NUCLEIC ACIDS FOR INHIBITING EXPRESSION OF PROS1 IN A CELL
20220135974 · 2022-05-05
Assignee
Inventors
- Ute Schaeper (Berlin, DE)
- Sibylle Dames (Berlin, DE)
- Eliot MORRISON (Berlin, DE)
- Raja PRINCE ELADNANI (Bern, CH)
- Anne Angelillo-Scherrer (Fribourg, CH)
Cpc classification
C12N15/113
CHEMISTRY; METALLURGY
C12N2310/317
CHEMISTRY; METALLURGY
A61P7/04
HUMAN NECESSITIES
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The invention relates to nucleic acid products that interfere with or inhibit PROS1 gene expression. It further relates to therapeutic uses of PROS1 inhibition for the treatment of bleeding disorders.
Claims
1. A double-stranded nucleic acid for inhibiting expression of PROS1, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences selected from SEQ ID NOs: 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, and 255.
2. A double-stranded nucleic acid that is capable of inhibiting expression of PROS1 for use as a medicament, wherein the nucleic acid comprises a first strand and a second strand.
3. The nucleic acid of claim 1, wherein the first strand and the second strand form a duplex region of 17-25 nucleotides in length.
4. The nucleic acid of claim 1, wherein the nucleic acid mediates RNA interference.
5. The nucleic acid of claim 1, wherein at least one nucleotide of the first and/or second strand is a modified nucleotide, particularly a non-naturally occurring nucleotide such as a 2′-F modified nucleotide.
6. The nucleic acid of claim 1, 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 claim 1, wherein the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide at its 5′ end.
8. The nucleic acid of claim 1, 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 particularly wherein the linkages between the remaining nucleotides are phosphodiester linkages.
9. The nucleic acid of claim 1, 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 claim 1, 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 the nucleic acid of claim 1 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. The nucleic acid of claim 1 for use as a medicament.
14. A nucleic acid of claim 1 for use in the prevention, decrease of the risk of suffering from, or treatment of a bleeding disorder, particularly haemophilia A or haemophilia B.
15. Use of a nucleic acid of claim 1 in the prevention, decrease of the risk of suffering from, or treatment of a bleeding disorder.
16. A method of preventing, decreasing the risk of suffering from, or treating a blood disorder comprising administering a pharmaceutically effective amount of a nucleic acid of claim 1 to an individual in need of treatment.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0541] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
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EXAMPLES
Example 1—Synthesis of Building Blocks
[0567] The synthesis route for DMT-Serinol (GalNAc)-CEP and CPG as described below is outlined in
[0568] DMT-Serinol(GalNAc) (3)
[0569] 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.
[0570] DMT-Serinol(GalNAc)-CEP (5)
[0571] 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% Et.sub.3N, 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.
[0572] DMT-Serinol(GalNAc)-succinate (6)
[0573] 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).
[0574] DMT-Serinol(GalNAc)-succinyl-Icaa-CPG (7)
[0575] 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 2—Oligonucleotide Synthesis
[0576] 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.
[0577] Downstream cleavage, deprotection and purification followed standard procedures that are known in the art.
[0578] 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 1.
[0579] 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.
[0580] 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.
[0581] 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.
[0582] 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.
[0583] Product containing fractions were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilised until further use.
[0584] 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 3—Double-Strand Formation
[0585] 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 4—Reduction of Human PROS1 mRNA Level in Human Hep3B Cells by Transfection of PROS1 siRNAs
[0586] In vitro testing shows over 70% reduction of PROS1 mRNA levels in human Hep3B cells by transfection of any of PROS1 siRNA molecules EU060 to EU083. Hep3B cells were seeded at a density of 12 000 cells per well in 96-well plates. The following day the cells were transfected with 10 nM, 1 nM or 0.1 nM PROS1 siRNA or non-targeting control siRNA (EU012) and 1 μg/ml AtuFECT. 24 hours thereafter cells were lysed for RNA extraction and PROS1 and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for PROS1 mRNA were normalized to values generated for the house keeping gene Actin and related to the mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean+/−SD from three biological replicates. siRNA duplexes used in this study are listed in Table 2. Results are shown in
Example 5—Dose Dependent Reduction of PROS1 mRNA Level in Human Cells by Transfection of PROS1 siRNAs
[0587] In vitro testing shows dose dependent reduction of PROS1 mRNA levels in human Hep3B cells by a number of PROS1 siRNA molecules. Hep3B cells were seeded at a density of 12 000 cells per well in 96-well plates. The following day the cells were transfected with 0.1 nM, 0.01 nM or 0.001 nM PROS1 siRNA or 0.1 nM non-targeting control siRNA (EU012) and 1 μg/ml AtuFECT. 24 hours thereafter cells were lysed for RNA extraction and PROS1 and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for PROS1 mRNA were normalized to values generated for the house keeping gene Actin and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean+/−SD from three biological replicates. siRNA duplexes used in this study are listed in Table 2. Results are shown in
Example 6—Inhibition of PROS1 Target Gene Expression in Primary Mouse Hepatocytes by Receptor Mediated Uptake of PROS1 siRNA Conjugates
[0588] The example shows dose dependent reduction of PROS1 mRNA levels in primary hepatocytes by receptor mediated uptake of EU140 to EU148. Primary mouse hepatocytes were seeded in a 96-well plate at a density of 25 000 cells per well. After attachment, they were incubated with PROS1 siRNA conjugates in the cell culture medium at 100 nM, 10 nM, 1 nM and 0.1 nM as indicated below, or they were incubated with 100 nM non-targeting control conjugates (EU110). The following day, cells were lysed for RNA extraction and PROS1 and ApoB mRNA levels were determined by Taqman qRT-PCR. Values obtained for PROS1 mRNA were normalized to values generated for the house keeping gene ApoB and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean+/−SD from three biological replicates. siRNA conjugates used in this study are listed in Table 2. Results are shown in
Example 7—Inhibition of Human PROS1 Gene Expression in Primary Human Hepatocytes by Receptor Mediated Uptake of PROS1 siRNA Coniugates
[0589] The example shows dose dependent reduction of human PROS1 mRNA levels by EU140 to 147 in primary human hepatocytes. Primary human hepatocytes (Life Technologies) were seeded in a 96-well plate at a density of 35 000 cells per well in plating medium and were subsequently incubated with PROS1 siRNA conjugates EU140 to EU147, in concentrations of 100 nM, 10 nM, 1 nM and 0.1 nM as shown in
Example 8—Loss of X-Ase Activity Rescues Pros1.SUP.−/− Mice
[0590] Pros1.sup.−/− females crossed with F8.sup.−/− males produced 25% F8.sup.+/− Pros1.sup.+/− progeny. F8.sup.+/− Pros1.sup.+/− females bred with F8.sup.−/− males resulted in 25% F8.sup.−/− Pros1.sup.+/− progeny (
[0591] Of 295 pups from F8.sup.−/− Pros1.sup.+/− breeding pairs, 72 (24%) were F8.sup.−/− Pros1.sup.+/+, 164 (56%) were F8.sup.−/− Pros1.sup.+/− and 59 (20%) were F8.sup.−/− Pros1.sup.−/− (χ.sup.2=4.8, P=0.09). Thus, F8.sup.−/− Pros1.sup.−/− mice were present at the expected Mendelian ratio. In contrast, of 219 pups from F9.sup.−/− Pros1.sup.+/− breeding pairs, 56 (26%) were F9.sup.−/− Pros1.sup.+/+, 132 (60%) were F9.sup.−/− Pros1.sup.+/− and 31 (14%) were F9.sup.−/− Pros1.sup.−/− (χ.sup.2=14.95, P=0.001). This is compatible with a transmission ratio distortion for F9.sup.−/− Pros1.sup.−/− mice consistent with the decreased litter sizes compared to those of matings from F9.sup.+/+ Pros1.sup.+/+ mice (5.2±0.7 versus 9.8±1.8, n=4 matings/over 3.sup.t generations, P=0.046).
[0592] F8.sup.−/− Pros1.sup.−/− and F9.sup.−/− Pros1.sup.−/− mice appeared completely normal. Their viability was monitored up to 20 (n=4) and 16 months (n=2), respectively, without showing any difference compared to F8.sup.−/− Pros1.sup.+/+ and F9.sup.−/− Pros1.sup.+/+ mice, respectively.
[0593] As a complete Pros1 deficiency in mice leads to consumptive coagulopathy, we assessed whether F8.sup.−/− Pros1.sup.−/− and F9.sup.−/− Pros1.sup.+/− mice developed DIC. DIC parameters were comparable in F8.sup.−/− Pros1.sup.+/+, F8.sup.−/− Pros1.sup.+/− and F8.sup.−/− Pros1.sup.+/− mice (
[0594] Therefore, loss of X-ase activity rescues the embryonic lethality of complete Pros1 deficiency. However, the rescue was only partial with the loss of FIX activity. A possible explanation is that severe HB appears to be a less serious condition compared to severe HA. Consequently, F9 disruption in Pros1.sup.−/− mice was less efficient in rebalancing coagulation than F8 disruption.
[0595] To explore whether restoring intrinsic X-ase activity by FVIII infusion induces DIC, thrombosis and purpura fulminans in F8.sup.−/− Pros1.sup.−/− mice, we administered recombinant FVIII (rFVIII) intravenously. No mouse died following rFVIII injection. Thrombi in numerous blood vessels and bleeding in the lungs were found in F8.sup.−/− Pros1.sup.−/− mice 24 h after a single injection of an overdose of rFVIII (
Example 9—Loss of X-Ase Activity does not Prevent Lethality Caused by TF-Induced Thromboembolism in Pros1.SUP.−/− Mice
[0596] We demonstrated previously that, although 88% of Pros1.sup.+/+ mice survived to a TF-induced thromboembolism model, only 25% of Pros1.sup.+/− mice were still alive 20 min after a low TF dose injection (˜1.1 nM). When using a higher TF dosage (˜4.3 nM), both Pros1.sup.+/+ and Pros1.sup.+/− mice died within 20 min. However, Pros1.sup.+/− died earlier than Pros1.sup.+/+. HA and WT mice were equally sensitive to this high TF-dose with more than 85% of them succumbing within 15 min (
[0597] Fibrin clots were detected in lung arteries of F8 Pros1.sup.+/+ and F8.sup.−/− Pros1.sup.−/− mice that died during the TF-induced thromboembolic challenge (
[0598] None of the F8.sup.−/− Pros1.sup.−/− mice that succumbed during the TF-induced thromboembolic-challenge developed purpura fulminans. Similar data were obtained with F9.sup.−/− Pros1.sup.+/+, F9.sup.−/− Pros1.sup.+/− and F9.sup.−/− Pros1.sup.−/− mice (not shown).
Example 10—Loss of FVIII Partially Protects Pros1.SUP.−/− Mice Against Thrombosis in Mesenteric Arterioles
[0599] We then recorded thrombus formation in mesenteric arterioles, a model sensitive to defects in the intrinsic pathway of coagulation. In F8.sup.+/+ Pros1.sup.+/+ mice, thrombi grew to occlusive size in 20 min, and all injured arterioles were occluded (
[0600] Emboli were generated during thrombus formation in F8.sup.+/+ Pros1.sup.+/+ mice, but not in F8.sup.−/− Pros1.sup.+/+ mice. In F8.sup.−/− Pros1.sup.−/− mice, multiple micro-emboli detached during partial thrombus growth, preventing the formation of occlusive thrombi.
Example 11—Pros1 Targeting Limits but does not Abrogate Tail Bleeding in Mice with HA
[0601] The bleeding phenotype was assessed by tail transection using a mild or a severe bleeding model.
[0602] In both models, blood loss was reduced in F8.sup.−/− Pros1.sup.−/− compared to F8.sup.−/− Pros1.sup.+/+ mice (
[0603] Then, an PS-neutralizing antibody was used to investigate how inhibition of PS activity alters tail bleeding in F8.sup.−/− Pros1.sup.+/− mice. This antibody limited blood loss in F8.sup.−/− Pros1.sup.+/− mice (
Example 12—Pros1 Targeting or PS Inhibition Fully Protects HA or HB Mice from Acute Hemarthrosis (AH)
[0604] Although bleeding may appear anywhere in haemophilia patients, most of haemorrhages occur in the joints. To determine whether Pros1 loss prevents hemarthrosis in haemophilic mice, we applied an AH model to F8.sup.−/− Pros1.sup.+/+, F8.sup.−/− Pros1.sup.−/−, F8.sup.−/− Pros1.sup.−/− and F8.sup.+/+ Pros1.sup.+/+ mice. Knee swelling after injury was reduced in F8.sup.−/− Pros1.sup.+/+ and F8.sup.+/+ Pros1.sup.+/+ mice compared to F8.sup.−/− Pros1.sup.+/+ and F8.sup.−/− Pros1.sup.+/− mice (
[0605] These results were confirmed by the continuous subcutaneous infusion during 4 days of a PS-neutralizing antibody or a control antibody in F8.sup.−/− Pros1.sup.+/− mice (starting 1 day before AH induction) (knee swelling in PS-neutralizing antibody group was 0.43±0.07 versus 0.69±0.09 mm in control group, n=9, P=0.04). PS plasma level in PS-neutralizing antibody group was 26±6% versus 45±3% in the controls (n=5, P=0.017). In addition, PS inhibition was alternatively achieved by intravenous injection of a murine PS (mPS) siRNA prior to the AH challenge in F8.sup.−/− Pros1.sup.+/− and F8.sup.−/− Pros1.sup.+/+ mice (
Example 13—Both PS and TFPI are Expressed in the Synovium of Mice
[0606] To understand the prominent intra-articular haemostatic effect of the genetic loss of Pros1 and PS inhibition in haemophilic mice, knee sections were immunostained for PS and TFPI. PS was mainly present at the lining layer of the synovial tissue of F8.sup.−/− Pros1.sup.+/+ mice with AH treated with control siRNA, whereas synovial staining for PS was remarkably reduced in F8.sup.−/− Pros1.sup.+/+ mice with AH that received mPS siRNA (
[0607] To demonstrate further that PS is expressed by fibroblast-like synoviocytes (FLS), we performed western blots on conditioned media collected from F8.sup.+/+ Pros1.sup.+/+, F8.sup.−/− Pros1.sup.+/+ and F8.sup.−/− Pros1.sup.−/− FLS. As shown in
[0608] We also studied TFPI expression in F8.sup.−/− Pros1.sup.+/+ and F8.sup.−/− Pros1.sup.−/− FLS conditioned media (
Example 14—Both PS and TFPI are Expressed in the Synovium of Patients with HA or HB
[0609] Human HA, HB and osteoarthritis knee synovial tissues were then analysed for both PS and TFPI (
Example 15—Loss of Pros1 is Responsible for the Lack of TFPI-Dependent PS Activity and Resistance to APC in HA Mice
[0610] The full protection against AH in HA or HB mice lacking Pros1 or in which PS was inhibited could be explained at least partly by the lack of PS cofactor activity for APC and TFPI in the joint. However, the reason for a partial haemostatic effect of the lack of Pros1 or PS inhibition in HA mice challenged in the tail bleeding models needs to be further investigated.
[0611] Ex vivo TF-initiated thrombin generation testing has shown a correlation between the capacity of plasma to generate thrombin and the clinical severity of haemophilia. Therefore, we investigated the impact of Pros1 loss on thrombin generation in plasma of HA mice. TFPI-dependent PS activity was not assessed in platelet-free plasma (PFP) but in platelet-rich plasma (PRP) because TFPI-cofactor activity of PS cannot be demonstrated in mouse plasma using thrombin generation tests. This is explained by the lack of TFPIα in mouse plasma and its presence in mouse platelets.
[0612] Both thrombin peak and endogenous thrombin potential (ETP) were significantly higher in F8.sup.−/− Pros1.sup.−/− than in F8.sup.−/− Pros1.sup.+/+ PRP in response to 1 μM TF (1072±160 vs 590±10 nmol/L.Math.min, n=3/group, P=0.04), suggesting the lack of PS TFPI-cofactor activity in F8.sup.−/− Pros1.sup.−/− PRP (
[0613] To assess whether F8.sup.−/− Pros1.sup.−/− mice exhibited defective functional APC-dependent PS activity, we used thrombin generation testing in Ca.sup.2+ ionophore-activated PRP in the absence of APC, in the presence of wild-type (WT) recombinant APC, or in the presence of a mutated (L38D) recombinant mouse APC (L38D APC, a variant with ablated PS cofactor activity). In this assay, APC titration showed that the addition of 8 nM WT APC was able to reduce ETP by 90% in activated PRP of WT mice whereas the same concentration of L38D APC diminished ETP by only 30% (data not shown). Based on these data, thrombin generation curves were recorded for activated PRP (3 mice/assay). The calculated APC ratio (ETP.sub.+APC WT/ETP.sub.+APC L38D) indicated an APC resistance in F8.sup.−/− Pros1.sup.−/− plasma but not in F8.sup.−/− Pros1.sup.+/+ plasma (0.87±0.13 versus 0.23±0.08, respectively, P=0.01) (
[0614] APC-dependent PS activity was also tested in PFP from F8.sup.−/− Pros1.sup.+/+ and F8.sup.−/− Pros1.sup.−/− mice (2 mice/assay) in the presence of 2 nM WT APC and L38D APC. Calculated APC ratio showed an APC resistance in F8.sup.−/− Pros1.sup.−/− but not in F8.sup.−/− Pros1.sup.+/+ mice (1.08±0.04 versus 0.25±0.09, respectively, P=0.0003) (
Example 16—Improved Fibrin Network in HA Mice Lackinq Pros1 Mice
[0615] Tail bleeding mouse models are not only sensitive to platelet dysfunction but also to coagulation and fibrinolysis alterations. To understand the differences between studied genotypes regarding tail bleeding, we used scanning electron microscopic imaging to investigate fibrin structure (
[0616] Fibrin fibres from F8.sup.−/− Pros1.sup.−/− and F8.sup.−/− Pros1.sup.+/+ mice, and from F9.sup.−/− Pros1 and F9.sup.−/− Pros1.sup.+/+ mice, displayed a larger diameter compared to fibres from F8.sup.+/+ Pros1.sup.+/+ mice or F9.sup.+/+ Pros1.sup.+/+ mice, respectively. Nevertheless, the fibre surface of F8.sup.−/− Pros1.sup.−/− and F9.sup.−/− Pros1.sup.−/− mice showed less porosity as compared to F8.sup.−/− Pros1.sup.+/+ or F9.sup.−/− Pros1.sup.+/+ mice, respectively, suggesting that F8.sup.−/− Pros1.sup.−/− and F9.sup.−/− Pros1.sup.−/−-derived fibres might be less permeable and thereby more resistant to fibrinolysis than F8.sup.−/− Pros1.sup.+/+ or F9.sup.−/− Pros1.sup.+/+ derived fibers. These data, in complement to both TFPI and APC cofactor activity results (
Example 17—PS Inhibition in Plasma Restores Thrombin Generation in Patients with HA
[0617] We then examined the effect of PS inhibition on thrombin generation in human HA plasma. ETP in PFP increased 2-4-fold in presence of a PS-neutralizing antibody. Similar results were obtained using an anti-human TFPI antibody against the C-terminal domain for efficient FXa inhibition, even in the presence of FVIII inhibitor (
Example 18—Materials and Methods for Examples 6-17
[0618] Mice
[0619] F8.sup.−/− mice (B6; 129S4-F8.sup.tm1Kaz/J) and F9.sup.−/− mice (B6.129P2-F9.sup.tm1Dws/J) with C57BL/6J background were obtained from The Jackson Laboratory. Pros1.sup.+/− mice were progeny of the original colony. The Swiss Federal Veterinary Office approved the experiments.
[0620] TF-Induced Pulmonary Embolism
[0621] Anesthetized mice, aged 6-9 weeks, received human recombinant TF (hrTF, Dade Innovin, Siemens) intravenously (2 μL/g) at 4.25 nM (1:2 dilution) or 2.1 nM (1:4 dilution). Two minutes after the onset of respiratory arrest or at the completion of the 20-min observation period, lungs were harvested and fixed in 4% PFA. Lung sections were stained with hematoxylin and eosin, and for fibrin. The extent of fibrin clots in the lungs was assessed as number of intravascular thrombi in 10 randomly chosen non overlapping fields (xl0 magnification).
[0622] Tail Clipping Model in HA Mice
[0623] Two different tail clipping models to evaluate bleeding phenotype were assessed as described.sup.14. Briefly, the distal tail of 8-10 week old mice was transected at 2 mm (mild injury) and the bleeding was venous or at 4 mm (severe injury) and the bleeding was arterial and venous. Bleeding was quantified as blood lost after 30 or 10 min, respectively. In the severe injury model some F8.sup.−/− Pros1.sup.+/− mice received a rabbit anti-human PS-IgG (Dako) or rabbit isotype IgG (R&D Systems) intravenously at a dose of 2.1 mg/kg 2 min before tail transection.
[0624] Acute Hemarthrosis Model
[0625] Joint diameters were measured at 0 and 72 h with a digital calliper (Mitutoyo 547-301, Kanagawa). At 72 h, mice were sacrificed, knees were isolated, fixed in 4% PFA, decalcified and embedded in paraffin. The intra-articular bleeding score (IBS) was assessed as described.
[0626] In Vivo PS Inhibition
[0627] 10-week-old mice received a continuous infusion of rabbit anti-human PS-IgG (Dako Basel, Switzerland) or rabbit isotype IgG (R&D Systems) at 1 mg/kg/day through subcutaneous osmotic minipumps (model2001, Alzet).
[0628] Alternatively, 10-week-old mice were treated with a single dose of mouse specific siRNA (s72206, Life Technologies) or control siRNA (4459405, In vivo Negative Control #1 Ambion, Life Technologies) at 1 mg/kg using a transfection agent (Invivofectamine 3.0, Invitrogen, Life Technologies) following the manufacturer's instructions. Acute hemarthrosis model was applied 2.5 days after PS inhibition.
[0629] Statistical Methods
[0630] Values were expressed as mean±sem. Chi-square for non-linked genetic loci was used to assess the Mendelian allele segregation. Survival data in the TF-induced venous thromboembolism model were plotted using the of Kaplan-Meier method. A log-rank test was used to statistically compare the curves (Prism 6.0d; GraphPad). The other data were analysed by t-test, one-way and two-way ANOVA test with GraphPad Prism 6.0d. A P-value of less than 0.05 was considered statistically significant.
[0631] Preparation of Murine Plasma
[0632] Mice aged 6-9 weeks were anesthetized with pentobarbital (40 mg/kg), and whole blood was drawn from the inferior vena cava into 3.13% citrate (1 vol anticoagulant/9 vol blood). Blood was centrifuged at 1031 g for 10 min with the centrifuge pre-warmed to 26° C. to obtain platelet rich plasma (PRP). Alternatively, blood was centrifuged at 2400 g for 10 min at room temperature (RT), to obtain platelet-poor plasma (PPP). To obtain platelet-free plasma (PFP), an additional centrifugation at 10000 g for 10 min was performed.
[0633] Platelet Count and Measurement of Coagulation Parameters
[0634] Platelet counts were carried out with an automated cell counter (Procyte Dx Hematology Analyzer, IDEXX). Fibrinogen, FVIII and FIX activity were measured on an automated Sysmex CA-7000 coagulation analyser (Sysmex Digitana). Prothrombin time (PT) and activated partial thromboplastin time (APTT) were measured on a coagulometer (MC4plus, Merlin Medical).
[0635] Measurement of Murine PS Antigen and TAT Complexes by ELISA
[0636] Wells from 96-well plates (Maxisorb, Thermo) were coated with 50 μL per well of 10 μg/mL of rabbit polyclonal anti-human PS (DAKO Cytomation) and incubated overnight at 4° C. After 3 washes with TBS buffer (0.05 M tris(hydroxymethyl)aminomethane, 0.15 M NaCl, pH 7.5, 0.05% Tween 20), the plate was blocked with TBS-BSA 2%. Diluted plasma samples (dilution range: 1:300-1:600) were added to the wells and incubated at RT for 2 h. After 3 washed, 50 μL of 1 μg/mL biotinylated chicken polyclonal anti-murine protein S were added and incubated for 2 h at RT. Signal was amplified by streptavidin-HRP conjugated horseradish peroxidase (Thermo) was added and plates incubated for 1 h. The plates were washed 3 times and 100 μL TMB substrate (KPL) was added. Reactions were stopped by adding 100 μL HCl (1M). Absorbance was measure at 450 nm. Standard curves were set up by using serial dilution of pooled normal plasma obtained from 14 healthy mice (8 males and 6 females, 7-12 weeks old). Results were expressed in percentage relative to the pooled normal plasma.
[0637] TAT level was measured in duplicate for each plasma sample using a commercially available ELISA (Enzygnost TAT micro, Siemens), according to the manufacturer's instructions.
[0638] Mouse Tissue Processing and Sectioning, Immunohistochemistry and Microscopy
[0639] Tissue sections (4 μm) with no pre-treatment were stained with haematoxylin/eosin or Masson Trichrome or immunostained for insoluble fibrin, PS or TFPI. The following antibodies were used: fibrin (mAb clone 102-10).sup.1 final concentration 15.6 μg/mL, incubation for 30 min at RT, secondary antibody rabbit anti-human, (ab7155 Abcam, Cambridge, UK) 1:200 dilution, incubation for 30 min at RT; PS (MAB 4976, R&D, dilution 1:50) incubation for 30 min at RT, secondary antibody rabbit anti-rat, (ab7155 Abcam)-1:200 dilution, incubation for 30 min at RT; TFPI (PAHTFPI-S, Hematological Technologies) final concentration 18.6 μg/mL, incubation for 30 min at RT, secondary antibody rabbit anti-sheep IgG (ab7106, Abcam) 1:200 dilution, incubation for 30 min at RT. All the stainings were performed with the immunostainer BOND RX (Leica Biosystems, Muttenz, Switzerland) following manufacturer's instructions. Whole slides were scanned using 3D HISTECH Panoramic 250 Flash II, with 20× (NA 0.8), 40× (NA 0.95) air objectives. Images processing was done using Panoramic Viewer software.
[0640] In Vivo Administration of FVIII to Mice with Complete Genetic Loss of F8
[0641] Mice, aged 6-9 week, were anesthetized with ketamine (80 mg/kg) and xylazine (16 mg/kg). We administered intravenously either 0.3 U/kg of recombinant FVIII (Advate®, Baxalta) to reach a FVIII level of 100% at 1 h (normal dose) or an overdose of recombinant FVIII (2 U/kg) to reach >200% at 1 h. Either the normal dose or the overdose was injected 1 h before and 1 h after the introduction of a jugular vein catheter (Mouse JVC 2Fr PU 10 cm, Instech) and then 4 h, 8 h and 16 h after the placement of the central line. Mice were sacrificed 24 h after the first injection. Blood was drawn and organs were harvested. FVIII, fibrinogen and thrombin-antithrombin complexes (TAT) were measured as described in the examples. Lungs were isolated, fixed in 4% paraformaldehyde (PFA) and embedded in paraffin.
[0642] FeCl.sub.3 Injury Thrombosis Model in Mesenteric Arteries
[0643] A model of thrombosis in mesenteric arteries using intravital microscopy was performed according to reference.sup.2 with minor modifications. Mice were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg/kg) and xylazine (16 mg/kg). Platelets were directly labelled in vivo by the injection of 100 μL rhodamine 6G (1.0 mM). After selection of the studied field, vessel wall injury was generated by a filter paper (1 mm diameter patch of 1M Whatman paper) saturated with 10% FeCl.sub.3 applied topically for 1 min. Thrombus formation was monitored in real time under a fluorescent microscope (IV-500, Micron instruments, San Diego, Calif.) with an FITC filter set, equipped with an affinity corrected water-immersion optics (Zeiss, Germany). The bright fluorescent labelled platelets and leucocytes allowed the observation of 1355 μm×965 μm field of view through video triggered stroboscopic epi-illumination (Chadwick Helmuth, El Monte, Calif.). A 10× objective Zeiss Plan-Neofluar with NA0.3. was used. All scenes were recorded on video-tape using a customized low-lag silicon-intensified target camera (Dage MTI, Michigan city, IN), a time base generator and a Hi-8 VCR (EV, C-100, Sony, Japan). Time to vessel wall occlusion was measured, as determined by cessation of the blood cell flow.
[0644] Fibroblast-Like Synoviocytes (FLS) Isolation, Culture and Flow Cytometry
[0645] Murine FLS from 8-10 weeks old mice were isolated and cultured according to.sup.3. After three passages, phase contrast images of cells were taken, and cells were incubated with FITC-conjugated rat anti-mouse CD11b antibody (M1/70, Pharmingen, BD Biosciences), PE-conjugated rat anti-mouse CD90.2 antibody (30-H12, Pharmingen, BD Biosciences), FITC-conjugated rat anti-mouse CD106 antibody (429 MVCAM.A, Pharmingen, BD Biosciences), PE-conjugated hamster anti-mouse CD54 antibody (3E2, Pharmingen, BD Biosciences), and fluorochrome-conjugated isotype control antibodies for 30 min at 4° C. in the dark. After a final washing and centrifugation step, all incubated cells were analysed on an LSR II flow cytometer (BD Biosciences) and FACS Diva 7.0 software (BD Biosciences). Human FLS from healthy individual and OA patient were purchased from Asterand, Bioscience and cultured according to manufacture instructions.
[0646] Western Blotting
[0647] PS and TFPI were detected in human and mouse samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% gradient SDS-PAGE, Bio-Rad) under reducing conditions. The proteins were transferred to nitrocellulose membranes (Bio-Rad), and then visualized using: 2 ug/mL monoclonal MAB-4976 (R&D system) for murine PS, 1 μg/mL polyclonal AF2975 for murine TFPI (R&D system). Recombinant murine PS.sup.4 (30 ng), recombinant human TFPI full length (provided by T. Hamuro, Kaketsuken, Japan), lysate of washed platelets, PFP from F8.sup.−/− Pros1.sup.+/+ mice and placenta lysates from F8.sup.+/+ Pros1.sup.+/+ mice were used as PS, TFPIα controls. Samples from confluent murine and human FLS conditioned media were collected after 24 h-incubation in a serum-free media (OptiMem) and concentrated 40 times using Amicon filter devices (Millipore, 10 kDa cut-off). For TFPI western blotting, samples were treated with a mixture of five protein deglycosidases (PNGase F, O-Glycosidase, Neuraminidase, β1-4 Galactosidase, p-N-Acetylglucosaminidase, Deglycosylation kit, V4931, Promega) for 12 h at 37° C. before being loaded on the gel. Final detection was completed by using a horseradish peroxidase-conjugated secondary antibody (Dako) and the Supersignal West Dura Extended Duration Chemiluminescence Substrate (Pierce), monitored with a Fuji LAS 30001R CCD camera.
[0648] Immunohistochemistry on Human Knee Synovium
[0649] Paraffin-embedded specimens of synovial tissue from twelve HA patients and four HB patients who underwent arthroplasty for severe knee arthropathy were collected at the archives of the Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence. Seven HA patients were treated on demand and five with secondary prophylaxis. All four HB patients were treated on demand. Synovial samples from seven osteoarthritis (OA) patients were used as controls. For immunohistochemistry analysis, synovial tissue sections (5 μm thick) were deparaffinized, rehydrated, boiled for 10 minutes in sodium citrate buffer (10 mM, pH 6.0) for antigen retrieval and subsequently treated with 3% H.sub.2O.sub.2 in methanol for 15 min at room temperature to block endogenous peroxidase activity. Sections were then washed in PBS and incubated with Ultra V block (UltraVision Large Volume Detection System Anti-Polyvalent, HRP, catalogue number TP-125-HL, LabVision) for 10 min at RT according to the manufacturer's protocol. After blocking non-specific site binding, slides were incubated overnight at 4° C. with rabbit polyclonal anti-human Protein S/PROS1 antibody (1:50 dilution, catalogue number NBP1-87218, Novus Biologicals) or sheep polyclonal anti-human Tissue Factor Pathway Inhibitor (TFPI) antibody (1:500 dilution, catalogue number PAHTFPI-S, Haematologic Technologies) diluted in PBS. For PS immunostaining, tissue sections were then incubated with biotinylated secondary antibodies followed by streptavidin peroxidase (UltraVision Large Volume Detection System Anti-Polyvalent, HRP; LabVision) according to the manufacturer's protocol. For TFPI immunostaining, tissue sections were instead incubated with HRP-conjugated donkey anti-sheep IgG (1:1000 dilution; catalogue number ab97125; Abcam) for 30 min. Immunoreactivity was developed using 3-amino-9-ethylcarbazole (AEC kit, catalogue number TA-125-SA; LabVision) as chromogen. Synovial sections were finally counterstained with Mayer's haematoxylin (Bio-Optica), washed, mounted in an aqueous mounting medium and observed under a Leica DM4000 B microscope (Leica Microsystems). Sections not exposed to primary antibodies or incubated with isotype-matched and concentration-matched non-immune IgG (Sigma-Aldrich) were included as negative controls for antibody specificity. Light microscopy images were captured with a Leica DFC310 FX 1.4-megapixel digital colour camera equipped with the Leica software application suite LAS V3.8 (Leica Microsystems).
[0650] Fibrin Clot Ultrastructure Investigation
[0651] Fibrin clots were prepared at 37° C. from PFP by the addition of ˜5 nM TF (Dade Innovin, Siemens). They were then fixed in 2% glutaraldehyde, dehydrated, dried and sputter-coated with gold palladium for visualization using scanning electron microscopy. Semi quantitative evaluation of network density and fibers branching were performed using STEPanizer software (www.stepanizer.com).
[0652] Calibrated Automated Thrombography Assays in Murine Samples
[0653] Thrombin generation in PFP and PRP was determined using the calibrated automated thrombogram (CAT) method.
[0654] TFPI dependent PS activity was assessed in PRP (150 G/L), as follows. Briefly, 10 μL mouse PRP (150 G/L) was mixed with 10 μL PRP reagent (Diagnostica Stago), and 30 μL of buffer A (25 mm Hepes, 175 mm NaCl, pH 7.4, 5 mg/mL BSA). Thrombin generation was initiated at 37° C. with 10 μL of a fluorogenic substrate/CaCl.sub.2 mixture. Final concentrations were as follows: 16.6% mouse plasma, 1 μM hrTF, 4 μM phospholipids, 16 mM CaCl.sub.2, and 0.42 mM fluorogenic substrate.
[0655] APC dependent PS activity was assessed in a CAT-based APC resistance test in mouse PFP and PRP. PRP (150 G/L) was previously activated using 40 μM Ca.sup.2+ ionophore (A23187) for 5 min at 37 C. Final concentrations were as follows: 16.6% mouse plasma, 22 μM A23187, 1 μM hrTF, 4 μM phospholipids, 2 nM (for PFP) or 8 nM (for PRP) wild type recombinant mouse APC (wt-rmAPC) or mutated recombinant mouse APC (rmAPC L38D), 16 mM CaCl.sub.2, and 0.42 mM fluorogenic substrate.
[0656] For TF titration on PFP, the following reagents were used: PPP reagent and MP reagent (Diagnostica Stago).
[0657] Fluorescence was measured using a Fluoroscan Ascent® fluorometer, equipped with a dispenser. Fluorescence intensity was detected at wavelengths of 390 nm (excitation filter) and 460 nm (emission filter). A dedicated software program, Thrombinoscope® version 3.0.0.29 (Thrombinoscope bv) enabled the calculation of thrombin activity against the calibrator (Thrombinoscope bv) and displayed thrombin activity with the time. All experiences were carried out in duplicate at 37° C. and the measurements usually lasted 60 min.
[0658] CAT Assay in Human Samples
[0659] Written informed consent was obtained from patients. Venous blood was drawn by venipuncture in 3.2% sodium citrate (vol/vol) and centrifuged at 2000 g for 5 min. Platelet-poor plasma (PPP) was then centrifuged at 10000 g for 10 min to obtain PFP. PFP was aliquoted, snap-frozen, and stored at −80° C. until use. For PRP, blood was centrifuged at 180 g×10 min. All subjects gave informed consent to participation. Thrombin generation was assessed in human PFP and PRP, according to ref.sup.13 with minor changes. Briefly, 68 μL PFP or PRP (150 G/L) was incubated for 15 min at 37° C. with 12 μL of either a polyclonal rabbit anti-human PS-IgG antibody (0.42 mg/mL, Dako) or monoclonal antibodies against TFPI (0.66 μm, MW1848, Sanquin) or buffer A. Coagulation was initiated with 20 μL of a 7:1 mixture of the PPP low and PPP 5 μm reagents (Diagnostica Stago) for PFP samples or with PRP reagent (Diagnostica stago) for PRP samples. After addition of 20 μL of CaCl.sub.2 and fluorogenic substrate (1-1140; Bachem), the thrombin generation was followed in a Fluoroskan Ascent reader (Thermo Labsystems).
Discussion of Examples 6-17
[0660] As PS is a key regulator of thrombin generation, we considered that targeting PS could constitute a potential therapy for haemophilia.
[0661] Extensive studies in mice provide proof of concept data supporting a central role for PS and TFPI as contributing to bleeding and serious joint damage in haemophilic mice. Targeting Pros1 or inhibiting PS has the ability to ameliorate haemophilia in mice as judged by the in vivo improvement of the bleeding phenotype in the tail bleeding assays and the full protection against hemarthrosis (
[0662] Observations using clinical samples from haemophilic patients are consistent with the lessons learned from murine studies. In humans, blocking PS in plasma from patients with HA with or without inhibitors normalizes the ETP (
[0663] The extensive findings in this report lead us to propose that targeting PS may potentially be translated to therapies useful for haemophilia. PS in human and murine joints is a novel pathophysiological contributor to hemarthrosis and constitutes an attractive potential therapeutic target especially because of its dual cofactor activity for both APC and TFPIα within the joints. In the presence of PS, hemarthrosis increases TFPIα expression in the synovia. Targeting PS in mice protects them from hemarthrosis. Thus, we propose that TFPIα and its cofactor PS, both produced by FLS, together with the TM-EPCR-PC pathway, comprise a potent intra-articular anticoagulant system that has an important pathologic impact on hemarthrosis. The murine PS silencing RNA that we successfully used in haemophilic mice (
Example 19—Inhibition of PROS1 Target Gene Expression in Primary Hepatocytes by PROS1 siRNA Conjugates
[0664] The example shows dose dependent reduction of PROS1 mRNA levels in primary hepatocytes by EU149 to EU160 by receptor mediated uptake.
[0665] Primary mouse hepatocytes were seeded in a 96 well plate at a density of 25 000 cells per well. After attachment they were incubated with PROS1 siRNA conjugates in the cell culture medium at 100 nM, 10 nM, 1 nM, 0.1 nM and 0.01 nM as indicated in
Example 20—Inhibition of Human PROS1 Gene Expression in Primary Human Hepatocytes by Receptor Mediated Uptake
[0666] The example shows dose dependent reduction of human PROS1 mRNA levels by EU149 to EU152, EU156, EU159 and EU160 in primary human hepatocytes by receptor mediated uptake.
[0667] Primary human hepatocytes (Life Technologies) were seeded in a 96 well plate at a density of 35 000 cells per well in plating medium and were subsequently incubated with PROS1 siRNA conjugates EU149 to EU152, EU156, EU159 and EU160, in concentrations of 100 nM, 10 nM, 1 nM, 0.1 nM or 0.01 nM as shown in
Example 21—Inhibition of PROS1 Gene Expression In Vivo by Single Administration of PROS1 siRNA Conjugates
[0668] The example shows dose dependent in vivo reduction of PROS1 mRNA levels in the liver of mice treated with EU140 to EU145, EU150 to EU152 or by EU159.
[0669] 9 to 12-week old C57BL/6 mice were treated by subcutaneous injection with a dose of 1 or 5 mg conjugate (EU140 to EU145, EU150 to EU152 or EU159) per kg body weight or with the vehicle PBS as indicated in
[0670] siRNA conjugates used in this study are listed in Table 2. The dose-dependent reduction of PROS1 mRNA in mouse liver after treatment with PROS1 siRNA conjugates is shown in
Example 22—Inhibition of PROS1 Gene Expression in Haemophilic Mice by Single Administration of PROS1 siRNA Conjugate
[0671] The example shows the reduction of PROS1 mRNA levels in the liver and of PROS1 levels in serum of haemophilia A mouse model treated with EU152.
[0672] 9 to 12-week old Factor 8 knock-out mice (F8.sup.−/− mice; Prince et al. Blood (2018) 131 (12): 1360-1371) were treated by subcutaneous injection with 3 mg EU152 per kg body weight or with the vehicle PBS as indicated in
[0673] Each bar (A) or line (B) in the scatter dot plot represents the mean value with standard deviation from 8-9 animals.
[0674] siRNA conjugates used in this study are listed in Table 2. The reduction of PROS1 mRNA in mouse liver after treatment with PROS1 siRNA conjugates is shown in
Example 23—Treatment with PROS1 siRNA Conjugate Reduces Knee Swelling in an Acute Hemarthrosis Model
[0675] The example shows the difference between knee diameter before and 72 hours after knee injury of F8.sup.−/− mice. Joint swelling is reduced in the cohort of mice treated prophylactically with EU152.
[0676] 9 to 12 week old Factor 8 knock-out mice (F8.sup.−/− mice; Prince et al. 2018) were treated by subcutaneous injection with 3 mg, 5 mg or 10 mg EU152 per kg body weight or with the vehicle PBS as indicated in
[0677] The scatter dot plot represents the median value from 7-10 animals. Statistics: Kruskal-Wallis test with Dunn's multiple comparisons test against control group (PBS).
[0678] The siRNA conjugate used in this study is listed in Table 2. The difference in knee diameter before and 72 hours after knee injury of F8.sup.−/− mice is shown in
Example 24—Treatment with PROS1 siRNA Conjugate Improves the Haemostatic Profile of Haemophilia a Animal Model
[0679] The example shows clotting time, clot formation time and the alpha angle of whole blood samples collected from wild type mice, haemophilia A mouse model (F8.sup.−/−) or from haemophilia A mouse model treated with PROS1 siRNA (F8.sup.−/− EU152). Clot formation was assessed by Rotational Thromboelastometry (ROTEM), a viscoelastic assay of haemostasis which allows the measurement of global clot formation in real time (Gorlinger et al, Ann Card Anaesth (2016), 19:516-20). In haemophilic mice clotting time and clot formation time is reduced while alpha angle is increased compared to the assessment of these haemostatic parameters in wild type mice. Treatment of haemophilic mice with PROS1 siRNA reduces clotting time, clot formation time and increases the alpha angle.
[0680] 9 to 12 week old Factor 8 knock-out mice (F8.sup.−/− mice; Prince et al. 2018) were treated by subcutaneous injection with 5 mg EU152 per kg body weight or with the vehicle PBS as indicated in
[0681] The scatter dot plot represents the median value from 6-11 animals. Statistic: Welch's Anova with Dunnett's T3 post-hoc test on log-transformed values.
[0682] The siRNA conjugate used in this study is listed in Table 2. The blood clotting time of blood samples collected from wild type mice (WT), haemophilia A mice treated with PBS (F8.sup.−/− PBS) or haemophilia A mice treated with PROS1 siRNA EU152 (F8.sup.−/− EU152) is shown in
Example 25—Reduction of Human Protein S mRNA Level in Human Hep3B Cells by Transfection of Protein S siRNAs
[0683] In vitro test shows dose-dependent reduction of Protein S mRNA levels in human Hep3B cells by transfection of Protein S siRNA molecules (EU199 to EU222).
[0684] Hep3B cells were seeded at a density of 12 000 cells per well in the 96-well plate. The following day, the cells were transfected with 0.1 nM, 0.01 nM or 0.001 nM Protein S siRNA or with non-targeting control siRNA (EU198) and 1 μg/ml AtuFECT. 24 hours later, cells were lysed for RNA extraction and Protein S and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for Protein S mRNA normalised to values generated for the house keeping gene Actin and related to mean of untreated sample (ut) set at 1-fold target gene expression are listed in Table A.+/−SD represents standard deviation from three biological replicates. siRNA duplexes used in this study are listed in Table 2.
TABLE-US-00002 TABLE A ProS mRNA level normalized to actin siRNA siRNA mRNA level and relative duplex conc. to ut set to 1 +/−SD Ut 1.00 0.07 EU199 0.1 nM 0.34 0.05 0.01 nM 0.29 0.06 0.001 nM 0.40 0.11 EU200 0.1 nM 0.28 0.09 0.01 nM 0.38 0.04 0.001 nM 0.53 0.15 EU201 0.1 nM 0.46 0.13 0.01 nM 0.66 0.21 0.001 nM 0.89 0.11 EU202 0.1 nM 0.46 0.10 0.01 nM 0.46 0.14 EU203 0.001 nM 0.62 0.09 0.1 nM 0.20 0.01 0.01 nM 0.29 0.03 0.001 nM 0.54 0.06 EU204 0.1 nM 0.40 0.07 0.01 nM 0.67 0.10 0.001 nM 0.71 0.12 EU205 0.1 nM 0.44 0.03 0.01 nM 0.58 0.11 0.001 nM 0.57 0.03 EU206 0.1 nM 0.26 0.04 0.01 nM 0.35 0.13 0.001 nM 0.44 0.11 EU207 0.1 nM 0.27 0.12 0.01 nM 0.45 0.05 0.001 nM 0.79 0.17 EU208 0.1 nM 0.41 0.02 0.01 nM 0.35 0.05 0.001 nM 0.41 0.01 EU209 0.1 nM 0.40 0.08 0.01 nM 0.44 0.02 0.001 nM 0.74 0.11 EU210 0.1 nM 0.76 0.27 0.01 nM 1.28 0.20 0.001 nM 1.32 0.00 EU211 0.1 nM 0.34 0.05 0.01 nM 0.33 0.04 0.001 nM 0.39 0.02 EU212 0.1 nM 0.33 0.09 0.01 nM 0.43 0.09 0.001 nM 0.63 0.19 EU213 0.1 nM 0.31 0.11 0.01 nM 0.65 0.13 0.001 nM 1.27 0.29 EU214 0.1 nM 0.51 0.13 0.01 nM 0.70 0.15 0.001 nM 0.98 0.08 EU215 0.1 nM 0.23 0.05 0.01 nM 0.34 0.03 0.001 nM 0.58 0.15 EU216 0.1 nM 0.29 0.05 0.01 nM 0.68 0.08 0.001 nM 1.14 0.19 EU217 0.1 nM 0.52 0.11 0.01 nM 0.74 0.13 0.001 nM 0.85 0.20 EU218 0.1 nM 0.29 0.08 0.01 nM 0.35 0.01 0.001 nM 0.45 0.07 EU219 0.1 nM 0.20 0.08 0.01 nM 0.51 0.11 0.001 nM 1.09 0.27 EU220 0.1 nM 0.34 0.13 0.01 nM 0.36 0.09 0.001 nM 0.48 0.07 EU221 0.1 nM 0.19 0.01 0.01 nM 0.15 0.02 0.001 nM 0.28 0.05 EU222 0.1 nM 0.57 0.05 0.01 nM 1.19 0.03 0.001 nM 1.57 0.02 EU198 0.1 nM 0.81 0.03 0.1 nM 0.81 0.15 0.1 nM 0.85 0.02
Example 26—Dose Dependent Reduction of Protein S mRNA Level in Human Cells by Transfection of Protein S siRNAs at Concentration Between 1 nM and 0.00001 nM
[0685] In vitro test shows dose-dependent reduction of Protein S mRNA levels in human Hep3B cells by transfection of Protein S siRNA molecules.
[0686] Hep3B cells were seeded at a density of 12 000 cells per well in the 96-well plate. The following day, the cells were transfected with 1 nM, 0.01 nM, 0.001 nM, 0.0001 nM or 0.00001 nM Protein S siRNA or 1 nM non-targeting control siRNA (EU0198) and 1 μg/ml AtuFECT. 24 hours later, cells were lysed for RNA extraction and Protein S and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for Protein S mRNA were normalized to values generated for the house keeping gene Actin and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean+/−SD from three biological replicates. siRNA duplexes used in this study are listed in Table 2. Results are shown in
Example 27—Inhibition of Human Protein S Gene Expression in Primary Human Hepatocytes by Receptor Mediated Uptake
[0687] In vitro test shows dose-dependent reduction of human Protein S mRNA levels by conjugated siRNAs EU161 to EU171 in primary human hepatocytes.
[0688] Primary human hepatocytes (Life Technologies) were seeded in a 96-well plate at a density of 35 000 cells per well in plating medium and were subsequently incubated with Protein S siRNA conjugates EU161 to EU171 in concentrations of 100 nM, 10 nM, 1 nM or 0.1 nM as shown in
Example 28—Inhibition of Protein S Gene Expression in Primary Cynomolqus Hepatocytes by Receptor Mediated Uptake
[0689] In vitro test shows dose dependent reduction of cynomolgus Protein S mRNA levels by conjugated siRNAs EU161 to EU171 in primary cynomolgus hepatocytes.
[0690] Primary cynomolgus hepatocytes (Life Technologies) were seeded in a 96-well plate at a density of 45 000 cells per well in plating medium and were subsequently incubated with Protein S siRNA conjugates EU161 to EU171 in concentrations of 100 nM, 10 nM, 1 nM or 0.1 nM as shown in
[0691] Summary Tables
TABLE-US-00003 TABLE 2 Summary duplex table Single Duplex Strands EU012 EU012A EU012B EU060 EU060A EU060B EU061 EU061A EU061B EU062 EU062A EU062B EU063 EU063A EU063B EU064 EU064A EU064B EU065 EU065A EU065A EU066 EU066A EU066B EU067 EU067A EU067B EU068 EU068A EU068B EU069 EU069A EU069B EU070 EU070A EU070B EU071 EU071A EU071B EU072 EU072A EU072B EU073 EU073A EU073B EU074 EU074A EU074B EU075 EU075A EU075B EU076 EU076A EU076B EU077 EU077A EU077B EU078 EU078A EU078B EU079 EU079A EU079B EU080 EU080A EU080B EU081 EU081A EU081B EU082 EU082A EU082B EU083 EU083A EU083B EU110 EU109A EU110B EU140 EU140A EU140B EU141 EU141A EU141B EU142 EU142A EU142B EU143 EU143A EU143B EU144 EU144A EU144B EU145 EU145A EU145B EU146 EU146A EU146B EU147 EU147A EU147B EU148 EU148A EU148B EU149 EU149A EU140B EU150 EU150A EU141B EU151 EU151A EU142B EU152 EU152A EU143B EU153 EU153A EU145B EU154 EU151A EU154B EU155 EU155A EU155B EU156 EU155A EU156B EU157 EU152A EU157B EU158 EU158A EU158B EU159 EU158A EU159B EU160 EU158A EU160B EU161 EU161A EU161B EU162 EU162A EU162B EU163 EU163A EU163B EU164 EU164A EU164B EU165 EU165A EU165B EU166 EU166A EU166B EU167 EU167A EU167B EU168 EU168A EU168B EU169 EU169A EU169B EU170 EU170A EU170B EU171 EU171A EU171B EU198 EU198A EU198B EU199 EU199A EU199B EU200 EU200A EU200B EU201 EU201A EU201B EU202 EU202A EU202B EU203 EU203A EU203B EU204 EU204A EU204B EU205 EU205A EU205B EU206 EU206A EU206B EU207 EU207A EU207B EU208 EU208A EU208B EU209 EU209A EU209B EU210 EU210A EU210B EU211 EU211A EU211B EU212 EU212A EU212B EU213 EU213A EU213B EU214 EU214A EU214B EU215 EU215A EU215B EU216 EU216A EU216B EU217 EU217A EU217B EU218 EU218A EU218B EU219 EU219A EU219B EU220 EU220A EU220B EU221 EU221A EU221B EU222 EU222A EU222B
TABLE-US-00004 TABLE 3 Summary abbreviations table Abbreviation Meaning mA, mU, mC, 2′-O-Methyl RNA nucleotides mG 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 (vp)-mU-phos ivA, ivC, ivU, inverted RNA (3′-3′) nucleotides ivG ST23 ST23-phos ST43 (or C6XLT)
[0692] 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-00005 TABLE 4 Summary sequence table Name (A = 1.sup.st SEQ strand; Unmodified ID B = 2.sup.nd sequence 5′- NO: strand) Sequence 5′-3′ 3′ counterpart 1 EU060Aun UGCUUUCAUUGCUUUGUCC UGCUUUCAUUGCUUUGUCC 2 EU060Bun GGACAAAGCAAUGAAAGCA GGACAAAGCAAUGAAAGCA 3 EU061Aun UUCCACAGACACCAUAUUC UUCCACAGACACCAUAUUC 4 EU061Bun GAAUAUGGUGUCUGUGGAA GAAUAUGGUGUCUGUGGAA 5 EU062Aun UAUUCCAGAAGCUCCUUGC UAUUCCAGAAGCUCCUUGC 6 EU062Bun GCAAGGAGCUUCUGGAAUA GCAAGGAGCUUCUGGAAUA 7 EU063Aun UUUGUGUCAAGGUUCAAGG UUUGUGUCAAGGUUCAAGG 8 EU063Bun CCUUGAACCUUGACACAAA CCUUGAACCUUGACACAAA 9 EU064Aun AUUGACACAGCUUCUUAGG AUUGACACAGCUUCUUAGG 10 EU064Bun CCUAAGAAGCUGUGUCAAU CCUAAGAAGCUGUGUCAAU 11 EU065Aun UUCUAAUUCUUCCACAGAC UUCUAAUUCUUCCACAGAC 12 EU065Aun GUCUGUGGAAGAAUUAGAA GUCUGUGGAAGAAUUAGAA 13 EU066Aun AUAUCCAUCUUCAUUGCAU AUAUCCAUCUUCAUUGCAU 14 EU066Bun AUGCAAUGAAGAUGGAUAU AUGCAAUGAAGAUGGAUAU 15 EU067Aun UUUUCAAAGACCUCCCUGG UUUUCAAAGACCUCCCUGG 16 EU067Bun CCAGGGAGGUCUUUGAAAA CCAGGGAGGUCUUUGAAAA 17 EU068Aun AGUUUGAAUCCUUUCUUCC AGUUUGAAUCCUUUCUUCC 18 EU068Bun GGAAGAAAGGAUUCAAACU GGAAGAAAGGAUUCAAACU 19 EU069Aun UUUCAUUGCUUUGUCCAAG UUUCAUUGCUUUGUCCAAG 20 EU069Bun CUUGGACAAAGCAAUGAAA CUUGGACAAAGCAAUGAAA 21 EU070Aun CAUUGCUUUGUCCAAGACG CAUUGCUUUGUCCAAGACG 22 EU070Bun CGUCUUGGACAAAGCAAUG CGUCUUGGACAAAGCAAUG 23 EU071Aun UAUGUUUAGAAAUGGCUUC UAUGUUUAGAAAUGGCUUC 24 EU071Bun GAAGCCAUUUCUAAACAUA GAAGCCAUUUCUAAACAUA 25 EU072Aun UGUUCUUGCACACAGCUGU UGUUCUUGCACACAGCUGU 26 EU072Bun ACAGCUGUGUGCAAGAACA ACAGCUGUGUGCAAGAACA 27 EU073Aun AUCUUGGGCAAGUUUGAAU AUCUUGGGCAAGUUUGAAU 28 EU073Bun AUUCAAACUUGCCCAAGAU AUUCAAACUUGCCCAAGAU 29 EU074Aun AACUCUUCUGAUCUUGGGC AACUCUUCUGAUCUUGGGC 30 EU074Bun GCCCAAGAUCAGAAGAGUU GCCCAAGAUCAGAAGAGUU 31 EU075Aun UUCUUCCACAGACACCAUA UUCUUCCACAGACACCAUA 32 EU075Bun UAUGGUGUCUGUGGAAGAA UAUGGUGUCUGUGGAAGAA 33 EU076Aun GUCAGGAUAAGCAUUAGUU GUCAGGAUAAGCAUUAGUU 34 EU076Bun AACUAAUGCUUAUCCUGAC AACUAAUGCUUAUCCUGAC 35 EU077Aun ACAGACACCAUAUUCCAUA ACAGACACCAUAUUCCAUA 36 EU077Bun UAUGGAAUAUGGUGUCUGU UAUGGAAUAUGGUGUCUGU 37 EU078Aun UUUGGAUAAAAAUAAUCCG UUUGGAUAAAAAUAAUCCG 38 EU078Bun CGGAUUAUUUUUAUCCAAA CGGAUUAUUUUUAUCCAAA 39 EU079Aun CUCACAACUCUUCUGAUCU CUCACAACUCUUCUGAUCU 40 EU079Bun AGAUCAGAAGAGUUGUGAG AGAUCAGAAGAGUUGUGAG 41 EU080Aun GCAUUCACUGGUGUGGCAC GCAUUCACUGGUGUGGCAC 42 EU080Bun GUGCCACACCAGUGAAUGC GUGCCACACCAGUGAAUGC 43 EU081Aun UAGGUCAGGAUAAGCAUUA UAGGUCAGGAUAAGCAUUA 44 EU081Bun UAAUGCUUAUCCUGACCUA UAAUGCUUAUCCUGACCUA 45 EU082Aun AGCACACAUGUUCUCAGAG AGCACACAUGUUCUCAGAG 46 EU082Bun CUCUGAGAACAUGUGUGCU CUCUGAGAACAUGUGUGCU 47 EU083Aun UCCACAGACACCAUAUUCC UCCACAGACACCAUAUUCC 48 EU083Bun GGAAUAUGGUGUCUGUGGA GGAAUAUGGUGUCUGUGGA 49 EU146Aun UCAUUCACUGGUGUGGCAC UCAUUCACUGGUGUGGCAC 50 EU012A mU fC mG fA mA fG mU fA mU fU mC fC mG fC mG fU mA fC mG UCGAAGUAUUCCGCGUACG 51 EU012B fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mU fC mG fA CGUACGCGGAAUACUUCGA 52 EU060A mU fG mC fU mU fU mC fA mU fU mG fC mU fU mU fG mU fC mC UGCUUUCAUUGCUUUGUCC 53 EU060B mG mG mA mC mA mA fA fG fC mA mA mU mG mA mA mA mG mC mA GGACAAAGCAAUGAAAGCA 54 EU061A mU fU mC fC mA fC mA fG mA fC mA fC mC fA mU fA mU fU mC UUCCACAGACACCAUAUUC 55 EU061B mG mA mA mU mA mU fG fG fU mG mU mC mU mG mU mG mG mA mA GAAUAUGGUGUCUGUGGAA 56 EU062A mU fA mU fU mC fC mA fG mA fA mG fC mU fC mC fU mU fG mC UAUUCCAGAAGCUCCUUGC 57 EU062B mG mC mA mA mG mG fA fG fC mU mU mC mU mG mG mA mA mU mA GCAAGGAGCUUCUGGAAUA 58 EU063A mU fU mU fG mU fG mU fC mA fA mG fG mU fU mC fA mA fG mG UUUGUGUCAAGGUUCAAGG 59 EU063B mC mC mU mU mG mA fA fC fC mU mU mG mA mC mA mC mA mA mA CCUUGAACCUUGACACAAA 60 EU064A mA fU mU fG mA fC mA fC mA fG mC fU mU fC mU fU mA fG mG AUUGACACAGCUUCUUAGG 61 EU064B mC mC mU mA mA mG fA fA fG mC mU mG mU mG mU mC mA mA mU CCUAAGAAGCUGUGUCAAU 62 EU065A mU fU mC fU mA fA mU fU mC fU mU fC mC fA mC fA mG fA mC UUCUAAUUCUUCCACAGAC 63 EU065A mG mU mC mU mG mU fG fG fA mA mG mA mA mU mU mA mG mA mA GUCUGUGGAAGAAUUAGAA 64 EU066A mA fU mA fU mC fC mA fU mC fU mU fC mA fU mU fG mC fA mU AUAUCCAUCUUCAUUGCAU 65 EU066B mA mU mG mC mA mA fU fG fA mA mG mA mU mG mG mA mU mA mU AUGCAAUGAAGAUGGAUAU 66 EU067A mU fU mU fU mC fA mA fA mG fA mC fC mU fC mC fC mU fG mG UUUUCAAAGACCUCCCUGG 67 EU067B mC mC mA mG mG mG fA fG fG mU mC mU mU mU mG mA mA mA mA CCAGGGAGGUCUUUGAAAA 68 EU068A mA fG mU fU mU fG mA fA mU fC mC fU mU fU mC fU mU fC mC AGUUUGAAUCCUUUCUUCC 69 EU068B mG mG mA mA mG mA fA fA fG mG mA mU mU mC mA mA mA mC mU GGAAGAAAGGAUUCAAACU 70 EU069A mU fU mU fC mA fU mU fG mC fU mU fU mG fU mC fC mA fA mG UUUCAUUGCUUUGUCCAAG 71 EU069B mC mU mU mG mG mA fC fA fA mA mG mC mA mA mU mG mA mA mA CUUGGACAAAGCAAUGAAA 72 EU070A mC fA mU fU mG fC mU fU mU fG mU fC mC fA mA fG mA fC mG CAUUGCUUUGUCCAAGACG 73 EU070B mC mG mU mC mU mU fG fG fA mC mA mA mA mG mC mA mA mU mG CGUCUUGGACAAAGCAAUG 74 EU071A mU fA mU fG mU fU mU fA mG fA mA fA mU fG mG fC mU fU mC UAUGUUUAGAAAUGGCUUC 75 EU071B mG mA mA mG mC mC fA fU fU mU mC mU mA mA mA mC mA mU mA GAAGCCAUUUCUAAACAUA 76 EU072A mU fG mU fU mC fU mU fG mC fA mC fA mC fA mG fC mU fG mU UGUUCUUGCACACAGCUGU 77 EU072B mA mC mA mG mC mU fG fU fG mU mG mC mA mA mG mA mA mC mA ACAGCUGUGUGCAAGAACA 78 EU073A mA fU mC fU mU fG mG fG mC fA mA fG mU fU mU fG mA fA mU AUCUUGGGCAAGUUUGAAU 79 EU073B mA mU mU mC mA mA fA fC fU mU mG mC mC mC mA mA mG mA mU AUUCAAACUUGCCCAAGAU 80 EU074A mA fA mC fU mC fU mU fC mU fG mA fU mC fU mU fG mG fG mC AACUCUUCUGAUCUUGGGC 81 EU074B mG mC mC mC mA mA fG fA fU mC mA mG mA mA mG mA mG mU mU GCCCAAGAUCAGAAGAGUU 82 EU075A mU fU mC fU mU fC mC fA mC fA mG fA mC fA mC fC mA fU mA UUCUUCCACAGACACCAUA 83 EU075B mU mA mU mG mG mU fG fU fC mU mG mU mG mG mA mA mG mA mA UAUGGUGUCUGUGGAAGAA 84 EU076A mG fU mC fA mG fG mA fU mA fA mG fC mA fU mU fA mG fU mU GUCAGGAUAAGCAUUAGUU 85 EU076B mA mA mC mU mA mA fU fG fC mU mU mA mU mC mC mU mG mA mC AACUAAUGCUUAUCCUGAC 86 EU077A mA fC mA fG mA fC mA fC mC fA mU fA mU fU mC fC mA fU mA ACAGACACCAUAUUCCAUA 87 EU077B mU mA mU mG mG mA fA fU fA mU mG mG mU mG mU mC mU mG mU UAUGGAAUAUGGUGUCUGU 88 EU078A mU fU mU fG mG fA mU fA mA fA mA fA mU fA mA fU mC fC mG UUUGGAUAAAAAUAAUCCG 89 EU078B mC mG mG mA mU mU fA fU fU mU mU mU mA mU mC mC mA mA mA CGGAUUAUUUUUAUCCAAA 90 EU079A mC fU mC fA mC fA mA fC mU fC mU fU mC fU mG fA mU fC mU CUCACAACUCUUCUGAUCU 91 EU079B mA mG mA mU mC mA fG fA fA mG mA mG mU mU mG mU mG mA mG AGAUCAGAAGAGUUGUGAG 92 EU080A mG fC mA fU mU fC mA fC mU fG mG fU mG fU mG fG mC fA mC GCAUUCACUGGUGUGGCAC 93 EU080B mG mU mG mC mC mA fC fA fC mC mA mG mU mG mA mA mU mG mC GUGCCACACCAGUGAAUGC 94 EU081A mU fA mG fG mU fC mA fG mG fA mU fA mA fG mC fA mU fU mA UAGGUCAGGAUAAGCAUUA 95 EU081B mU mA mA mU mG mC fU fU fA mU mC mC mU mG mA mC mC mU mA UAAUGCUUAUCCUGACCUA 96 EU082A mA fG mC fA mC fA mC fA mU fG mU fU mC fU mC fA mG fA mG AGCACACAUGUUCUCAGAG 97 EU082B mC mU mC mU mG mA fG fA fA mC mA mU mG mU mG mU mG mC mU CUCUGAGAACAUGUGUGCU 98 EU083A mU fC mC fA mC fA mG fA mC fA mC fC mA fU mA fU mU fC mC UCCACAGACACCAUAUUCC 99 EU083B mG mG mA mA mU mA fU fG fG mU mG mU mC mU mG mU mG mG mA GGAAUAUGGUGUCUGUGGA 100 EU109A mU (ps) fC (ps) mG fA mA fG mU fA mU fU mC fC mG fC mG fU mA UCGAAGUAUUCCGCGUACG (ps) fC (ps) mG 101 EU110B [ST23 (ps)]3 ST43 (ps) fC mG fU mA fC mG fC mG fG mA fA mU fA mC CGUACGCGGAAUACUUCGA fU mU fC (ps) mG (ps) fA 102 EU140A mU (ps) fU (ps) mC fC mA fC mA fG mA fC mA fC mC fA mU fA mU UUCCACAGACACCAUAUUC (ps) fU (ps) mC 103 EU140B [ST23 (ps)]3 ST43 (ps) mG mA mA mU mA mU fG fG fU mG mU mC mU mG GAAUAUGGUGUCUGUGGAA mU mG mG (ps) mA (ps) mA 104 EU141A mU (ps) fU (ps) mC fU mA fA mU fU mC fU mU fC mC fA mC fA mG UUCUAAUUCUUCCACAGAC (ps) fA (ps) mC 105 EU141B [ST23 (ps)]3 ST43 (ps) mG mU mC mU mG mU fG fG fA mA mG mA mA mU GUCUGUGGAAGAAUUAGAA mU mA mG (ps) mA (ps) mA 106 EU142A mU (ps) fU (ps) mU fU mC fA mA fA mG fA mC fC mU fC mC fC mU UUUUCAAAGACCUCCCUGG (ps) fG (ps) mG 107 EU142B [ST23 (ps)]3 ST43 (ps) mC mC mA mG mG mG fA fG fG mU mC mU mU mU CCAGGGAGGUCUUUGAAAA mG mA mA (ps) mA (ps) mA 108 EU143A mU (ps)fU (ps) mU fC mA fU mU fG mC fU mU fU mG fU mC fC mA (ps) UUUCAUUGCUUUGUCCAAG fA (ps) mG 109 EU143B [ST23 (ps)]3 ST43 (ps) mC mU mU mG mG mA fC fA fA mA mG mC mA mA CUUGGACAAAGCAAUGAAA mU mG mA (ps) mA (ps) mA 110 EU144A mU (ps) fG (ps) mU fU mC fU mU fG mC fA mC fA mC fA mG fC mU UGUUCUUGCACACAGCUGU (ps) fG (ps) mU 111 EU144B [ST23 (ps)]3 ST43 (ps) mA mC mA mG mC mU fG fU fG mU mG mC mA mA ACAGCUGUGUGCAAGAACA mG mA mA (ps) mC (ps) mA 112 EU145A mA (ps) fC (ps) mA fG mA fC mA fC mC fA mU fA mU fU mC fC mA ACAGACACCAUAUUCCAUA (ps) fU (ps) mA 113 EU145B [ST23 (ps)]3 ST43 (ps) mU mA mU mG mG mA fA fU fA mU mG mG mU mG UAUGGAAUAUGGUGUCUGU mU mC mU (ps) mG (ps) mU 114 EU146A mU (ps) fC (ps) mA fU mU fC mA fC mU fG mG fU mG fU mG fG mC UCAUUCACUGGUGUGGCAC (ps) fA (ps) mC 115 EU146B [ST23 (ps)]3 ST43 (ps) mG mU mG mC mC mA fC fA fC mC mA mG mU mG GUGCCACACCAGUGAAUGC mA mA mU (ps) mG (ps) mC 116 EU147A mA (ps) fG (ps) mC fA mC fA mC fA mU fG mU fU mC fU mC fA mG AGCACACAUGUUCUCAGAG (ps) fA (ps) mG 117 EU147B [ST23 (ps)]3 ST43 (ps) mC mU mC mU mG mA fG fA fA mC mA mU mG mU CUCUGAGAACAUGUGUGCU mG mU mG (ps) mC (ps) mU 118 EU148A mU (ps) fC (ps) mC fA mC fA mG fA mC fA mC fC mA fU mA fU mU UCCACAGACACCAUAUUCC (ps) fC (ps) mC 119 EU148B [ST23 (ps)]3 ST43 (ps) mG mG mA mA mU mA fU fG fG mU mG mU mC mU GGAAUAUGGUGUCUGUGGA mG mU mG (ps) mG (ps) mA 120 EU149A (vp) mU fU mC fC mA fC mA fG mA fC mA fC mC fA mU fA mU (ps) fU UUCCACAGACACCAUAUUC (ps) mC 121 EU150A (vp) mU fU mC fU mA fA mU fU mC fU mU fC mC fA mC fA mG (ps) fA UUCUAAUUCUUCCACAGAC (ps) mC 122 EU151A (vp) mU fU mU fU mC fA mA fA mG fA mC fC mU fC mC fC mU (ps) fG UUUUCAAAGACCUCCCUGG (ps) mG 123 EU152A (vp) mU fU mU fC mA fU mU fG mC fU mU fU mG fU mC fC mA (ps) fA UUUCAUUGCUUUGUCCAAG (ps) mG 124 EU153A (vp) mU fC mA fG mA fC mA fC mC fA mU fA mU fU mC fC mA (ps) fU UCAGACACCAUAUUCCAUA (ps) mA 125 EU154B Ser (GN) (ps) mC (ps) mC (ps) mA mG mG mG fA fG fG mU mC mU mU CCAGGGAGGUCUUUGAAAA mU mG mA mA (ps) mA (ps) mA (ps) Ser (GN) 126 EU155A (vp) mU fUmU fU mC fA mA fA mG fA mC fC mU fC mC fC mU fG (ps2) UUUUCAAAGACCUCCCUGG mG 127 EU155B Ser (GN) mC (ps2) mC mA mG mG mG fA fG fG mU mC mU mU mU mG mA CCAGGGAGGUCUUUGAAAA mA mA (ps2) mA Ser (GN) 128 EU155A (vp) mU fU mU fU mC fA mA fA mG fA mC fC mU fC mC fC mU fG (ps2) UUUUCAAAGACCUCCCUGG mG 129 EU156B [ST23]3 5T43 mC (ps2) mC mA mG mG mG fA fG fG mU mC mU mU mU mG CCAGGGAGGUCUUUGAAAA mA mA mA (ps2) mA 130 EU157B Ser (GN) (ps) mC (ps) mU (ps) mU mG mG mA fC fA fA mA mG mC mA CUUGGACAAAGCAAUGAAA mA mU mG mA (ps) mA (ps) mA (ps) Ser (GN) 131 EU158A (vp) mU fU mU fC mA fU mU fG mC fU mU fU mG fU mC fC mA fA (ps2) UUUCAUUGCUUUGUCCAAG mG 132 EU158B Ser (GN) mC (ps2) mU mU mG mG mA fC fA fA mA mG mC mA mA mU mG CUUGGACAAAGCAAUGAAA mA mA (ps2) mA Ser (GN) 133 EU159B [ST23]3 ST43 mC (ps2) mU mU mG mG mA fC fA fA mA mG mC mA mA mU CUUGGACAAAGCAAUGAAA mG mA mA (ps2) mA 134 EU160B [ST23]3 5T43 mC (ps2) mU mU mG mG mA fC fA fA mA mG mC mA mA mU CUUGGACAAAGCAAUGAAA mG mA mA irA 135 EU142B mC mC mA mG mG mG fA fG fG mU mC mU mU mU mG mA mA (ps) mA (ps) CCAGGGAGGUCUUUGAAAA without mA ligand 136 EU143B mC mU mU mG mG mA fC fA fA mA mG mC mA mA mU mG mA (ps) mA (ps) CUUGGACAAAGCAAUGAAA without mA ligand 137 EU198A mU fC mG fA mA fG mU fA mU fU mC fC mG fC mG fU mA fC mG UCGAAGUAUUCCGCGUACG 138 EU198B fC mG fU mA fC mG fC mG fG mA fA mU fA mC fU mU fC mG fA CGUACGCGGAAUACUUCGA 139 EU199A mU (ps) fU (ps) mU fU mC fA mA fA mG fA mC fC mU fC mC fC mU UUUUCAAAGACCUCCCUGG (ps) fG (ps) mG 140 EU199B mC (ps) mC (ps) mA mG mG mG fA fG fG mU mC mU mU mU mG mA mA CCAGGGAGGUCUUUGAAAA (ps) mA (ps) mA 141 EU200A mU (ps) fU (ps) mA fU mA fA mA fA mG fG mC fA mU fU mC fA mC UUAUAAAAGGCAUUCACUG (ps) fU (ps) mG 142 EU200B mC (ps) mA (ps) mG mU mG mA fA fU fG mC mC mU mU mU mU mA mU CAGUGAAUGCCUUUUAUAA (ps) mA (ps) mA 143 EU201A mU (ps) fU (ps) mU fU mG fU mA fA mU fG mU fA mG fA mC fC mU UUUUGUAAUGUAGACCUUG (ps) fU (ps) mG 144 EU201B mC (ps) mA (ps) mA mG mG mU fC fU fA mC mA mU mU mA mC mA mA CAAGGUCUACAUUACAAAA (ps) mA (ps) mA 145 EU202A mA (ps) fU (ps) mU fA mA fU mA fU mU fC mA fC mU fU mC fC mA AUUAAUAUUCACUUCCAUG (ps) fU (ps) mG 146 EU202B mC (ps) mA (ps) mU mG mG mA fA fG fU mG mA mA mU mA mU mU mA CAUGGAAGUGAAUAUUAAU (ps) mA (ps) mU 147 EU203A mU (ps) fU (ps) mG fU mA fC mU fU mC fA mA fC mA fA mU fC mA UUGUACUUCAACAAUCACA (ps) fC (ps) mA 148 EU203B mU (ps) mG (ps) mU mG mA mU fU fG fU mU mG mA mA mG mU mA mC UGUGAUUGUUGAAGUACAA (ps) mA (ps) mA 149 EU204A mC (ps) fU (ps) mU fU mA fU mU fG mC fA mC fA mG fU mU fC mU CUUUAUUGCACAGUUCUUC (ps) fU (ps) mC 150 EU204B mG (ps) mA (ps) mA mG mA mA fC fU fG mU mG mC mA mA mU mA mA GAAGAACUGUGCAAUAAAG (ps) mA (ps) mG 151 EU205A mU (ps) fA (ps) mU fU mU fG mA fG mG fG mA fU mC fU mU fU mG UAUUUGAGGGAUCUUUGCA (ps) fC (ps) mA 152 EU205B mU (ps) mG (ps) mC mA mA mA fG fA fU mC mC mC mU mC mA mA mA UGCAAAGAUCCCUCAAAUA (ps) mU (ps) mA 153 EU206A mA (ps) fU (ps) mA fU mU fC mA fC mU fU mC fC mA fU mG fC mA AUAUUCACUUCCAUGCAGC (ps) fG (ps) mC 154 EU206B mG (ps) mC (ps) mU mG mC mA fU fG fG mA mA mG mU mG mA mA mU GCUGCAUGGAAGUGAAUAU (ps) mA (ps) mU 155 EU207A mA (ps) fG (ps) mU fA mU fA mA fU mU fA mC fA mC fA mC fA mA AGUAUAAUUACACACAAGG (ps) fG (ps) mG 156 EU207B mC (ps) mC (ps) mU mU mG mU fG fU fG mU mA mA mU mU mA mU mA CCUUGUGUGUAAUUAUACU (ps) mC (ps) mU 157 EU208A mU (ps) fA (ps) mA fU mA fG mA fC mC fA mC fC mA fU mC fU mC UAAUAGACCACCAUCUCUU (ps) fU (ps) mU 158 EU208B mA (ps) mA (ps) mG mA mG mA fU fG fG mU mG mG mU mC mU mA mU AAGAGAUGGUGGUCUAUUA (ps) mU (ps) mA 159 EU209A mA (ps) fA (ps) mA fU mG fC mA fU mC fA mC fA mG fU mA fC mC AAAUGCAUCACAGUACCAG (ps) fA (ps) mG 160 EU209B mC (ps) mU (ps) mG mG mU mA fC fU fG mU mG mA mU mG mC mA mU CUGGUACUGUGAUGCAUUU (ps) mU (ps) mU 161 EU210A mG (ps) fU (ps) mC fA mU fU mU fU mC fA mA fA mG fA mC fC mU GUCAUUUUCAAAGACCUCC (ps) fC (ps) mC 162 EU210B mG (ps) mG (ps) mA mG mG mU fC fU fU mU mG mA mA mA mA mU mG GGAGGUCUUUGAAAAUGAC (ps) mA (ps) mC 163 EU211A mU (ps) fU (ps) mG fA mA fA mA fG mA fG mC fG mA fA mG fA mC UUGAAAAGAGCGAAGACAA (ps) fA (ps) mA 164 EU211B mU (ps) mU (ps) mG mU mC mU fU fC fG mC mU mC mU mU mU mU mC UUGUCUUCGCUCUUUUCAA (ps) mA (ps) mA 165 EU212A mU (ps) fG (ps) mU fA mU fG mU fU mC fA mU fU mC fU mU fA mA UGUAUGUUCAUUCUUAAGC (ps) fG (ps) mC 166 EU212B mG (ps) mC (ps) mU mU mA mA fG fA fA mU mG mA mA mC mA mU mA GCUUAAGAAUGAACAUACA (ps) mC (ps) mA 167 EU213A mU (ps) fU (ps) mA fA mU fG mA fG mU fU mC fA mC fU mU fU mC UUAAUGAGUUCACUUUCCA (ps) fC (ps) mA 168 EU213B mU (ps) mG (ps) mG mA mA mA fG fU fG mA mA mC mU mC mA mU mU UGGAAAGUGAACUCAUUAA (ps) mA (ps) mA 169 EU214A mU (ps) fU (ps) mU fU mA fC mA fG mG fA mA fC mA fG mU fG mG UUUUACAGGAACAGUGGUA (ps) fU (ps) mA 170 EU214B mU (ps) mA (ps) mC mC mA mC fU fG fU mU mC mC mU mG mU mA mA UACCACUGUUCCUGUAAAA (ps) mA (ps) mA 171 EU215A mC (ps) fA (ps) mU fU mC fU mU fA mA fG mC fU mG fA mA fC mU CAUUCUUAAGCUGAACUUC (ps) fU (ps) mC 172 EU215B mG (ps) mA (ps) mA mG mU mU fC fA fG mC mU mU mA mA mG mA mA GAAGUUCAGCUUAAGAAUG (ps) mU (ps) mG 173 EU216A mC (ps) fA (ps) mU fU mA fU mU fA mU fA mA fU mC fU mA fU mG CAUUAUUAUAAUCUAUGUG (ps) fU (ps) mG 174 EU216B mC (ps) mA (ps) mC mA mU mA fG fA fU mU mA mU mA mA mU mA mA CACAUAGAUUAUAAUAAUG (ps) mU (ps) mG 175 EU217A mC (ps) fG (ps) mA fA mU fA mU fU mC fA mA fG mG fU mC fA mC CGAAUAUUCAAGGUCACAU (ps) fA (ps) mU 176 EU217B mA (ps) mU (ps) mG mU mG mA fC fC fU mU mG mA mA mU mA mU mU AUGUGACCUUGAAUAUUCG (ps) mC (ps) mG 177 EU218A mC (ps) fA (ps) mC fU mG fA mA fU mG fG mA fA mC fA mU fC mU CACUGAAUGGAACAUCUGG (ps) fG (ps) mG 178 EU218B mC (ps) mC (ps) mA mG mA mU fG fU fU mC mC mA mU mU mC mA mG CCAGAUGUUCCAUUCAGUG (ps) mU (ps) mG 179 EU219A mU (ps) fC (ps) mU fG mG fA mA fU mG fG mC fA mU fU mG fA mC UCUGGAAUGGCAUUGACAC (ps) fA (ps) mC 180 EU219B mG (ps) mU (ps) mG mU mC mA fA fU fG mC mC mA mU mU mC mC mA GUGUCAAUGCCAUUCCAGA (ps) mG (ps) mA 181 EU220A mA (ps) fA (ps) mG fU mU fU mG fC mC fU mC fU mG fA mG fA mC AAGUUUGCCUCUGAGACGG (ps) fG (ps) mG 182 EU220B mC (ps) mC (ps) mG mU mC mU fC fA fG mA mG mG mC mA mA mA mC CCGUCUCAGAGGCAAACUU (ps) mU (ps) mU 183 EU221A mU (ps) fU (ps) mC fG mU fA mU fA mC fA mU fC mC fA mU fC mU UUCGUAUACAUCCAUCUAG (ps) fA (ps) mG 184 EU221B mC (ps) mU (ps) mA mG mA mU fG fG fA mU mG mU mA mU mA mC mG CUAGAUGGAUGUAUACGAA (ps) mA (ps) mA 185 EU222A mC (ps) fU (ps) mU fA mG fG mG fC mC fU mG fU mA fU mC fC mG CUUAGGGCCUGUAUCCGAU (ps) fA (ps) mU 186 EU222B mA (ps) mU (ps) mC mG mG mA fU fA fC mA mG mG mC mC mC mU mA AUCGGAUACAGGCCCUAAG (ps) mA (ps) mG 187 EU200Aun UUAUAAAAGGCAUUCACUG UUAUAAAAGGCAUUCACUG 188 EU200Bun CAGUGAAUGCCUUUUAUAA CAGUGAAUGCCUUUUAUAA 189 EU201Aun UUUUGUAAUGUAGACCUUG UUUUGUAAUGUAGACCUUG 190 EU201Bun CAAGGUCUACAUUACAAAA CAAGGUCUACAUUACAAAA 191 EU202Aun AUUAAUAUUCACUUCCAUG AUUAAUAUUCACUUCCAUG 192 EU202Bun CAUGGAAGUGAAUAUUAAU CAUGGAAGUGAAUAUUAAU 193 EU203Aun UUGUACUUCAACAAUCACA UUGUACUUCAACAAUCACA 194 EU203Bun UGUGAUUGUUGAAGUACAA UGUGAUUGUUGAAGUACAA 195 EU204Aun CUUUAUUGCACAGUUCUUC CUUUAUUGCACAGUUCUUC 196 EU204Bun GAAGAACUGUGCAAUAAAG GAAGAACUGUGCAAUAAAG 197 EU205Aun UAUUUGAGGGAUCUUUGCA UAUUUGAGGGAUCUUUGCA 198 EU205Bun UGCAAAGAUCCCUCAAAUA UGCAAAGAUCCCUCAAAUA 199 EU206Aun AUAUUCACUUCCAUGCAGC AUAUUCACUUCCAUGCAGC 200 EU206Bun GCUGCAUGGAAGUGAAUAU GCUGCAUGGAAGUGAAUAU 201 EU207Aun AGUAUAAUUACACACAAGG AGUAUAAUUACACACAAGG 202 EU207Bun CCUUGUGUGUAAUUAUACU CCUUGUGUGUAAUUAUACU 203 EU208Aun UAAUAGACCACCAUCUCUU UAAUAGACCACCAUCUCUU 204 EU208Bun AAGAGAUGGUGGUCUAUUA AAGAGAUGGUGGUCUAUUA 205 EU209Aun AAAUGCAUCACAGUACCAG AAAUGCAUCACAGUACCAG 206 EU209Bun CUGGUACUGUGAUGCAUUU CUGGUACUGUGAUGCAUUU 207 EU210Aun GUCAUUUUCAAAGACCUCC GUCAUUUUCAAAGACCUCC 208 EU210Bun GGAGGUCUUUGAAAAUGAC GGAGGUCUUUGAAAAUGAC 209 EU211Aun UUGAAAAGAGCGAAGACAA UUGAAAAGAGCGAAGACAA 210 EU211Bun UUGUCUUCGCUCUUUUCAA UUGUCUUCGCUCUUUUCAA 211 EU212Aun UGUAUGUUCAUUCUUAAGC UGUAUGUUCAUUCUUAAGC 212 EU212Bun GCUUAAGAAUGAACAUACA GCUUAAGAAUGAACAUACA 213 EU213Aun UUAAUGAGUUCACUUUCCA UUAAUGAGUUCACUUUCCA 214 EU213Bun UGGAAAGUGAACUCAUUAA UGGAAAGUGAACUCAUUAA 215 EU214Aun UUUUACAGGAACAGUGGUA UUUUACAGGAACAGUGGUA 216 EU214Bun UACCACUGUUCCUGUAAAA UACCACUGUUCCUGUAAAA 217 EU215Aun CAUUCUUAAGCUGAACUUC CAUUCUUAAGCUGAACUUC 218 EU215Bun GAAGUUCAGCUUAAGAAUG GAAGUUCAGCUUAAGAAUG 219 EU216Aun CAUUAUUAUAAUCUAUGUG CAUUAUUAUAAUCUAUGUG 220 EU216Bun CACAUAGAUUAUAAUAAUG CACAUAGAUUAUAAUAAUG 221 EU217Aun CGAAUAUUCAAGGUCACAU CGAAUAUUCAAGGUCACAU 222 EU217Bun AUGUGACCUUGAAUAUUCG AUGUGACCUUGAAUAUUCG 223 EU218Aun CACUGAAUGGAACAUCUGG CACUGAAUGGAACAUCUGG 224 EU218Bun CCAGAUGUUCCAUUCAGUG CCAGAUGUUCCAUUCAGUG 225 EU219Aun UCUGGAAUGGCAUUGACAC UCUGGAAUGGCAUUGACAC 226 EU219Bun GUGUCAAUGCCAUUCCAGA GUGUCAAUGCCAUUCCAGA 227 EU220Aun AAGUUUGCCUCUGAGACGG AAGUUUGCCUCUGAGACGG 228 EU220Bun CCGUCUCAGAGGCAAACUU CCGUCUCAGAGGCAAACUU 229 EU221Aun UUCGUAUACAUCCAUCUAG UUCGUAUACAUCCAUCUAG 230 EU221Bun CUAGAUGGAUGUAUACGAA CUAGAUGGAUGUAUACGAA 231 EU222Aun CUUAGGGCCUGUAUCCGAU CUUAGGGCCUGUAUCCGAU 232 EU222Bun AUCGGAUACAGGCCCUAAG AUCGGAUACAGGCCCUAAG 233 EU161A mA (ps) fU (ps) mA fU mU fC mA fC mU fU mC fC mA fU mG fC mA AUAUUCACUUCCAUGCAGC (ps) fG (ps) mC 234 EU161B [ST23 (ps)]3 ST41 (ps) mG mC mU mG mC mA fU fG fG mA mA mG mU mG GCUGCAUGGAAGUGAAUAU mA mA mU (ps) mA (ps) mU 235 EU162A (vp)-mU fU mA fU mU fC mA fC mU fU mC fC mA fU mG fC mA (ps) fG UUAUUCACUUCCAUGCAGC (ps) mC 236 EU162B [ST23 (ps)]3 ST41 (ps) mG mC mU mG mC mA fU fG fG mA mA mG mU mG GCUGCAUGGAAGUGAAUAU mA mA mU (ps) mA (ps) mU 237 EU163A mU (ps) fA (ps) mA fU mA fG mA fC mC fA mC fC mA fU mC fU mC UAAUAGACCACCAUCUCUU (ps) fU (ps) mU 238 EU163B [ST23 (ps)]3 ST41 (ps) mA mA mG mA mG mA fU fG fG mU mG mG mU mC AAGAGAUGGUGGUCUAUUA mU mA mU (ps) mU (ps) mA 239 EU164A (vp)-mU fA mA fU mA fG mA fC mC fA mC fC mA fU mC fU mC (ps) fU UAAUAGACCACCAUCUCUU (ps) mU 240 EU164B [ST23 (ps)]3 ST41 (ps) mA mA mG mA mG mA fU fG fG mU mG mG mU mC AAGAGAUGGUGGUCUAUUA mU mA mU (ps) mU (ps) mA 241 EU165A (vp)-mU fA mA fU mA fG mA fC mC fA mC fC mA fU mC fU mC fU (ps2) UAAUAGACCACCAUCUCUU mU 242 EU165B [ST23]3 ST41 mA (ps2) mA mG mA mG mA fU fG fG mU mG mG mU mC mU AAGAGAUGGUGGUCUAUUA mA mU mU (ps2) mA 243 EU166A mU (ps) fU (ps) mG fA mA fA mA fG mA fG mC fG mA fA mG fA mC UUGAAAAGAGCGAAGACAA (ps) fA (ps) mA 244 EU166B [ST23 (ps)]3 ST41 (ps) mU mU mG mU mC mU fU fC fG mC mU mC mU mU UUGUCUUCGCUCUUUUCAA mU mU mC (ps) mA (ps) mA 245 EU167A (vp)-mU fU mG fA mA fA mA fG mA fG mC fG mA fA mG fA mC (ps) fA UUGAAAAGAGCGAAGACAA (ps) mA 246 EU167B [ST23 (ps)]3 ST41 (ps) mU mU mG mU mC mU fU fC fG mC mU mC mU mU UUGUCUUCGCUCUUUUCAA mU mU mC (ps) mA (ps) mA 247 EU168A (vp)-mU fU mG fA mA fA mA fG mA fG mC fG mA fA mG fA mC fA (ps2) UUGAAAAGAGCGAAGACAA mA 248 EU168B [ST23]3 ST41 mU (ps2) mU mG mU mC mU fU fC fG mC mU mC mU mU mU UUGUCUUCGCUCUUUUCAA mU mC mA (ps2) mA 249 EU169A mU (ps) fU (ps) mC fG mU fA mU fA mC fA mU fC mC fA mU fC mU UUCGUAUACAUCCAUCUAG (ps) fA (ps) mG 250 EU169B [ST23 (ps)]3 ST41 (ps) mC mU mA mG mA mU fG fG fA mU mG mU mA mU CUAGAUGGAUGUAUACGAA mA mC mG (ps) mA (ps) mA 251 EU170A (vp)-mU fU mC fG mU fA mU fA mC fA mU fC mC fA mU fC mU (ps) fA UUCGUAUACAUCCAUCUAG (ps) mG 252 EU170B [ST23 (ps)]3 ST41 (ps) mC mU mA mG mA mU fG fG fA mU mG mU mA mU CUAGAUGGAUGUAUACGAA mA mC mG (ps) mA (ps) mA 253 EU171A (vp)-mU fU mC fG mU fA mU fA mC fA mU fC mC fA mU fC mU fA (ps2) UUCGUAUACAUCCAUCUAG mG 254 EU171B [ST23]3 ST41 mC (ps2) mU mA mG mA mU fG fG fA mU mG mU mA mU mA CUAGAUGGAUGUAUACGAA mC mG mA (ps2) mA 255 EU162Aun UUAUUCACUUCCAUGCAGC UUAUUCACUUCCAUGCAGC 256 EU161B mG mC mU mG mC mA fU fG fG mA mA mG mU mG mA mA mU (ps) mA (ps) GCUGCAUGGAAGUGAAUAU without mU ligand 257 EU163B mA mA mG mA mG mA fU fG fG mU mG mG mU mC mU mA mU (ps) mU (ps) AAGAGAUGGUGGUCUAUUA without mA ligand 258 EU170B mC mU mA mG mA mU fG fG fA mU mG mU mA mU mA mC mG (ps) mA (ps) CUAGAUGGAUGUAUACGAA without mA ligand