand sequence comprises, essentially consists of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 1. (m) the unmodified equivalent of the first strand and the unmodified equivalent of the second strand of any one of the nucleic acid molecules of subparagraphs (a)-(l) above are present on a single strand, and the unmodified equivalent of the first strand and the unmodified equivalent of the second strand are capable of hybridizing to one another, thereby forming a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; or (n) the unmodified equivalent of the first strand and the unmodified equivalent of the second strand of any one of the nucleic acid molecules of subparagraphs (a)-(l) above are on two separate strands that are capable of hybridizing to each other to thereby form a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; A nucleic acid according to any one of statements 1 to 8.

[0589] 10. The nucleic acid of any one of statements 1 to 9, wherein at least one nucleotide of the first and / or second strand is a modified nucleotide.

[0590] 11. The nucleic acid according to statement 10, wherein at least nucleotides 2 and 14 of the first strand are modified by a first modification, and the nucleotides are numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand having a given SEQ ID NO.

[0591] 12. The nucleic acid of statement 10 or statement 11, wherein each of the even-numbered nucleotides of the first strand is modified by a first modification, and the nucleotides are numbered consecutively starting with nucleotide number 1 at the 5' end of the first strand having a given SEQ ID NO.

[0592] 13. The nucleic acid of statement 11 or statement 12, wherein the odd-numbered nucleotides of the first strand having a given SEQ ID NO are modified by a second modification, the second modification being different from the first modification.

[0593] 14. The nucleic acid according to any one of statements 11 to 13, wherein a nucleotide of the second strand having a given SEQ ID NO that is at a position corresponding to an even-numbered nucleotide of the first strand having a given SEQ ID NO is modified by a third modification, the third modification being different from the first modification.

[0594] 15. The nucleic acid of any one of statements 11 to 14, wherein a nucleotide of the second strand having a given SEQ ID NO that is at a position corresponding to an odd-numbered nucleotide of the first strand having a given SEQ ID NO is modified by a fourth modification, which, if the second and / or third modification are present, is different from the second modification and different from the third modification.

[0595] 16. The nucleic acid according to any one of statements 11 to 13, wherein nucleotide 11, or nucleotide 13, or nucleotides 11 and 13, or a nucleotide or nucleotides of a second strand having a given SEQ ID NO that are located at a position corresponding to nucleotides 11 to 13 of a first strand having a given SEQ ID NO, are modified by a fourth modification, and preferably, a nucleotide of the second strand that is not modified by a fourth modification is modified by a third modification.

[0596] 17. The nucleic acid according to any one of statements 11 to 16, wherein if both modifications are present in the nucleic acid, the first modification is the same as the fourth modification, and preferably, if both modifications are present in the nucleic acid, the second modification is the same as the third modification.

[0597] 18. The nucleic acid according to any one of statements 11 to 17, wherein the first modification is a 2'-F modification, the second modification, if present in the nucleic acid, is preferably a 2'-OMe modification, the third modification, if present in the nucleic acid, is preferably a 2'-OMe modification, and the fourth modification, if present in the nucleic acid, is preferably a 2'-F modification.

[0598] 19. The nucleic acid of any one of statements 10 to 18, wherein each of the nucleotides of the first strand and the second strand is a modified nucleotide.

[0599] 20. The nucleic acid according to any one of statements 10 to 19, wherein the first strand having a given SEQ ID NO has a terminal 5' (E)-vinyl phosphonate nucleotide at its 5' end, and the terminal 5' (E)-vinyl phosphonate nucleotide is preferably linked to a second nucleotide of the first strand by a phosphodiester linkage.

[0600] 21. The nucleic acid according to any one of statements 1 to 20, wherein the nucleic acid comprises phosphorothioate linkages between the two or three 3' and / or 5' nucleotides at the terminus of the first strand having the given SEQ ID NO and / or the second strand having the given SEQ ID NO, and preferably the linkages between the remaining nucleotides are phosphodiester linkages.

[0601] 22. The nucleic acid of any one of statements 1 to 20, comprising a phosphorodithioate linkage between each of the two, three, or four terminal nucleotides at the 3' end of a first strand having a given SEQ ID NO, and / or a phosphorodithioate linkage between each of the two, three, or four terminal nucleotides at the 3' end of a second strand having a given SEQ ID NO and / or a phosphorodithioate linkage between each of the two, three, or four terminal nucleotides at the 5' end of a second strand having a given SEQ ID NO, and comprising linkages other than phosphorodithioate linkages between the two, three, or four terminal nucleotides at the 5' end of the first strand.

[0602] 23. The nucleic acid of any one of statements 1 to 20, comprising phosphorothioate linkages between each of the two, three, or four terminal nucleotides at the 3' end of a first strand having a given SEQ ID NO, and / or between each of the two, three, or four terminal nucleotides at the 5' end of a first strand having a given SEQ ID NO, and / or between each of the two, three, or four terminal nucleotides at the 3' end of a second strand having a given SEQ ID NO, and / or between each of the two, three, or four terminal nucleotides at the 5' end of a second strand having a given SEQ ID NO.

[0603] 24. The nucleic acid of any one of statements 1 to 20, wherein all linkages between nucleotides of both strands other than the linkages between the two terminal nucleotides at the 3'-end of the first strand and the linkages between the two terminal nucleotides at the 3'-end and 5'-end of the second strand are phosphodiester linkages.

[0604] 25. (a) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than three nucleotides from any one of the first strand sequences in Table 5b (Table 9), and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than three nucleotides from the corresponding second strand sequence in Table 5b (Table 9); (b) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than two nucleotides from any one of the first strand sequences in Table 5b (Table 9), and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than two nucleotides from the corresponding second strand sequence in Table 5b (Table 9); (c) the unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than one nucleotide from any one of the first strand sequences in Table 5b (Table 9), and optionally the unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than one nucleotide from the corresponding second strand sequence in Table 5b (Table 9); (d) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5b (Table 9), and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5b (Table 9); (e) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5b (Table 9), and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5b (Table 9); (f) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5b (Table 9), and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 5b (Table 9); (g) an unmodified equivalent of a first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 5b (Table 9), and optionally an unmodified equivalent of a second strand sequence having a given SEQ ID NO: shown in Table 5b (Table 9) comprises a sequence corresponding to nucleotides 1-18 from the 5' end of the corresponding second strand sequence; (h) the unmodified equivalent of the first strand sequence comprises any one of the sequences of the first strand sequences having a given SEQ ID NO shown in Table 5b (Table 9), and optionally the unmodified equivalent of the second strand sequence comprises the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5b (Table 9); or (i) the unmodified equivalent of the first strand sequence consists essentially of any one of the first strand sequences having a given SEQ ID NO shown in Table 5b (Table 9), and optionally the unmodified equivalent of the second strand sequence consists essentially of the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5b (Table 9); (j) the unmodified equivalent of the first strand sequence consists of any one of the first strand sequences having a given SEQ ID NO shown in Table 5b (Table 9), and optionally the unmodified equivalent of the second strand sequence consists of the corresponding second strand sequence having a given SEQ ID NO shown in Table 5b (Table 9); A nucleic acid according to any one of statements 10 to 24.

[0605] 26. (a) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than three nucleotides from any one of the first strand sequences in Table 2, and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than three nucleotides from the corresponding second strand sequence in Table 2; (b) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than two nucleotides from any one of the first strand sequences in Table 2, and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than two nucleotides from the corresponding second strand sequence in Table 2; (c) an unmodified equivalent of the first strand sequence comprises a sequence that differs by no more than one nucleotide from any one of the first strand sequences in Table 2, and optionally an unmodified equivalent of the second strand sequence comprises a sequence that differs by no more than one nucleotide from the corresponding second strand sequence in Table 2; (d) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-17 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (e) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (f) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (g) the unmodified equivalent of the first strand sequence comprises a sequence corresponding to nucleotides 2-19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2, and optionally the unmodified equivalent of the second strand sequence comprises a sequence corresponding to nucleotides 1-18 from the 5' end of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (h) the unmodified equivalent of the first strand sequence comprises the sequence of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2, and optionally the unmodified equivalent of the second strand sequence comprises the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; or (i) the unmodified equivalent of the first strand sequence consists essentially of any one of the first strand sequences having a given SEQ ID NO shown in Table 2, and optionally the unmodified equivalent of the second strand sequence consists essentially of the sequence of the corresponding second strand sequence having a given SEQ ID NO shown in Table 2; (j) the unmodified equivalent of the first strand sequence consists of any one of the first strand sequences having a given SEQ ID NO shown in Table 2, and optionally the unmodified equivalent of the second strand sequence consists of the corresponding second strand sequence having a given SEQ ID NO shown in Table 2; (k) the first strand sequence consists of a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2, said first strand sequence further consisting of one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the first strand sequences having a given SEQ ID NO: shown in Table 2, and optionally the second strand sequence comprises, consists essentially of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (l) the sequence of the first strand comprises a sequence corresponding to nucleotides 1 to 19 from the 5' end of any one of the sequences of the first strand having a given sequence number shown in Table 2, and the sequence of the first strand further comprises one (nucleotide 20 counting from the 5' end), two (nucleotides 20 and 21), three (nucleotides 20, 21, and 22), four (nucleotides 20, 21, 22, and 23), five (nucleotides 20, 21, 22, 23, and 24), or six (nucleotides 20, 21, 22, 23, 24, and 25) additional nucleotides at the 3' end of any one of the sequences of the first strand having a given sequence number shown in Table 2, and the sequence of the first strand comprises a continuous region 17 to 25 nucleotides in length, preferably 18 to 24 nucleotides in length, that is complementary to the MASP-2 transcript of SEQ ID NO: 821; Optionally, the second strand sequence comprises, consists essentially of, or consists of the sequence of the corresponding second strand sequence having a given SEQ ID NO: shown in Table 2; (m) the first strand and the second strand of any one of the nucleic acid molecules of subparagraphs (a)-(l) above are present on a single strand, and the first strand and the second strand are capable of hybridizing to each other, thereby forming a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; or (n) the first strand and the second strand of any one of the nucleic acid molecules of subparagraphs (a) through (l) above are on two separate strands that are capable of hybridizing to one another to thereby form a double-stranded nucleic acid having a duplex region of 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length; 26. A nucleic acid according to any one of statements 10 to 25.

[0606] 27. The nucleic acid according to any one of statements 1 to 26, which is conjugated to a heterologous moiety.

[0607] 28. The nucleic acid of statement 27, wherein the heterologous moiety comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates at least one GalNAc moiety or derivative thereof to the nucleic acid.

[0608] 29. The nucleic acid has the formula (II): [SX 1 -PX 2 ]3-AX 3 - (II) is conjugated to a heterologous moiety comprising a compound of During the ceremony, S represents a functional moiety, e.g., a ligand, e.g., a sugar, preferably the sugar is N-acetylgalactosamine; X 1 is C3-C6 alkylene or (-CH2-CH2-O) m (-CH2)2-, where m is 1, 2, or 3; P is a phosphate or a modified phosphate, preferably a thiophosphate; X2 is an alkylene or a group of the formula (-CH2) n -O-CH2- alkylene ether, where n=1 to 6; A is a branching unit, X3 represents a cross-linking unit; A nucleic acid as defined in any of statements 1 to 27, wherein the nucleic acid is linked to X via a phosphate or modified phosphate, preferably a thiophosphate. 3 Conjugated to A nucleic acid according to statement 27 or statement 28.

[0609] 30. The first strand of the nucleic acid has the formula (V):

[0610] [ka]

[0611] is a compound of In the formula, b is 0 or 1; The second chain has formula (VI):

[0612] [ka]

[0613] is a compound of During the ceremony, c and d are independently 0 or 1; Z1 and Z2 are the first and second strands of a nucleic acid, respectively; Y is independently O or S; n is independently 0, 1, 2, or 3; L1 is a linker to which the ligand is attached, L1 is the same or different in formulas (V) and (VI), and when L1 occurs more than once in the same formula, it is the same or different in formulas (V) and (VI); b+c+d is 2 or 3; 30. A nucleic acid according to any one of statements 27 to 29.

[0614] 31. The nucleic acid of any one of statements 27 to 30, which is one of the duplexes having a given duplex identification number shown in Table 5c (Table 10).

[0615] 32. A composition comprising a nucleic acid according to any of the preceding statements and a solvent, and / or a delivery vehicle, and / or a physiologically acceptable excipient, and / or a carrier, and / or a salt, and / or a diluent, and / or a buffer, and / or a preservative.

[0616] 33. A composition comprising a nucleic acid according to any one of statements 1 to 31 and a further therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody, and a peptide.

[0617] 34. A nucleic acid according to any one of statements 1 to 31 or a composition according to statement 32 or 33 for use as a therapeutic agent.

[0618] 35. A nucleic acid according to any one of statements 1 to 31 or a composition according to statement 32 or 33 for use in the prevention or treatment of a disease, disorder, or syndrome.

[0619] 36. The nucleic acid or composition for use according to statement 35, wherein the disease, disorder, or syndrome is a complement-mediated disease, disorder, or syndrome.

[0620] 37. A nucleic acid or composition for use according to statement 35 or 36, wherein the disease, disorder or syndrome is associated with aberrant or overactivation of the complement pathway and / or overexpression, or ectopic expression, or localization, or accumulation of MASP-2.

[0621] 38. The disease, disorder, or syndrome is: a) selected from the group comprising C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), lupus nephritis, IgA nephropathy (IgAN), cold agglutinin disease (CAD), myasthenia gravis (MG), primary membranous nephropathy, immune complex-mediated glomerulonephritis (IC-mediated GN), post-infectious glomerulonephritis (PIGN), systemic lupus erythematosus (SLE), ischemia / reperfusion injury, age-related macular degeneration (AMD), rheumatoid arthritis (RA), antineutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), dysbiotic periodontitis, malarial anemia, neuromyelitis optica, post-HCT / solid organ transplant (TMA), Guillain-Barre syndrome, membranous glomerulonephritis, thrombotic thrombocytopenic purpura, and sepsis; b) selected from the group consisting of C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), lupus nephritis, IgA nephropathy (IgAN), and primary membranous nephropathy; c) selected from the group including C3 glomerulopathy (C3G), antineutrophil cytoplasmic autoantibody-associated vasculitis (ANCA-AV), atypical hemolytic uraemic syndrome (aHUS), cold agglutinin disease (CAD), myasthenia gravis (MG), IgA nephropathy (IgA N), and paroxysmal nocturnal hemoglobinuria (PNH); d) selected from the group consisting of C3 glomerulopathy (C3G), cold agglutinin disease (CAD), myasthenia gravis (MG), neuromyelitis optica, atypical hemolytic uraemic syndrome (aHUS), antineutrophil cytoplasmic autoantibody associated vasculitis (ANCA-AV), IgA nephropathy (IgAN), post-HCT / solid organ transplant (TMA), Guillain-Barre syndrome, paroxysmal nocturnal hemoglobinuria (PNH), membranous glomerulonephritis, lupus nephritis, and thrombotic thrombocytopenic purpura; e) selected from the group consisting of C3 glomerulopathy (C3G), cold agglutinin (CAD), and IgA nephropathy (IgAN), or f) C3 glomerulopathy (C3G), 38. A nucleic acid or composition for use according to any one of statements 35 to 37.

[0622] 39. Use of a nucleic acid according to any one of statements 1 to 31, or a composition according to statement 32 or 33, in the preparation of a medicament for the prevention or treatment of a disease, disorder, or syndrome.

[0623] 40. A method of prevention or treatment of a disease, disorder, or syndrome, comprising administering to an individual in need of treatment a pharma- ceutical effective dose of a nucleic acid according to any one of statements 1 to 31, or a composition according to statement 32 or 33, preferably wherein the nucleic acid or composition is administered to the subject subcutaneously, intravenously, or by oral, rectal, or intraperitoneal administration.

Claims

1. A double-stranded nucleic acid for inhibiting expression of MASP-2, comprising a first strand and a second strand, wherein an unmodified equivalent of the sequence of the first strand consists of a sequence derived from any one of the first strand sequences of SEQ ID NOs: 245, 147, 385, 199, 235, 237, 309, 381, 386, 387, 388, 389, 289.

2. 2. The nucleic acid of claim 1, wherein the unmodified equivalent of the second strand sequence comprises a sequence derived from any one of the corresponding second strand sequences of SEQ ID NOs: 246, 148, 178, 200, 236, 238, 310, 346, 382, ​​384, 290.

3. 3. The nucleic acid of claim 1 or 2, wherein the first strand and the second strand form a duplex region of 17 to 25 nucleotides in length.

4. A nucleic acid according to any one of claims 1 to 3, which mediates RNA interference.

5. 5. The nucleic acid of any one of claims 1 to 4, wherein the sequence of the first strand consists of SEQ ID NO: 245 and the second strand consists of SEQ ID NO:

246.

6. 6. The nucleic acid of claim 1, wherein at least one nucleotide of the first and / or second strand is a modified nucleotide.

7. 7. The nucleic acid of claim 1, wherein the first strand has a terminal 5'(E)-vinylphosphonate nucleotide at its 5' end.

8. 8. The nucleic acid of claim 1, wherein the nucleic acid comprises a phosphorothioate linkage between each of the three 3' nucleotides at the end of the first strand.

9. 9. The nucleic acid of any one of claims 1 to 8, wherein the nucleic acid comprises a phosphorothioate linkage between each of the three 3' nucleotides at the end of the second strand.

10. 10. The nucleic acid of claim 8 or 9, wherein the linkages between the remaining nucleotides of the first strand and / or the second strand are phosphodiester linkages.

11. 11. The nucleic acid of any one of claims 1 to 10, wherein the sequence of the first strand comprises: (vp)-mU fC mG fG mA fG mC fG mG fA mA fG mG fU mA fA mU (ps) fG (ps) mU (SEQ ID NO: 804), and optionally the sequence of the second strand comprises: mA mC mA mU mU mA fC fC fU mU mC mC mG mC mU mC mC (ps) mG (ps) mA (SEQ ID NO: 820).

12. 12. The nucleic acid of any one of claims 1 to 11, conjugated to a heterologous moiety.

13. 13. The nucleic acid of claim 12, wherein the heterologous moiety comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates at least one GalNAc moiety or derivative thereof to the nucleic acid.

14. The sequence of the first strand comprises: (vp)-mU fC mG fG mA fG mC fG mG fA mA fG mG fU mA fA mU (ps) fG (ps) mU (SEQ ID NO: 804), and optionally the sequence of the second strand comprises: [ST23 (ps)] 3 14. The nucleic acid of claim 12 or 13, comprising ST41 (ps) mA mC mA mU mU mA fC fC fU mU mC mC mG mC mU mC mC (ps) mG (ps) mA (sequence number 805).

15. 15. A composition comprising a nucleic acid according to any one of claims 1 to 14 and a solvent, and / or a delivery vehicle, and / or a physiologically acceptable excipient, and / or a carrier, and / or a salt, and / or a diluent, and / or a buffer, and / or a preservative, and / or a further therapeutic agent selected from the group comprising oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies, and peptides.

16. 16. A nucleic acid according to any one of claims 1 to 14 or a composition according to claim 15 for use as a therapeutic agent.

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