US20220195436A1 - Nucleic acids for inhibiting expression of c3 in a cell - Google Patents

Nucleic acids for inhibiting expression of c3 in a cell Download PDF

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US20220195436A1
US20220195436A1 US17/612,120 US202017612120A US2022195436A1 US 20220195436 A1 US20220195436 A1 US 20220195436A1 US 202017612120 A US202017612120 A US 202017612120A US 2022195436 A1 US2022195436 A1 US 2022195436A1
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Verena Aumiller
Lucas Bethge
Judith HAUPTMANN
Marie Wikström Lindholm
Adrien WEINGÄRTNER
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Silence Therapeutics GmbH
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Definitions

  • the invention relates to nucleic acid products that interfere with or inhibit complement component C3 gene expression. It further relates to therapeutic uses of such inhibition such as for the treatment of diseases and disorders associated with complement pathway deregulation and/or over-activation or with ectopic expression or localisation or accumulation of the complement component C3 in the body.
  • RNA interference Double-stranded RNAs (dsRNA) able to bind through complementary base pairing to expressed mRNAs have been shown to block gene expression (Fire et al., 1998, Nature. 1998 Feb. 19; 391(6669):806-11 and Elbashir et al., 2001, Nature. 2001 May 24; 411(6836):494-88) by a mechanism that has been termed “RNA interference (RNAi)”.
  • RNAi RNA interference
  • RNAi is mediated by the RNA induced silencing complex (RISC), a sequence specific, multi component nuclease that degrades messenger RNAs having sufficient complementary or homology to the silencing trigger loaded into the RISC complex.
  • RISC RNA induced silencing complex
  • Interfering RNAs such as siRNAs, antisense RNAs, and micro RNAs, are oligonucleotides that prevent the formation of proteins by gene silencing, i.e., inhibiting gene translation of the protein through degradation of mRNA molecules.
  • Gene silencing agents are becoming increasingly important for therapeutic applications in medicine.
  • the complement system or pathway is part of the innate immune system of host defence against invading pathogens. It mainly consists of a number of proteins that circulate in the bloodstream in the form of precursors. Most of the proteins that form the complement system, including the complement component protein C3 (also referred to herein simply as C3), are largely synthesised and secreted into the bloodstream by hepatocytes in the liver. Activation of the system leads to inflammatory responses resulting in phagocyte attraction and opsonization and consequently clearance of pathogens, immune complexes and cellular debris (Janeway's Immunobiology 9th Edition).
  • the complement system consists of 3 pathways (Classical, Leptin and Alternative pathways), which all converge at the formation of so-called complement component 3 convertase enzyme complexes. These enzyme complexes cleave the complement component C3 protein into C3a and C3b. Once cleaved, C3b forms part of a complex that in turn cleaves C5 into C5a and C5b. After cleavage, C5b is one of the key components of the main complement pathway effectors, the membrane attack complex. C3 is therefore a key component of the complement system activation pathway.
  • C3 Glomerulopathy C3 Glomerulopathy
  • aHUS atypical Hemolytic Uremic Syndrome
  • IC-mediated GN Immune Complex-mediated Glomerulonephritis
  • PIGN post-Infectious Glomerulonephritis
  • IgA N IgA nephropathy
  • diseases of other organs are also known to be related to complement dysfunction, such as age-related macular degeneration (AMD), Rheumatoid arthritis (RA), antineutrophil Cytoplasmic Autoantibodies-associated Vasculitis (ANCA-AV), dysbiotic periodontal Disease, Malarial Anaemia, Paroxysmal Nocturnal Hemoglobinuria (PNH) and sepsis.
  • AMD age-related macular degeneration
  • RA Rheumatoid arthritis
  • ANCA-AV antineutrophil Cytoplasmic Autoantibodies-associated Vasculitis
  • PNH Paroxysmal Nocturnal Hemoglobinuria
  • sepsis sepsis.
  • C3 In C3G, C3 accumulates in the glomeruli in the kidney and clogs them. The accumulation of C3 also leads to kidney damage.
  • aHUS atypical Hemolytic Uremic Syndrome
  • the complement system targets red blood cells, which leads to lysis of the red blood cells.
  • Eculizumab There are currently only few treatments for complement system mediated diseases, disorders and syndromes.
  • the monoclonal humanized antibody Eculizumab is one of them. It is known to bind complement protein C5, thereby blocking the membrane attack complex at the end of the complement cascade (Hillmen et al., 2006 NEJM).
  • C5 is a pivotal factor in the complement pathway activation. Inhibiting C3 expression therefore presents a promising therapeutic strategy for many complement-mediated diseases.
  • One aspect of the invention is a double-stranded nucleic acid for inhibiting expression of complement component C3, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences SEQ ID NO: 370, 364, 365, 366, 368, 372, 377, 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123,
  • One aspect relates to a double-stranded nucleic acid that is capable of inhibiting expression of complement component C3, preferably in a cell, for use as a medicament or in associated diagnostic or therapeutic methods, wherein the nucleic acid preferably comprises or consists of a first strand and a second strand and preferably wherein the first strand comprises sequences sufficiently complementary to a complement component C3 mRNA so as to mediate RNA interference.
  • compositions 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.
  • compositions comprising a nucleic acid as disclosed herein and a further therapeutic agent selected from e.g., an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.
  • a further therapeutic agent selected from e.g., an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.
  • One aspect relates to a nucleic acid or a composition comprising it as disclosed herein for use as a medicament or in associated methods.
  • One aspect relates to a nucleic acid or a composition comprising it as disclosed herein for use in the prevention, decrease of the risk of suffering from, or treatment of a disease, disorder or syndrome.
  • One aspect relates to the use of a nucleic acid or a composition comprising it as disclosed herein in the prevention, decrease of the risk of suffering from, or treatment of a disease, disorder or syndrome, wherein the disease, disorder or syndrome is preferably C3 Glomerulopathy (C3G).
  • C3G C3 Glomerulopathy
  • One aspect relates to a method of preventing, decreasing the risk of suffering from, or treating a disease, disorder or syndrome comprising administering a pharmaceutically effective dose or amount of a nucleic acid or composition comprising it as disclosed herein to an individual in need of treatment, preferably wherein the nucleic acid or composition is administered to the subject subcutaneously, intravenously or by oral, rectal, pulmonary, intramuscular or intraperitoneal administration.
  • the present invention relates to a nucleic acid which is double-stranded and which comprises a sequence homologous to an expressed RNA transcript of the complement component C3, and compositions thereof.
  • These nucleic acids, or conjugates or compositions thereof may be used in the treatment and prevention of a variety of diseases, disorders and syndromes in which reduced expression of the C3 gene product is desirable.
  • a first aspect of the invention is a double-stranded nucleic acid for inhibiting expression of C3, preferably in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences selected from SEQ ID NO: 370, 364, 365, 366, 368, 372, 377, 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119
  • nucleic acids among others have the advantage of being active in various species that are relevant for pre-clinical and clinical development and/or of having few relevant off-target effects. Having few relevant off-target effects means that a nucleic acid specifically inhibits the intended target and does not significantly inhibit other genes or inhibits only one or few other genes at a therapeutically acceptable level.
  • the first strand sequence comprises a sequence of at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all 19 nucleotides differing by no more than 3 nucleotides, preferably by no more than 2 nucleotides, more preferably by no more than 1 nucleotide, and most preferably not differing by any nucleotide from any one of the sequences selected from SEQ ID NO: 370, 364, 365, 366, 368, 372, 377, 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109,
  • the first strand sequence of the nucleic acid consists of one of the sequences selected from SEQ ID NOs: 370, 364, 365, 366, 368, 372, 377, 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 127, 129, 133 or 416.
  • sequence may however be modified by a number of nucleic acid modifications that do not change the identity of the nucleotide.
  • modifications of the backbone or sugar residues of the nucleic acid do not change the identity of the nucleotide because the base itself remains the same as in the reference sequence.
  • a nucleic acid that comprises a sequence according to a reference sequence herein means that the nucleic acid comprises a sequence of contiguous nucleotides in the order as defined in the reference sequence.
  • nucleotide sequence comprising or consisting of nucleotides
  • this reference is not limited to the sequence with unmodified nucleotides.
  • the same reference also encompasses the same nucleotide sequence in which one, several, such as two, three, four, five, six, seven or more, including all, nucleotides are modified by modifications such as 2′-OMe, 2′-F, a ligand, a linker, a 3′ end or 5′ end modification or any other modification. It also refers to sequences in which two or more nucleotides are linked to each other by the natural phosphodiester linkage or by any other linkage such as a phosphorothioate or a phosphorodithioate linkage.
  • a double-stranded nucleic acid is a nucleic acid in which the first strand and the second strand hybridise to each other over at least part of their lengths and are therefore capable of forming a duplex region under physiological conditions, such as in PBS at 37° C. at a concentration of 1 ⁇ M of each strand.
  • the first and second strand are preferably able to hybridise to each other and therefore to form a duplex region over a region of at least 15 nucleotides, preferably 16, 17, 18 or 19 nucleotides.
  • This duplex region comprises nucleotide base parings between the two strands, preferably based on Watson-Crick base pairing and/or wobble base pairing (such as GU base pairing).
  • the double-stranded nucleic acid is preferably a stable double-stranded nucleic acid under physiological conditions, and preferably has a melting temperature (Tm) of 45° C. or more, preferably 50° C. or more, and more preferably 55° C. or more for example in PBS at a concentration of 1 ⁇ M of each strand.
  • Tm melting temperature
  • a stable double-stranded nucleic acid under physiological conditions is a double-stranded nucleic acid that has a Tm of 45° C. or more, preferably 50° C. or more, and more preferably 55° C. or more, for example in PBS at a concentration of 1 ⁇ M of each strand.
  • the first strand and the second strand are preferably capable of forming a duplex region (i.e., are complementary to each other) over i) at least a portion of their lengths, preferably over at least 15 nucleotides of both of 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 the second strand.
  • Strands being complementary to each other over a certain length means that the strands are able to base pair to each other, either via Watson-Crick or wobble base pairing, over that length.
  • Each nucleotide of the length does not necessarily have to be able to base pair with its counterpart in the other strand over the entire given length as long as a stable double-stranded nucleotide under physiological conditions can be formed. It is however, preferred, in certain embodiments, if each nucleotide of the length can base pair with its counterpart in the other strand over the entire given length.
  • 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 can be tolerated in the context of the siRNA and even have the potential in certain cases to increase RNA interference (e.g., inhibition) activity.
  • the inhibition activity of the nucleic acids according to the present invention relies on the formation of a duplex region between all or a portion of the first strand and a portion of a target nucleic acid.
  • the portion of the target nucleic acid that forms a duplex region with the first strand defined as beginning 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, target sequence.
  • the duplex region formed between the first strand and the second strand need not be the same as the duplex region formed between the first strand and the target sequence. That is, the second strand may have a sequence different from the target sequence; however, 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.
  • the complementarity between the first strand and the target sequence may be perfect (i.e., 100% identity with no nucleotide mismatches or insertions or deletions in the first strand as compared to the target sequence).
  • the complementarity between the first strand and the target sequence may not be perfect.
  • the complementarity may be from about 70% to about 100%. More specifically, the complementarity may be at least 70%, 80%, 85%, 90% or 95% and intermediate values.
  • the identity between the first strand and the complementary sequence of the target sequence may range from about 75% to about 100%. More specifically, the complementarity may be at least 75%, 80%, 85%, 90% or 95% and intermediate values, provided a nucleic acid is capable of reducing or inhibiting the expression of the complement component C3.
  • a nucleic acid having less than 100% complementarity between the first strand and the target sequence may be able to reduce the expression of the complement component C3 to the same level as a nucleic acid having perfect complementarity between the first strand and target sequence.
  • it may be able to reduce expression of the complement component C3 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 the nucleic acid with perfect complementarity.
  • a nucleic acid of the present disclosure is a nucleic acid wherein
  • Table 1 is:
  • Second strand sequence (SEQ ID NO:) (SEQ ID NO:) 364 363 or 375 365 363 366 367 or 376 368 369 370 379 or 371, preferably 379 372 373 362 374 377 378 361 112 95 96 111 112 125 126 131 132 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 97 98 99 100 101 102 103 104 105 106 107 108 109 110 113 114 115 116 117 118 119 120 121 122
  • the nucleic acid is a nucleic acid wherein:
  • the 5′-most nucleotide of the first strand is a nucleotide other than A or U, this nucleotide is replaced by an A or U.
  • this nucleotide is replaced by U, and more preferably by U with a 5′ vinylphosphonate.
  • nucleic acid of the invention does not comprise the entire sequence of a reference first strand and/or second strand sequence, as for example given in Table 1, or one or both strands differ from the corresponding reference sequence by one, two or three nucleotides
  • 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%, yet more preferably at least 95% and most preferably at least 100% of the C3 inhibition activity compared to the inhibition activity of the corresponding nucleic acid that comprises the entire first strand and second strand reference sequences in a comparable experiment.
  • the nucleic acid is a nucleic acid wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 361 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 112; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 95 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 96; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 111 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably
  • the nucleic acid is a double-stranded nucleic acid for inhibiting expression of C3, preferably in a cell, wherein the nucleic acid comprises a first nucleic acid strand and a second nucleic acid strand, wherein the first strand is capable of hybridising under physiological conditions to a nucleic acid of sequence selected from SEQ ID NO: 379, 363, 375, 367, 376, 369, 371, 373, 380, 374, 378, 112, 96, 126, 132, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104, 106, 108, 110, 114, 116, 118,
  • the second strand is capable of hybridising under physiological conditions to the first strand to form a duplex region.
  • Nucleic acids that are capable of hybridising under physiological conditions are nucleic acids that are capable of forming base pairs, preferably Watson-Crick or wobble base-pairs, between at least a portion of the opposed nucleotides in the strands so as to form at least a duplex region.
  • Such a double-stranded nucleic acid is preferably a stable double-stranded nucleic acid under physiological conditions (for example in PBS at 37° C. at a concentration of 1 ⁇ M of each strand), meaning that under such conditions, the two strands stay hybridised to each other.
  • the Tm of the double-stranded nucleotide is preferably 45° C. or more, preferably 50° C. or more and more preferably 55° C. or more.
  • nucleic acid for inhibiting expression of the complement component C3, wherein the nucleic acid comprises a first sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides differing by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide and most preferably not differing by any nucleotide from any of the sequences of Table 5, the first sequence being able to hybridise to a target gene transcript (such as an mRNA) under physiological conditions.
  • a target gene transcript such as an mRNA
  • the nucleic acid further comprises a second sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides differing by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide and most preferably not differing by any nucleotide from any of the sequences of Table 5, the second sequence being able to hybridise to the first sequence under physiological conditions and preferably the nucleic acid being an siRNA that is capable of inhibiting C3 expression via the RNAi pathway.
  • One aspect relates to any double-stranded nucleic acid as disclosed in Table 3, preferably for inhibiting expression of the complement component C3, provided that the double-stranded nucleic acid is able to inhibit expression of complement component C3.
  • These nucleic acids are all siRNAs with various nucleotide modifications. Some of them are conjugates comprising GalNAc moieties that can be specifically targeted to cells with GalNAc receptors, such as hepatocytes.
  • One aspect relates to a double-stranded nucleic acid that is capable of inhibiting expression of complement component C3, preferably in a cell, for use as a medicament or in associated diagnostic or therapeutic methods, wherein the nucleic acid preferably comprises or consists of a first strand and a second strand and preferably wherein the first strand comprises sequences sufficiently complementary to a complement component C3 mRNA so as to mediate RNA interference.
  • the nucleic acids described herein may be capable of inhibiting the expression of the complement component C3. Inhibition may be complete, i.e., 0% remaining expression. Inhibition of C3 expression may be partial, i.e., it may be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more, or intermediate values of inhibition of the level of C3 expression in the absence of a nucleic acid of the invention. The level of inhibition may be measured by comparing a treated sample with an untreated sample or with a sample treated with a control such as for example a siRNA that does not target C3.
  • Inhibition may be measured by measuring C3 mRNA and/or protein levels or levels of a biomarker or indicator that correlates with C3 presence or activity. It may be measured in cells that may have been treated in vitro with a nucleic acid described herein. Alternatively, or in addition, inhibition may be measured in cells, such as hepatocytes, or tissue, such as liver tissue, or an organ, such as the liver, or in a body fluid such as blood, serum, lymph or any other body part or fluid that has been taken from a subject previously treated with a nucleic acid disclosed herein.
  • inhibition of C3 expression is determined by comparing the C3 mRNA level measured in C3-expressing cells after 24 or 48 hours in vitro treatment with a double-stranded RNA disclosed herein under ideal conditions (see the examples for appropriate concentrations and conditions) to the C3 mRNA level measured in control cells that were untreated or mock treated or treated with a control double-stranded RNA under the same conditions.
  • One aspect of the present invention relates to a nucleic acid, wherein the first strand and the second strand are present on a single strand of a nucleic acid that loops around so that the first strand and the second strand are able to hybridise to each other and to thereby form a double-stranded nucleic acid with a duplex region.
  • the first strand and the second strand of the nucleic acid are separate strands.
  • the two separate strands are preferably each 17-25 nucleotides in length, more preferably 18-25 nucleotides in length.
  • the two strands may be of the same or different lengths.
  • the first strand may be 17-25 nucleotides in length, preferably it may be 18-24 nucleotides in length, it may be 18, 19, 20, 21, 22, 23 or 24 nucleotides in length. Most preferably, the first strand is 19 nucleotides in length.
  • the second strand may independently be 17-25 nucleotides in length, preferably it may be 18-24 nucleotides in length, it may be 18, 19, 20, 21, 22, 23 or 24 nucleotides in length. More preferably, the second strand is 18 or 19 or 20 nucleotides in length, and most preferably it is 19 nucleotides in length.
  • the first strand and the second strand of the nucleic acid form a duplex region of 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 here as the region between and including the 5′-most nucleotide of the first strand that is base paired to a nucleotide of the second strand to the 3′-most nucleotide of the first strand that is base paired to a nucleotide of the second strand.
  • the duplex region may comprise nucleotides in either or both strands that are not base-paired to a nucleotide in the other strand. It may comprise one, two, three or four such nucleotides on the first strand and/or on the second strand. However, preferably, the duplex region consists of 17-25 consecutive nucleotide base pairs. That is to say that it preferably comprises 17-25 consecutive nucleotides on both of the strands that all base pair to a nucleotide in the other strand. More preferably, the duplex region consists of 18 or 19 consecutive nucleotide base pairs, most preferably 18.
  • the nucleic acid may be blunt ended at both ends; have an overhang at one end and a blunt end at the other end; or have an overhang at both ends.
  • the nucleic acid may have an overhang at one end and a blunt end at the other end.
  • the nucleic acid may have an overhang at both ends.
  • the nucleic acid may be blunt ended at both ends.
  • the nucleic acid may be blunt ended at the end with 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.
  • the nucleic acid may comprise an overhang at a 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 at both the 5′ end and 3′ end of the first strand.
  • the nucleic acid may have an overhang at both the 5′ end and 3′ end 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.
  • An overhang at the 3′ end or 5′ end of the second strand or the first strand may consist of 1, 2, 3, 4 and 5 nucleotides in length.
  • an overhang may consist of 1 or 2 nucleotides, which may or may not be modified.
  • the 5′ end of the first strand is a single-stranded overhang of one, two or three nucleotides, preferably of one nucleotide.
  • the nucleic acid is an siRNA.
  • siRNAs are short interfering or short silencing RNAs that are able to inhibit the expression of a target gene through the RNA interference (RNAi) pathway. Inhibition occurs through targeted degradation of mRNA transcripts of the target gene after transcription.
  • RNAi RNA interference
  • the siRNA forms part of the RISC complex.
  • the RISC complex specifically targets the target RNA by sequence complementarity of the first (antisense) strand with the target sequence.
  • the nucleic acid mediates RNA interference (RNAi).
  • RNAi RNA interference
  • the nucleic acid mediates RNA interference with an efficacy of at least 50% inhibition, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, yet more preferably at least 95% and most preferably 100% inhibition.
  • the inhibition efficacy is preferably measured by comparing the C3 mRNA level in cells, such as hepatocytes, treated with a C3 specific siRNA to the C3 mRNA level in cells treated with a control in a comparable experiment.
  • the control can be a treatment with a non-C3 targeting siRNA or without a siRNA.
  • the nucleic acid, or at least the first strand of the nucleic acid is therefore preferably able to be incorporated into the RISC complex.
  • the nucleic acid, or at least the first strand of the nucleic acid is therefore able to guide the RISC complex to a specific target RNA with 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 and as a result leads to inhibition of the expression of the gene from which the RNA stems.
  • Nucleic acids discussed herein include unmodified RNA as well as RNA which has been modified, e.g., to improve efficacy or stability.
  • Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as those which occur in nature, for example as occur naturally in the human body.
  • modified nucleotide refers to a nucleotide in which one or more of the components of the nucleotide, namely the sugar, base, and phosphate moiety, is/are different from those which occur in nature.
  • modified nucleotide also refers in certain cases to molecules that are not nucleotides in the strict sense of the term because they lack, or have a substitute of, an essential component of a nucleotide, such as the sugar, base or phosphate moiety.
  • a nucleic acid comprising such modified nucleotides is still to be understood as being a nucleic acid, even if one or more of the nucleotides of the nucleic acid has been replaced by a modified nucleotide that lacks, or has a substitution of, an essential component of a nucleotide.
  • Modifications of the nucleic acid of the present invention generally provide a powerful tool in overcoming potential limitations including, but not limited to, in vitro and in vivo stability and bioavailability inherent to native RNA molecules.
  • the nucleic acids according to the invention may be modified by chemical modifications. Modified nucleic acids can also minimise the possibility of inducing interferon activity in humans. Modifications can further enhance the functional delivery of a nucleic acid to a target cell.
  • the modified nucleic acids of the present invention may comprise one or more chemically modified ribonucleotides of either or both of the first strand or the second strand.
  • a ribonucleotide may comprise a chemical modification of the base, sugar or phosphate moieties.
  • the ribonucleic acid may be modified by substitution with or insertion of analogues of nucleic acids or bases.
  • “same or common modification” means the same modification to any nucleotide, be that A, G, C or U modified with a group such as a methyl group (2′-OMe) or a fluoro group (2′-F).
  • 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.
  • a 2′-F modification is a different modification compared to a 2′-OMe modification.
  • 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.
  • a modified nucleotide can be a nucleotide with a modification of the sugar group.
  • the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified nucleotide may contain a sugar such as arabinose.
  • Modified nucleotides can also include “abasic” sugars, which lack a nucleobase at C-1′. These abasic sugars can further contain modifications at one or more of the constituent sugar atoms.
  • the 2′ modifications may be used in combination with one or more phosphate internucleoside linker modifications (e.g., phosphorothioate or phosphorodithioate).
  • phosphate internucleoside linker modifications e.g., phosphorothioate or phosphorodithioate.
  • nucleic acid of the present 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 a single-stranded region.
  • the modified nucleotide may be on the first strand and may be outside the duplex region.
  • the modified nucleotide may be on the second strand and may be 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.
  • a nucleic acid of the invention may have 1 modified nucleotide or a nucleic acid of the invention may have about 2-4 modified nucleotides, or a nucleic acid may 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.
  • nucleic acid comprising said modified nucleotides retains at least 50% of its activity as compared to the same nucleic acid but without said modified nucleotides or vice versa.
  • the nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% and intermediate values of its activity as compared to the same nucleic acid but without said modified nucleotides, or may have more than 100% of the activity of the same nucleic acid without said modified nucleotides.
  • 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 nucleotides may be selected from the group consisting of a 3′ terminal deoxy thymine (dT) nucleotide, a 2′-O-methyl (2′-OMe) modified nucleotide, a 2′ modified nucleotide, a 2′ deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′ amino modified nucleotide, a 2′ alkyl modified nucleotide, a 2′-deoxy-2′-fluoro (2′-F) modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic and a terminal nucleotide linked to a cholesteryl derivative or a do
  • the nucleic acid may comprise a nucleotide comprising a modified base, wherein the base is selected from 2-aminoadenosine, 2,6-diaminopurine, inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g.
  • modifications described herein and that occur within a nucleic acid will be repeated within a polynucleotide molecule, such as a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the possible positions/nucleotides in the polynucleotide but in many cases it will not.
  • a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, such as at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand.
  • a modification may occur in a double-strand region, a single-strand region, or in both.
  • a modification may occur only in the double-strand region of a nucleic acid of the invention or may only occur in a single-strand region of a nucleic acid of the invention.
  • a phosphorothioate or phosphorodithioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4 or 5 nucleotides of a strand, or may occur in duplex and/or in single-strand regions, particularly at termini.
  • the 5′ end and/or 3′ end may be phosphorylated.
  • Stability of a nucleic acid of the invention may be increased by including particular bases in overhangs, or by including modified nucleotides, in single-strand overhangs, e.g., in a 5′ or 3′ overhang, or in both.
  • Purine nucleotides may be included in overhangs. All or some of the bases in a 3′ or 5′ overhang may 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 in the phosphate group, such as phosphorothioate or phosphorodithioate modifications. Overhangs need not be homologous with the target sequence.
  • Nucleases can hydrolyse nucleic acid phosphodiester bonds. However, chemical modifications to nucleic acids can confer improved properties, and, can render oligoribonucleotides more stable to nucleases.
  • Modified nucleic acids can include one or more of:
  • the nucleic acid may comprise one or more nucleotides on the second and/or first strands that are modified. Alternating nucleotides may be modified, to form modified nucleotides.
  • Alternating as described herein means to occur one after another in a regular way. In other words, alternating means to occur in turn repeatedly. For example, if one nucleotide is modified, the next contiguous nucleotide is not modified and the following contiguous nucleotide is modified and so on. One nucleotide may be modified with a first modification, the next contiguous nucleotide may be modified with a second modification and the following contiguous nucleotide is modified with the first modification and so on, where the first and second modifications are different.
  • nucleic acid at least nucleotides 2 and 14 of the first strand are modified, preferably by 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 2′-F.
  • At least one, several or preferably all the even-numbered nucleotides of the first strand are modified, preferably by 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 2′-F.
  • At least one, several or preferably all the 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.
  • they are modified by a second modification. This second modification is preferably different from the first modification if the nucleic acid also comprises a first modification, for example of nucleotides 2 and 14 or of all the even-numbered nucleotides of the first strand.
  • the first modification is preferably any 2′ ribose modification that is of 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′-Fluoroarabino Nucleic Acid (FANA) modification.
  • a 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group can for example be a 2′-F, 2′-H, 2′-halo, or 2′-NH 2 .
  • the second 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 for example be a 2′-OMe, 2′-O-MOE (2′-O-methoxyethyl), 2′-O-allyl or 2′-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions.
  • the first modification is preferably 2′-F and/or the second modification is preferably 2′-OMe.
  • the size or volume of a substituent is preferably measured as the van der Waals volume.
  • At least one, several or preferably all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified, preferably by a third modification.
  • a third modification Preferably in the same nucleic acid nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification.
  • the odd-numbered nucleotides of the first strand are modified with a second modification.
  • 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 of 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′-Fluoroarabino Nucleic Acid (FANA) modification.
  • a 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group can for example be a 2′-F, 2′-H, 2′-halo, or 2′-NH 2 .
  • 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 for example be a 2′-OMe, 2′-O-MOE (2′-O-methoxyethyl), 2′-O-allyl or 2′-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions.
  • the first modification is preferably 2′-F and/or the second and/or third modification is/are preferably 2′-OMe.
  • the nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand.
  • a nucleotide of the second strand that is in a position corresponding, for example, to an even-numbered nucleotide of the first strand is a nucleotide of the second strand that is base-paired to an even-numbered nucleotide of the first strand.
  • At least one, several or preferably all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand are modified, preferably by a fourth modification.
  • Preferably in the same nucleic acid nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification.
  • the odd-numbered nucleotides of the first strand are modified with a second modification.
  • all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified with a third modification.
  • the fourth modification is preferably different from the second modification and 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 of 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′-Fluoroarabino Nucleic Acid (FANA) modification.
  • a 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group can for example be a 2′-F, 2′-H, 2′-halo, or 2′-NH 2 .
  • 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 for example be a 2′-OMe, 2′-O-MOE (2′-O-methoxyethyl), 2′-O-allyl or 2′-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions.
  • the first and/or the fourth modification is/are preferably a 2′-OMe modification and/or the second and/or third modification is/are preferably a 2′-F modification.
  • the nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand.
  • the nucleotide/nucleotides of the second strand in a position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11-13 of the first strand is/are modified by a fourth modification.
  • all the nucleotides of the second strand other than the nucleotide/nucleotides in a position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11-13 of the first strand is/are modified by a third modification.
  • nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification.
  • the odd-numbered nucleotides of the first strand are modified with a second modification.
  • the fourth modification is preferably different from the second modification and 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 of 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′-Fluoroarabino Nucleic Acid (FANA) modification.
  • a 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group can for example be a 2′-F, 2′-H, 2′-halo, or 2′-NH 2 .
  • 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 for example be a 2′-OMe, 2′-O-MOE (2′-O-methoxyethyl), 2′-O-allyl or 2′-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions.
  • the first and/or the fourth modification is/are preferably a 2′-OMe modification and/or the second and/or third modification is/are preferably a 2′-F modification.
  • the nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand.
  • all the even-numbered nucleotides of the first strand are modified by a first modification
  • all the odd-numbered nucleotides of the first strand are modified by a second modification
  • all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified by a third modification
  • all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand are modified by a fourth modification, wherein the first and/or fourth modification is/are 2′-F and/or the second and/or third modification is/are 2′-OMe.
  • all the even-numbered nucleotides of the first strand are modified by a first modification
  • all the odd-numbered nucleotides of the first strand are modified by a second modification
  • all the nucleotides of the second strand in positions corresponding to nucleotides 11-13 of the first strand are modified by a fourth modification
  • all the nucleotides of the second strand other than the nucleotides corresponding to nucleotides 11-13 of the first strand are modified by a third modification, wherein the first and fourth modification are 2′-F and the second and third modification are 2′-OMe.
  • 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 would normally be the case).
  • the inverted RNA nucleotide is preferably an unmodified nucleotide in the sense that it does not comprise any modifications compared to the natural nucleotide counterpart.
  • the inverted RNA nucleotide is preferably a 2′-OH nucleotide.
  • the nucleic acid is blunt-ended at least at the end that comprises the 5′ end of the first strand.
  • One aspect of the present invention is a nucleic acid as disclosed herein for inhibiting expression of the C3 gene, preferably in a cell, wherein said first strand includes modified nucleotides or unmodified nucleotides at a plurality of positions in order to facilitate processing of the nucleic acid by RISC.
  • “facilitate processing by RISC” means that the nucleic acid can be processed by RISC, for example any modification present will permit the nucleic acid to be processed by RISC and preferably, will be beneficial to processing by RISC, suitably such that siRNA activity can take place.
  • the nucleotide on the second strand which corresponds to position 13 of the first strand is the nucleotide that forms a base pair with position 13 (from the 5′ end) of the first strand.
  • the nucleotide on the second strand which corresponds to position 11 of the first strand is the nucleotide that forms a base pair with position 11 (from the 5′ end) of the first strand.
  • the nucleotide on the second strand which corresponds to position 12 of the first strand is the nucleotide that forms a base pair with position 12 (from the 5′ end) of the first strand.
  • position 13 (from the 5′ end) of the first strand would pair with position 7 (from the 5′ end) of the second strand.
  • Position 11 (from the 5′ end) of the first strand would pair with position 9 (from the 5′ end) of the second strand.
  • This nomenclature may be applied to other positions of the second strand.
  • the nucleotide on the second strand that “corresponds to” a position on the first strand may not necessarily form a base pair if that position is the position in which there is a mismatch, but the principle of the nomenclature still applies.
  • One aspect 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 which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2′-F modification.
  • One aspect 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 which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are not modified with a 2′-OMe modification.
  • One aspect 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 which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2′-F modification.
  • One aspect is a nucleic acid as disclosed herein wherein greater than 50% of the nucleotides of the first and/or second strand comprise a 2′-OMe modification, such as greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more, of the first and/or second strand comprise a 2′-OMe modification, preferably measured as a percentage of the total nucleotides of both the first and second strands.
  • a 2′-OMe modification such as greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more
  • nucleic acid as disclosed herein wherein greater than 50% of the nucleotides of the first and/or second strand comprise a naturally occurring RNA modification, such as wherein greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and/or second strands comprise such a modification, preferably measured as a percentage of the total nucleotides of both the first and second strands.
  • Suitable naturally occurring modifications include, as well as 2′-OMe, other 2′ sugar modifications, in particular a 2′-H modification resulting in a DNA nucleotide.
  • nucleic acid as disclosed herein comprising no more than 20%, such as no more than 15% such as no more than 10%, of nucleotides which have 2′ modifications that are not 2′-OMe modifications on the first and/or second strand, preferably as a percentage of the total nucleotides of both the first and second strands.
  • One aspect is a nucleic acid as disclosed herein, wherein the number of nucleotides in the first and/or second strand with a 2′-modification that is not a 2′-OMe modification is no more than 7, more preferably no more than 5, and most preferably no more than 3.
  • One aspect is a nucleic acid as disclosed herein comprising no more than 20%, (such as no more than 15% or no more than 10%) of 2′-F modifications on the first and/or second strand, preferably as a percentage of the total nucleotides of both strands.
  • One aspect is a nucleic acid as disclosed herein, wherein the number of nucleotides in the first and/or second strand with a 2′-F modification is no more than 7, more preferably no more than 5, and most preferably no more than 3.
  • nucleic acid as disclosed herein, wherein all nucleotides are modified with a 2′-OMe modification except positions 2 and 14 from the 5′ end of the first strand and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand.
  • nucleotides that are not modified with 2′-OMe are modified with fluoro at the 2′ position (2′-F modification).
  • nucleic acid as disclosed herein wherein all nucleotides of the nucleic acid are modified at the 2′ position of the sugar.
  • these nucleotides are modified with a 2′-F modification where the modification is not a 2′-OMe modification.
  • the nucleic acid is modified on the first strand with alternating 2′-OMe modifications and 2-F modifications, and positions 2 and 14 (starting from the 5′ end) are modified with 2′-F.
  • the second strand is modified with 2′-F modifications at nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand.
  • 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, and the remaining modifications are naturally occurring modifications, preferably 2′-OMe.
  • the complementary region at least in this case starts at the first position of the second strand that has a corresponding nucleotide in the first strand, regardless of whether the two nucleotides are able to base pair to each other.
  • each of the nucleotides of the first strand and of the second strand is a modified nucleotide.
  • odd numbered means a number not divisible by two. Examples of odd numbers are 1, 3, 5, 7, 9, 11 and so on.
  • even numbered as described herein means a number which is evenly divisible by two. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14 and so on.
  • nucleotides of the first strand are numbered contiguously starting with nucleotide number 1 at the 5′ end of the first strand.
  • nucleotides of the second strand are numbered contiguously starting with nucleotide number 1 at the 3′ end of the second strand.
  • One or more nucleotides on 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, wherein the at least second modification is different from the modification on the one or more odd 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.
  • a plurality of odd-numbered nucleotides in the first strand may be modified in the nucleic acid of the 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 that are modified by a common modification.
  • the first strand may also comprise adjacent nucleotides that are modified by a second different modification (i.e. the first strand may comprise nucleotides that are adjacent to each other and modified by a first modification as well as other nucleotides that are adjacent to each other and modified by a second modification that is different to the first modification).
  • One or more of the odd-numbered nucleotides of the second strand may be modified by a modification that is different to the modification of the odd-numbered nucleotides on the first strand (wherein the nucleotides are numbered contiguously 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 of 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 the 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 that is present on the first stand odd-numbered nucleotides.
  • a plurality of odd-numbered nucleotides on the second strand may be modified by a modification that is different from the modification of the first strand odd-numbered nucleotides.
  • the second strand may comprise adjacent nucleotides that are modified by a common modification, which may be a modification that is different from the modification of the odd-numbered nucleotides of the first strand.
  • each of the odd-numbered nucleotides in the first strand and each of the even-numbered nucleotides in the second strand may be modified with a common modification and, each of the even-numbered nucleotides may be modified in the first strand with a different modification and each of the odd-numbered nucleotides may be modified in the second strand with the different modification.
  • the nucleic acid of the invention may have the modified nucleotides of the first strand shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.
  • 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 on 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 on 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 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 on 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 in the second strand by a common modification.
  • One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.
  • the nucleic acid of the invention may comprise single- or double-stranded constructs that comprise at least two regions of alternating modifications in one or both of the strands. These alternating regions can 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 acid of the invention.
  • the nucleic acid 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 from about 5 to about 12 contiguous unmodified or differently or commonly modified nucleotides.
  • 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 comprise adjacent nucleotides that are 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 the second modification.
  • One or more nucleotides with the second modification may be adjacent to each other and to nucleotides having a modification that is the same as the modification of the odd-numbered nucleotides of the first strand.
  • the first strand may also comprise phosphorothioate linkages between the two nucleotides at the 3′ end and at 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 5′ end.
  • the second strand may also be conjugated to a ligand at the 5′ end.
  • the nucleic acid of the invention may comprise a first strand comprising adjacent nucleotides that are modified with a common modification.
  • One or more such nucleotides may be adjacent to one or more nucleotides which may be modified with a second modification.
  • One or more nucleotides with the second modification may be adjacent.
  • the second strand may comprise adjacent nucleotides that are 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 the second modification.
  • One or more nucleotides with the second modification may be adjacent.
  • the first strand may also comprise phosphorothioate linkages between the two nucleotides at the 3′ end and at 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 numbered from 5′ to 3′ on the first strand and 3′ to 5′ on the second strand, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 may be modified by a modification on the first strand.
  • the nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the first strand.
  • the nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a modification on the second strand.
  • the nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand.
  • Nucleotides are numbered for the sake of the nucleic acid of the present invention from 5′ to 3′ on the first strand and 3′ to 5′ on the second strand.
  • the nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a modification on the first strand.
  • the nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a second modification on the first strand.
  • the nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a modification on the second strand.
  • the nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand.
  • first and/or the second strand are shorter than 25 nucleotides in length, such as 19 nucleotides in length, there are no nucleotides numbered 20, 21, 22, 23, 24 and 25 to be modified.
  • nucleotides numbered 20, 21, 22, 23, 24 and 25 there are no nucleotides numbered 20, 21, 22, 23, 24 and 25 to be modified. The skilled person understands the description above to apply to shorter strands, accordingly.
  • One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a common modification.
  • One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a different modification.
  • One or more modified nucleotides on the first strand may be paired with unmodified nucleotides on the second strand.
  • One or more modified nucleotides on the second strand may be paired with unmodified nucleotides on the first strand.
  • the alternating nucleotides can be aligned on the two strands such as, for example, all the modifications in the alternating regions of the second strand are paired with identical modifications in the first strand or alternatively the modifications can be offset by one nucleotide with the common modifications in the alternating regions of one strand pairing with dissimilar modifications (i.e. a second or further modification) in the other strand.
  • dissimilar modifications i.e. a second or further modification
  • the modifications on the first strand may be shifted by one nucleotide relative to the modified nucleotides on the second strand, such that common modified nucleotides are not paired with each other.
  • the modification and/or modifications may each and individually be selected from the group consisting of 3′ terminal deoxy thymine, 2′-OMe, a 2′ deoxy modification, a 2′ amino modification, a 2′ alkyl modification, a morpholino modification, a phosphoramidate modification, 5′-phosphorothioate group modification, a 5′ phosphate or 5′ phosphate mimic modification and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification and/or the modified nucleotide may be any one of a locked nucleotide, an abasic nucleotide or a non-natural base comprising nucleotide.
  • 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 on the first and/or second strand.
  • the nucleic acid of the invention may comprise an inverted RNA nucleotide at one or several of the strand ends.
  • Such inverted nucleotides provide stability to the nucleic acid.
  • the nucleic acid comprises at least an inverted nucleotide at the 3′ end of the first and/or the 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.
  • the nucleic acid comprises an inverted RNA nucleotide at the 3′ end of the second strand and this nucleotide is preferably an inverted A.
  • An inverted nucleotide is a nucleotide that is linked to the 3′ end of a nucleic acid through its 3′ carbon, rather than its 5′ carbon as would normally be the case or is linked to the 5′ end of a nucleic acid through its 5′ carbon, rather than its 3′ carbon as would normally be the case.
  • the inverted nucleotide is preferably present at an end of a strand not as an overhang but opposite a corresponding nucleotide in the other strand.
  • the nucleic acid is preferably blunt-ended at the end that comprises the inverted RNA nucleotide.
  • An inverted RNA nucleotide being present at the end of a strand preferably means that the last nucleotide at this end of the strand is the inverted RNA nucleotide.
  • a nucleic acid with such a nucleotide is stable and easy to synthesise.
  • the inverted RNA nucleotide is preferably an unmodified nucleotide in the sense that it does not comprise any modifications compared to the natural nucleotide counterpart.
  • the inverted RNA nucleotide is preferably a 2′-OH nucleotide.
  • Nucleic acids of the invention may comprise one or more nucleotides modified at the 2′ position with a 2′-H, and therefore having a DNA nucleotide within the nucleic acid.
  • Nucleic acids of the invention may comprise DNA nucleotides at positions 2 and/or 14 of the first strand counting from the 5′ end of the first strand.
  • Nucleic acids may comprise DNA nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand.
  • Nucleic acids of the invention may comprise one or more LNA nucleotides. Nucleic acids of the invention may comprise LNA nucleotides at positions 2 and/or 14 of the first strand counting from the 5′ end of the first strand. Nucleic acids may comprise LNA on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand.
  • the nucleic acid may comprise a first modification and a second or further modification which are each and individually selected from the group comprising 2′-OMe modification and 2′-F modification.
  • the nucleic acid may comprise a modification that is 2′-OMe that may be a first modification, and a second modification that is 2′-F.
  • the nucleic acid of the invention may also include a phosphorothioate or phosphorodithioate modification and/or a deoxy modification which may be present in or between the terminal 2 or 3 nucleotides of each or any end of each or both strands.
  • At least one nucleotide of the first and/or second strand is a modified nucleotide, wherein if the first strand comprises at least one modified nucleotide:
  • the second strand comprises at least one modified nucleotide:
  • nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand;
  • nucleic acid is conjugated to a ligand.
  • One aspect is a double-stranded nucleic acid for inhibiting expression of C3, preferably in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences SEQ ID NO: 370, 364, 365, 366, 368, 372, 377, 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123
  • One aspect is a double-stranded nucleic acid for inhibiting expression of C3, preferably in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences SEQ ID NO: 370, 364, 365, 366, 368, 372, 377, 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123
  • the 3′ and 5′ ends of an oligonucleotide can be modified. Such modifications can be at the 3′ end or the 5′ end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group.
  • the 3′ and 5′ ends of an oligonucleotide can be conjugated to other functional molecular entities such as labelling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester).
  • labelling moieties e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester).
  • the functional molecular entities can be attached to the sugar through a phosphate group and/or a linker.
  • the terminal atom of the linker can 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.
  • the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs).
  • the 3′ end can be an —OH group.
  • terminal modifications include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, 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-pyrene butyric 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-
  • Terminal modifications can also be useful for monitoring distribution, and in such cases the groups to be added may include fluorophores, e.g., fluorescein or an Alexa dye. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety.
  • fluorophores e.g., fluorescein or an Alexa dye.
  • Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety.
  • Terminal modifications can be added for a number of reasons, including to modulate activity or to modulate resistance to degradation.
  • Terminal modifications useful for modulating activity include modification of the 5′ end with phosphate or phosphate analogues.
  • Nucleic acids of the invention, on the first or second strand, may be 5′ phosphorylated or include a phosphoryl analogue at the 5′ prime terminus.
  • 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing.
  • Suitable modifications include: 5′-monophosphate ((HO) 2 (O)P—O-5′); 5′-diphosphate ((HO) 2 (O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO) 2 (O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO) 2 (S)P—O-5′); 5′
  • moieties may be linked to the 5′ terminus of the first strand or the second strand. These include abasic ribose moiety, abasic deoxyribose moiety, modifications abasic ribose and abasic deoxyribose moieties including 2′-O alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and modifications thereof, C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5′OMe nucleotide; and nucleotide analogues including 4′,5′-methylene nucleotide; 1-( ⁇ -D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropy
  • a C-terminal “—OH” moiety may be substituted for a C-terminal “—NH 2 ” moiety, and vice-versa.
  • 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.
  • This terminal 5′ (E)-vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage.
  • the first strand of the nucleic acid may comprise formula (I):
  • ‘(vp)-’ is the 5′ (E)-vinylphosphonate
  • ‘N’ is a nucleotide
  • ‘po’ is a phosphodiester linkage
  • n is from 1 to (the total number of nucleotides in the first strand—2), preferably wherein n is from 1 to (the total number of nucleotides in the first strand—3), more preferably wherein n is from 1 to (the total number of nucleotides in the first strand—4).
  • the terminal 5′ (E)-vinylphosphonate nucleotide is an RNA nucleotide, preferably a (vp)-U.
  • a terminal 5′ (E)-vinylphosphonate nucleotide is a nucleotide wherein the natural phosphate group at the 5′-end has been replaced with a E-vinylphosphonate, in which the bridging 5′-oxygen atom of the terminal nucleotide of the 5′ phosphorylated strand is replaced with a methynyl (—CH ⁇ ) group:
  • a 5′ (E)-vinylphosphonate is a 5′ phosphate mimic.
  • a biological mimic is a molecule that is capable of carrying out the same function as and is structurally very similar to the original molecule that is being mimicked.
  • 5′ (E)-vinylphosphonate mimics the function of a normal 5′ phosphate, e.g. enabling efficient RISC loading.
  • 5′ (E) vinylphosphonate is capable of stabilizing the 5′-end nucleotide by protecting it from dephosphorylation by enzymes such as phosphatases.
  • the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide at its 5′ end, the terminal 5′ (E)-vinylphosphonate nucleotide is linked to the second nucleotide in the first strand by a phosphodiester linkage and the first strand comprises a) more than 1 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.
  • 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.
  • the first strand and/or the second strand of the nucleic acid comprises more than one phosphorothioate and/or more than one phosphorodithioate linkage.
  • the first strand and/or the second strand of the nucleic acid comprises a phosphorothioate or phosphorodithioate linkage between the terminal two 3′ nucleotides or phosphorothioate or phosphorodithioate linkages between the terminal three 3′ nucleotides.
  • the linkages between the other nucleotides in the first strand and/or the second strand are phosphodiester linkages.
  • the first strand and/or the second strand of the nucleic acid comprises a phosphorothioate linkage between the terminal two 5′ nucleotides or a phosphorothioate linkages between the terminal three 5′ nucleotides.
  • the nucleic acid of the present invention comprises one or more phosphorothioate or phosphorodithioate modifications on one or more of the terminal ends of the first and/or the second strand.
  • each or either end of the first strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleoside linkage).
  • each or either end of the second strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleoside linkage).
  • the nucleic acid comprises a phosphorothioate linkage between the terminal two or three 3′ nucleotides and/or 5′ nucleotides of the first and/or the second strand.
  • the nucleic acid comprises a phosphorothioate linkage between each of the terminal three 3′ nucleotides and the terminal three 5′ nucleotides of the first strand and of the second strand.
  • all remaining linkages between nucleotides of the first and/or of the second strand are phosphodiester linkages.
  • the nucleic acid comprises a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3′ end of the first strand and/or comprises a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3′ end of the second strand and/or a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 5′ end of the second strand and comprises a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5′ end of the first strand.
  • the nucleic acid comprises a phosphorothioate linkage between the terminal three 3′ nucleotides and the terminal three 5′ nucleotides of the first strand and of the second strand.
  • all remaining linkages between nucleotides of the first and/or of the second strand are phosphodiester linkages.
  • nucleic acid in one aspect, the nucleic acid:
  • nucleic acid in one aspect, the nucleic acid:
  • a phosphorodithioate linkage in the nucleic acid of the invention reduces the variation in the stereochemistry of a population of nucleic acid molecules compared to molecules comprising a phosphorothioate in that same position.
  • Phosphorothioate linkages introduce chiral centres and it is difficult to control which non-linking oxygen is substituted for sulphur.
  • the use of a phosphorodithioate ensures that no chiral centre exists in that linkage and thus reduces or eliminates any variation in the population of nucleic acid molecules, depending on the number of phosphorodithioate and phosphorothioate linkages used in the nucleic acid molecule.
  • the nucleic acid comprises a phosphorodithioate linkage between the two terminal nucleotides at the 3′ end of the first strand and a phosphorodithioate linkage between the two terminal nucleotides at the 3′ end of the second strand and a phosphorodithioate linkage between the two terminal nucleotides at the 5′ end of the second strand and comprises a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5′ end of the first strand.
  • the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide at its 5′ end.
  • This terminal 5′ (E)-vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage.
  • all the linkages between the nucleotides of both strands other than the linkage 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 at the 5′ end of the second strand are phosphodiester linkages.
  • the nucleic acid comprises a phosphorothioate linkage between each of the three terminal 3′ nucleotides and/or between each of the three terminal 5′ nucleotides on the first strand, and/or between each of the three terminal 3′ nucleotides and/or between each of the three terminal 5′ nucleotides of the second strand when there is no phosphorodithioate linkage present at that end.
  • No phosphorodithioate linkage being present at an end means that the linkage between the two terminal nucleotides, or preferably between the three terminal nucleotides of the nucleic acid end in question are linkages other than phosphorodithioate linkages.
  • all the linkages of the nucleic acid between the nucleotides of both strands other than the linkage 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 at the 5′ end of the second strand are phosphodiester linkages.
  • the phosphate linker can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates).
  • the replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.
  • the phosphate groups can also individually be replaced by non-phosphorus containing connectors.
  • moieties which 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.
  • replacements may include the methylenecarbonylamino and methylenemethylimino groups.
  • the phosphate linker and ribose sugar may be replaced by nuclease resistant nucleotides.
  • nuclease resistant nucleotides examples include the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
  • PNA surrogates may be used.
  • the nucleic acid which is preferably an siRNA that inhibits expression of the complement component C3, preferably via RNAi, and preferably in a cell, comprises one or more or all of:
  • the nucleic acids of the invention may be conjugated to a ligand.
  • Efficient delivery of oligonucleotides, in particular double-stranded nucleic acids of the invention, to cells in vivo is important and requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins.
  • One method of achieving specific targeting is to conjugate a ligand to the nucleic acid.
  • the ligand helps in targeting the nucleic acid to a target cell which has a cell surface receptor that binds to and internalizes the conjugated ligand.
  • a ligand which can mediate internalization of the nucleic acid into a target cell by mechanisms other than receptor mediated endocytosis may alternatively be conjugated to a nucleic acid of the invention for cell or tissue specific targeting.
  • ASGP-R asialoglycoprotein receptor complex
  • the ASGP-R complex is composed of varying ratios of multimers of membrane ASGR1 and ASGR2 receptors, which are highly abundant on 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 having three GalNAc ligands and comprising phosphate groups are known and are described in Dubber et al. (Bioconjug. Chem. 2003 January-February; 14(1):239-46).
  • the ASGP-R complex shows a 50-fold higher affinity for N-Acetyl-D-Galactosamine (GalNAc) than D-Gal.
  • the ASGP-R complex recognizes specifically terminal ⁇ -galactosyl subunits of glycosylated proteins or other oligosaccharides (Weigel, P. H. et. al., Biochim. Biophys. Acta. 2002 Sep. 19; 1572(2-3):341-63) and can be used for delivering a drug to the liver's hepatocytes expressing the receptor complex by covalent coupling of 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 significantly increased by the multi-valency effect, which is achieved by the repetition of the targeting moiety (Biessen E A, et al., J Med Chem. 1995 Apr. 28; 38(9):1538-46).
  • the ASGP-R complex is a mediator for an active uptake of terminal ⁇ -galactosyl containing glycoproteins to the cell's endosomes.
  • the ASGPR is highly suitable for targeted delivery of drug candidates conjugated to such ligands like, e.g., nucleic acids into receptor-expressing cells (Akinc et al., Mol Ther. 2010 July; 18(7):1357-64).
  • the ligand can comprise a saccharide that is selected to have an affinity for at least one type of receptor on a target cell.
  • the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex described before (ASGP-R).
  • the saccharide may be selected from N-acetyl galactosamine, mannose, galactose, glucose, glucosamine and fucose.
  • the saccharide may be N-acetyl galactosamine (GalNAc).
  • a ligand for use in the present invention may therefore comprise (i) one or more N-acetyl galactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GalNAc moieties to a nucleic acid as defined in any preceding aspects.
  • the linker may be a monovalent structure or bivalent or trivalent or tetravalent branched structure.
  • the nucleotides may be modified as defined herein.
  • the ligand may therefore comprise GalNAc.
  • the nucleic acid is conjugated to a ligand comprising a compound of formula (II):
  • the branching unit “A” preferably branches into three in order to accommodate three saccharide ligands.
  • the branching unit is preferably covalently attached to the remaining tethered portions of the ligand and the nucleic acid.
  • the branching unit may comprise a branched aliphatic group comprising groups selected from alkyl, amide, disulphide, polyethylene glycol, ether, thioether and hydroxyamino groups.
  • the branching unit may comprise groups selected from alkyl and ether groups.
  • the branching unit A may have a structure selected from:
  • each A 1 independently represents O, S, C ⁇ O or NH; and each n independently represents an integer from 1 to 20.
  • the branching unit may have a structure selected from:
  • each A 1 independently represents O, S, C ⁇ O or NH; and each n independently represents an integer from 1 to 20.
  • the branching unit may have a structure selected from:
  • the branching unit may have the structure:
  • the branching unit may have the structure:
  • the branching unit may have the structure:
  • the branching unit A may have a structure selected from:
  • R1 is hydrogen or C1-C10 alkylene
  • R2 is C1-C10 alkylene.
  • the branching unit consists of only a carbon atom.
  • the “X 3 ” portion is a bridging unit.
  • the bridging unit is linear and is covalently bound to the branching unit and the nucleic acid.
  • X 3 may be selected from —C 1 -C 20 alkylene-, —C 2 -C 20 alkenylene-, an alkylene ether of formula —(C 1 -C 20 alkylene)-O—(C 1 -C 20 alkylene)-, —C(O)—C 1 -C 20 alkylene-, —C 0 -C 4 alkylene(Cy)C 0 -C 4 alkylene- wherein Cy represents a substituted or unsubstituted 5 or 6 membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, —C 1 -C 4 alkylene-NHC(O)—C 1 -C 4 alkylene-, —C 1 -C 4 alkylene-C(O)NH—C 1 -C 4 alkylene-, —C 1 -C 4 alkylene-SC(O)—C 1 -C 4 alkylene-, —C 1 -C 4 al
  • X 3 may be an alkylene ether of formula —(C 1 -C 20 alkylene)-O—(C 1 -C 20 alkylene)-.
  • X 3 may be an alkylene ether of formula —(C 1 -C 20 alkylene)-O—(C 4 -C 20 alkylene)-, wherein said (C 4 -C 20 alkylene) is linked to Z.
  • X 3 may be selected from the group consisting of —CH 2 —O—C 3 H 6 —, —CH 2 —O—C 4 H 8 —, —CH 2 —O—C 6 H 12 — and —CH 2 —O—C 8 H 16 —, especially —CH 2 —O—C 4 H 8 —, —CH 2 —O—C 6 H 12 — and —CH 2 —O—C 8 H 16 —, wherein in each case the —CH 2 — group is linked to A.
  • the nucleic acid is conjugated to a ligand comprising a compound of formula (III):
  • the branching unit A may have the structure:
  • the branching unit A may have the structure:
  • X 3 may be C 1 -C 20 alkylene.
  • X 3 is selected from the group consisting of —C 3 H 6 —, —C 4 H 8 —, —C 6 H 12 — and —C 8 H 16 —, especially —C 4 H 8 —, —C 6 H 12 — and —C 8 H 16 —.
  • the nucleic acid is conjugated to a ligand comprising a compound of formula (IV):
  • the branching unit may comprise carbon.
  • the branching unit is a carbon.
  • X 3 may be selected from the group consisting of —CH 2 —O—C 4 H 8 —, —CH 2 —O—C 5 H 10 —, —CH 2 —O—C 6 H 12 —, —CH 2 —O—C 7 H 14 —, and —CH 2 —O—C 3 H 16 —.
  • X 3 is selected from the group consisting of —CH 2 —O—C 4 H 8 —, —CH 2 —O—C 6 H 12 — and —CH 2 —O—CH 16 .
  • X 1 may be (—CH 2 —CH 2 —O)(—CH 2 ) 2 —.
  • X 1 may be (—CH 2 —CH 2 —O) 2 (—CH 2 ) 2 —.
  • X 1 may be (—CH 2 —CH 2 —O) 3 (—CH 2 ) 2 —.
  • X 1 is (—CH 2 —CH 2 —O) 2 (—CH 2 ) 2 —.
  • X 1 represents C 3 -C 6 alkylene.
  • X 1 may be propylene.
  • X 1 may be butylene.
  • X 1 may be pentylene.
  • X 1 may be hexylene.
  • the alkyl is a linear alkylene. In particular, X 1 may be butylene.
  • X 2 represents an alkylene ether of formula —C 3 H 6 —O—CH 2 — i.e. C 3 alkoxy methylene, or —CH 2 CH 2 CH 2 OCH 2 —.
  • P when P represents a modified phosphate group, P can be represented by:
  • Y 1 and Y 2 each independently represent ⁇ O, ⁇ S, —O ⁇ , —OH, —SH, —BH 3 , —OCH 2 CO 2 , —OCH 2 CO 2 R x , —OCH 2 C(S)OR x , and —OR x , wherein R x represents C 1 -C 6 alkyl and wherein indicates attachment to the remainder of the compound.
  • modified phosphate it is meant a phosphate group wherein one or more of the non-linking oxygens is replaced.
  • modified phosphate groups include phosphorothioate, phosphorodithioates, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulphur.
  • One, each or both non-linking oxygens in the phosphate group can be independently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).
  • the phosphate can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates).
  • the replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.
  • Y 1 may represent —OH and Y 2 may represent ⁇ O or ⁇ S; or
  • Y 1 may represent —O ⁇ and Y 2 may represent ⁇ O or ⁇ S;
  • Y 1 may represent ⁇ O and Y 2 may represent —CH 3 , —SH, —OR x , or —BH 3
  • Y 1 may represent ⁇ S and Y 2 may represent —CH 3 , OR x or —SH.
  • the modified phosphate group is a thiophosphate group.
  • Thiophosphate groups include bithiophosphate (i.e. where Y 1 represents ⁇ S and Y 2 represents —S ⁇ ) and monothiophosphate (i.e. where Y 1 represents —O ⁇ and Y 2 represents ⁇ S, or where Y 1 represents ⁇ O and Y 2 represents —S ⁇ ).
  • P is a monothiophosphate.
  • conjugates having thiophosphate groups in replacement of phosphate groups have improved potency and duration of action in vivo.
  • P may also be an ethylphosphate (i.e. where Y 1 represents ⁇ O and Y 2 represents OCH 2 CH 3 ).
  • the saccharide may be selected to have an affinity for at least one type of receptor on a target cell.
  • the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex (ASGP-R).
  • the saccharide may be selected from N-acetyl with one or more of galactosamine, mannose, galactose, glucose, glucosamine and fructose.
  • a ligand to be used in the present invention may include N-acetyl galactosamine (GalNAc).
  • the compounds of the invention may have 3 ligands, which will each preferably include N-acetyl galactosamine.
  • GalNAc refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetyl galactosamine.
  • Reference to “GalNAc” or “N-acetyl galactosamine” includes both the ⁇ -form: 2-(Acetylamino)-2-deoxy- ⁇ -D-galactopyranose and the ⁇ -form: 2-(Acetylamino)-2-deoxy- ⁇ -D-galactopyranose.
  • both the ⁇ -form: 2-(Acetylamino)-2-deoxy- ⁇ -D-galactopyranose and ⁇ -form: 2-(Acetylamino)-2-deoxy- ⁇ -D-galactopyranose may be used interchangeably.
  • the compounds of the invention comprise the ⁇ -form, 2-(Acetylamino)-2-deoxy- ⁇ -D-galactopyranose.
  • the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a triantennary ligand with one of the following structures:
  • Z is any nucleic acid as defined herein.
  • the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a triantennary ligand with the following structures:
  • Z is any nucleic acid as defined herein.
  • a ligand of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein can be attached at the 3′-end of the first (antisense) strand and/or at any of the 3′ and/or 5′ end of the second (sense) strand.
  • the nucleic acid can comprise more than one ligand of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein.
  • a single ligand of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein is preferred because a single such ligand is sufficient for efficient targeting of the nucleic acid to the target cells.
  • at least the last two, preferably at least the last three and more preferably at least the last four nucleotides at the end of the nucleic acid to which the ligand is attached are linked by a phosphodiester linkage.
  • the 5′-end of the first (antisense) strand is not attached to a ligand of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein, since a ligand in this position can potentially interfere with the biological activity of the nucleic acid.
  • a nucleic acid with a single ligand of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein at the 5′ end of a strand is easier and therefore cheaper to synthesise than the same nucleic acid with the same ligand at the 3′ end.
  • a single ligand of any of formulae (II), (III) or (IV) or any one of the triantennary ligands disclosed herein is covalently attached to (conjugated with) the 5′ end of the second strand of the nucleic acid.
  • the first strand of the nucleic acid is a compound of formula (V):
  • the second strand is a compound of formula (VI):
  • L 1 in formulae (V) and (VI) is of formula (VII):
  • the first strand is a compound of formula (VIII)
  • the second strand is a compound of formula (IX):
  • the first strand of the nucleic acid is a compound of formula (X):
  • the second strand is a compound of formula (XI):
  • b is 0, c is 1 and d is 1; b is 1, c is 0 and d is 1; b is 1, c is 1 and d is 0; or b is 1, c is 1 and d is 1 in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI).
  • b is 0, c is 1 and d is 1; b is 1, c is 0 and d is 1; or b is 1, c is 1 and d is 1.
  • b is 0, c is 1 and d is 1.
  • Y is O in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI).
  • Y is S.
  • Y is independently selected from O or S in the different positions in the formulae.
  • R 1 is H or methyl in any of the nucleic acids of formulae (VIII) and (IX). In one aspect, R 1 is H. In another aspect, R 1 is methyl.
  • n is 0, 1, 2 or 3 in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI).
  • n is 0.
  • F moieties in any of the nucleic acids of formulae (X) and (XI) include (CH 2 ) 1-6 e.g. (CH 2 ) 1-4 e.g. CH 2 , (CH 2 ) 4 , (CH 2 ) 5 or (CH 2 ) 6 , or CH 2 O(CH 2 ) 2-3 , e.g. CH 2 O(CH 2 )CH 3 .
  • L 2 in formulae (X) and (XI) is:
  • L 2 is:
  • L 2 is:
  • L 2 is:
  • Y is O or S.
  • L in the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI), is selected from the group comprising, or preferably consisting of:
  • L is:
  • L 2 in the nucleic acids of formulae (X) and (XI) is typically the same. Between moieties bracketed by b, c and d, L 2 may be the same or different.
  • L 2 in the moiety bracketed by c is the same as the L 2 in the moiety bracketed by d. In an embodiment, L 2 in the moiety bracketed by c is not the same as L 2 in the moiety bracketed by d. In an embodiment, the L 2 in the moieties bracketed by b, c and d is the same, for example when the linker moiety is a serinol-derived linker moiety.
  • Serinol derived linker moieties may be based on serinol in any stereochemistry i.e. derived from L-serine isomer, D-serine isomer, a racemic serine or other combination of isomers.
  • the serinol-GalNAc moiety (SerGN) has the following stereochemistry:
  • 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:
  • 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:
  • 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:
  • the nucleic acid is conjugated to a triantennary ligand with the following structure:
  • nucleic acid is conjugated to the ligand via the phosphate group of the ligand a) to the last nucleotide at the 5′ end of the second strand; b) to the last nucleotide at the 3′ end of the second strand; or c) to the last nucleotide at the 3′ end of the first strand.
  • the cells that are targeted by the nucleic acid with a ligand are hepatocytes.
  • the GalNAc may be substituted for any other targeting ligand, such as those mentioned herein, in particular mannose, galactose, glucose, glucosamine and fucose.
  • a particularly preferred embodiment is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO: 409 and the second strand optionally comprises or consists of SEQ ID NO: 423.
  • This nucleic acid can be further conjugated to a ligand.
  • One aspect of the invention is EJ0020.
  • An alternative particularly preferred embodiment is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO: 420 and the second strand optionally comprises or consists of SEQ ID NO: 424.
  • This nucleic acid can be further conjugated to a ligand.
  • One aspect of the invention is EV0212.
  • An alternative particularly preferred embodiment is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO: 417 and the second strand optionally comprises or consists of SEQ ID NO: 425.
  • This nucleic acid can be further conjugated to a ligand.
  • Most preferred is an siRNA that consists of SEQ ID NO: 417 and SEQ ID NO: 418.
  • One aspect of the invention is EV0210. Preliminary NHP data show that this siRNA is surprisingly potent in vivo in higher species.
  • the nucleic acid is conjugated to a ligand that comprises a lipid, and more preferably, a ligand that comprises a cholesterol.
  • the present invention also provides compositions comprising a nucleic acid of the invention.
  • the nucleic acids and compositions may be used as medicaments or as diagnostic agents, alone or in combination with other agents.
  • one or more nucleic acid(s) of the invention can be combined with a delivery vehicle (e.g., liposomes) and/or excipients, such as carriers, diluents.
  • a delivery vehicle e.g., liposomes
  • excipients such as carriers, diluents.
  • Other agents such as preservatives and stabilizers can also be added.
  • Pharmaceutically acceptable salts or solvates of any of the nucleic acids of the invention are likewise within the scope of the present invention.
  • Methods for the delivery of nucleic acids are known in the art and within the knowledge of the person skilled in the art.
  • compositions disclosed herein are particularly pharmaceutical compositions. Such compositions are suitable for administration to a subject.
  • the composition comprises a nucleic acid disclosed herein, or a pharmaceutically 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.
  • a solvent preferably water
  • a delivery vehicle preferably water
  • a physiologically acceptable excipient and/or a carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative.
  • Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Subcutaneous or transdermal modes of administration may be particularly suitable for the compounds described herein.
  • the therapeutically effective amount of a nucleic acid of the present invention will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts.
  • the dosage sizes and dosing regimen most appropriate for human use may be guided by the results obtained by the present invention, and may be confirmed in properly designed clinical trials.
  • An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the skilled person.
  • Nucleic acids of the present invention, or salts thereof may be formulated as pharmaceutical compositions prepared for storage or administration, which typically comprise a therapeutically effective amount of a nucleic acid of the invention, or a salt thereof, in a pharmaceutically acceptable carrier.
  • the nucleic acid or conjugated nucleic acid of the present invention can also be administered in combination with other therapeutic compounds, either administrated separately or simultaneously, e.g., as a combined unit dose.
  • the invention also includes a composition comprising one or more nucleic acids according to the present invention in a physiologically/pharmaceutically acceptable excipient, such as a stabilizer, preservative, diluent, buffer, and the like.
  • the composition comprises a nucleic acid disclosed herein and a further therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.
  • the further therapeutic agent is an agent that targets, preferably inhibits the expression or the activity, of the complement component C3 or of another element, such as a protein, of the immune system or more specifically of the complement pathway.
  • the further therapeutic agent is one of the following: a) a peptide that inhibits the expression or activity of one of the components of the complement pathway, preferably either C3 or C5 or one of their subunits; b) an antibody that specifically binds under physiological conditions to one of the components of the complement pathway, preferably either C3 or C5 or one of their subunits; c) Eculizumab or an antigen-binding derivative thereof.
  • Eculizumab is a humanised monoclonal antibody that specifically binds to the complement component C5 and is commercialised under the trade name SOLIRIS®. It specifically binds the complement component C5 with high affinity and inhibits cleavage of C5 to C5a and C5b.
  • the antibody is for example described in the patent EP 0 758 904 B1 and its family members.
  • two or more nucleic acids of the invention with different sequences may be administered simultaneously or sequentially.
  • the present invention provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of different nucleic acids of the invention and at least one pharmaceutically acceptable carrier.
  • a unit dose may contain between about 0.01 mg/kg and about 100 mg/kg body weight of nucleic acid or conjugated nucleic acid.
  • the dose can be from 10 mg/kg to 25 mg/kg body weight, or 1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 1 mg/kg body weight, or 0.1 mg/kg to 0.5 mg/kg body weight, or 0.5 mg/kg to 1 mg/kg body weight.
  • the dose can be from 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, wherein “about” is a deviation of up to 30%, preferably up to 20%, more preferably up to 10%, yet more preferably up to 5% and most preferably 0% from the indicated value. Dosage levels may also be calculated via other parameters such as, e.g., body surface area.
  • a particularly preferred embodiment is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO: 409 and the second strand optionally comprises or consists of SEQ ID NO: 423.
  • This nucleic acid can be further conjugated to a ligand.
  • One aspect of the invention is EJ0020.
  • a dose unit of these nucleic acids preferably comprises about 1 mg/kg to about 5 mg/kg body weight, or 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 C3 mRNA level in the liver and/or the C3 protein level in the plasma or blood of a subject treated by a dose unit of the nucleic acid is preferably decreased at the time point of maximum effect by at least 30%, at least 40%, at least 50%, at least 60% or at least 70% as compared to a control that was not treatment with the nucleic acid or treated with a control nucleic acid under comparable conditions.
  • the 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.
  • a relatively low dosage is administered at relatively infrequent intervals over a relatively long period of time. Some subjects may continue to receive treatment over their lifetime.
  • a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient may be switched to a suitable prophylactic dosing regimen.
  • compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing deleterious side effects to the subject or patient.
  • a selected dosage level will depend upon a variety of factors, such as pharmacokinetic factors, including the activity of the particular nucleic acid or composition employed, the route of administration, the time of administration, the rate of excretion of the particular nucleic acid being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject or patient being treated, and similar factors well known in the medical arts.
  • factors such as pharmacokinetic factors, including the activity of the particular nucleic acid or composition employed, the route of administration, the time of administration, the rate of excretion of the particular nucleic acid being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject or patient being treated, and similar factors well known in the medical arts.
  • the pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in a lyophilized form.
  • the pharmaceutical compositions can be in unit dosage form.
  • the composition is divided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of a pen.
  • Compositions may be formulated for any suitable route and means of administration.
  • compositions and medicaments of the present invention may be administered to a mammalian subject in a pharmaceutically effective dose.
  • the mammal may be selected from a human, a non-human primate, a simian or prosimian, a dog, a cat, a horse, cattle, a pig, a goat, a sheep, a mouse, a rat, a hamster, a hedgehog and a guinea pig, or other species of relevance.
  • C3 as used herein denotes nucleic acid or protein in any of the above-mentioned species, if expressed therein naturally or artificially, but preferably this wording denotes human nucleic acids or proteins.
  • compositions of the invention may be administered alone or in combination with one or more other therapeutic or diagnostic agents.
  • a combination therapy may include a nucleic acid of the present invention combined with at least one other therapeutic agent selected based on the particular patient, disease or condition to be treated.
  • other such agents include, inter alia, a therapeutically active small molecule or polypeptide, a single chain antibody, a classical antibody or fragment thereof, or a nucleic acid molecule which modulates gene expression of one or more additional genes, and similar modulating therapeutics which may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen.
  • compositions are typically sterile and stable under the conditions of manufacture and storage.
  • the composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier may be a solvent or dispersion medium containing, for example, water, alcohol such as ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixtures.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by use of surfactants according to formulation chemistry well known in the art.
  • isotonic agents e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be desirable in the composition.
  • Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatine.
  • nucleic acid or a composition disclosed herein for use as a medicament.
  • the nucleic acid or composition is preferably for use in the prevention, decrease of the risk of suffering from, or treatment of a disease, disorder or syndrome.
  • the present invention provides a nucleic acid for use, alone or in combination with one or more additional therapeutic agents in a pharmaceutical composition, for treatment or prophylaxis of conditions, diseases and disorders responsive to inhibition of C3 expression.
  • One aspect of the invention is the use of a nucleic acid or a composition as disclosed herein in the prevention, decrease of the risk of suffering from, or treatment of a disease, disorder or syndrome.
  • Nucleic acids and pharmaceutical compositions of the invention may be used in the treatment of a variety of conditions, disorders or diseases. Treatment with a nucleic acid of the invention preferably leads to in vivo C3 depletion, preferably in the liver and/or in blood. As such, nucleic acids of the invention, and compositions comprising them, will be useful in methods for treating a variety of pathological disorders in which inhibiting the expression of C3 may be beneficial.
  • the present invention provides methods for treating a disease, disorder or syndrome comprising the step of administering to a subject in need thereof a therapeutically effective amount of a nucleic acid of the invention.
  • the invention thus provides methods of treatment or prevention of a disease, disorder or syndrome, the method comprising the step of administering to a subject (e.g., a patient) in need thereof a therapeutically effective amount of a nucleic acid or pharmaceutical composition comprising a nucleic acid of the invention.
  • the most desirable therapeutically effective amount is an amount that will produce a desired efficacy of a particular treatment selected by one of skill in the art for a given subject in need thereof. This amount will vary depending upon a variety of factors understood by the skilled worker, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
  • nucleic acids and pharmaceutical compositions of the invention may be used to treat or prevent a disease, disorder or syndrome.
  • the present invention provides methods for treating a disease, disorder or syndrome in a mammalian subject, such as a human, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a nucleic acid as disclosed herein.
  • Administration of a “therapeutically effective dosage” of a nucleic acid of the invention may result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • Nucleic acids of the invention may be beneficial in treating or diagnosing a disease, disorder or syndrome that may be diagnosed or treated using the methods described herein. Treatment and diagnosis of other diseases, disorders or syndromes are also considered to fall within the scope of the present invention.
  • One aspect of the invention is a method of preventing, decreasing the risk of suffering from, or treating a disease, disorder or syndrome comprising administering a pharmaceutically effective dose or amount a nucleic acid or a composition disclosed herein to an individual in need of treatment, preferably wherein the nucleic acid or composition is administered to the subject subcutaneously, intravenously or by oral, rectal, pulmonary, intramuscular or intraperitoneal administration. Preferably, it is administered subcutaneously.
  • the disease, disorder or syndrome to be prevented, or treated with a nucleic acid or composition disclosed herein is preferably a complement-mediated disease, disorder or syndrome or a disease disorder or syndrome associated with the complement pathway.
  • the disease, disorder or syndrome to be prevented or treated with a nucleic acid or composition disclosed herein is preferably associated with aberrant activation and/or over-activation (hyper-activation) of the complement pathway and/or with over-expression or ectopic expression or localisation or accumulation of the complement component C3.
  • C3 Glomerulopathy 3G
  • C3 accumulates in the kidney glomeruli.
  • the aberrant or over activation of the complement pathway to be prevented or treated can have genetic causes or can be acquired.
  • the disease, disorder or syndrome to be prevented or treated is C3 Glomerulopathy (C3G).
  • the disease, disorder or syndrome to be prevented or treated with a nucleic acid or composition disclosed herein is preferably a) selected from the group comprising, and preferably consisting of 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 Autoantibodies-associated Vasculitis (ANCA-AV), dysbiotic periodontal Disease, Mal
  • a nucleic acid or compositions disclosed herein may be for use in a regimen comprising treatments once or twice weekly, every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, every twelve weeks, every three months, every four months, every five months, every six months or in regimens with varying dosing frequency such as combinations of the before-mentioned intervals.
  • the nucleic acid or composition may be for use subcutaneously, intravenously or using any other application routes such as oral, rectal, pulmonary, or intraperitoneal. Preferably, it is for use subcutaneously.
  • the C3 expression may be inhibited compared to untreated cells and/or subjects by a range from 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 intermediate values.
  • the level of inhibition may allow treatment of a disease associated with C3 expression or overexpression or complement over-activation, or may serve to further investigate the functions and physiological roles of the C3 gene products.
  • the level of inhibition is preferably measured in the liver or in the blood or in the kidneys, preferably in the blood, of the subject treated with the nucleic acid or composition.
  • a nucleic acid or composition as disclosed herein in the manufacture of a medicament for treating a disease, disorder or syndromes, such as those as listed above or additional pathologies associated with elevated levels of C3, preferably in the blood or in the kidneys, or over activation of the complement pathway, or additional therapeutic approaches where inhibition of C3 expression is desired.
  • a medicament is a pharmaceutical composition.
  • nucleic acids of the invention and pharmaceutically acceptable salts and solvates thereof constitutes an individual embodiment of the invention.
  • nucleic acid or composition may be administered in a regimen comprising treatments twice every week, once every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight to twelve or more weeks or in regimens with varying dosing frequency such as combinations of the before-mentioned intervals.
  • the nucleic acid or conjugated nucleic acid may be for use subcutaneously or intravenously or other application routes such as oral, rectal or intraperitoneal.
  • a nucleic acid of the invention may be administered by any appropriate administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g., topical administration of a cream, gel or ointment, or by means of a transdermal patch).
  • any appropriate administration pathway known in the art including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g., topical administration of a cream, gel or ointment, or by means of a transdermal patch).
  • Parental administration is typically associated with injection at or in communication with the intended site of action, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration.
  • nucleic acids confers nuclease stability in serum and makes for example subcutaneous application route feasible.
  • Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and/or tonicity adjusting agents such as, e.g., sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like.
  • acids or bases such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like.
  • Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Sterile injectable solutions may be prepared by incorporating a nucleic acid in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by sterilization microfiltration.
  • Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a dispersion medium and optionally other ingredients, such as those described above.
  • the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient in addition to any additional desired ingredient from a sterile-filtered solution thereof.
  • nucleic acid of the invention When a therapeutically effective amount of a nucleic acid of the invention is administered by, e.g., 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 consideration appropriate pH, isotonicity, stability, and the like, are within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to a 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 vehicle as known in the art.
  • a pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those of skill in the art.
  • nucleic acid which can be combined with a carrier material 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. In general, it will be an amount of the composition that produces an appropriate therapeutic effect under the particular circumstances. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of nucleic acid, from about 0.1% to about 70%, or from about 1% to about 30% of nucleic acid in combination with a pharmaceutically acceptable carrier.
  • the nucleic acid may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.
  • a controlled release formulation 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 the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
  • Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a dose may be administered, several divided doses may be administered overtime, or the dose may be proportionally reduced or increased as indicated by the particular circumstances of the therapeutic situation, on a case by case basis. It is especially advantageous to formulate parenteral compositions in dosage unit forms for ease of administration and uniformity of dosage when administered to the subject or patient.
  • a dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce a desired therapeutic effect.
  • the specification for the dosage unit forms of the invention depend on the specific characteristics of the active compound and the particular therapeutic effect(s) to be achieved and the treatment and sensitivity of any individual patient.
  • nucleic acid or composition of the present invention can be produced using routine methods in the art including chemical synthesis, such as solid phase chemical synthesis.
  • Nucleic acids or compositions of the invention may be administered with one or more of a variety of medical devices known in the art.
  • a nucleic acid of the invention may be administered with a needleless hypodermic injection device.
  • implants and modules useful in the present invention are in the art, including e.g., implantable micro-infusion pumps for controlled rate delivery; devices for administering through the skin; infusion pumps for delivery at a precise infusion rate; 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.
  • the nucleic acid or composition of the invention may be formulated to ensure a desired distribution in vivo.
  • a therapeutic compound or composition of the invention to a particular in vivo location, they can be formulated, for example, in liposomes which may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery.
  • the invention is characterized by high specificity at the molecular and tissue-directed delivery level.
  • the sequences of the nucleic acids of the invention are highly specific for their target, meaning that they do not inhibit the expression of genes that they are not designed to target or only minimally inhibit the expression of genes that they are not designed to target and/or only inhibit the expression of a low number of genes that they are not designed to target.
  • a further level of specificity is achieved when nucleic acids are linked to a ligand that is specifically recognised and internalised by a particular cell type. This is for example the case when a nucleic acid is linked to a ligand comprising GalNAc moieties, which are specifically recognised and internalised by hepatocytes.
  • the present invention thus provides nucleic acids of the invention linked to a ligand comprising one or more GalNAc moieties, or comprising one or more other moieties that confer cell-type or tissue-specific internalisation of the nucleic acid thereby conferring additional specificity of target gene knockdown by RNA interference.
  • the nucleic acid as described herein may be formulated with a lipid in the form of a liposome. Such a formulation may be described in the art as a lipoplex.
  • the composition with a lipid/liposome may be used to assist with delivery of the nucleic acid of the invention to the target cells.
  • the lipid delivery system herein described may be used as an alternative to a conjugated ligand.
  • the modifications herein described may be present when using the nucleic acid of the invention with a lipid delivery system or with a ligand conjugate delivery system.
  • Such a lipoplex may comprise a lipid composition comprising:
  • the cationic lipid may be an amino cationic lipid.
  • the cationic lipid may have the formula (XII):
  • X represents O, S or NH
  • R 1 and R 2 each independently represents a C 4 -C 22 linear or branched alkyl chain or a C 4 -C 22 linear or branched alkenyl chain with one or more double bonds, wherein the alkyl or alkenyl chain optionally contains an intervening ester, amide or disulfide;
  • R 3 and R 4 each independently represent hydrogen, methyl, ethyl, a mono- or polyamine moiety, or R 3 and R 4 together form a heterocyclyl ring;
  • R3 and R4 each independently represent hydrogen, methyl, ethyl, a mono- or polyamine moiety, or R 3 and R 4 together form a heterocyclyl ring, or R 3 represents hydrogen and R 4 represents C(NH)(NH 2 ).
  • the cationic lipid may have the formula (XIII):
  • the cationic lipid may have the formula (XIV):
  • the content of the cationic lipid component may be from about 55 mol % to about 65 mol % of the overall lipid content of the composition.
  • the cationic lipid component is about 59 mol % of the overall lipid content of the composition.
  • compositions can further comprise a steroid.
  • the steroid may be cholesterol.
  • the content of the steroid may be from about 26 mol % to about 35 mol % of the overall lipid content of the lipid composition. More particularly, the content of steroid may be about 30 mol % of the overall lipid content of the lipid composition.
  • the phosphatidylethanolamine phospholipid may be selected from the group consisting of 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 (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
  • 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 to 5 mol % of the overall lipid content of the composition.
  • the content of the cationic lipid component in the composition may be from about 55 mol % to about 65 mol % of the overall lipid content of the lipid composition, preferably about 59 mol % of the overall lipid content of the lipid composition.
  • the composition may have a molar ratio of the components of 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.
  • composition may comprise a cationic lipid having the structure
  • Neutral liposome compositions may be formed from, for example, dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).
  • Anionic liposome compositions may be formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes may be formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
  • Another type of liposomal composition may be formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
  • a positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells.
  • DOTMA analogues can also be used to form liposomes.
  • lipids described herein may also be used to form liposomes.
  • a liposome containing a nucleic acid can be prepared by a variety of methods.
  • the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component.
  • the lipid component can be an amphipathic cationic lipid or lipid conjugate.
  • the detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine.
  • the nucleic acid preparation is then added to the micelles that include the lipid component.
  • the cationic groups on the lipid interact with the nucleic acid and condense around the nucleic acid to form a liposome.
  • the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of nucleic acid.
  • a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition.
  • the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favour condensation.
  • Nucleic acid formulations of the present invention may include a surfactant.
  • the nucleic acid is formulated as an emulsion that includes a surfactant.
  • a surfactant that is not ionized is a non-ionic surfactant.
  • non-ionic 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 polymers.
  • a surfactant that carries a negative charge when dissolved or dispersed in water is an anionic surfactant.
  • anionic surfactant 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, acyl taurates and sulfosuccinates, and phosphates.
  • 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, acyl taurates and
  • a surfactant that carries a positive charge when dissolved or dispersed in water is a cationic surfactant.
  • examples include quaternary ammonium salts and ethoxylated amines.
  • a surfactant that has the ability to carry either a positive or negative charge is an amphoteric surfactant.
  • amphoteric surfactant examples include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • Micelles are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
  • a micelle may be formed by mixing an aqueous solution of the nucleic acid, an alkali metal alkyl sulphate, and at least one micelle forming compound.
  • Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.
  • Phenol and/or m-cresol may be added to the mixed micellar composition to act as a stabiliser and preservative.
  • An isotonic agent such as glycerine may as be added.
  • a nucleic acid preparation may be incorporated into a particle such as a microparticle.
  • Microparticles can be produced by spray-drying, lyophilisation, evaporation, fluid bed drying, vacuum drying, or a combination of these methods.
  • the terms “inhibit”, “down-regulate”, or “reduce” with respect to gene expression mean that the expression of the gene, or the level of RNA molecules or equivalent RNA molecules 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 either in the absence of the nucleic acid or conjugated nucleic acid of the invention or as compared to that obtained with an siRNA molecule with no known homology to the human transcript (herein termed non-silencing control). Such control may be conjugated and modified in an analogous manner to the molecule of the invention and delivered into the target cell by the same route.
  • the expression after treatment with the nucleic acid of the invention may be reduced to 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5% or 0% or to intermediate values, or less than that observed in the absence of the nucleic acid or conjugated nucleic acid.
  • the expression may be measured in the cells to which the nucleic acid is applied. Alternatively, especially if the nucleic acid is administered to a subject, the level can be measured in a different group of cells or in a tissue or an organ or in a body fluid such as blood or plasma.
  • the level of inhibition is preferably measured in conditions that have been selected because they show the greatest effect of the nucleic acid on the target mRNA level in cells treated with the nucleic acid in vitro.
  • the level of inhibition may for example be measured after 24 hours or 48 hours of treatment with a nucleic acid at a concentration of between 0.038 nM-10 ⁇ M, preferably 1 nM, 10 nM or 100 nM.
  • a nucleic acid at a concentration of between 0.038 nM-10 ⁇ M, preferably 1 nM, 10 nM or 100 nM.
  • These conditions may be different for different nucleic acid sequences or for different types of nucleic acids, such as for nucleic acids that are unmodified or modified or conjugated to a ligand or not. Examples of suitable conditions for determining levels of inhibition are described in the examples.
  • nucleic acid it is meant a nucleic acid comprising two strands comprising nucleotides, that is able to interfere with gene expression. Inhibition may be complete or partial and results in down regulation of gene expression in a targeted manner.
  • the nucleic acid comprises two separate polynucleotide strands; the first strand, which may also be a guide strand; and a second strand, which may also be a passenger strand.
  • the first strand and the second strand may be part of the same polynucleotide molecule that is self-complementary which ‘folds’ back to form a double-stranded molecule.
  • the nucleic acid may be an siRNA molecule.
  • the nucleic acid may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogues non-nucleotides that are able to mimic nucleotides such that they may ‘pair’ with the corresponding base on the target sequence or complementary strand.
  • the nucleic acid may further comprise a double-stranded nucleic acid portion or duplex region formed by all or a portion of the first strand (also known in the art as a guide strand) and all or a portion of the second strand (also known in the art as a passenger strand).
  • the duplex region is defined as beginning with the first base pair formed between the first strand and the second strand and ending with the last base pair formed between the first strand and the second strand, inclusive.
  • duplex region it is meant the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary.
  • an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 nucleotides on each strand are complementary or substantially complementary, such that the “duplex region” consists of 19 base pairs.
  • the remaining base pairs may exist as 5′ and 3′ overhangs, or as single-stranded regions.
  • ⁇ complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well known in the art. Alternatively, two strands can be synthesised and added together under biological conditions to determine if they anneal to one another. The portion of the first strand and second strand that forms at least one duplex region may be fully complementary and is at least partially complementary to each other. Depending on the length of a 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 hybridise under physiological conditions.
  • non-pairing nucleotide analogue means a nucleotide analogue which includes a non-base pairing moiety including but not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, and N3-Me dC.
  • the non-base pairing nucleotide analogue is a ribonucleotide. In other embodiments it is a deoxyribonucleotide.
  • terminal functional group includes without limitation a halogen, alcohol, amine, carboxylic, ester, amide, aldehyde, ketone, and ether groups.
  • an “overhang” as used herein has its normal and customary meaning in the art, i.e. a single-stranded portion of a nucleic acid that extends beyond the terminal nucleotide of a complementary strand in a double-strand nucleic acid.
  • the term “blunt end” includes double-stranded nucleic acid whereby both strands terminate at the same position, regardless of whether the terminal nucleotide(s) are base-paired.
  • the terminal nucleotide of a first strand and a second strand at a blunt end may be base paired.
  • the terminal nucleotide of a first strand and a second strand at a blunt end may not be paired.
  • the terminal two nucleotides of a first strand and a second strand at a blunt end may be base-paired.
  • the terminal two nucleotides of a first strand and a second strand at a blunt end may not be paired.
  • An O atom of said structure typically links to an RNA strand and the N atom typically links to the targeting ligand.
  • patient may be used interchangeably and refer to either a human or a non-human animal.
  • mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).
  • livestock animals e.g., bovines, porcines
  • companion animals e.g., canines, felines
  • rodents e.g., mice and rats.
  • treating or “treatment” and grammatical variants thereof refer to an approach for obtaining beneficial or desired clinical results.
  • the term may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above.
  • beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival relative to expected survival time if not receiving treatment.
  • a subject e.g., a human
  • treatment includes inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant disease, disorder or condition.
  • prevention refers to an approach for preventing the development of, or altering the pathology of, a condition, disease or disorder.
  • prevention may refer to prophylactic or preventive measures.
  • beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable.
  • a subject e.g., a human
  • prevention includes slowing the onset of disease relative to the absence of treatment, and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition.
  • preventing or “prevention” of a condition may in certain contexts refer to reducing the risk of developing the condition, or preventing or delaying the development of symptoms associated with the condition.
  • an “effective amount,” “therapeutically effective amount” or “effective dose” is an amount of a composition (e.g., a therapeutic composition or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition.
  • the term “pharmaceutically acceptable salt” refers to a salt that is not harmful to a patient or subject to which the salt in question is administered. It may be a salt chosen, e.g., among acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts and acetate salts.
  • Examples of basic salts include salts wherein the cation is selected from alkali metal cations, such as sodium or potassium ions, alkaline earth metal cations, such as calcium or magnesium ions, as well as substituted ammonium ions, such as ions of the type N(R 1 )(R 2 )(R 3 )(R 4 )+, wherein R 1 , R 2 , R 3 and R 4 independently will typically designate hydrogen, optionally substituted C1-6-alkyl groups or optionally substituted C2-6-alkenyl groups. Examples of relevant C1-6-alkyl groups include methyl, ethyl, 1-propyl and 2-propyl groups.
  • C2-6-alkenyl groups of possible relevance include ethenyl, 1-propenyl and 2-propenyl.
  • Other examples of pharmaceutically acceptable salts are described in “Remington's Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 (and more recent editions thereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977).
  • a “pharmaceutically acceptable salt” retains qualitatively a desired biological activity of the parent compound without imparting any undesired effects relative to the compound.
  • Acid addition salts include salts derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphorous, phosphoric, sulfuric, hydrobromic, hydroiodic and the like, or from nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • nontoxic inorganic acids such as hydrochloric, nitric, phosphorous, phosphoric, sulfuric, hydrobromic, hydroiodic and the like
  • nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like.
  • Base addition salts include salts derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N, N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
  • pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers.
  • Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • sterile saline and phosphate-buffered saline at slightly acidic or physiological pH may be used.
  • Exemplary pH buffering agents include phosphate, citrate, acetate, tris/hydroxymethyl)aminomethane (TRIS), N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, which is a preferred buffer, arginine, lysine, or acetate or mixtures thereof.
  • TIS tris/hydroxymethyl)aminomethane
  • TAPS N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid
  • ammonium bicarbonate diethanolamine
  • histidine which is a preferred buffer
  • arginine arginine
  • lysine lysine
  • a “pharmaceutically acceptable carrier” includes any and all physiologically acceptable, i.e., compatible, solvents, dispersion media, coatings, antimicrobial agents, isotonic and absorption delaying agents, and the like.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
  • the nucleic acid may be coated in 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.
  • solvate in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (in casu, a nucleic acid compound or pharmaceutically acceptable salt thereof according to the invention) and a solvent.
  • the solvent in this connection may, for example, be water or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid.
  • a solvate is normally referred to as a hydrate.
  • FIGS. 1A, 1B and 1C show concentration-response-curves of selected siRNAs.
  • FIGS. 2A and 2B show concentration-response-curves of selected siRNA GalNAc conjugates in human primary hepatocytes.
  • FIGS. 3A and 3B show concentration-response-curves of selected siRNA GalNAc conjugates in mouse primary hepatocytes.
  • FIGS. 4A, 4B and 4C show concentration-dependent C3 mRNA inhibition of selected siRNA GalNAc conjugates respectively in primary mouse, human and cynomolgus hepatocytes.
  • FIG. 5 shows a possible synthesis route to DMT-Serinol(GalNAc)-CEP and CPG.
  • FIGS. 6A and 6B show in vivo C3 mRNA levels in hepatocytes as well as C3 protein levels in serum in response to sc treatment of mice with selected siRNA GalNAc conjugates.
  • FIGS. 7A, 7B and 7C show relative C3 mRNA expression in primary mouse (A), cynomolgus (B) and human (C) hepatocytes after incubation with selected siRNA GalNac conjugates (1 nM, 10 nM and 100 nM) normalized to ACTIN mRNA.
  • FIGS. 9A-E show relative C3 protein serum levels in % from mouse serum samples taken before (BL), at day 4, at day 10, day 14, day 21, day 28, day 35 and day 42 of the study after dosing of 5 or 10 mg/kg siRNA.
  • Data points depict serum C3 level of individual animals determined using a standard C3 ELISA. Data was normalized to each group's baseline mean and then to the time matched PBS control, which was set as 100%. The plotted line connects the individual group means at the respective timepoints.
  • FIG. 10 shows C3 mRNA knockdown efficiency by various siRNAs in vitro.
  • FIG. 11 shows human C3 mRNA knockdown efficiency by various siRNAs in vitro.
  • FIG. 12 shows mouse C3 mRNA knockdown efficiency by various siRNAs in vitro.
  • FIG. 13 shows C3 mRNA knockdown efficiency by various siRNA conjugates in mouse hepatocytes.
  • FIG. 14 shows C3 mRNA knockdown efficiency by various siRNA conjugates in cynomolgus hepatocytes.
  • FIG. 15 shows C3 mRNA knockdown efficiency by siRNA conjugate variants in mouse hepatocytes.
  • FIG. 16 shows C3 mRNA knockdown efficiency by siRNA conjugate variants in cynomolgus hepatocytes.
  • FIG. 17 shows relative C3 mRNA expression in % in murine liver 43 days after a single dosing of GalNAc conjugated C3 siRNA EJ0020.
  • FIG. 18 shows relative C3 protein serum levels in % from mouse serum samples taken before, at day 7, day 14, day 28 and day 43 of the study after dosing of 1 or 3 mg/kg GalNAc conjugated C3 siRNA EJ0020.
  • FIG. 19 shows expression of C3 mRNA in primary human hepatocytes after incubation with the GalNAc siRNA conjugates EV0210, EV0211, EV0212 and EV0213 at 1, 10 and 100 nM.
  • FIG. 20 shows the effect of treatment with different doses of a conjugated C3 siRNA on C3 tubular deposition in wild-type and C3G disease model mice ten days post treatment.
  • FIG. 21 shows the effect of treatment with different doses of a conjugated C3 siRNA on Complement Factor B fragmentation in wild-type and C3G disease model mice ten days post treatment.
  • FIGS. 22A and 22B show the effect of treatment with multiple doses of a conjugated C3 siRNA on levels of C3 ⁇ -chain and C3 ⁇ -chain fragments in C3G disease model mice.
  • FIG. 23 shows the effect of treatment with multiple doses of a conjugated C3 siRNA on Complement Factor B fragmentation in C3G disease model mice.
  • C3 knockdown efficacy of siRNAs EV0001-EV0100 was determined after transfection of 10 nM siRNA in HepG2 cells. The results are shown in Table 2 below. Remaining C3 mRNA levels after knockdown were in the range of 6 to 83%. The most potent siRNAs were EV0001, EV0007, EV0008, EV0009, EV0012, EV0013, EV0018, EV0020, EV0030, EV0033, and EV0004.
  • siRNAs For transfection of HepG2 cells with siRNAs, cells were seeded at a density of 15,000 cells/well into collagen-coated 96-well tissue culture plates (#655150, GBO, Germany). Transfection of siRNAs was carried out with Lipofectamine RNAiMax (Invitrogen/Life Technologies, Düsseldorf, Germany) according to the manufacturer's instructions directly after seeding. The screen was performed with C3 siRNAs in quadruplicates at 10 nM, with siRNAs targeting Aha1, Firefly-Luciferase and Factor VII as unspecific controls and a mock transfection.
  • Lipofectamine RNAiMax Invitrogen/Life Technologies, Düsseldorf, Germany
  • Luminescence was read using 1420 Luminescence Counter (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Jügesheim, Germany) following 30 minutes of incubation at RT in the dark.
  • the C3 mRNA level was normalized to the respective GAPDH mRNA level.
  • the activity of a given C3 siRNA was expressed as percent remaining C3 mRNA concentration (normalized to GAPDH mRNA) in treated cells, relative to the C3 mRNA concentration (normalized to GAPDH mRNA) averaged across control wells.
  • siRNAs showed a dose-dependent knockdown of C3 mRNA after transfection.
  • the most potent siRNAs were EV0008, EV0033 and EV0081, with a residual C3 expression of 4.3, 5.3 and 5.3% at 100 nM siRNA, respectively.
  • the activity of a given C3 siRNA was expressed as percent remaining C3 mRNA (normalized to GAPDH mRNA) in treated cells, relative to the C3 mRNA concentration (normalized to GAPDH mRNA) averaged across control wells.
  • Concentration-response-curves were fitted with GraphPad Prism version 7.05 using a four-parameter logistic (4PL) model without further constraints.
  • siRNA GalNAc conjugates were able to decrease C3 mRNA level in a concentration-dependent fashion with maximal inhibition at 10 ⁇ M between 32 and 70%, respectively.
  • the most potent siRNAs were EV0102 with 61%, EV0109 with 67%, EV0111 with 69% and EV0312 with 70% reduction of the C3 mRNA level at 10 ⁇ M.
  • Human cryopreserved primary hepatocytes were purchased from Primacyt (Schwerin, Germany, cat #GuCPI, Lot #BHum16061-P). Directly before treatment, cells were thawed, transferred to a tube with thawing medium (Primacyt, cat #HTM), centrifuged and washed with washing Medium (Primacyt, cat #HWM). Cells were seeded at a density of 90,000 cells per well in plating medium (Primacyt, cat #MPM-cryo) on collagen coated 96-well plates (Greiner-Bio-One, #655150). Directly after seeding, cells were treated with the siRNAs as they adhered as a monolayer in plating medium.
  • Each siRNA was applied to the cells for the concentration-response-curve at concentrations starting with 10 ⁇ M, sequentially diluted in 4-fold dilution steps down to 38 pM. Each concentration was applied as quadruplicate. After 5 hours, the medium was changed to maintenance medium (Primacyt cat #HHMM). The medium was changed every 24 hours and the cells were harvested for analysis by Quantigene bDNA assay 48 hours after seeding. The C3 mRNA concentrations were normalised to GAPDH mRNA. Concentration-response-curves were fitted with GraphPad Prism version 7.05 using a four-parameter logistic (4PL) model without further constraints.
  • Cryopreserved murine hepatocytes were purchased from Thermo Fisher (#MSCP10, Lot #MC817) and plated in plating medium (Thermo Fisher Sci, Cat. No. CM3000 supplement pack added to William's E Medium, no phenol red—to 500 ml total, Thermo Fisher Sci, Cat. No. A12176-01).
  • Thermo Fisher Sci Cat. No. CM3000 supplement pack added to William's E Medium, no phenol red—to 500 ml total, Thermo Fisher Sci, Cat. No. A12176-01.
  • the cells were thawed and plated at a density of 60,000 cells per well into a collagen-coated 96-well plate (Greiner-Bio-One, #655150). Directly after seeding, cells were treated with the siRNAs as they adhered as a monolayer in plating medium.
  • Each siRNA was applied to the cells as concentration-response-curve at concentrations starting with 10 ⁇ M, sequentially diluted in 4-fold dilution steps down to 0.038 nM. Each concentration was applied as quadruplicate. After 5 hours, the medium was changed to maintenance medium (Thermo Fisher Sci, Cat. No. CM4000 supplement pack added to William's E Medium, no phenol red—to 500 ml total, Thermo Fisher Sci, Cat. No. A12176-01). The medium was changed every 24 hours and the cells were harvested for analysis by Quantigene bDNA assay 48 hours after seeding.
  • FIGS. 3A and 3B depict % remaining C3 mRNA expression in primary mouse hepatocytes after incubation with siRNA GalNAc conjugates (0.038 nM-10 ⁇ M) normalized to GAPDH mRNA. Concentration-response curves were fitted with GraphPad Prism version 7.05 using a four-parameter logistic (4PL) model without further constraints.
  • 40,000 (human), 30,000 (mouse) or 45,000 (cynomolgus) cells were seeded on collagen-coated 96-well plates.
  • siRNAs in indicated concentrations were added immediately after seeding. 24 hours post treatment, cells were lysed using InviTrap RNA Cell HTS96 Kit/C (Stratec). qPCR was performed using mRNA-specific primers and probes against C3 and Actin.
  • FIGS. 4A, 4B and 4C The results are shown in FIGS. 4A, 4B and 4C .
  • DMT-Serinol(GalNAc)-CEP and CPG The synthesis route for DMT-Serinol(GalNAc)-CEP and CPG as described below is outlined in FIG. 5 .
  • Starting material DMT-Serinol(H) (1) was made according to literature published methods (Hoevelmann et al. Chem. Sci., 2016, 7, 128-135) from commercially available L-Serine.
  • GalNAc(Ac 3 )—C 4 H 8 —COOH (2) was prepared according to literature published methods (Nair et al. J. Am. Chem. Soc., 2014, 136 (49), pp 16958-1696), starting from commercially available per-acetylated galactose amine.
  • Phosphitylation reagent 2-Cyanoethyl-N,N-diisopropylchlorophosphor-amidite (4) is commercially available. Synthesis of (vp)-mU-phos was performed as described in Prakash, Nucleic Acids Res. 2015, 43(6), 2993-3011 and Haraszti, Nucleic Acids Res. 2017, 45(13), 7581-7592. Synthesis of the phosphoramidite derivatives of ST43 (ST43-phos) as well as ST23 (ST23-phos) can be performed as described in WO2017/174657.
  • HBTU (9.16 g, 24.14 mmol) was added to a stirring solution of GalNAc(Ac 3 )—C 4 H 8 —COOH (2) (11.4 g, 25.4 mmol) and DIPEA (8.85 ml, 50.8 mmol). After 2 minutes activation time a solution of DMT-Serinol(H) (1) (10 g, 25.4 mmol) in Acetonitrile (anhydrous) (200 ml) was added to the stirring mixture. After 1 h LCMS showed good conversion. The reaction mixture was concentrated in vacuo. The residue was dissolved up in EtOAc, washed subsequently with water (2 ⁇ ) and brine. The organic layer was dried over Na 2 SO 4 , filtered and concentrated under reduced pressure.
  • DMAP (1.11 g, 9.11 mmol) was added to a stirring solution of DMT-Serinol(GalNAc) (3) (7.5 g, 9.11 mmol) and succinic anhydride (4.56 g, 45.6 mmol) in a mixture of Dichloromethane (50 ml) and Pyridine (50 ml) under argon atmosphere. After 16 h of stirring the reaction mixture was concentrated in vacuo and the residue was taken up in EtOAc and washed with 5% citric acid (aq). The aqueous layer was extracted with EtOAc.
  • the DMT-Serinol(GalNAc)-succinate (6) (1 eq.) and HBTU (1.1 eq.) were dissolved in CH 3 CN (10 ml).
  • Diisopropylethylamine (2 eq.) was added to the solution, and the mixture was swirled for 2 min followed by addition native amino-Icaa-CPG (500 A, 88 ⁇ mol/g, 1 eq.).
  • the suspension was gently shaken at room temperature on a wrist-action shaker for 16 h, then filtered and washed with acetonitrile.
  • the solid support was dried under reduced pressure for 2 h.
  • Example compounds were synthesised according to methods described below and known to the person skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed by solid phase synthesis applying phosphoramidite methodology.
  • Oligonucleotide syntheses was performed on an AKTA oligopilot 10 using commercially available 2′O-Methyl RNA and 2′Fluoro-2′Deoxy RNA base loaded CPG solid support and phosphoramidites (all standard protection, ChemGenes, LinkTech) were used. Synthesis of DMT-(S)-Serinol(GalNAc)-succinyl Icaa CPG (7) and DMT-(S)-Serinol(GalNAc)-CEP (5) are described in example 6.
  • Ancillary reagents were purchased from EMP Biotech. Synthesis was performed using a 0.1 M solution of the phosphoramidite in dry acetonitrile ( ⁇ 20 ppm H 2 O) and benzylthiotetrazole (BTT) was used as activator (0.3M in acetonitrile). Coupling time was 10 min. A Cap/OX/Cap or Cap/Thio/Cap cycle was applied (Cap: Ac 2 O/NMI/Lutidine/Acetonitrile, Oxidizer: 0.05M I 2 in pyridine/H 2 O). Phosphorothioates were introduced using commercially available thiolation reagent 50 mM EDITH in acetonitrile (Link technologies).
  • DMT cleavage was achieved by treatment with 3% dichloroacetic acid in toluene. Upon completion of the programmed synthesis cycles a diethylamine (DEA) wash was performed. All oligonucleotides were synthesized in DMT-off mode.
  • DEA diethylamine
  • the CPG carrying the fully assembled oligonucleotide was dried under reduced pressure and transferred into a 20 ml PP syringe reactor for solid phase peptide synthesis equipped with a disc frit (Carl Roth GmbH).
  • the CPG was then brought into contact with a solution of 250 ⁇ L TMSBr and 177 ⁇ L pyridine in CH 2 Cl 2 (0.5 ml/ ⁇ mol solid support bound oligonucleotide) at room temperature and the reactor was sealed with a Luer cap.
  • the reaction vessels were slightly agitated over a period of 2 ⁇ 15 min, the excess reagent discarded, and the residual CPG washed 2 ⁇ with 10 ml acetonitrile. Further downstream processing did not alter from any other example compound.
  • the single strands were cleaved off the CPG by 40% aq. methylamine treatment (90 min, RT).
  • the resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6 ml, GE Healthcare) on a AKTA Pure HPLC System using a sodium chloride gradient. Product containing fractions were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilized until further use.
  • mice with an age of 8 weeks were obtained from CHARLES RIVER, Sulzfeld, Germany. Animal experiments were performed according to ethical guidelines of the German Protection of Animals Act in its version of July 2013. Mice were randomized according to weight into groups of 4 mice. On day 0 of the study animals received a single subcutaneous dose of 1 or 5 mg/kg siRNA dissolved in phosphate buffered saline (PBS) or PBS only as control. The viability, body weight and behaviour of the mice was monitored during the study without pathological findings. Serum samples were taken before the application, at day 4, day 10 and day 14. At day 14 the study was terminated, animals were euthanized, and liver samples were snap frozen and stored at ⁇ 80° C. until further analysis.
  • PBS phosphate buffered saline
  • RNAs were isolated using the InviTrap Spin Tissue RNA Mini Kit from Stratec according to the manufacturer's protocol.
  • QPCR was performed using C3 and Actin specific primer probe sets and TakyonTM One-Step Low Rox Probe 5 ⁇ MasterMix dTTP on the QuantStudio6 device from Applied Biosystems in single-plex 384 well format. Expression differences were calculated using the delta delta Ct method and relative expression of C3 versus the house keeping gene actin normalized to the PBS control experiment was used for comparison of the different siRNAs.
  • EV0107, EV0313 and EV0110 induced a dose dependent knockdown of liver C3 mRNA.
  • Serum samples were analysed using commercially available C3 ELISA Kits. The analyses were carried out according to the manufacturer's protocol, and C3 serum levels were calculated relative to the respective pre dose levels. Results are shown in FIG. 6B .
  • siRNA sequences and modifications are listed in Tables 3 and 5.
  • the mRNA level of the house keeping gene ACTIN served as housekeeping control.
  • Human and cynomolgus primary hepatocytes were seeded into collagen I-coated 96-well plates (Life Technologies) at a density of 40,000 cells per well.
  • Mouse hepatocytes were seeded at a density of 25,000 cells per well.
  • GalNAc-conjugated siRNAs were added immediately after plating in the previously defined media to final siRNA concentrations of 100, 10 and 1 nM. Plates were then incubated at 37° C. in a 5% CO2 atmosphere for 24 hours. Subsequently, cells were lysed and RNA was isolated using InviTrap RNA Cell HTS96 Kit/C (Stratec).
  • RNA-solution 10 ⁇ l of RNA-solution was used for gene expression analysis by reverse transcription quantitative polymerase chain reaction (RT-qPCR) performed with amplicon sets/sequences for ACTB (Eurogentec) and C3 (BioTez GmbH, Berlin, Germany), respectively.
  • the RT-qPCR reactions were carried out with an ABI StepOne Plus (Applied Biosystems, part of Thermo Fisher Scientific, Massachusetts, USA) using standard protocols for RT-PCR (48° C. 30 min, 95° C. 10 min, 40 cycles at 95° C. 15 s followed by 60° C. 1 min).
  • the data were calculated by using the comparative CT method also known as the 2-deltadelta Ct method.
  • SiRNAs EV0201, EV0203, EV0204, EV0205 and EV0207 show dose-dependent inhibition of C3 mRNA expression in primary hepatocytes.
  • siRNA sequences and modifications are listed in Tables 3 and 5.
  • the mRNA level of the house keeping gene ACTIN served as housekeeping control.
  • Male C57BL/6N mice with an age of about 8 weeks were obtained from CHARLES RIVER, Sulzfeld, Germany.
  • Animal experiments were conducted in compliance with the principles of the Hungarian Act 1998: XXVIII regulating animal protection (latest modified by Act 2011 CLVIII) and in Government Decree 40/2013 on animal experiments. Mice were assigned into groups of 4 mice. On day 0 of the study, the animals received a single subcutaneous dose of 5 or 10 mg/kg siRNA dissolved in phosphate buffered saline (PBS) or PBS only as control.
  • PBS phosphate buffered saline
  • mice The viability, body weight and behaviour of the mice was monitored during the study without pathological findings. Serum samples were taken before the application, at day 4, day 10, day 14, day 21, day 28, day 35 and day 42. At day 14 and at day 42 half of the groups, respectively, were terminated, the animals were euthanized, and liver samples were snap frozen and stored at ⁇ 80° C. until further analysis.
  • RNAs were isolated using the InviTrap Spin Tissue RNA Mini Kit from Stratec according to the manufacturer's protocol. RT-qPCR was performed using C3 and ACTIN specific primer probe sets and TakyonTM One-Step Low Rox Probe 5 ⁇ MasterMix dTTP on the QuantStudio6 device from Applied Biosystems in single-plex 384 well format. Expression differences were calculated using the delta delta Ct method and relative expression of C3 versus the house keeping gene ACTIN normalized to the PBS group was used for comparison of the different siRNAs.
  • siRNAs (EV0201, EV0203, EV0204, EV0205 and EV0207) inhibit C3 mRNA expression by more than 70% after 14 days after a single dose of 5 or 10 mg/kg ( FIG. 8A ). After 42 days, the inhibition of C3 expression by EV0203, EV0204, EV0205 and EV0207 was still more than 80% knockdown with a 10 mg/kg siRNA dose ( FIG. 8B ).
  • C3 protein level analysis serum samples were measured using commercially available C3 ELISA Kits. The analyses were carried out according the manufacturer's protocol, and % C3 serum levels were calculated relative to the group means at baseline/before the application and relative to the time matched PBS control group's means. The data for the C3 protein analyses mirror the results from the RNA analyses ( FIG. 9 ). EV0203, EV0204, EV0205 and EV0207 were able to induce a long lasting C3 serum decrease. A reduction of up to 80% 42 days after a single application of 10 mg/kg was obtained for EV0203 ( FIG. 9B ).
  • siRNAs targeting C3 are active in vitro.
  • C3 knockdown efficacy of siRNAs EJ0001, EJ0002, EJ0003 and EJ0004 was determined after transfection of 0.1-10 nM siRNA in Hep3B cells. The results are depicted in FIG. 10 . After transfection with EJ0001, a dose dependent reduction of C3 mRNA levels with a maximum of ⁇ 90% knockdown is observed. EJ0002, EJ0003 and EJ0004 are in a similar activity range.
  • siRNAs For transfection of Hep3B cells with siRNAs, cells were seeded at a density of 12,000 cells/well into 96-well tissue culture plates. Transfection of siRNA was carried out with Atufect liposomal transfection reagent 24 h after seeding. The screen was performed with siRNAs targeting C3 in triplicates at 0.1, 1 and 10 nM. An siRNA targeting Firefly Luciferase (“Luc”) was used as control. After 24 h of incubation with siRNAs, medium was removed, cells were lysed, and total RNA was extracted. C3 and ApoB mRNA levels were determined by TaqMan qRT-PCR. Each bar represents mean ⁇ SD from three technical replicates.
  • siRNAs targeting human C3 are active in vitro.
  • C3 knockdown efficacy of siRNAs EJ0001, EJ0005, EJ0006 and EJ0007 was determined after transfection of 0.01-1 nM siRNA in Hep3B cells. The results are depicted in FIG. 11 . After transfection with 1 nM, C3 mRNA knockdown is around 90% for all tested siRNAs and at 0.1 nM, knockdown is around 50% for EJ0001, EJ0006 and EJ0007. EJ0005 performs better with 80% knockdown at 0.1 nM.
  • siRNAs For transfection of Hep3B cells with siRNAs, cells were seeded at a density of 8,000 cells/well into 96-well tissue culture plates. Transfection of siRNA was carried out with Atufect liposomal transfection reagent 24 h after seeding. The screen was performed in triplicates at 0.01, 0.1 and 1 nM siRNA concentration. An siRNA targeting Firefly Luciferase (“Luc”) was used as control. After 24 h of incubation with siRNAs, medium was removed, cells were lysed and total RNA was extracted. C3 and PTEN mRNA levels were determined by TaqMan qRT-PCR. Each bar represents mean ⁇ SD from three technical replicates.
  • siRNAs targeting mouse C3 are active in vitro.
  • C3 knockdown efficacy of siRNAs EJ0001, EJ005, EJ0006 and EJ0007 was determined after transfection of 0.01-10 nM siRNA in AML12 cells. The results are depicted in FIG. 12 . After siRNA transfection, a dose-dependent C3 mRNA knockdown with a maximum of around 60% is reached.
  • siRNA For transfection of AML12 cells, cells were seeded at a density of 6,000 cells/well into 96-well tissue culture plates. Transfection of siRNA was carried out with Atufect liposomal transfection reagent 24 h after seeding. The screen was performed with siRNAs targeting C3 in triplicates at 0.01, 0.1, 1 and 10 nM. An siRNA targeting Firefly Luciferase (“Luc”) was used as control. After 24 h of incubation with siRNAs, medium was removed, cells were lysed, and total RNA was extracted. C3 and PTEN mRNA levels were determined by TaqMan qRT-PCR. Each bar represents mean ⁇ SD from three technical replicates.
  • GalNAc-conjugated siRNAs targeting mouse C3 are active in vitro.
  • Mouse primary hepatocytes were seeded at a density of 25,000 cells/well into 96-well tissue culture plates and treated with 100, 10, 1 and 0.1 nM GalNAc-conjugated siRNAs directly upon plating.
  • a GalNAc-conjugated, scrambled sequence was used as non-targeting control (NTC).
  • NTC non-targeting control
  • the cells were lysed after 24 h of incubation with GalNAc-conjugates and total RNA was extracted.
  • C3 and APOB mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ⁇ SD from three technical replicates.
  • GalNAc-conjugated siRNAs targeting cynomolgus C3 are active in vitro.
  • Cynomolgus primary hepatocytes were seeded at a density of 36,500 cells/well into 96-well tissue culture plates and treated with 100 and 10 nM GalNAc-conjugated siRNAs directly upon plating.
  • a GalNAc-conjugated, scrambled sequence was used as non-targeting control (NTC).
  • NTC non-targeting control
  • the cells were lysed after 24 h of incubation with GalNAc-conjugates and total RNA was extracted.
  • C3 and APOB mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ⁇ SD from three technical replicates.
  • Variants of GalNAc-conjugated siRNAs repress C3 in primary mouse hepatocytes.
  • Mouse primary hepatocytes were seeded at a density of 25,000 cells/well into 96-well tissue culture plates and treated with 100, 10 and 1 nM GalNAc-conjugated siRNAs directly upon plating.
  • a GalNAc-conjugated, scrambled sequence was used as non-targeting control (NTC).
  • NTC non-targeting control
  • the cells were lysed after 24 h of incubation with GalNAc conjugates and total RNA was extracted.
  • C3 and APOB mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ⁇ SD from three technical replicates.
  • Cynomolgus primary hepatocytes were seeded at a density of 40,000 cells/well into 96-well tissue culture plates and treated with 100, 10 and 1 nM GalNAc-conjugated siRNAs directly upon plating.
  • a GalNAc-conjugated, scrambled sequence was used as non-targeting control (NTC).
  • NTC non-targeting control
  • the cells were lysed after 24 h of incubation with GalNAc conjugates and total RNA was extracted.
  • C3 and ACTB mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ⁇ SD from three technical replicates.
  • mice Male C57BL/6N mice aged 8 weeks were obtained from Janvier, France. Animal experiments were performed according to ethical guidelines of the German Protection of Animals Act in its version of July 2013. Mice were randomized according to weight into groups of 4 mice. On day 0 of the study, animals received a single subcutaneous dose of 1 or 3 mg/kg siRNA dissolved in phosphate buffered saline (PBS) or PBS only as control. The viability, body weight and behaviour of the mice was monitored during the study without pathological findings. Serum samples were taken before the application, at day 7, day 14, day 28 and day 43. At day 43, the study was terminated, animals were euthanized, and liver samples were snap frozen and stored at ⁇ 80° C. until further analysis.
  • PBS phosphate buffered saline
  • FIG. 19 Expression of C3 mRNA after incubation with the GalNAc siRNA conjugates EV0210, EV0211, EV0212 and EV0213 at 1, 10 and 100 nM is shown in FIG. 19 .
  • the siRNA GalNAc conjugates are listed in Table 3. mRNA level of the gene APOB served as housekeeping control.
  • hepatocytes Human primary hepatocytes were seeded into collagen 1-coated 96-well plates (Life Technologies) at a density of 40,000 cells per well. GalNAc-conjugated siRNAs were added immediately after plating to final siRNA concentrations of 100, 10 and 1 nM. Plates were then incubated at 37° C. in a 5% CO 2 atmosphere for 24 hours. Subsequently, cells were lysed, and RNA was isolated using InviTrap RNA Cell HTS96 Kit/C (Stratec).
  • RNA-solution Ten ⁇ l of RNA-solution was used for gene expression analysis by reverse transcription quantitative polymerase chain reaction (RT-qPCR) performed with amplicon sets/sequences for APOB (Eurogentec) and C3 (BioTez GmbH, Berlin, Germany), respectively.
  • the RT-qPCR reactions were carried out with an ABI StepOne Plus (Applied Biosystems, part of Thermo Fisher Scientific, Massachusetts, USA) using standard protocols for RT-PCR (48° C. 30 min, 95° C. 10 min, 40 cycles at 95° C. 15 s followed by 60° C. 1 min).
  • the data were calculated by using the comparative CT method also known as the 2-deltadelta Ct method.
  • SiRNAs EV0210, EV0211, EV0212 and EV0213 were able to inhibit C3 mRNA expression in primary human hepatocytes in a dose-dependent manner.
  • EV0203 was tested in a murine disease model of C3 Glomerulopathy and in wild-type mice. Heterozygous complement factor H deficient mice (Cfh def.) were used as the C3 Glomerulopathy disease model. The animals were treated with a single dose of EV0203 or with controls (PBS or non-targeting siRNAs). The mice were sacrificed 10 days after treatment and tubular deposition of C3 was measured in the kidneys of the mice by C3 staining with an anti-C3 antibody and quantified (results are shown in FIG. 20 ).
  • Complement factor B (FB) fragmentation which is determined as the ratio of Ba fragments to full length FB, was also measured in the plasma by western blot and quantified—results are shown in FIG. 21 .
  • the results of the C3 staining show that tubular C3 deposits are significantly decreased in a dose-dependent manner by treatment with a conjugated C3 siRNA.
  • the FB fragmentation data show that Cfh def. mice have increased levels of FB fragmentation compared to wild-type mice, but that this increased fragmentation level can be reduced by treatment with a conjugated C3 siRNA. Conjugated C3 siRNAs are therefore expected to be a powerful treatment for C3-related diseases and in particular for C3 Glomerulopathy and/or at least its symptoms.
  • EV0203 was tested in a murine disease model of C3 Glomerulopathy.
  • Heterozygous complement factor H deficient mice (Cfh def.) were used as the C3 Glomerulopathy disease model.
  • Seven- to eight-month-old mice were treated on the first day of the study and then monthly with 5 mg/kg of EV0203 or with PBS or a none-targeting siRNA as a control. Wild type mice treated with PBS were also used as a control.
  • the mice were sacrificed three months after the start of the study (i.e., after three treatments with a conjugated C3 siRNA or a control).
  • Conjugated C3 siRNAs from the above examples were tested in vivo in healthy cynomolgus monkeys. The animals were treated once or multiple times with different doses of conjugated C3 siRNAs. Preliminary data from monkeys treated with the tested conjugated C3 siRNAs show reduced C3 protein levels in serum. These in vivo experiments are currently ongoing. Additional testing of conjugated C3 siRNAs in vivo in healthy cynomolgus monkeys is planned. After treatment once or multiple times with different doses of conjugated C3 siRNAs, C3 protein levels are measured in serum and C3 mRNA levels are measured in liver tissues. The C3 protein levels in serum and the C3 mRNA levels in the liver are both expected to be reduced after treatment with an effective dose of a conjugated C3 siRNA.
  • Second strand sequence (SEQ ID NO:) (SEQ ID NO:) 364 363 or 375 365 363 366 367 or 376 368 369 370 379 or 371, preferably 379 372 373 362 374 377 378 361 112 95 96 111 112 125 126 131 132 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 97 98 99 100 101 102 103 104 105 106 107 108 109 110 113 114 115 116 117 118 119 120 121 122 123

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