NUCLEIC ACIDS FOR INHIBITING EXPRESSION OF C3 IN A CELL

Abstract

The invention relates to nucleic acid products that interfere with complement component C3 gene expression or inhibit its expression. The nucleic acids are preferably for use as treatment, prevention or reduction of risk of suffering from complement component C3 associated diseases, disorders or syndromes, particularly 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), and Primary Membranous Nephropathy.

Claims

1. 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 or 416.

2. A double-stranded nucleic acid that is capable of inhibiting expression of complement component C3 for use as a medicament, wherein the nucleic acid comprises a first strand and a second strand.

3. The nucleic acid of any of the preceding claims, wherein the first strand and the second strand form a duplex region of from 17-25 nucleotides in length.

4. The nucleic acid of any of the preceding claims, wherein the nucleic acid mediates RNA interference.

5. The nucleic acid of any of the preceding claims, wherein at least one nucleotide of the first and/or second strand is a modified nucleotide, preferably the modified nucleotide is a non-naturally occurring nucleotide such as a 2′-F modified nucleotide.

6. The nucleic acid of any of the preceding claims, wherein at least nucleotides 2 and 14 of the first strand are modified by a first modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand.

7. The nucleic acid of any of the previous claims, wherein the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide at its 5′ end.

8. The nucleic acid of any of the preceding claims, wherein 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 and preferably wherein the linkages between the remaining nucleotides are phosphodiester linkages.

9. The nucleic acid of any of the preceding claims, comprising a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3′ end of the first strand and/or comprising 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 comprising a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5′ end of the first strand.

10. The nucleic acid of any of the preceding claims, wherein the nucleic acid is conjugated to a ligand.

11. The nucleic acid of claim 10, wherein the ligand comprises (i) one or more N-acetyl galactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates the at least one GalNAc moiety or derivative thereof to the nucleic acid.

12. A composition comprising a nucleic acid of any of the previous claims and a solvent and/or a delivery vehicle and/or a physiologically acceptable excipient and/or a carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative and/or a further therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.

13. A nucleic acid of any of claims 1 and 3-11 or a composition of claim 12 for use as a medicament.

14. A nucleic acid of any of claims 1 and 3-11 or a composition of claim 12 for use 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 a complement-mediated disease, disorder or syndrome.

15. Use of a nucleic acid of any of claims 1 and 3-11 or a composition of claim 12 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 selected from the group comprising C3 Glomerulopathy (C3G), Cold Agglutinin Disease (CAD) and IgA nephropathy (IgA N).

16. Method of preventing, decreasing the risk of suffering from, or treating a disease, disorder or syndrome comprising administering a pharmaceutically effective amount of a nucleic acid of any of claims 1 and 3-11 or a composition of claim 12 to an individual in need of treatment, wherein the disease, disorder or syndrome is preferably selected from the group comprising C3 Glomerulopathy (C3G), Cold Agglutinin Disease (CAD) and IgA nephropathy (IgA N).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0565] FIGS. 1A, 1B and 1C show concentration-response-curves of selected siRNAs.

[0566] FIGS. 2A and 2B show concentration-response-curves of selected siRNA GalNAc conjugates in human primary hepatocytes.

[0567] FIGS. 3A and 3B show concentration-response-curves of selected siRNA GalNAc conjugates in mouse primary hepatocytes.

[0568] FIGS. 4A, 4B and 4C show concentration-dependent C3 mRNA inhibition of selected siRNA GalNAc conjugates respectively in primary mouse, human and cynomolgus hepatocytes.

[0569] FIG. 5 shows a possible synthesis route to DMT-Serinol(GalNAc)-CEP and CPG.

[0570] 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.

[0571] 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.

[0572] FIGS. 8A and 8B show relative C3 mRNA expression in % in murine liver 14 days (A) or 42 days (B) after a single dosing of GalNAc conjugated siRNAs EV0201, EV0203, EV0204, EV0205 and EV0207. Data is shown in bar charts as mean±SD (n=4 per group).

[0573] 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. [0574] A) Normalised C3 serum levels after dosing of 5 and 10 mg/kg EV0201 [0575] B) Normalised C3 serum levels after dosing of 5 and 10 mg/kg EV0203 [0576] C) Normalised C3 serum levels after dosing of 5 mg/kg EV0204 [0577] D) Normalised C3 serum levels after dosing of 5 and 10 mg/kg EV0205 [0578] E) Normalised C3 serum levels after dosing of 5 and 10 mg/kg EV0207.

[0579] FIG. 10 shows C3 mRNA knockdown efficiency by various siRNAs in vitro.

[0580] FIG. 11 shows human C3 mRNA knockdown efficiency by various siRNAs in vitro.

[0581] FIG. 12 shows mouse C3 mRNA knockdown efficiency by various siRNAs in vitro.

[0582] FIG. 13 shows C3 mRNA knockdown efficiency by various siRNA conjugates in mouse hepatocytes.

[0583] FIG. 14 shows C3 mRNA knockdown efficiency by various siRNA conjugates in cynomolgus hepatocytes.

[0584] FIG. 15 shows C3 mRNA knockdown efficiency by siRNA conjugate variants in mouse hepatocytes.

[0585] FIG. 16 shows C3 mRNA knockdown efficiency by siRNA conjugate variants in cynomolgus hepatocytes.

[0586] FIG. 17 shows relative C3 mRNA expression in % in murine liver 43 days after a single dosing of GalNAc conjugated C3 siRNA EJ0020.

[0587] 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.

[0588] 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.

[0589] 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.

[0590] 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.

[0591] FIGS. 22A and 22B show the effect of treatment with multiple doses of a conjugated C3 siRNA on levels of C3α-chain and C3α-chain fragments in C3G disease model mice.

[0592] 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.

EXAMPLES

Example 1

[0593] In vitro study in HepG2 cells showing C3 mRNA knockdown efficacy of tested siRNAs after transfection of 10 nM siRNA.

[0594] 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.

[0595] 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, Karlsruhe, 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. After 24 h of incubation with siRNAs, medium was removed, and cells were lysed in 150 μl Medium-Lysis Mixture (1 volume lysis mixture, 2 volumes cell culture medium) and then incubated at 53° C. for 30 minutes. bDNA assay was performed according to the manufacturer's instructions.

[0596] 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. For each well, 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.

TABLE-US-00002 TABLE 2 Duplex (% remaining mRNA ID Mean SD EV0001 8.87 0.61 EV0002 10.61 0.72 EV0003 10.68 0.82 EV0004 9.59 0.43 EV0005 22.27 0.97 EV0006 13.10 1.16 EV0007 8.89 0.90 EV0008 6.02 0.47 EV0009 6.80 0.45 EV0010 20.18 1.32 EV0011 10.09 0.34 EV0012 9.48 1.06 EV0013 7.40 1.08 EV0014 9.75 1.25 EV0015 13.08 1.52 EV0016 16.68 0.66 EV0017 47.72 1.80 EV0018 7.86 0.77 EV0019 18.83 1.08 EV0020 8.80 0.87 EV0021 13.88 0.98 EV0022 79.91 4.20 EV0023 13.32 1.29 EV0024 11.11 0.76 EV0025 16.35 0.50 EV0026 10.03 0.88 EV0027 10.11 1.03 EV0028 11.83 0.53 EV0029 9.71 1.02 EV0030 9.05 0.49 EV0031 10.70 0.64 EV0032 12.93 0.55 EV0033 9.29 0.59 EV0034 15.20 0.50 EV0035 16.87 0.62 EV0036 14.37 1.05 EV0037 15.36 2.68 EV0038 16.50 1.29 EV0039 9.69 0.71 EV0040 25.55 1.40 EV0041 12.79 1.34 EV0042 14.63 0.51 EV0043 13.05 0.83 EV0044 16.29 0.87 EV0045 18.63 0.98 EV0046 21.78 1.18 EV0047 20.78 1.04 EV0048 17.59 1.31 EV0049 15.27 0.70 EV0050 20.75 1.03 EV0051 11.84 0.60 EV0052 18.23 1.26 EV0053 15.40 0.60 EV0054 12.80 0.69 EV0055 44.97 4.50 EV0056 83.23 5.03 EV0057 55.45 3.76 EV0058 22.12 0.84 EV0059 12.74 0.48 EV0060 14.38 0.79 EV0061 12.92 0.83 EV0062 13.26 0.83 EV0063 21.32 1.61 EV0064 15.14 0.86 EV0065 12.01 0.42 EV0066 13.14 1.32 EV0067 13.57 0.61 EV0068 24.89 0.87 EV0069 15.36 2.68 EV0070 16.50 1.29 EV0071 9.69 0.71 EV0072 25.55 1.40 EV0073 12.79 1.34 EV0074 14.63 0.51 EV0075 13.05 0.83 EV0076 16.29 0.87 EV0077 18.63 0.98 EV0078 21.78 1.18 EV0079 20.78 1.04 EV0080 17.59 1.31 EV0081 15.27 0.70 EV0082 20.75 1.03 EV0083 11.84 0.60 EV0084 18.23 1.26 EV0085 15.40 0.60 EV0086 12.80 0.69 EV0087 44.97 4.50 EV0088 83.23 5.03 EV0089 55.45 3.76 EV0090 22.12 0.84 EV0091 12.74 0.48 EV0092 14.38 0.79 EV0093 12.92 0.83 EV0094 13.26 0.83 EV0095 21.32 1.61 EV0096 15.14 0.86 EV0097 12.01 0.42 EV0098 13.14 1.32 EV0099 13.57 0.61 EV0100 24.89 0.87 Results of screening of C3 siRNAs—the identity of the single strands forming each of the siRNA duplexes as well as their sequences and modifications are to be found in the tables at the end of the description.

Example 2

[0597] In vitro study in HepG2 cells showing C3 mRNA knockdown efficacy of tested siRNAs after transfection of 0.01 pM-100 nM siRNA (concentration-response-curve experiment).

[0598] C3 knockdown efficacy of siRNAs EV0001, EV0008, EV0013, EV0030, EV0033, EV0039, EV0043, EV0053, EV0054, EV0059, EV0060, EV0061, EV0066, EV0072, EV0075, EV0081, EV0091 and EV0098 was determined after transfection of 0.01 pM-100 nM siRNA in HepG2 cells. The identity of the single strands forming each of the siRNA duplexes as well as their sequences are to be found in the tables at the end of the description. Results are presented in FIGS. 1A, 1B and 1C. All 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.

[0599] 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 siRNA was carried out with Lipofectamine RNAiMax (Invitrogen/Life Technologies, Karlsruhe, Germany) according to the manufacturer's instructions directly after seeding. Concentration-response experiments were done with C3 siRNA in 10 concentrations transfected in quadruplicates, starting at 100 nM in 6-fold dilution steps down to 0.01 pM. Mock transfected cells served as control in CRC experiments. After 24 h of incubation with siRNAs, medium was removed and cells were lysed in 150 μl Medium-Lysis Mixture (1 volume lysis mixture, 2 volumes cell culture medium) and then incubated at 53° C. for 30 minutes. bDNA assay was performed according to the manufacturer's instructions. 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. For each well, 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 (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.

Example 3

[0600] In vitro study in primary human hepatocytes showing C3 mRNA knockdown efficacy of tested siRNA-GalNAc conjugates in concentration-response-curve format (0.038 nM-10 μM siRNA conjugate).

[0601] Expression of C3 mRNA after incubation with the GalNAc siRNA conjugates EV0101, EV0102, EV0103, EV0104, EV0105, EV0106, EV0107, EV0108, EV0109, EV0110, EV0111 and EV0312 was analysed in a concentration-response format. The identity of the single strands forming each of the siRNA duplexes as well as their sequences are to be found in the tables at the end of the description. Results are shown in FIGS. 2A and 2B. The mRNA level of the house keeping gene GAPDH served as control for all experiments. All siRNA GalNAc conjugates were able to decrease C3 mRNA level in a concentration-dependent fashion with maximal inhibition at 10 μM 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 μM.

[0602] 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 μM, 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.

Example 4

[0603] In vitro study in primary mouse hepatocytes showing C3 mRNA knockdown efficacy of tested siRNA-GalNAc conjugates in concentration-response-curve format (0.038 nM-10 μM siRNA conjugate).

[0604] Expression of C3 mRNA after incubation with the GalNAc siRNA conjugates EV0104, EV0105, EV0107, EV0108, EV0109, EV0110, EV0111 and EV0312 in a concentration-response format was analysed. The identity of the single strands forming each of the siRNA duplexes as well as their sequences are to be found in the tables at the end of the description. The mRNA level of the house keeping gene GAPDH served as control. The siRNA GalNAc conjugates were able to decrease C3 mRNA levels in a concentration dependent fashion with maximal inhibition at 10 μM between 56 and 72%. The most potent siRNAs were EV0104 with 68%, EV0109 with 67% and EV0111 with 72% reduction of C3 mRNA at 10 μM, respectively.

[0605] 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). On the day of seeding, 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 μM, 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.

[0606] The results are shown in FIGS. 3A and 3B. They depict % remaining C3 mRNA expression in primary mouse hepatocytes after incubation with siRNA GalNAc conjugates (0.038 nM-10 μM) 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.

Example 5

[0607] In vitro study in primary mouse, human and cynomolgus hepatocytes showing C3 mRNA knockdown efficacy of tested siRNA-GalNAc conjugates at 1, 10 and 100 nM.

[0608] The expression of C3 mRNA after incubation with the GalNAc siRNA conjugates EV0312 and EV0313 at 1, 10 and 100 nM was analysed. The identity of the single strands forming each of the siRNA duplexes as well as their sequences are to be found in the tables at the end of the description. The mRNA level of the house keeping gene Actin served as control.

[0609] 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.

[0610] The results are shown in FIGS. 4A, 4B and 4C.

Example 6—Synthesis of Building Blocks

[0611] 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.sub.3)—C.sub.4H.sub.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.

[0612] DMT-Serinol(GalNAc) (3)

[0613] HBTU (9.16 g, 24.14 mmol) was added to a stirring solution of GalNAc(Ac.sub.3)—C.sub.4H.sub.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.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was further purified by column chromatography (3% MeOH in CH.sub.2Cl.sub.2+1% Et.sub.3N, 700 g silica). Product containing fractions were pooled, concentrated and stripped with CH.sub.2Cl.sub.2 (2×) to yield to yield 10.6 g (51%) of DMT-Serinol(GalNAc) (3) as an off-white foam.

[0614] DMT-Serinol(GalNAc)-CEP (5)

[0615] 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (4) (5.71 ml, 25.6 mmol) was added slowly to a stirring mixture of DMT-Serinol(GalNAc) (3) (15.0 g, 17.0 mmol), DIPEA (14.9 ml, 85 mmol) and 4 Å molecular sieves in Dichloromethane (dry) (150 ml) at 0° C. under argon atmosphere. The reaction mixture was stirred at 0° C. for 1 h. TLC indicated complete conversion. The reaction mixture was filtered and concentrated in vacuo to give a thick oil. The residue was dissolved in Dichloromethane and was further purified by flash chromatography (0-50% acetone in toluene 1% Et3N, 220 g silica). Product containing fractions were pooled and concentrated in vacuo. The resulting oil was stripped with MeCN (2×) to yield 13.5 g (77%) of the colorless DMT-Serinol(GalNAc)-CEP (5) foam.

[0616] DMT-Serinol(GalNAc)-succinate (6)

[0617] 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 combined organic layers were washed subsequently with sat NaHCO.sub.3 (aq.) and brine, dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. Further purification was achieved by flash chromatography (0-5% MeOH in CH.sub.2Cl.sub.2+1% Et.sub.3N, 120 g silica). Product containing fractions were pooled and concentrated in vacuo. The residue was stripped with MeCN (3×) to yield 5.9 g (70%) DMT-Serinol(GalNAc)-succinate (6).

[0618] DMT-Serinol(GalNAc)-succinyl-Icaa-CPG (7)

[0619] The DMT-Serinol(GalNAc)-succinate (6) (1 eq.) and HBTU (1.1 eq.) were dissolved in CH.sub.3CN (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 μmol/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. The unreacted amines on the support were capped by stirring with Ac.sub.2O/2,6-lutidine/NMI at room temperature (2×15 min). The washing of the support was repeated as above. The solid was dried under vacuum to yield DMT-Serinol(GalNAc)-succinyl-Icaa-CPG (7) (loading: 34 μmol/g, determined by detritylation assay).

Example 7—Oligonucleotide Synthesis

[0620] 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.

[0621] Downstream cleavage, deprotection and purification followed standard procedures that are known in the art.

[0622] 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.

[0623] 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.sub.2O) 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.sub.2O/NMI/Lutidine/Acetonitrile, Oxidizer: 0.05M I.sub.2 in pyridine/H.sub.2O). 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.

[0624] Attachment of the Serinol(GalNAc) moiety was achieved by use of either base-loaded (S)-DMT-Serinol(GalNAc)-succinyl-Icaa-CPG (7) or a (S)-DMT-Serinol(GalNAc)-CEP (5). Triantennary GalNAc clusters (ST23/ST43) were introduced by successive coupling of the branching trebler amidite derivative (C6XLT-phos) followed by the GalNAc amidite (ST23-phos). Attachment of (vp)-mU moiety was achieved by use of (vp)-mU-phos in the last synthesis cycle. The (vp)-mU-phos does not provide a hydroxy group suitable for further synthesis elongation and therefore, does not possess an DMT-group. Hence coupling of (vp)-mU-phos results in synthesis termination.

[0625] For the removal of the methyl esters masking the vinylphosphonate, 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 μL TMSBr and 177 μL pyridine in CH.sub.2Cl.sub.2 (0.5 ml/μmol 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.

[0626] 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.

[0627] All final single-stranded products were analysed by AEX-HPLC to prove their purity. Identity of the respective single-stranded products was proved by LC-MS analysis.

Example 8—Double-Strand Formation

[0628] Individual single strands were dissolved in a concentration of 60 OD/ml in H.sub.2O. Both individual oligonucleotide solutions were added together in a reaction vessel. For easier reaction monitoring a titration was performed. The first strand was added in 25% excess over the second strand as determined by UV-absorption at 260 nm. The reaction mixture was heated to 80° C. for 5 min and then slowly cooled to RT. Double-strand formation was monitored by ion pairing reverse phase HPLC. From the UV-area of the residual single strand the needed amount of the second strand was calculated and added to the reaction mixture. The reaction was heated to 80° C. again and slowly cooled to RT. This procedure was repeated until less than 10% of residual single strand was detected.

Example 9

[0629] In vivo study showing knockdown of C3 mRNA in murine liver tissue and serum protein after single subcutaneous dosing of 1 or 5 mg/kg GalNAc conjugated siRNAs.

[0630] Female C57BL/6N 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. For analysis, 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 Takyon™ 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. The maximum achieved knockdown was observed using siRNA EV0107 (57%) and EV0110 (61%) using 5 mg/kg siRNA, respectively. Results are shown in FIG. 6A. The figure shows relative C3 mRNA expression in % in murine liver 14 days after a single dosing of GalNAc conjugated siRNAs EV0107, EV0313 and EV0110. The identity of the single strands forming each of the siRNA duplexes as well as their sequences are to be found in the tables at the end of the description. Data is shown in bar charts as mean±SD (n=4 per group).

[0631] 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. The figure shows relative C3 protein serum levels in % from mouse serum samples taken before, at day 4, at day 10 and at day 14 of the study after dosing of 5 mg/kg EV0313, EV0110 and EV0107 GalNAc conjugated siRNAs. Data is shown as means±SD (n=3 or 4 per group).

Example 10

[0632] In vitro study in primary mouse, human and cynomolgus hepatocytes showing C3 knockdown efficacy of tested siRNA-GalNAc conjugates at 1, 10 an 100 nM.

[0633] Expression of C3 mRNA after incubation with the GalNAc siRNA conjugates EV0201, EV0203, EV0204, EV0205 and EV0207 at 1, 10 and 100 nM was measured (FIG. 7). 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).

[0634] 10 μl 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.

Example 11

[0635] In vivo study showing knockdown of C3 mRNA in murine liver tissue and serum protein after a single subcutaneous dosing of 5 or 10 mg/kg GalNAc conjugated modified siRNAs.

[0636] 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. 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.

[0637] For 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 Takyon™ 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.

[0638] All tested 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).

[0639] For 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).

Example 12

[0640] Various siRNAs targeting C3 are active in vitro.

[0641] 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.

[0642] 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.

Example 13

[0643] Various siRNAs targeting human C3 are active in vitro.

[0644] 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.

[0645] 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.

Example 14

[0646] Various siRNAs targeting mouse C3 are active in vitro.

[0647] 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.

[0648] 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.

Example 15

[0649] Various GalNAc-conjugated siRNAs targeting mouse C3 are active in vitro.

[0650] C3 mRNA knockdown efficiency of GalNAc siRNA conjugates EV0201, EJ0010, EJ0011, EJ0012, EJ0014 and EJ0015 was determined after receptor-mediated uptake in mouse primary hepatocytes. The results are depicted in FIG. 13. A dose-dependent knockdown with a maximum of around 75% was achieved.

[0651] 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). 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.

Example 16

[0652] Various GalNAc-conjugated siRNAs targeting cynomolgus C3 are active in vitro.

[0653] C3 mRNA knockdown efficiency of GalNAc siRNA conjugates EV0201, EJ0010, EJ0011, EJ0012, EJ0014 and EJ0015 was determined after receptor-mediated uptake in cynomolgus primary hepatocytes. The results are depicted in FIG. 14. A dose-dependent knockdown with a maximum of around 90% was achieved.

[0654] 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). 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.

Example 17

[0655] Variants of GalNAc-conjugated siRNAs repress C3 in primary mouse hepatocytes.

[0656] C3 mRNA knockdown efficiency of GalNAc-siRNA conjugates EV0201 and variants thereof (EJ0009, EV0203), EJ0014 and variants thereof (EJ0019, EJ0020), EJ0015 and variants thereof (EJ0021-23) as well as EJ0012 and variants thereof (EJ0016-18) was determined after receptor-mediated uptake in mouse primary hepatocytes. The results are depicted in FIG. 15. A dose-dependent knockdown with a maximum of around 85% was achieved with some of the variants.

[0657] 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). 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.

Example 18

[0658] Variants of GalNAc-conjugated siRNAs repress C3 in primary cynomolgus hepatocytes

[0659] C3 mRNA knockdown efficiency of GalNAc-siRNA conjugates EV0201 and variants thereof (EJ0009, EV0203), EJ0014 and variants thereof (EJ0019, EJ0020), EJ0015 and variants thereof (EJ0021-23) as well as EJ0012 and variants thereof (EJ0016-18) was determined after receptor-mediated uptake in cynomolgus primary hepatocytes. The results are depicted in FIG. 16. A dose-dependent knockdown with a maximum of around 90% was achieved with some of the variants.

[0660] 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). 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.

Example 19

[0661] In vitro study showing knockdown of C3 mRNA in murine liver tissue and serum protein after single subcutaneous dosing of 1 or 3 mg/kg of GalNAc conjugated C3 siRNA EJ0020.

[0662] 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.

[0663] For analysis, 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 Takyon™ One-Step Low Rox Probe 5× MasterMix dTTP on the QuantStudio6 device from Applied Biosystems in single-plex 384 well format. Expression was calculated using the delta delta Ct method and relative expression of C3 versus the house keeping gene normalized to PBS was used for comparisons. 38% C3 mRNA knock down was observed using 1 mg/kg siRNA EJ0020 and 73% C3 knock down using 3 mg/kg of the same siRNA. Results are shown in FIG. 17 (data are shown in bar charts as mean±SD (n=4 per group)).

[0664] C3 protein levels in serum were obtained 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. 18 (data are shown as means±SD (n=3 or 4 per group)).

Example 20

[0665] In vitro study in primary human hepatocytes showing C3 knock down efficacy of tested siRNA-GalNAc conjugates at 1, 10 and 100 nM.

[0666] 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.

[0667] 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.sub.2 atmosphere for 24 hours. Subsequently, cells were lysed, and RNA was isolated using InviTrap RNA Cell HTS96 Kit/C (Stratec).

[0668] Ten μl 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.

[0669] SiRNAs EV0210, EV0211, EV0212 and EV0213 were able to inhibit C3 mRNA expression in primary human hepatocytes in a dose-dependent manner.

Example 21

[0670] In vivo study of the efficacy of C3 siRNA GalNAc conjugates in a murine disease model of C3 Glomerulopathy (C3G).

[0671] 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.

Example 22

[0672] In vivo study of the efficacy of multiple doses of a C3 siRNA GalNAc conjugate in a murine disease model of C3 Glomerulopathy (C3G).

[0673] 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). The levels of C3α-chain and C3α-chain fragments (activated C3 fragments) in the plasma were measured by western blot and quantified (results are shown in FIGS. 22A and 22B). 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. 23. These data confirm that conjugated C3 siRNAs are effective in reducing C3 fragment levels and FB fragmentation in aged mice.

[0674] Glomerular C3d deposits were also measured, by C3d staining. Three doses of the conjugated C3 siRNA were able to reduce glomerular C3d deposits of Cfh def. mice to levels similar to those of age-matched wild-type mice. 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.

Example 23

[0675] Conjugated C3 siRNAs from the above examples, including EV0210, EV0212 and EJ0020, 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.

[0676] Statements

[0677] The following statements represent aspects of the invention. [0678] 1. 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, 127, 129, 133 or 416. [0679] 2. The nucleic acid of statement 1, wherein [0680] (a) the first strand sequence comprises a sequence of at least 18 nucleotides differing by no more than 3 nucleotides from any one of the first strand sequences of Table 1 and optionally wherein the second strand sequence comprises a sequence of at least 18 nucleotides differing by no more than 3 nucleotides from the second strand sequence in the same line of the table; [0681] (b) the first strand sequence comprises a sequence of at least 18 nucleotides differing by no more than 1 nucleotide from any one of the first strand sequences of Table 1 and optionally wherein the second strand sequence comprises a sequence of at least 18 nucleotides differing by no more than 1 nucleotide from the second strand sequence in the same line of the table; [0682] (c) the first strand sequence comprises a sequence of at least 18 nucleotides of any one of the first strand sequences of Table 1 and optionally wherein the second strand sequence comprises a sequence of at least 18 nucleotides of the second strand sequence in the same line of the table; or [0683] (d) the first strand sequence consists of any one of the first strand sequences of Table 1 and optionally wherein the second strand sequence consists of the sequence of the second strand sequence in the same line of the table; [0684] wherein Table 1 is:

TABLE-US-00003 First strand sequence 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 99 100 101 102 103 104 105 106 107 108 109 110 113 114 115 116 117 118 119 120 121 122 123 124 127 128 129 130 133 134 416 26 [0685] 3. The nucleic acid of any of the preceding statements, wherein the first strand sequence comprises the sequence of SEQ ID NO: 361 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 112; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 95 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 96; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 125 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 126; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 131 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 132; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 364 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 363 or 375; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 365 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 363; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 366 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO:367 or 376; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 368 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 369; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 370 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 371 or 379, preferably 379; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 372 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 373 or 380; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 362 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 374; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 377 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 378; or wherein the first strand sequence comprises the sequence of SEQ ID NO: 416 and optionally wherein the second strand sequence comprises a sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 26. [0686] 4. A double-stranded nucleic acid that is capable of inhibiting expression of complement component C3 for use as a medicament, wherein the nucleic acid comprises a first strand and a second strand. [0687] 5. The nucleic acid of any of the preceding statements, wherein the first strand and the second strand are separate strands and are each 18-25 nucleotides in length. [0688] 6. The nucleic acid of any of the preceding statements, wherein the first strand and the second strand form a duplex region of from 17-25 nucleotides in length. [0689] 7. The nucleic acid of any of the preceding statements, wherein the duplex region consists of 17-25 consecutive nucleotide base pairs. [0690] 8. The nucleic acid of any of the preceding statements, wherein said nucleic acid: [0691] a) is blunt ended at both ends; [0692] b) has an overhang at one end and a blunt end at the other end; or [0693] c) has an overhang at both ends. [0694] 9. The nucleic acid of any of the preceding statements, wherein the nucleic acid is a siRNA. [0695] 10. The nucleic acid of any of the preceding statements, wherein the nucleic acid mediates RNA interference. [0696] 11. The nucleic acid of any of the preceding statements, wherein at least one nucleotide of the first and/or second strand is a modified nucleotide. [0697] 12. The nucleic acid of any of the preceding statements, wherein at least nucleotides 2 and 14 of the first strand are modified by a first modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand. [0698] 13. The nucleic acid of any of the preceding statements, wherein each of the even-numbered nucleotides of the first strand are modified by a first modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand. [0699] 14. The nucleic acid of any of statements 12-13, wherein the odd-numbered nucleotides of the first strand are modified by a second modification, wherein the second modification is different from the first modification. [0700] 15. The nucleic acid of statements 12-14, wherein 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, wherein the third modification is different from the first modification. [0701] 16. The nucleic acid of statements 12-15, wherein 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 fourth modification is different from the second modification and different from the third modification when a second and/or a third modification are present. [0702] 17. The nucleic acid of statements 12-14, wherein 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 and preferably wherein the nucleotides of the second strand that are not modified by a fourth modification are modified by a third modification. [0703] 18. The nucleic acid of statements 12-17, wherein the first modification is the same as the fourth modification if both modifications are present in the nucleic acid and preferably wherein the second modification is the same as the third modification if both modifications are present in the nucleic acid. [0704] 19. The nucleic acid of statements 12-18, wherein the first modification is a 2′-F modification; the second modification, if present in the nucleic acid, is preferably a 2′-OMe modification; the third modification, if present in the nucleic acid, is preferably a 2′-OMe modification; and the fourth modification, if present in the nucleic acid, is preferably a 2′-F modification. [0705] 20. The nucleic acid of any of the preceding statements, wherein each of the nucleotides of the first strand and of the second strand is a modified nucleotide. [0706] 21. The nucleic acid of any of the previous statements, wherein the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide at its 5′ end and wherein the terminal 5′ (E)-vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage. [0707] 22. The nucleic acid of any of the preceding statements, wherein 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 and preferably wherein the linkages between the remaining nucleotides are phosphodiester linkages. [0708] 23. The nucleic acid of any of statements 1-21, comprising a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3′ end of the first strand and/or comprising 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 comprising a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5′ end of the first strand. [0709] 24. The nucleic acid of statement 23, wherein 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. [0710] 25. The nucleic acid of statement 23, wherein 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. [0711] 26. The nucleic acid of any of the preceding statements, wherein the nucleic acid is conjugated to a ligand. [0712] 27. The nucleic acid of statement 26, wherein the ligand comprises (i) one or more N-acetyl galactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates the at least one GalNAc moiety or derivative thereof to the nucleic acid. [0713] 28. The nucleic acid of any of statements 1-27, wherein the nucleic acid is conjugated to a ligand comprising a compound of formula (II):

[S—X.sup.1—P—X.sup.2].sub.3-A-X.sup.3—  (II) [0714] wherein: [0715] S represents a saccharide, preferably wherein the saccharide is N-acetyl galactosamine; [0716] X.sup.1 represents C.sub.3-C.sub.6 alkylene or (—CH.sub.2—CH.sub.2—O).sub.m(—CH.sub.2).sub.2— wherein m is 1, 2, or 3; [0717] P is a phosphate or modified phosphate, preferably a thiophosphate; [0718] X.sup.2 is alkylene or an alkylene ether of the formula (—CH.sub.2).sub.n—O—CH.sub.2— where n=1-6; [0719] A is a branching unit; [0720] X.sub.3 represents a bridging unit; [0721] wherein a nucleic acid as defined in any of statements 1 to 27 is conjugated to X.sup.3 via a phosphate or modified phosphate, preferably a thiophosphate. [0722] 29. The nucleic acid of any of statements 1-27, wherein the first strand of the nucleic acid is a compound of formula (V):

##STR00051## [0723] wherein b is 0 or 1; and [0724] wherein the second strand is a compound of formula (VI):

##STR00052## [0725] wherein: [0726] c and d are independently 0 or 1; [0727] Z.sub.1 and Z.sub.2 are respectively the first and second strand of the nucleic acid; [0728] Y is independently O or S; [0729] n is independently 0, 1, 2 or 3; and [0730] L.sub.1 is a linker to which a ligand is attached, wherein L.sub.1 is the same or different in formulae (V) and (VI), and is the same or different within formulae (V) and (VI) when L.sub.1 is present more than once within the same formula; [0731] and wherein b+c+d is 2 or 3. [0732] 30. A composition comprising a nucleic acid of any of the previous statements and a solvent and/or a delivery vehicle and/or a physiologically acceptable excipient and/or a carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative. [0733] 31. A composition comprising a nucleic acid of any of statements 1-29 and a further therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide. [0734] 32. A nucleic acid of any of statements 1-3 or 5-29 or a composition of any of statements 30-31 for use as a medicament. [0735] 33. A nucleic acid of any of statements 1-29 or a composition of any of statements 30-32 for use in the prevention, decrease of the risk of suffering from, or treatment of a disease, disorder or syndrome. [0736] 34. The nucleic acid or composition of statement 33, wherein the disease, disorder or syndrome is a complement-mediated disease, disorder or syndrome. [0737] 35. The nucleic acid or composition of any of statements 33-34, wherein the disease, disorder or syndrome is associated with aberrant activation or over-activation of the complement pathway and/or with over-expression or ectopic expression or localisation or accumulation of C3. [0738] 36. The nucleic acid or composition of any of statements 33-35, wherein the disease, disorder or syndrome is: [0739] a) selected from the group comprising 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, Malarial Anaemia, Neuromyelitis Optica, Post-HCT/Solid Organ Transplant (TMAs), Guillain-Barre Syndrome, Membranous Glomerulonephritis, Thrombotic Thrombocytopenic Purpura and sepsis; [0740] b) selected from the group comprising C3 Glomerulopathy (C3G), Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical Hemolytic Uremic Syndrome (aHUS), Lupus nephritis, IgA nephropathy (IgA N) and Primary Membranous Nephropathy; [0741] c) selected from the group comprising C3 Glomerulopathy (C3G), antineutrophil Cytoplasmic Autoantibodies-associated Vasculitis (ANCA-AV), atypical Hemolytic Uremic Syndrome (aHUS), Cold Agglutinin Disease (CAD), Myasthenia gravis (MG), IgA nephropathy (IgA N), Paroxysmal Nocturnal Hemoglobinuria (PNH); [0742] d) selected from the group comprising C3 Glomerulopathy (C3G), Cold Agglutinin Disease (CAD), Myasthenia gravis (MG), Neuromyelitis Optica, atypical Hemolytic Uremic Syndrome (aHUS), antineutrophil Cytoplasmic Autoantibodies-associated Vasculitis (ANCA-AV), IgA nephropathy (IgA N), Post-HCT/Solid Organ Transplant (TMAs), Guillain-Barre Syndrome, Paroxysmal Nocturnal Hemoglobinuria (PNH), Membranous Glomerulonephritis, Lupus nephritis and Thrombotic Thrombocytopenic Purpura [0743] e) selected from the group comprising C3 Glomerulopathy (C3G), Cold Agglutinin Disease (CAD) and IgA nephropathy (IgA N); or [0744] f) C3 Glomerulopathy (C3G). [0745] 37. Use of a nucleic acid of any of statements 1-29 or a composition of any of statements 30-31 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). [0746] 38. A method of preventing, decreasing the risk of suffering from, or treating a disease, disorder or syndrome comprising administering a pharmaceutically effective dose of a nucleic acid of any of statements 1-29 or 32-36 or a composition of any of statements 30-36 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 or intraperitoneal administration.

[0747] Summary Tables

TABLE-US-00004 Summary duplex table Table 3 Single Duplex Strands EV0001 EV0001A EV0001B EV0002 EV0002A EV0002B EV0003 EV0003A EV0003B EV0004 EV0004A EV0004B EV0005 EV0005A EV0005B EV0006 EV0006A EV0006B EV0007 EV0007A EV0007B EV0008 EV0008A EV0008B EV0009 EV0009A EV0009B EV0010 EV0010A EV0010B EV0011 EV0011A EV0011B EV0012 EV0012A EV0012B EV0013 EV0013A EV0013B EV0014 EV0014A EV0014B EV0015 EV0015A EV001513 EV0016 EV0016A EV0016B EV0017 EV0017A EV0017B EV0018 EV0018A EV0018B EV0019 EV0019A EV0019B EV0020 EV0020A EV0020B EV0021 EV0021A EV0021B EV0022 EV0022A EV0022B EV0023 EV0023A EV0023B EV0024 EV0024A EV0024B EV0025 EV0025A EV0025B EV0026 EV0026A EV0026B EV0027 EV0027A EV0027B EV0028 EV0028A EV0028B EV0029 EV0029A EV0029B EV0030 EV0030A EV0030B EV0031 EV0031A EV0031B EV0032 EV0032A EV0032B EV0033 EV0033A EV0033B EV0034 EV0034A EV0034B EV0035 EV0035A EV0035B EV0036 EV0036A EV0036B EV0037 EV0037A EV0037B EV0038 EV0038A EV0038B EV0039 EV0039A EV0039B EV0040 EV0040A EV0040B EV0041 EV0041A EV0041B EV0042 EV0042A EV0042B EV0043 EV0043A EV0043B EV0044 EV0044A EV0044B EV0045 EV0045A EV0045B EV0046 EV0046A EV0046B EV0047 EV0047A EV0047B EV0048 EV0048A EV0048B EV0049 EV0049A EV0049B EV0050 EV0050A EV0050B EV0051 EV0051A EV0051B EV0052 EV0052A EV0052B EV0053 EV0053A EV0053B EV0054 EV0054A EV0054B EV0055 EV0055A EV0055B EV0056 EV0056A EV0056B EV0057 EV0057A EV0057B EV0058 EV0058A EV0058B EV0059 EV0059A EV0059B EV0060 EV0060A EV0060B EV0061 EV0061A EV0061B EV0062 EV0062A EV0062B EV0063 EV0063A EV0063B EV0064 EV0064A EV0064B EV0065 EV0065A EV0065B EV0066 EV0066A EV0066B EV0067 EV0067A EV0067B EV0068 EV0068A EV0068B EV0069 EV0037A EV0069B EV0070 EV0038A EV0070B EV0071 EV0039A EV0071B EV0072 EV0040A EV0072B EV0073 EV0041A EV0073B EV0074 EV0042A EV0074B EV0075 EV0043A EV0075B EV0076 EV0044A EV0076B EV0077 EV0045A EV0077B EV0078 EV0046A EV0078B EV0079 EV0047A EV0079B EV0080 EV0048A EV0080B EV0081 EV0049A EV0081B EV0082 EV0050A EV0082B EV0083 EV0051A EV0083B EV0084 EV0052A EV0084B EV0085 EV0053A EV0085B EV0086 EV0054A EV0086B EV0087 EV0055A EV0087B EV0088 EV0056A EV0088B EV0089 EV0057A EV0089B EV0090 EV0058A EV0090B EV0091 EV0059A EV0091B EV0092 EV0060A EV0092B EV0093 EV0061A EV0093B EV0094 EV0062A EV0094B EV0095 EV0063A EV0095B EV0096 EV0064A EV0096B EV0097 EV0065A EV0097B EV0098 EV0066A EV0098B EV0099 EV0067A EV0099B EV0100 EV0068A EV0100B EV0101 EV0101A EV0101B EV0102 EV0102A EV0102B EV0103 EV0103A EV0103B EV0104 EV0104A EV0104B EV0105 EV0105A EV0105B EV0106 EV0106A EV0106B EV0107 EV0107A EV0107B EV0108 EV0108A EV0108B EV0109 EV0109A EV0109B EV0110 EV0110A EV0110B EV0111 EV0111A EV0111B EV0201 EV0201A EV0201B EV0202 EV0201A EV0202B EV0203 EV0203A EV0201B EV0204 EV0203A EV0202B EV0205 EV0110A EV0205B EV0206 EV0110A EV0206B EV0207 EV0207A EV0205B EV0208 EV0207A EV0206B EV0209 EV0209A EV0205B EV0312 EV0312A EV0112B EV0313 EV0313A EV0313B EJ0001 EJ0001A EJ0001B EJ0002 EJ0002A EJ0001B EJ0003 EJ0003A EJ0001B EJ0004 EJ0004A EJ0004B EJ0005 EJ0005A EJ0005B EJ0006 EJ0006A EJ0006B EJ0007 EJ0007A EJ0007B EJ0008 EJ0001A EJ0008B EJ0009 EJ0001A EJ0009B EJ0010 EJ0002A EJ0010B EJ0011 EJ0004A EJ0011B EJ0012 EJ0012A EJ0012B EJ0013 EJ0005A EJ0013B EJ0014 EJ0006A EJ0014B EJ0015 EJ0007A EJ0015B EJ0016 EJ0016A EJ0012B EJ0017 EJ0017A EJ0017B EJ0018 EJ0018A EJ0017B EJ0019 EJ0019A EJ0014B EJ0020 EJ0020A EJ0020B EJ0021 EJ0021A EJ0015B EJ0022 EJ0022A EJ0022B EJ0023 EJ0023A EJ0022B EV0210 EV0210A EV0210B EV0211 EV0211A EV0210B EV0212 EV0212A EV0211B EV0213 EV0213A EV0211B

TABLE-US-00005 TABLE 4 Summary abbreviations table Abbreviation Meaning mA, mU, mC, mG 2′-O-Methyl RNA nucleotides 2′-OMe 2′-O-Methyl modification fA, fU, fC, fG 2′ deoxy-2′-F RNA nucleotides 2′-F 2′-fluoro modification (ps) phosphorothioate (ps2) phosphorodithioate (vp) Vinyl-(E)-phosphonate (vp)-mU [00053] (vp)-mU-phos [00054] ivA, ivC, ivU, ivG inverted RNA (3′-3′) nucleotides ST23 [00055] ST23-phos [00056] ST43 (or C6XLT) [00057] ST43-phos (or C6XLT-phos) [00058] Ser(GN) (when at the end of a chain, one of the O--- is OH) [00059] [ST23 (ps)]3 ST43 (ps) [00060] [ST23]3 ST43 [00061]

[0748] The abbreviations as shown in the above abbreviation table may be used herein. The list of abbreviations may not be exhaustive and further abbreviations and their meaning may be found throughout this document.

TABLE-US-00006 TABLE 5 Summary sequence table SEQ Name ID (A = l.sup.st strand; Unmodified sequence NO: B = 2.sup.nd strand) Sequence 5′-3′ 5′-3′ counterpart 1 EV0001Aun UUUCAUAGUAGGCUCGGAU UUUCAUAGUAGGCUCGGAU 2 EV0001Bun AUCCGAGCCUACUAUGAAA AUCCGAGCCUACUAUGAAA 3 EV0002Aun UUUCUCUGUAGGCUCCACU UUUCUCUGUAGGCUCCACU 4 EV0002Bun AGUGGAGCCUACAGAGAAA AGUGGAGCCUACAGAGAAA 5 EV0003Aun UUAUAGAUGUAGUAGAAUU UUAUAGAUGUAGUAGAAUU 6 EV0003Bun AAUUCUACUACAUCUAUAA AAUUCUACUACAUCUAUAA 7 EV0004Aun AUGACAAAGGCAGUUCCCU AUGACAAAGGCAGUUCCCU 8 EV0004Bun AGGGAACUGCCUUUGUCAU AGGGAACUGCCUUUGUCAU 9 EV0005Aun AUCUGGUAGGGAGAGGUCA AUCUGGUAGGGAGAGGUCA 10 EV0005Bun UGACCUCUCCCUACCAGAU UGACCUCUCCCUACCAGAU 11 EV0006Aun UGUGUGUUGAUGCUGAGUU UGUGUGUUGAUGCUGAGUU 12 EV0006Bun AACUCAGCAUCAACACACA AACUCAGCAUCAACACACA 13 EV0007Aun UAAUUGUUGGAGUUGCCCA UAAUUGUUGGAGUUGCCCA 14 EV0007Bun UGGGCAACUCCAACAAUUA UGGGCAACUCCAACAAUUA 15 EV0008Aun AGGAAGUUGACGUUGAGGG AGGAAGUUGACGUUGAGGG 16 EV0008Bun CCCUCAACGUCAACUUCCU CCCUCAACGUCAACUUCCU 17 EV0009Aun AUGAAGUCGGUGGUGAUGG AUGAAGUCGGUGGUGAUGG 18 EV0009Bun CCAUCACCACCGACUUCAU CCAUCACCACCGACUUCAU 19 EV0010Aun UUUACCACCAGCGAGCCCA UUUACCACCAGCGAGCCCA 20 EV0010Bun UGGGCUCGCUGGUGGUAAA UGGGCUCGCUGGUGGUAAA 21 EV0011Aun UCUAUCUUCAGGGUCAUCU UCUAUCUUCAGGGUCAUCU 22 EV0011Bun AGAUGACCCUGAAGAUAGA AGAUGACCCUGAAGAUAGA 23 EV0012Aun AUGUAGUUGCAGCAGUCCA AUGUAGUUGCAGCAGUCCA 24 EV0012Bun UGGACUGCUGCAACUACAU UGGACUGCUGCAACUACAU 25 EV0013Aun AAUAUAUUCAUGAGCUUCG AAUAUAUUCAUGAGCUUCG 26 EV0013Bun CGAAGCUCAUGAAUAUAUU CGAAGCUCAUGAAUAUAUU 27 EV0014Aun AAAUAUAUUCAUGAGCUUC AAAUAUAUUCAUGAGCUUC 28 EV0014Bun GAAGCUCAUGAAUAUAUUU GAAGCUCAUGAAUAUAUUU 29 EV0015Aun UCCUGCAUUACUGUGACCU UCCUGCAUUACUGUGACCU 30 EV0015Bun AGGUCACAGUAAUGCAGGA AGGUCACAGUAAUGCAGGA 31 EV0016Aun CAACAGAGUAGGGUAGCCG CAACAGAGUAGGGUAGCCG 32 EV0016Bun CGGCUACCCUACUCUGUUG CGGCUACCCUACUCUGUUG 33 EV0017Aun UCGAACAACAGAGUAGGGU UCGAACAACAGAGUAGGGU 34 EV0017Bun ACCCUACUCUGUUGUUCGA ACCCUACUCUGUUGUUCGA 35 EV0018Aun UUUCGAACAACAGAGUAGG UUUCGAACAACAGAGUAGG 36 EV0018Bun CCUACUCUGUUGUUCGAAA CCUACUCUGUUGUUCGAAA 37 EV0019Aun GUUUCAUCCAGGUAAUGCA GUUUCAUCCAGGUAAUGCA 38 EV0019Bun UGCAUUACCUGGAUGAAAC UGCAUUACCUGGAUGAAAC 39 EV0020Aun UUAUCUUUGGCUGUGGUCA UUAUCUUUGGCUGUGGUCA 40 EV0020Bun UGACCACAGCCAAAGAUAA UGACCACAGCCAAAGAUAA 41 EV0021Aun UGUUCAUUGAGCCAACGCA UGUUCAUUGAGCCAACGCA 42 EV0021Bun UGCGUUGGCUCAAUGAACA UGCGUUGGCUCAAUGAACA 43 EV0023Aun UUGGUAUUGAGCCAAGGCU UUGGUAUUGAGCCAAGGCU 44 EV0023Bun AGCCUUGGCUCAAUACCAA AGCCUUGGCUCAAUACCAA 45 EV0024Aun UUUAUUACAGGUGAGUUGA UUUAUUACAGGUGAGUUGA 46 EV0024Bun UCAACUCACCUGUAAUAAA UCAACUCACCUGUAAUAAA 47 EV0025Aun UUUCUGUUUCCGGUGCUGG UUUCUGUUUCCGGUGCUGG 48 EV0025Bun CCAGCACCGGAAACAGAAA CCAGCACCGGAAACAGAAA 49 EV0026Aun UCAAGGAUCAUAGUGUUCU UCAAGGAUCAUAGUGUUCU 50 EV0026Bun AGAACACUAUGAUCCUUGA AGAACACUAUGAUCCUUGA 51 EV0027Aun AUCUCAAGGAUCAUAGUGU AUCUCAAGGAUCAUAGUGU 52 EV0027Bun ACACUAUGAUCCUUGAGAU ACACUAUGAUCCUUGAGAU 53 EV0028Aun UCAUACUUGGAGAUGUAUC UCAUACUUGGAGAUGUAUC 54 EV0028Bun GAUACAUCUCCAAGUAUGA GAUACAUCUCCAAGUAUGA 55 EV0029Aun UAUCGGAGAAGGCUUUGUC UAUCGGAGAAGGCUUUGUC 56 EV0029Bun GACAAAGCCUUCUCCGAUA GACAAAGCCUUCUCCGAUA 57 EV0030Aun AUGAUGAGGGUGUUCCUAU AUGAUGAGGGUGUUCCUAU 58 EV0030Bun AUAGGAACACCCUCAUCAU AUAGGAACACCCUCAUCAU 59 EV0031Aun UAUUGGUGAACUUUGAAAG UAUUGGUGAACUUUGAAAG 60 EV0031Bun CUUUCAAAGUUCACCAAUA CUUUCAAAGUUCACCAAUA 61 EV0032Aun AGCUUGUUCAGCUUUCCAU AGCUUGUUCAGCUUUCCAU 62 EV0032Bun AUGGAAAGCUGAACAAGCU AUGGAAAGCUGAACAAGCU 63 EV0033Aun UUUGUAUGAAGCAAUUCUC UUUGUAUGAAGCAAUUCUC 64 EV0033Bun GAGAAUUGCUUCAUACAAA GAGAAUUGCUUCAUACAAA 65 EV0034Aun GUCUUGUACACAUAGUCCA GUCUUGUACACAUAGUCCA 66 EV0034Bun UGGACUAUGUGUACAAGAC UGGACUAUGUGUACAAGAC 67 EV0035Aun UGCUCAAUGGCCAUGAUGU UGCUCAAUGGCCAUGAUGU 68 EV0035Bun ACAUCAUGGCCAUUGAGCA ACAUCAUGGCCAUUGAGCA 69 EV0036Aun UUGGCAUUCGUCCUCCUCG UUGGCAUUCGUCCUCCUCG 70 EV0036Bun CGAGGAGGACGAAUGCCAA CGAGGAGGACGAAUGCCAA 71 EV0037Aun GUCUGGAUGAAGAGGUACC GUCUGGAUGAAGAGGUACC 72 EV0037Bun GGUACCUCUUCAUCCAGAC GGUACCUCUUCAUCCAGAC 73 EV0038Aun UGUCUGGAUGAAGAGGUAC UGUCUGGAUGAAGAGGUAC 74 EV0038Bun GUACCUCUUCAUCCAGACA GUACCUCUUCAUCCAGACA 75 EV0039Aun UCUGUCUGGAUGAAGAGGU UCUGUCUGGAUGAAGAGGU 76 EV0039Bun ACCUCUUCAUCCAGACAGA ACCUCUUCAUCCAGACAGA 77 EV0040Aun CUUGUCUGUCUGGAUGAAG CUUGUCUGUCUGGAUGAAG 78 EV0040Bun CUUCAUCCAGACAGACAAG CUUCAUCCAGACAGACAAG 79 EV0041Aun AUGGUCUUGUCUGUCUGGA AUGGUCUUGUCUGUCUGGA 80 EV0041Bun UCCAGACAGACAAGACCAU UCCAGACAGACAAGACCAU 81 EV0042Aun AGAUGGUCUUGUCUGUCUG AGAUGGUCUUGUCUGUCUG 82 EV0042Bun CAGACAGACAAGACCAUCU CAGACAGACAAGACCAUCU 83 EV0043Aun GUAGAUGGUCUUGUCUGUC GUAGAUGGUCUUGUCUGUC 84 EV0043Bun GACAGACAAGACCAUCUAC GACAGACAAGACCAUCUAC 85 EV0044Aun GUGUAGAUGGUCUUGUCUG GUGUAGAUGGUCUUGUCUG 86 EV0044Bun CAGACAAGACCAUCUACAC CAGACAAGACCAUCUACAC 87 EV0045Aun GGGGUGUAGAUGGUCUUGU GGGGUGUAGAUGGUCUUGU 88 EV0045Bun ACAAGACCAUCUACACCCC ACAAGACCAUCUACACCCC 89 EV0046Aun UAGGCUCGGAUCUUCCACU UAGGCUCGGAUCUUCCACU 90 EV0046Bun AGUGGAAGAUCCGAGCCUA AGUGGAAGAUCCGAGCCUA 91 EV0047Aun CUCGAAACUGGGCAGCACG CUCGAAACUGGGCAGCACG 92 EV0047Bun CGUGCUGCCCAGUUUCGAG CGUGCUGCCCAGUUUCGAG 93 EV0048Aun UUGGUGAAGUGGAUCUGGU UUGGUGAAGUGGAUCUGGU 94 EV0048Bun ACCAGAUCCACUUCACCAA ACCAGAUCCACUUCACCAA 95 EV0049Aun UCUUGGUGAAGUGGAUCUG UCUUGGUGAAGUGGAUCUG 96 EV0049Bun CAGAUCCACUUCACCAAGA CAGAUCCACUUCACCAAGA 97 EV0050Aun GUCUUGGUGAAGUGGAUCU GUCUUGGUGAAGUGGAUCU 98 EV0050Bun AGAUCCACUUCACCAAGAC AGAUCCACUUCACCAAGAC 99 EV0051Aun GGUGUCUUGGUGAAGUGGA GGUGUCUUGGUGAAGUGGA 100 EV0051Bun UCCACUUCACCAAGACACC UCCACUUCACCAAGACACC 101 EV0052Aun AACACCAUGAGGUCAAAGG AACACCAUGAGGUCAAAGG 102 EV0052Bun CCUUUGACCUCAUGGUGUU CCUUUGACCUCAUGGUGUU 103 EV0053Aun GAACACCAUGAGGUCAAAG GAACACCAUGAGGUCAAAG 104 EV0053Bun CUUUGACCUCAUGGUGUUC CUUUGACCUCAUGGUGUUC 105 EV0054Aun GUCACGAACACCAUGAGGU GUCACGAACACCAUGAGGU 106 EV0054Bun ACCUCAUGGUGUUCGUGAC ACCUCAUGGUGUUCGUGAC 107 EV0055Aun ACACAGAUCCCUUUCUUGU ACACAGAUCCCUUUCUUGU 108 EV0055Bun ACAAGAAAGGGAUCUGUGU ACAAGAAAGGGAUCUGUGU 109 EV0058Aun CCUUGCAGGAGAAUUCUGG CCUUGCAGGAGAAUUCUGG 110 EV0058Bun CCAGAAUUCUCCUGCAAGG CCAGAAUUCUCCUGCAAGG 111 EV0059Aun AGAGAGAAGACCUUGACCA AGAGAGAAGACCUUGACCA 112 EV0059Bun UGGUCAAGGUCUUCUCUCU UGGUCAAGGUCUUCUCUCU 113 EV0060Aun GAGUCGAUGGCGAUGAGGU GAGUCGAUGGCGAUGAGGU 114 EV0060Bun ACCUCAUCGCCAUCGACUC ACCUCAUCGCCAUCGACUC 115 EV0061Aun GCUUGGAACACCAUGAAGG GCUUGGAACACCAUGAAGG 116 EV0061Bun CCUUCAUGGUGUUCCAAGC CCUUCAUGGUGUUCCAAGC 117 EV0062Aun UCAUUUUCCUUGGUCUCUU UCAUUUUCCUUGGUCUCUU 118 EV0062Bun AAGAGACCAAGGAAAAUGA AAGAGACCAAGGAAAAUGA 119 EV0063Aun UGUGUCUGGAGCAAAGCCA UGUGUCUGGAGCAAAGCCA 120 EV0063Bun UGGCUUUGCUCCAGACACA UGGCUUUGCUCCAGACACA 121 EV0064Aun AGGUAGAUGAUGAGGGUGU AGGUAGAUGAUGAGGGUGU 122 EV0064Bun ACACCCUCAUCAUCUACCU ACACCCUCAUCAUCUACCU 123 EV0065Aun UUGUAAUAGGCGUAGACCU UUGUAAUAGGCGUAGACCU 124 EV0065Bun AGGUCUACGCCUAUUACAA AGGUCUACGCCUAUUACAA 125 EV0066Aun GUUGUAAUAGGCGUAGACC GUUGUAAUAGGCGUAGACC 126 EV0066Bun GGUCUACGCCUAUUACAAC GGUCUACGCCUAUUACAAC 127 EV0067Aun GGGUCUUGUACACAUAGUC GGGUCUUGUACACAUAGUC 128 EV0067Bun GACUAUGUGUACAAGACCC GACUAUGUGUACAAGACCC 129 EV0068Aun CACUUGAUGGGGCUGAUGA CACUUGAUGGGGCUGAUGA 130 EV0068Bun UCAUCAGCCCCAUCAAGUG UCAUCAGCCCCAUCAAGUG 131 EV0312Aun AUUGUAAUAGGCGUAGACC AUUGUAAUAGGCGUAGACC 132 EV0112Bun GGUCUACGCCUAUUACAAU GGUCUACGCCUAUUACAAU 133 EV0313Aun AUGUAGAUGGUCUUGUCUG AUGUAGAUGGUCUUGUCUG 134 EV0313Bun CAGACAAGACCAUCUACAU CAGACAAGACCAUCUACAU 135 EV0069Bun GGUACCUCUUCAUCCAGAA GGUACCUCUUCAUCCAGAA 136 EV0072Bun CUUCAUCCAGACAGACAAA CUUCAUCCAGACAGACAAA 137 EV0073Bun UCCAGACAGACAAGACCAA UCCAGACAGACAAGACCAA 138 EV0074Bun CAGACAGACAAGACCAUCA CAGACAGACAAGACCAUCA 139 EV0075Bun GACAGACAAGACCAUCUAA GACAGACAAGACCAUCUAA 140 EV0076Bun CAGACAAGACCAUCUACAA CAGACAAGACCAUCUACAA 141 EV0077Bun ACAAGACCAUCUACACCCA ACAAGACCAUCUACACCCA 142 EV0079Bun CGUGCUGCCCAGUUUCGAA CGUGCUGCCCAGUUUCGAA 143 EV0082Bun AGAUCCACUUCACCAAGAA AGAUCCACUUCACCAAGAA 144 EV0083Bun UCCACUUCACCAAGACACA UCCACUUCACCAAGACACA 145 EV0084Bun CCUUUGACCUCAUGGUGUA CCOUUGACCUCAUGGUGUA 146 EV0085Bun CUUUGACCUCAUGGUGUUA CUUUGACCUCAUGGUGUUA 147 EV0086Bun ACCUCAUGGUGUUCGUGAA ACCUCAUGGUGUUCGUGAA 148 EV0087Bun ACAAGAAAGGGAUCUGUGA ACAAGAAAGGGAUCUGUGA 149 EV0088Bun AGGGAUCUGUGUGGCAGAA AGGGAUCUGUGUGGCAGAA 150 EV0089Bun AAUGCAGGACUUCUUCAUA AAUGCAGGACUUCUUCAUA 151 EV0090Bun CCAGAAUUCUCCUGCAAGA CCAGAAUUCUCCUGCAAGA 152 EV0091Bun UGGUCAAGGUCUUCUCUCA UGGUCAAGGUCUUCUCUCA 153 EV0092Bun ACCUCAUCGCCAUCGACUA ACCUCAUCGCCAUCGACUA 154 EV0093Bun CCUUCAUGGUGUUCCAAGA CCUUCAUGGUGUUCCAAGA 155 EV0096Bun ACACCCUCAUCAUCUACCA ACACCCUCAUCAUCUACCA 156 EV0098Bun GGUCUACGCCUAUUACAAA GGUCUACGCCUAUUACAAA 157 EV0099Bun GACUAUGUGUACAAGACCA GACUAUGUGUACAAGACCA 158 EV0100Bun UCAUCAGCCCCAUCAAGUA UCAUCAGCCCCAUCAAGUA 159 EV0001A mU fU mU fC mA fU mA fG mU fA mG fG mC fU mC fG mG fA mU UUUCAUAGUAGGCUCGGAU 160 EV0001B fA mU fC mC fG mA fG mC fC mU fA mC fU mA fU mG fA mA fA AUCCGAGCCUACUAUGAAA 161 EV0002A mU fU mU fC mU fC mU fG mU fA mG fG mC fU mC fC mA fC mU UUUCUCUGUAGGCUCCACU 162 EV0002B fA mG fU mG fG mA fG mC fC mU fA mC fA mG fA mG fA mA fA AGUGGAGCCUACAGAGAAA 163 EV0003A mU fU mA fU mA fG mA fU mG fU mA fG mU fA mG fA mA fU mU UUAUAGAUGUAGUAGAAUU 164 EV0003B fA mA fU mU fC mU fA mC fU mA fC mA fU mC fU mA fU mA fA AAUUCUACUACAUCUAUAA 165 EV0004A mA fU mG fA mC fA mA fA mG fG mC fA mG fU mU fC mC fC mU AUGACAAAGGCAGUUCCCU 166 EV0004B fA mG fG mG fA mA fC mU fG mC fC mU fU mU fG mU fC mA fU AGGGAACUGCCUUUGUCAU 167 EV0005A mA fU mC fU mG fG mU fA mG fG mG fA mG fA mG fG mU fC mA AUCUGGUAGGGAGAGGUCA 168 EV0005B fU mG fA mC fC mU fC mU fC mC fC mU fA mC fC mA fG mA fU UGACCUCUCCCUACCAGAU 169 EV0006A mU fG mU fG mU fG mU fU mG fA mU fG mC fU mG fA mG fU mU UGUGUGUUGAUGCUGAGUU 170 EV0006B fA mA fC mU fC mA fG mC fA mU fC mA fA mC fA mC fA mC fA AACUCAGCAUCAACACACA 171 EV0007A mU fA mA fU mU fG mU fU mG fG mA fG mU fU mG fC mC fC mA UAAUUGUUGGAGUUGCCCA 172 EV0007B fU mG fG mG fC mA fA mC fU mC fC mA fA mC fA mA fU mU fA UGGGCAACUCCAACAAUUA 173 EV0008A mA fG mG fA mA fG mU fU mG fA mC fG mU fU mG fA mG fG mG AGGAAGUUGACGUUGAGGG 174 EV0008B fC mC fC mU fC mA fA mC fG mU fC mA fA mC fU mU fC mC fU CCCUCAACGUCAACUUCCU 175 EV0009A mA fU mG fA mA fG mU fC mG fG mU fG mG fU mG fA mU fG mG AUGAAGUCGGUGGUGAUGG 176 EV0009B fC mC fA mU fC mA fC mC fA mC fC mG fA mC fU mU fC mA fU CCAUCACCACCGACUUCAU 177 EV0010A mU fU mU fA mC fC mA fC mC fA mG fC mG fA mG fC mC fC mA UUUACCACCAGCGAGCCCA 178 EV0010B fU mG fG mG fC mU fC mG fC mU fG mG fU mG fG mU fA mA fA UGGGCUCGCUGGUGGUAAA 179 EV0011A mU fC mU fA mU fC mU fU mC fA mG fG mG fU mC fA mU fC mU UCUAUCUUCAGGGUCAUCU 180 EV0011B fA mG fA mU fG mA fC mC fC mU fG mA fA mG fA mU fA mG fA AGAUGACCCUGAAGAUAGA 181 EV0012A mA fU mG fU mA fG mU fU mG fC mA fG mC fA mG fU mC fC mA AUGUAGUUGCAGCAGUCCA 182 EV0012B fU mG fG mA fC mU fG mC fU mG fC mA fA mC fU mA fC mA fU UGGACUGCUGCAACUACAU 183 EV0013A mA fA mU fA mU fA mU fU mC fA mU fG mA fG mC fU mU fC mG AAUAUAUUCAUGAGCUUCG 184 EV0013B fC mG fA mA fG mC fU mC fA mU fG mA fA mU fA mU fA mU fU CGAAGCUCAUGAAUAUAUU 185 EV0014A mA fA mA fU mA fU mA fU mU fC mA fU mG fA mG fC mU fU mC AAAUAUAUUCAUGAGCUUC 186 EV0014B fG mA fA mG fC mU fC mA fU mG fA mA fU mA fU mA fU mU fU GAAGCUCAUGAAUAUAUUU 187 EV0015A mU fC mC fU mG fC mA fU mU fA mC fU mG fU mG fA mC fC mU UCCUGCAUUACUGUGACCU 188 EV0015B fA mG fG mU fC mA fC mA fG mU fA mA fU mG fC mA fG mG fA AGGUCACAGUAAUGCAGGA 189 EV0016A mC fA mA fC mA fG mA fG mU fA mG fG mG fU mA fG mC fC mG CAACAGAGUAGGGUAGCCG 190 EV0016B fC mG fG mC fU mA fC mC fC mU fA mC fU mC fU mG fU mU fG CGGCUACCCUACUCUGUUG 191 EV0017A mU fC mG fA mA fC mA fA mC fA mG fA mG fU mA fG mG fG mU UCGAACAACAGAGUAGGGU 192 EV0017B fA mC fC mC fU mA fC mU fC mU fG mU fU mG fU mU fC mG fA ACCCUACUCUGUUGUUCGA 193 EV0018A mU fU mU fC mG fA mA fC mA fA mC fA mG LA mG fU mA fG mG UUUCGAACAACAGAGUAGG 194 EV0018B fC mC fU mA fC mU fC mU fG mU fU mG fU mU fC mG fA mA fA CCUACUCUGUUGUUCGAAA 195 EV0019A mG fU mU fU mC fA mU fC mC fA mG fG mU fA mA fU mG fC mA GUUUCAUCCAGGUAAUGCA 196 EV0019B fU mG fC mA fU mU fA mC fC mU fG mG fA mU fG mA fA mA fC UGCAUUACCUGGAUGAAAC 197 EV0020A mU fU mA fU mC fU mU fU mG fG mC fU mG fU mG fG mU fC mA UUAUCUUUGGCUGUGGUCA 198 EV0020B fU mG fA mC fC mA fC mA fG mC fC mA fA mA fG mA fU mA fA UGACCACAGCCAAAGAUAA 199 EV0021A mU fG mU fU mC fA mU fU mG fA mG fC mC fA mA fC mG fC mA UGUUCAUUGAGCCAACGCA 200 EV0021B fU mG fC mG fU mU fG mG fC mU fC mA fA mU fG mA fA mC fA UGCGUUGGCUCAAUGAACA 201 EV0022A mA fA mC fA mC fC mA fU mG fA mA fG mG fU mG fG mC fC mU AACACCAUGAAGGUGGCCU 202 EV0022B fA mG fG mC fC mA fC mC fU mU fC mA fU mG fG mU fG mU fU AGGCCACCUUCAUGGUGUU 203 EV0023A mU fU mG fG mU fA mU fU mG fA mG fC mC fA mA fG mG fC mU UUGGUAUUGAGCCAAGGCU 204 EV0023B fA mG fC mC fU mU fG mG fC mU fC mA fA mU fA mC fC mA fA AGCCUUGGCUCAAUACCAA 205 EV0024A mU fU mU fA mU fU mA fC mA fG mG fU mG fA mG fU mU fG mA UUUAUUACAGGUGAGUUGA 206 EV0024B fU mC fA mA fC mU fC mA fC mC fU mG fU mA fA mU fA mA fA UCAACUCACCUGUAAUAAA 207 EV0025A mU fU mU fC mU fG mU fU mU fC mC fG mG fU mG fC mU fG mG UUUCUGUUUCCGGUGCUGG 208 EV0025B fC mC fA mG fC mA fC mC fG mG fA mA fA mC fA mG fA mA fA CCAGCACCGGAAACAGAAA 209 EV0026A mU fC mA fA mG fG mA fU mC fA mU fA mG fU mG fU mU fC mU UCAAGGAUCAUAGUGUUCU 210 EV0026B fA mG fA mA fC mA fC mU fA mU fG mA fU mC fC mU fU mG fA AGAACACUAUGAUCCUUGA 211 EV0027A mA fU mC fU mC fA mA fG mG fA mU fC mA fU mA fG mU fG mU AUCUCAAGGAUCAUAGUGU 212 EV0027B fA mC fA mC fU mA fU mG fA mU fC mC fU mU fG mA fG mA fU ACACUAUGAUCCUUGAGAU 213 EV0028A mU fC mA fU mA fC mU fU mG fG mA fG mA fU mG fU mA fU mC UCAUACUUGGAGAUGUAUC 214 EV0028B fG mA fU mA fC mA fU mC fU mC fC mA fA mG fU mA fU mG fA GAUACAUCUCCAAGUAUGA 215 EV0029A mU fA mU fC mG fG mA fG mA fA mG fG mC fU mU fU mG fU mC UAUCGGAGAAGGCUUUGUC 216 EV0029B fG mA fC mA fA mA fG mC fC mU fU mC fU mC fC mG fA mU fA GACAAAGCCUUCUCCGAUA 217 EV0030A mA fU mG fA mU fG mA fG mG fG mU fG mU fU mC fC mU fA mU AUGAUGAGGGUGUUCCUAU 218 EV0030B fA mU fA mG fG mA fA mC fA mC fC mC fU mC fA mU fC mA fU AUAGGAACACCCUCAUCAU 219 EV0031A mU fA mU fU mG fG mU fG mA fA mC fU mU fU mG fA mA fA mG UAUUGGUGAACUUUGAAAG 220 EV0031B fC mU fU mU fC mA fA mA fG mU fU mC fA mC fC mA fA mU fA CUUUCAAAGUUCACCAAUA 221 EV0032A mA fC mC fU mU fG mU fU mC fA mG fC mU fU mU fC mC fA mU AGCUUGUUCAGCUUUCCAU 222 EV0032B fA mU fC mG fA mA fA mG fC mU fC mA fA mC fA mA fC mC fU AUGGAAAGCUGAACAAGCU 223 EV0033A mU fU mU fC mU fA mU fG mA fA mG fC mA fA mU fU mC fU mC UUUGUAUGAAGCAAUUCUC 224 EV0033B fG mA fG mA fA mU fU mG fC mU fU mC fA mU fA mC fA mA fA GAGAAUUGCUUCAUACAAA 225 EV0034A mG fU mC fU mU fG mU fA mC fA mC fA mU fA mG fU mC fC mA GUCUUGUACACAUAGUCCA 226 EV0034B fU mG fG mA fC mU fA mU fG mU fG mU fA mC LA mA fG mA fC UGGACUAUGUGUACAAGAC 227 EV0035A mU fG mC fU mC fA mA fU mG fG mC fC mA fU mG fA mU fG mU UGCUCAAUGGCCAUGAUGU 228 EV0035B fA mC fA mU fC mA fU mG fG mC fC mA fU mU fG mA fG mC fA ACAUCAUGGCCAUUGAGCA 229 EV0036A mU fU mG fG mC fA mU fU mC fG mU fC mC fU mC fC mU fC mG UUGGCAUUCGUCCUCCUCG 230 EV0036B fC mG fA mG fG mA fG mG fA mC fG mA fA mU fG mC fC mA fA CGAGGAGGACGAAUGCCAA 231 EV0037A mG fU mC fU mG fG mA fU mG fA mA fG mA fG mG fU mA fC mC GUCUGGAUGAAGAGGUACC 232 EV0037B fG mG fU mA fC mC fU mC fU mU fC mA fU mC fC mA fG mA fC GGUACCUCUUCAUCCAGAC 233 EV0038A mU fG mU fC mU fG mG fA mU fG mA fA mG fA mG fG mU fA mC UGUCUGGAUGAAGAGGUAC 234 EV0038B fG mU fA mC fC mU fC mU fU mC fA mU fC mC fA mG fA mC fA GUACCUCUUCAUCCAGACA 235 EV0039A mU fC mU fG mU fC mU fG mG fA mU fG mA fA mG fA mG fG mU UCUGUCUGGAUGAAGAGGU 236 EV0039B fA mC fC mU fC mU fU mC fA mU fC mC fA mG fA mC fA mG fA ACCUCUUCAUCCAGACAGA 237 EV0040A mC fU mU fG mU fC mU fG mU fC mU fG mG fA mU fG mA fA mG CUUGUCUGUCUGGAUGAAG 238 EV0040B fC mU fU mC fA mU fC mC fA mG fA mC fA mG fA mC fA mA fG CUUCAUCCAGACAGACAAG 239 EV0041A mA fU mG fG mU fC mU fU mG fU mC fU mG fU mC fU mG fG mA AUGGUCUUGUCUGUCUGGA 240 EV0041B fU mC fC mA fG mA fC mA fG mA fC mA fA mG fA mC fC mA fU UCCAGACAGACAAGACCAU 241 EV0042A mA fG mA fU mG fG mU fC mU fU mG fU mC fU mG fU mC fU mG AGAUGGUCUUGUCUGUCUG 242 EV0042B fC mA fG mA fC mA fG mA fC mA fA mG fA mC fC mA fU mC fU CAGACAGACAAGACCAUCU 243 EV0043A mG fU mA fG mA fU mG fG mU fC mU fU mG fU mC fU mG fU mC GUAGAUGGUCUUGUCUGUC 244 EV0043B fG mA fC mA fG mA fC mA fA mG fA mC fC mA fU mC fU mA fC GACAGACAAGACCAUCUAC 245 EV0044A mG fU mG fU mA fG mA fU mG fG mU fC mU fU mG fU mC fU mG GUGUAGAUGGUCUUGUCUG 246 EV0044B fC mA fG mA fC mA fA mG fA mC fC mA fU mC fU mA fC mA fC CAGACAAGACCAUCUACAC 247 EV0045A mG fG mG fG mU fG mU fA mG fA mU fG mG fU mC fU mU fG mU GGGGUGUAGAUGGUCUUGU 248 EV0045B fA mC fA mA fG mA fC mC fA mU fC mU fA mC fA mC fC mC fC ACAAGACCAUCUACACCCC 249 EV0046A mU fA mG fG mC fU mC fG mG fA mU fC mU fU mC fC mA fC mU UAGGCUCGGAUCUUCCACU 250 EV0046B fA mG fU mG fG mA fA mG fA mU fC mC fG mA fG mC fC mU fA AGUGGAAGAUCCGAGCCUA 251 EV0047A mC fU mC fG mA fA mA fC mU fG mG fG mC fA mG fC mA fC mG CUCGAAACUGGGCAGCACG 252 EV0047B fC mG fU mG fC mU fG mC fC mC fA mG fU mU fU mC fG mA fG CGUGCUGCCCAGUUUCGAG 253 EV0048A mU fU mG fG mU fG mA fA mG fU mG fG mA fU mC fU mG fG mU UUGGUGAAGUGGAUCUGGU 254 EV0048B fA mC fC mA fG mA fU mC fC mA fC mU fU mC fA mC fC mA fA ACCAGAUCCACUUCACCAA 255 EV0049A mU fC mU fU mG fG mU fG mA fA mG fU mG fG mA fU mC fU mG UCUUGGUGAAGUGGAUCUG 256 EV0049B fC mA fG mA fU mC fC mA fC mU fU mC fA mC fC mA fA mG fA CAGAUCCACUUCACCAAGA 257 EV0050A mG fU mC fU mU fG mG fU mG fA mA fG mU fG mG fA mU fC mU GUCUUGGUGAAGUGGAUCU 258 EV0050B fA mG fA mU fC mC fA mC fU mU fC mA fC mC fA mA fG mA fC AGAUCCACUUCACCAAGAC 259 EV0051A mG fG mU fG mU fC mU fU mG fG mU fG mA fA mG fU mG fG mA GGUGUCUUGGUGAAGUGGA 260 EV0051B fU mC fC mA fC mU fU mC fA mC fC mA fA mG fA mC fA mC fC UCCACUUCACCAAGACACC 261 EV0052A mA fA mC fA mC fC mA fU mG fA mG fG mU fC mA fA mA fG mG AACACCAUGAGGUCAAAGG 262 EV0052B fC mC fU mU fU mG fA mC fC mU fC mA fU mG fG mU fG mU fU CCUUUGACCUCAUGGUGUU 263 EV0053A mG fA mA fC mA fC mC fA mU fG mA fG mG fU mC fA mA fA mG GAACACCAUGAGGUCAAAG 264 EV0053B fC mU fU mU fG mA fC mC fU mC fA mU fG mG fU mG fU mU fC CUUUGACCUCAUGGUGUUC 265 EV0054A mG fU mC fA mC fG mA fA mC fA mC fC mA fU mG fA mG fG mU GUCACGAACACCAUGAGGU 266 EV0054B fA mC fC mU fC mA fU mG fG mU fG mU fU mC fG mU fG mA fC ACCUCAUGGUGUUCGUGAC 267 EV0055A mA fC mA fC mA fG mA fU mC fC mC fU mU fU mC fU mU fG mU ACACAGAUCCCUUUCUUGU 268 EV0055B fA mC fA mA fG mA fA mA fG mG fG mA fU mC fU mG fU mG fU ACAAGAAAGGGAUCUGUGU 269 EV0056A mG fU mC fU mG fC mC fA mC fA mC fA mG fA mU fC mC fC mU GUCUGCCACACAGAUCCCU 270 EV0056B fA mG fG mG fA mU fC mU fG mU fG mU fG mG fC mA fG mA fC AGGGAUCUGUGUGGCAGAC 271 EV0057A mG fA mU fG mA fA mG fA mA fG mU fC mC fU mG fC mA fU mU GAUGAAGAAGUCCUGCAUU 272 EV0057B fA mA fU mG fC mA fG mG fA mC fU mU fC mU fU mC fA mU fC AAUGCAGGACUUCUUCAUC 273 EV0058A mC fC mU fU mG fC mA fG mG fA mG fA mA fU mU fC mU fG mG CCUUGCAGGAGAAUUCUGG 274 EV0058B fC mC fA mG fA mA fU mU fC mU fC mC fU mG fC mA fA mG fG CCAGAAUUCUCCUGCAAGG 275 EV0059A mA fG mA fG mA fG mA fA mG fA mC fC mU fU mG fA mC fC mA AGAGAGAAGACCUUGACCA 276 EV0059B fU mG fG mU fC mA fA mG fG mU fC mU fU mC fU mC fU mC fU UGGUCAAGGUCOUCUCUCU 277 EV0060A mG fA mG fU mC fG mA fU mG fG mC fG mA fU mG fA mG fG mU GAGUCGAUGGCGAUGAGGU 278 EV0060B fA mC fC mU fC mA fU mC fG mC fC mA fU mC fG mA fC mU fC ACCUCAUCGCCAUCGACUC 279 EV0061A mG fC mU fU mG fG mA fA mC fA mC fC mA fU mG fA mA fG mG GCUUGGAACACCAUGAAGG 280 EV0061B fC mC fU mU fC mA fU mG fG mU fG mU fU mC fC mA fA mG fC CCUUCAUGGUGUUCCAAGC 281 EV0062A mU fC mA fU mU fU mU fC mC fU mU fG mG fU mC fU mC fU mU UCAUUUUCCUUGGUCOCUU 282 EV0062B fA mA fG mA fG mA fC mC fA mA fG mG fA mA fA mA fU mG fA AAGAGACCAAGGAAAAUGA 283 EV0063A mU fG mU fG mU fC mU fG mG fA mG fC mA fA mA fG mC fC mA UGUGUCUGGAGCAAAGCCA 284 EV0063B fU mG fG mC fU mU fU mG fC mU fC mC fA mG fA mC fA mC fA UGGCUUUGCUCCAGACACA 285 EV0064A mA fG mG fU mA fG mA fU mG fA mU fG mA fC mG fG mU fG mU AGGUAGAUGAUGAGGGUGU 286 EV0064B fA mC fA mC fC mC fU mC fA mU fC mA fU mC fU mA fC mC fU ACACCCUCAUCAUCUACCU 287 EV0065A mU fU mG fU mA fA mU fA mG fG mC fG mU fA mG fA mC fC mU UUGUAAUAGGCGUAGACCU 288 EV0065B fA mG fG mU fC mU fA mC fG mC fC mU fA mU fU mA fC mA fA AGGUCUACGCCUAUUACAA 289 EV0066A mG fU mU fG mU fA mA fU mA fG mG fC mG fU mA fG mA fC mC GUUGUAAUAGGCGUAGACC 290 EV0066B fG mG fU mC fU mA fC mG fC mC fU mA fU mU fA mC fA mA fC GGUCUACGCCUAUUACAAC 291 EV0067A mG fG mG fU mC fU mU fG mU fA mC fA mC fA mU fA mG fU mC GGGUCUUGUACACAUAGUC 292 EV0067B fG mA fC mU fA mU fG mU fG mU fA mC fA mA fG mA fC mC fC GACUAUGUGUACAAGACCC 293 EV0068A mC fA mC fU mU fG mA fU mG fG mG fG mC fU mG fA mU fG mA CACUUGAUGGGGCUGAUGA 294 EV0068B fU mC fA mU fC mA fG mC fC mC fC mA fU mC fA mA fG mU fG UCAUCAGCCCCAUCAAGUG 295 EV0069B mG mG mU mA mC mC fU fC fU mU mC mA mU mC mC mA mG mA irA GGUACCUCUUCAUCCAGAA 296 EV0070B mG mU mA mC mC mU fC fU fU mC mA mU mC mC mA mG mA mC irA GUACCUCUUCAUCCAGACA 297 EV0071B mA mC mC mU mC mU fU fC fA mU mC mC mA mG mA mC mA mG irA ACCUCUUCAUCCAGACAGA 298 EV0072B mC mU mU mC mA mU fC fC fA mG mA mC mA mG mA mC mA mA irA CUUCAUCCAGACAGACAAA 299 EV0073B mU mC mC mA mG mA fC fA fG mA mC mA mA mG mA mC mC mA irA UCCAGACAGACAAGACCAA 300 EV0074B mC mA mG mA mC mA fG fA fC mA mA mG mA mC mC mA mU mC irA CAGACAGACAAGACCAUCA 301 EV0075B mG mA mC mA mG mA fC fA fA mG mA mC mC mA mU mC mU mA irA GACAGACAAGACCAUCUAA 302 EV0076B mC mA mG mA mC mA fA fG fA mC mC mA mU mC mU mA mC mA irA CAGACAAGACCAUCUACAA 303 EV0077B mA mC mA mA mG mA fC fC fA mU mC mU mA mC mA mC mC mC irA ACAAGACCAUCUACACCCA 304 EV0078B mA mG mU mG mG mA fA fG fA mU mC mC mG mA mG mC mC mU irA AGUGGAAGAUCCGAGCCUA 305 EV0079B mC mG mU mG mC mU fG fC fC mC mA mG mU mU mU mC mG mA irA CGUGCUGCCCAGUUUCGAA 306 EV0080B mA mC mC mA mG mA fU fC fC mA mC mU mU mC mA mC mC mA irA ACCAGAUCCACUUCACCAA 307 EV0081B mC mA mG mA mU mC fC fA fC mU mU mC mA mC mC mA mA mG irA CAGAUCCACUUCACCAAGA 308 EV0082B mA mG mA mU mC mC fA fC fU mU mC mA mC mC mA mA mG mA irA AGAUCCACUUCACCAAGAA 309 EV0083B mU mC mC mA mC mU fU fC fA mC mC mA mA mG mA mC mA mC irA UCCACUUCACCAAGACACA 310 EV0084B mC mC mU mU mU mG fA fC fC mU mC mA mU mG mG mU mG mU irA CCUUUGACCUCAUGGUGUA 311 EV0085B mC mU mU mU mG mA fC fC fU mC mA mU mG mG mU mG mU mU irA CUUUGACCUCAUGGUGUUA 312 EV0086B mA mC mC mU mC mA fU fG fG mU mG mU mU mC mG mU mG mA irA ACCUCAUGGUGUUCGUGAA 313 EV0087B mA mC mA mA mG mA fA fA fG mG mG mA mU mC mU mG mU mG irA ACAAGAAAGGGAUCUGUGA 314 EV0088B mA mG mG mG mA mU fC fU fG mU mG mU mG mG mC mA mG mA irA AGGGAUCUGUGUGGCAGAA 315 EV0089B mA mA mU mG mC mA fG fG fA mC mU mU mC mU mU mC mA mU irA AAUGCAGGACUUCUUCAUA 316 EV0090B mC mC mA mG mA mA fU fU fC mU mC mC mU mG mC mA mA mG irA CCAGAAUUCUCCUGCAAGA 317 EV0091B mU mG mG mU mC mA fA fG fG mU mC mU mU mC mU mC mU mC irA UGGUCAAGGUCUUCUCUCA 318 EV0092B mA mC mC mU mC mA fU fC fG mC mC mA mU mC mG mA mC mU irA ACCUCAUCGCCAUCGACUA 319 EV0093B mC mC mU mU mC mA fU fG fG mU mG mU mU mC mC mA mA mG irA CCOUCAUGGUGUUCCAAGA 320 EV0094B mA mA mG mA mG mA fC fC fA mA mG mG mA mA mA mA mU mG irA AAGAGACCAAGGAAAAUGA 321 EV0095B mU mG mC mC mU mU fU fG fC mU mC mC mA mG mA mC mA mC irA UGGCUUUGCUCCAGACACA 322 EV0096B mA mC mA mC mC mC fU fC fA mU mC mA mU mC mU mA mC mC irA ACACCCUCAUCAUCUACCA 323 EV0097B mA mG mG mU mC mU fA fC fG mC mC mU mA mU mU mA mC mA irA AGGUCUACGCCUAUUACAA 324 EV0098B mG mG mU mC mU mA fC fG fC mC mU mA mU mU mA mC mA mA irA GGUCUACGCCUAUUACAAA 325 EV0099B mG mA mC mU mA mU fC fU fG mU mA mC mA mA mG mA mC mC irA GACUAUGUGUACAAGACCA 326 EV0100B mU mC mA mU mC mA fG fC fC mC mC mA mU mC mA mA mG mU irA UCAUCAGCCCCAUCAAGUA 327 EV0101A mA (ps) fG (ps) mG fA mA fG mU fU mG fA mC fG mU fU mG fA mG AGGAAGUUGACGUUGAGGG (ps) fG (ps) mG 328 EV0101B [ST23 (ps)]3 ST43 (ps) fC mC fC mU fC mA fA mC fG mU fC mA fA mC CCCUCAACGUCAACUUCCU fU mU fC (ps) mC (ps) fU 329 EV0102A mA (ps) fA (ps) mU fA mU fA mU fU mC fA mU fG mA fG mC fU mU AAUAUAUUCAUGAGCUUCG (ps) fC (ps) mG 330 EV0102B [ST23 (ps)]3 ST43 (ps) fC mG fA mA fC mC fU mC fA mU fG mA fA mU CGAAGCUCAUGAAUAUAUU fA mU fA (ps) mU (ps) fU 331 EV0103A mA (ps) fU (ps) mG fA mU fG mA fG mG fG mU fG mU fU mC fC mU AUGAUGAGGGUGUUCCUAU (ps) fA (ps) mU 332 EV0103B [ST23 (ps)]3 ST43 (ps) fA mU fA mG fG mA fA mC fA mC fC mC fU mC AUAGGAACACCCUCAUCAU fA mU fC (ps) mA (ps) fU 333 EV0104A mU (ps) fC (ps) mU fG mU fC mU fG mG fA mU fG mA fA mG fA mG UCUGUCUGGAUGAAGAGGU (ps) fG (ps) mU 334 EV0104B [ST23 (ps)]3 ST43 (ps) fA mC fC mU fC mU fU mC fA mU fC mC fA mG ACCUCUUCAUCCAGACAGA fA mC fA (ps) mG (ps) fA 335 EV0105A mG (ps) fU (ps) mA fC mA fU mG fG mU fC mU fU mG fU mC fU mG GUAGAUGGUCUUGUCUGUC (ps) fU (ps) mC 336 EV0105B [ST23 (ps)]3 ST43 (ps) fG mA fC mA fG mA fC mA fA mG fA mC fC mA GACAGACAAGACCAUCUAC fU mC fU (ps) mA (ps) fC 337 EV0106A mG (ps) fA (ps) mA fC mA fC mC fA mU fG mA fG mG fU mC fA mA GAACACCAUGAGGUCAAAG (ps) fA (ps) mG 338 EV0106B [ST23 (ps)]3 ST43 (ps) fC mU fU mU fG mA fC mC fU mC fA mU fG mG CUUUGACCUCAUGGUGUUC fU mG fU (ps) mU (ps) fC 339 EV0107A mA (ps) fG (ps) mA fG mA fG mA fA mG fA mC fC mU fU mG fA mC AGAGAGAAGACCUUGACCA (ps) fC (ps) mA 340 EV0107B [ST23 (ps)]3 ST43 (ps) mU fG mG fU mC fA mA fG mG fU mC fU mU fC UGGUCAAGGUCUUCUCUCU mU fC mU (ps) fC (ps) mU 341 EV0108A mC (ps) fU (ps) mU fG mU fC mU fG mU fC mU fG mG fA mU fG mA CUUGUCUGUCUGGAUGAAG (ps) fA (ps) mG 342 EV0108B [ST23 (ps)]3 ST43 (ps) mC mU mU mC mA mU fC fC fA mG mA mC mA mG CUUCAUCCAGACAGACAAA mA mC mA mA irA 343 EV0109A mG (ps) fU (ps) mA fG mA fU mG fG mU fC mU fU mG fU mC fU mG GUAGAUGGUCUUGUCUGUC (ps) fU (ps) mC 344 EV0109B [ST23 (ps)]ST43 (ps) mG mA mC mA mG mA fC fA fA mG mA mC mC mA GACAGACAAGACCAUCUAA mU mC mU mA irA 345 EV0110A mU (ps) fC (ps) mU fU mG fG mU fG mA fA mG fU mG fG mA fU mC UCUUGGUGAAGUGGAUCUG (ps) fU (ps) mG 346 EV0110B [ST23 (ps)]3 ST43 (ps) mC mA mG mA mU mC fC fA fC mU mU mC mA mC CAGAUCCACUUCACCAAGA mC mA mA mG irA 347 EV0111A mG (ps) fU (ps) mU fG mU fA mA fU mA fG mG fC mG fU mA fG mA GUUGUAAUAGGCGUAGACC (ps) fC (ps) mC 348 EV0111B [ST23 (ps)]3 ST43 (ps) mG mG mU mC mU mA fC fG fC mC mU mA mU mU GGUCUACGCCUAUUACAAA mA mC mA mA irA 349 EV0312A mA (ps) fU (ps) mU fG mU fA mA fU mA fG mG fC mG fU mA fG mA AUUGUAAUAGGCGUAGACC (ps) fC (ps) mC 350 EV0112B [ST23 (ps)]3 ST43 (ps) fG mG fU mC fU mA fC mG fC mC fU mA fU mU GGUCUACGCCUAUUACAAU fA mC fA (ps) mA (ps) fU 351 EV0313A mA (ps) fU (ps) mG fU mA fG mA fU mG fG mU fC mU fU mG fU mC AUGUAGAUGGUCUUGUCUG (ps) fU (ps) mG 352 EV0313B [ST23 (ps)]3 ST43 (ps) fC mA fG mA fC mA fA mG fA mC fC mA fU mC CAGACAAGACCAUCUACAU fU mA fC (ps) mA (ps) fU 353 EV0201A mU (ps) fG (ps) mA fG mA fG mA fA mG fA mC fC mU fU mG fA mC UGAGAGAAGACCUUGACCA (ps) fC (ps) mA 354 EV0201B [ST23 (ps)]3 ST43 (ps) mU mG mG mU mC mA fA fG fG mU mC mU mU mC UGGUCAAGGUCUUCUCUCU mU mC mU (ps) mC (ps) mU 355 EV0202B Ser(GN) (ps) mU (ps) mG (ps) mG mU mC mA fA fG fG mU mC mU mU mC UGGUCAAGGUCUUCUCUCU mU mC mU (ps) mC (ps) mU (ps) Ser(GN) 356 EV0203A (vp)-mU fG mA fG mA fG mA fA mG fA mC fC mU fU mG fA mC (ps) fC UGAGAGAAGACCUUGACCA (ps) mA 357 EV0205B [ST23 (ps)]3 ST43 (ps) mC mA mG mA mU mC fC fA fC mU mU mC mA mC CAGAUCCACUUCACCAAGA mC mA mA (ps) mG (ps) mA 358 EV0206B Ser(GN) (ps) mC (ps) mA (ps) mG mA mU mC fC fA fC mU mU mC mA mC CAGAUCCACUUCACCAAGA mC mA mA (ps) mG (ps) mA (ps) Ser(GN) 359 EV0207A (vp)-mU (ps) fC (ps) mU fU mG fG mU fG mA fA mG fU mG fG mA fU UCUUGGUGAAGUGGAUCUG mC (ps) fU (ps) mG 360 EV0209A (vp)-mU fC mU fU mG fG mU fG mA fA mG fU mG fG mA fU mC (ps) fU UCUUGGUGAAGUGGAUCUG (ps) mG 361 EV0201Aun UGAGAGAAGACCUUGACCA UGAGAGAAGACCUUGACCA 362 EJ0001Aun UGAGAGAAGACCUUGACCA UGAGAGAAGACCUUGACCA 363 EJ0001Bun UGGUCAAGGUCUUCUCUCU UGGUCAAGGUCUUCUCUCU 364 EJ0002Aun UGAGAGACGACCUUGACCA UGAGAGACGACCUUGACCA 365 EJ0003Aun UGAGAGAAUACCUUGACCA UGAGAGAAUACCUUGACCA 366 EJ0004Aun UGAGAGAAGACCUUGACCG UGAGAGAAGACCUUGACCG 367 EJ0004Bun UGGUCAAGGUCUUCUCUCU UGGUCAAGGUCUUCUCUCU 368 EJ0005Aun AUGAGCUUCGUAGAGAUUC AUGAGCUUCGUAGAGAUUC 369 EJ0005Bun GAAUCUCUACGAAGCUCAU GAAUCUCUACGAAGCUCAU 370 EJ0006Aun UUGUAGUAGCGGAUCUUGG UUGUAGUAGCGGAUCUUGG 371 EJ0006Bun CCAAGAUCCGCUACUACAC CCAAGAUCCGCUACUACAC 372 EJ0007Aun UUCUGUCUGGAUGAAGAGG UUCUGUCUGGAUGAAGAGG 373 EJ0007Bun CCUCUUCAUCCAGACAGAC CCUCUUCAUCCAGACAGAC 374 EJ0009Bun UGGUCAAGGUCUUCUCUCA UGGUCAAGGUCUUCUCUCA 375 EJ0010Bun UGGUCAAGGUCGUCUCUCA UGGUCAAGGUCGUCUCUCA 376 EJ0011Bun CGGUCAAGGUCUUCUCUCA CGGUCAAGGUCUUCUCUCA 377 EJ0012Aun UGAGAGACGACCUUGACCG UGAGAGACGACCUUGACCG 378 EJ0012Bun CGGUCAAGGUCGUCUCUCA CGGUCAAGGUCGUCUCUCA 379 EJ0014Bun CCAAGAUCCGCUACUACAA CCAAGAUCCGCUACUACAA 380 EJ0015Bun CCUCUUCAUCCAGACAGAA CCUCUUCAUCCAGACAGAA 381 EJ0001A mU (ps) fG (ps) mA fG mA fG mA fA mG fA mC fC mU fU mG fA mC UGAGAGAAGACCUUGACCA (ps) fC (ps) mA 382 EJ0001B mU (ps) mG (ps) mG mU mC mA fA fG fG mU mC mU mU mC mU mC mU UGGUCAAGGUCUUCUCUCU (ps) mC (ps) mU 383 EJ0002A mU (ps) fG (ps) mA fG mA fG mA fC mG fA mC fC mU fU mG fA mC UGAGAGACGACCUUGACCA (ps) fC (ps) mA 384 EJ0003A mU (ps) fG (ps) mA fG mA fG mA fA mU fA mC fC mU fU mG fA mC UGAGAGAAUACCUUGACCA (ps) fC (ps) mA 385 EJ0004A mU (ps) fG (ps) mA fG mA fG mA fA mG fA mC fC mU fU mG fA mC UGAGAGAAGACCUUGACCG (ps) fC (ps) mG 386 EJ0004B mC (ps) mG (ps) mG mU mC mA fA fG fG mU mC mU mU mC mU mC mU CGGUCAAGGUCUUCUCUCU (ps) mC (ps) mU 387 EJ0005A mA (ps) fU (ps) mG fA mG fC mU fU mC fG mU fA mG fA mG fA mU AUGAGCUUCGUAGAGAUUC (ps) fU (ps) mC 388 EJ0005B mG (ps) mA (ps) mA mU mC mU fC fU fA mC mG mA mA mG mC mU mC GAAUCUCUACGAAGCUCAU (ps) mA (ps) mU 389 EJ0006A mU (ps) fU (ps) mG fU mA fG mU fA mG fC mG fG mA fU mC fU mU UUGUAGUAGCGGAUCUUGG (ps) fG (ps) mG 390 EJ0006B mC (ps) mC (ps) mA mA mG mA fU fC fC mG mC mU mA mC mU mA mC CCAAGAUCCGCUACUACAC (ps) mA (ps) mC 391 EJ0007A mU (ps) fU (ps) mC fU mG fU mC fU mG fG mA fU mG fA mA fG mA UUCUGUCUGGAUGAAGAGG (ps) fG (ps) mG 392 EJ0007B mC (ps) mC (ps) mU mC mU mU fC fA fU mC mC mA mG mA mC mA mG CCUCUUCAUCCAGACAGAC (ps) mA (ps) mC 393 EJ0008B [ST23 (ps)]3 ST43 (ps) mU mG mG mU mC mA fA fG fG mU mC mU mU mC UGGUCAAGGUCUUCUCUCU mU mC mU (ps) mC (ps) mU 394 EJ0009B [ST23 (ps)]3 ST43 (ps) mU mG mG mU mC mA fA fG fG mU mC mU mU mC UGGUCAAGGUCUUCUCUCA mU mC mU (ps) mC (ps) mA 395 EJ0002A mU (ps) fG (ps) mA fG mA fG mA fC mG fA mC fC mU fU mG fA mC UGAGAGACGACCUUGACCA (ps) fC (ps) mA 396 EJ0010B [ST23 (ps)]3 ST43 (ps) mU mG mG mU mC mA fA fG fG mU mC mG mU mC UGGUCAAGGUCGUCUCUCA mU mC mU (ps) mC (ps) mA 397 EJ0011B [ST23 (ps)]3 ST43 (ps) mC mG mG mU mC mA fA fG fG mU mC mU mU mC CGGUCAAGGUCUUCUCUCA mU mC mU (ps) mC (ps) mA 398 EJ0012A mU (ps) fG (ps) mA fG mA fG mA fC mG fA mC fC mU fU mG fA mC UGAGAGACGACCUUGACCG (ps) fC (ps) mG 399 EJ0012B [ST23 (ps)]3 ST43 (ps) mC mG mG mU mC mA fA fG fG mU mC mG mU mC CGGUCAAGGUCGUCUCUCA mU mC mU (ps) mC (ps) mA  400 EJ0013B [ST23 (ps)]3 ST43 (ps) mG mA mA mU mC mU fC fU fA mC mG mA mA mG GAAUCUCUACGAAGCUCAU mC mU mC (ps) mA (ps) mU 401 EJ0014B [ST23 (ps)]3 ST43 (ps) mC mC mA mA mG mA fU fC fC mG mC mU mA mC CCAAGAUCCGCUACUACAA mU mA mC (ps) mA (ps) mA 402 EJ0015B [ST23 (ps)]3 ST43 (ps) mC mC mU mC mU mU fC fA fU mC mC mA mG mA CCUCUUCAUCCAGACAGAA mC mA mG (ps) mA (ps) mA 403 EJ0016A (vp)-mU fG mA fG mA fG mA fC mG fA mC fC mU fU mG fA mC (ps) fC UGAGAGACGACCUUGACCG (ps) mG 404 EJ0017A (vp)-mU fG mA fG mA fG mA fC mG fA mC fC mU fU mG fA mC fC (ps2) UGAGAGACGACCUUGACCG mG 405 EJ0017B [ST23]3 ST43 mC (ps2) mG mG mU mC mA fA fG fG mU mC mG mU mC mU CGGUCAAGGUCGUCUCUCA mC mU mC (ps2) mA 406 EJ0018A mU (ps) fG (ps) mA fG mA fG mA fC mG fA mC fC mU fU mG fA mC fC UGAGAGACGACCUUGACCG (ps2) mG 407 EJ0017B [ST23]3 ST43 mC (ps2) mG mG mU mC mA fA fG fG mU mC mG mU mC mU CGGUCAAGGUCGUCUCUCA mC mU mC (ps2) mA 408 EJ0019A (vp)-mU fU mG fU mA fG mU fA mG fC mG fG mA fU mC fU mU (ps) fG UUGUAGUAGCGGAUCUUGG (ps) mG 409 EJ0020A (vp)-mU fU mG fU mA fG mU fA mG fC mG fG mA fU mC fU mU fG (ps2) UUGUAGUAGCGGAUCUUGG mG 410 EJ0020B [ST23]3 ST43 mC (ps2) mC mA mA mG mA fU fC fC mG mC mU mA mC mU CCAAGAUCCGCUACUACAA mA mC mA (ps2) mA 411 EJ0021A (vp)-mU fU mC fU mG fU mC fU mG fG mA fU mG fA mA fG mA (ps) fG UUCUGUCUGGAUGAAGAGG (ps) mG 412 EJ0022A (vp)-mU fU mC fU mG fU mC fU mG fG mA fU mG fA mA fG mA fG (ps2) UUCUGUCUGGAUGAAGAGG mG 413 EJ0022B [ST23]3 ST43 mC (ps2) mC mU mC mU mU fC fA fU mC mC mA mG mA mC CCUCUUCAUCCAGACAGAA mA mG mA (ps2) mA 414 EJ0023A mU (ps) fU (ps) mC fU mG fU mC fU mG fG mA fU mG fA mA fG mA fG UUCUGUCUGGAUGAAGAGG (ps2) mG 415 EJ0022B [S123]3 ST43 mC (ps2) mC mU mC mU mU fC fA fU mC mC mA mG mA mC CCUCUUCAUCCAGACAGAA mA mG mA (ps2) mA 416 EV0210Aun UAUAUAUUCAUGAGCUUCG UAUAUAUUCAUGAGCUUCG 417 EV0210A mU (ps) fA (ps) mU fA mU fA mU fU mC fA mU fG mA fG mC fU mU UAUAUAUUCAUGAGCUUCG (ps) fC (ps) mG 418 EV0210B [ST23 (ps)]3 ST43 (ps) mC mG mA mA mG mC fU fC fA mU mG mA mA mU CGAAGCUCAUGAAUAUAUU mA mU mA (ps) mU (ps) mU 419 EV0211A (vp)-mU fA mU fA mU fA mU fU mC fA mU fG mA fG mC fU mU (ps) fC UAUAUAUUCAUGAGCUUCG (ps) mG 420 EV0212A (vp)-mU fA mU fA mU fA mU fU mC fA mU fG mA fG mC fU mU fC (ps2) UAUAUAUUCAUGAGCUUCG mG 421 EV0211B [ST23]3 ST43 mC (ps2) mG mA mA mG mC fU fC fA mU mG mA mA mU mA CGAAGCUCAUGAAUAUAUU mU mA mU (ps2) mU 422 EV0213A mU (ps) fA (ps) mU fA mU fA mU fU mC fA mU fG mA fG mC fU mU fC UAUAUAUUCAUGAGCUUCG (ps2) mG 423 EJ0020B mC (ps2) mC mA mA mG mA fU fC fC mG mC mU mA mC mU mA mC mA CCAAGAUCCGCUACUACAA without (ps2) mA ligand 424 EV0211B mC (ps2) mG mA mA mG mC fU fC fA mU mG mA mA mU mA mU mA mU CGAAGCUCAUGAAUAUAUU without (ps2) mU ligand 425 EV0210B mC mG mA mA mG mC fU fC fA mU mG mA mA mU mA mU mA (ps) mU (ps) CGAAGCUCAUGAAUAUAUU without mU ligand