NUCLEIC ACIDS FOR INHIBITING EXPRESSION OF CNNM4 IN A CELL

Abstract

The invention relates to nucleic acid products that interfere with or inhibit CNNM4 (Cyclin M4) gene expression. It further relates to therapeutic uses of CNNM4 inhibition for the treatment of diseases, such as liver diseases including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver steatosis, liver fibrosis, liver cirrhosis, liver cancer and other diseases associated with magnesium dysregulation.

Claims

1-16. (canceled)

17. A double-stranded nucleic acid for inhibiting expression of CNNM4, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences selected from SEQ ID NO: 371, 243, 267, 277, 279, 287, 317, 319, 325, 333, 345, 347, 349, 361, 367, 369, 377, 401, 411, 413, 415, 420, 421, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 549, 550, 551 and 552.

18. The nucleic acid of claim 17, wherein the first strand and the second strand form a duplex region of from 17-25 nucleotides in length.

19. The nucleic acid of claim 17, wherein the nucleic acid mediates RNA interference.

20. The nucleic acid of claim 17, wherein at least one nucleotide of the first and/or second strand is a modified nucleotide.

21. The nucleic acid of claim 17, 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.

22. The nucleic acid of claim 17, wherein the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide at its 5′ end.

23. The nucleic acid of claim 17, 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.

24. The nucleic acid of claim 17, 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.

25. The nucleic acid of claim 17, wherein the nucleic acid is conjugated to a ligand.

26. The nucleic acid of claim 25, 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.

27. A composition comprising a nucleic acid of claim 17 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.

28. A method of preventing, decreasing the risk of suffering from, or treating a disease, disorder or syndrome comprising administering a pharmaceutically effective amount of the nucleic acid of claim 17 to an individual in need of treatment.

29. The method of claim 28, wherein the disease, disorder or syndrome is a liver disease, a kidney disease or a lung disease.

30. The method of claim 28, wherein the disease is a liver disease selected from the group consisting of non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma (HCC), drug-induced liver injury (DILI), non-alcoholic fatty liver disease (NAFLD), fatty liver, liver cancer, liver fibrosis, veno-occlusive liver disease, hepatic sinusoidal obstruction syndrome (SOS), steatosis, Budd-Chiari syndrome, viral hepatitis B, viral hepatitis C, alcoholic hepatitis, hepatic ischemia reperfusion injury, primary biliary cirrhosis (PBC), a chronic liver disease, an acute liver disease, liver damage, a non-proliferative liver disease cholangiocarcinoma (bile duct cancer), a disease associated with hypomagnesemia in the liver.

31. The method of claim 30, wherein the liver disease is selected from the group consisting of non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma (HCC), drug-induced liver injury (DILI) and non-alcoholic fatty liver disease (NAFLD).

32. A method of preventing, decreasing the risk of suffering from, or treating a disease, disorder or syndrome comprising administering a pharmaceutically effective amount of the composition of claim 27 to an individual in need of treatment.

33. The method of claim 32, wherein the disease, disorder or syndrome is a liver disease, a kidney disease or a lung disease.

34. The method of claim 32, wherein the disease is a liver disease selected from the group consisting of non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma (HCC), drug-induced liver injury (DILI), non-alcoholic fatty liver disease (NAFLD), fatty liver, liver cancer, liver fibrosis, veno-occlusive liver disease, hepatic sinusoidal obstruction syndrome (SOS), steatosis, Budd-Chiari syndrome, viral hepatitis B, viral hepatitis C, alcoholic hepatitis, hepatic ischemia reperfusion injury, primary biliary cirrhosis (PBC), a chronic liver disease, an acute liver disease, liver damage, a non-proliferative liver disease cholangiocarcinoma (bile duct cancer), a disease associated with hypomagnesemia in the liver.

35. The method of claim 34, wherein the liver disease is selected from the group consisting of non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma (HCC), drug-induced liver injury (DILI) and non-alcoholic fatty liver disease (NAFLD).

36. The nucleic acid of claim 20, wherein the modified nucleotide is a non-naturally occurring nucleotide such as a 2′-F modified nucleotide.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0573] FIG. 1. CNNM4 expression in liver determined by IHC in human samples and mouse models from DILI and different pathologies of chronic liver disease. *p<0.05 vs Healthy; **p<0.01 vs Healthy; ***p<0.001 vs Healthy.

[0574] FIG. 2A. CNNM4 expression, determined by qPCR of CNNM4 mRNA levels in human liver samples from healthy subjects compared with samples from steatotic and NASH patients.

[0575] FIG. 2B. CNNM4 expression, determined by qPCR of CNNM4 mRNA levels in an in vivo mouse model.

[0576] FIG. 2C. CNNM4 expression determined by qPCR of CNNM4 mRNA levels in an in vitro mouse cell model. *p<0.05 vs Healthy.

[0577] FIG. 3A. Lipid content in NASH-induced primary hepatocytes return into healthy levels when treated with siRNA CNNM4.

[0578] FIG. 3B. Inflammation induced by ROS in treated mice.

[0579] FIG. 3C. DILI-induced cell death is decreased by siRNA therapy. *p<0.05 vs Healthy **p<0.01 vs Healthy; ***p<0.001 vs Healthy; #p<0.05 vs NASH; ##p<0.01 vs NASH; ###p<0.001 vs DILI.

[0580] FIG. 4A. The lipid content in NASH-induced human cells returns to healthy levels when treated with CNNM4 siRNA.

[0581] FIG. 4B. The lipid content in NASH-induced human cells returns to healthy levels when treated with CNNM4 shRNA. *p<0.05 vs Healthy **p<0.01 vs Healthy; #p<0.05 vs NASH control; ##p<0.01 vs NASH control.

[0582] FIG. 5A. The lipid content in NASH-induced primary hepatocytes does not return to healthy levels when treated with siRNAs against CNNM1, CNNM2 or CNNM3.

[0583] FIG. 5B. Magnesium supplementation does not reduce lipid content in primary hepatocytes when CNNM4 is overexpressed.

[0584] FIG. 5C. CNNM4 siRNA treatment of primary hepatocytes reduces lipid accumulation caused by magnesium deficiency. ***p<0.001 vs Healthy; ##p<0.01 vs NASH model/Without Mg2+ + siRNA Ø.

[0585] FIG. 6. Parameters analysed for observing NAFLD progression after CNNM4 siRNA therapy. A) Sudan Red as indicator of lipid content decrease, B) GPT in serum as indicator of liver damage, C) DHE as indicator of inflammation by ROS and D) αSMA as indicator of fibrosis. *p<0.05 vs siRNA Ø; **p<0.01 vs siRNA Ø.

[0586] FIG. 7. Pharmacological inhibition of CNNM4 by 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one. Treated hepatocytes have A) reduced lipid levels and B) an increase of intracellular magnesium levels. *p<0.05 vs Untreated; ***p<0.001 vs Untreated.

[0587] FIG. 8. CNNM4 expression determined by IHC in animal samples of renal fibrosis. ***p<0.001 vs Healthy.

[0588] FIG. 9. A) CNNM4 expression determined in TCGA (The Cancer Genome Atlas) primary tumour samples of Liver Hepatocellular Carcinoma (HHC) compared to normal tissue. B) CNNM4 expression determined in TCGA (The Cancer Genome Atlas) primary tumour samples of lung adenocarcinoma (LUAD) compared to normal tissue.

[0589] FIG. 10. Possible synthesis route to DMT-Serinol(GaINAc)-CEP and CPG.

[0590] FIG. 11. Reduction of human CNNM4 mRNA level in human HepG2 cells by transfection of CNNM4 siRNAs

[0591] FIG. 12. Reduction of CNNM4 mRNA level in murine Hepa 1-6 cells by transfection of CNNM4 siRNAs.

[0592] FIG. 13. Dose-dependent reduction of CNNM4 mRNA level in murine Hepa 1-6 cells by transfection of CNNM4 siRNAs

[0593] FIG. 14. Inhibition of human CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates

[0594] FIG. 15. Inhibition of mouse CNNM4 gene expression in primary mouse hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

[0595] FIG. 16. Inhibition of human CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

[0596] FIG. 17. Inhibition of CNNM4 gene expression in primary murine hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

[0597] FIG. 18. Dose-dependent inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

[0598] FIG. 19. Dose-dependent inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

[0599] FIG. 20. Long-lasting inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

[0600] FIG. 21. Inhibition of CNNM4 expression in rodent a NASH model treated with CNNM4 siRNA conjugates.

[0601] FIG. 22. Treatment with CNNM4 siRNA conjugates reduces lipid accumulation in hepatocytes.

[0602] FIG. 23. Treatment with CNNM4 siRNA conjugates reduces mitochondrial reactive oxygen species (ROS) production in hepatocytes.

[0603] FIG. 24. Treatment of rodent NASH model with CNNM4 siRNA conjugates reduces development of NASH.

[0604] FIG. 25. CNNM4 siRNA conjugates protect hepatocytes from apoptosis and cell death induced by acetaminophen (APAP).

[0605] FIG. 26. Reduction of human CNNM4 mRNA level in human Huh-7 cells by transfection of CNNM4 siRNAs.

[0606] FIG. 27. Dose-dependent reduction of CNNM4 mRNA level in human Huh-7 cells by transfection of CNNM4 siRNAs.

[0607] FIG. 28. Inhibition of CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake.

[0608] FIG. 29. Inhibition of CNNM4 gene expression in primary cynomolgus hepatocytes by receptor mediated uptake.

[0609] FIG. 30. Inhibition of CNNM4 gene expression in rodent model for NASH

EXAMPLES

Example 1

Materials and Methods

Human Samples

[0610] All the studies were performed in agreement with the Declaration of Helsinki and according to local national laws. The Human Ethics Committee of each hospital approved the study procedures and written informed consent was obtained from all patients before inclusion in the study.

[0611] Magnesium was quantified from human serum samples from a cohort of 8 healthy samples, 31 obese-diagnosed patients and 43 from a cohort of clinical trials. Patients were evaluated for non-alcoholic fatty liver disease (NAFLD) by different markers once discarded alcoholic disease and viral hepatitis infection.

[0612] Human CNNM4 expression in non-alcoholic fatty liver disease NAFLD was determined in a cohort of 42 patients: 10 healthy patients, 20 patients diagnosed with steatosis and 12 diagnosed with NAFLD. The CNNM4 levels in cirrhotic patients were determined in a cohort of 12 patients from which 5 were diagnosed as healthy and 7 as cirrhotic. 47 hepatocellular carcinoma (HCC) patients’ CNNM4 levels were also determined: 6 patients were healthy and 41 patients were diagnosed HCC. CNNM4 levels were determined in a cohort of 11 drug-induced liver injury (DILI) patients and compared to 3 healthy patients. Finally, renal fibrosis 14 human samples were analysed to determine CNNM4 expression. 7 samples were healthy and another 7 have been diagnoses with renal fibrosis.

Animal Experiments

[0613] All the animal experiments were conducted in accordance with the Spanish Guide for Care and use of Laboratory animals, and with the International Care and Use Committee Standards. All procedures were approved by the CIC bioGUNE’s Animal Care and Use Committee and the competent authority (Diputación de Bizkaia). Mice were housed in a temperature-controlled animal facility (AAALAC-accredited) within 12-hour light/dark cycles. G-They were fed a standard diet (Harlan Tekland) with water ad libitum.

[0614] NAFLD animal model: 0.1% methionine and choline-deficient diet (0.1% MCDD) for CNNM4 determination C57BL/6J wild-type mice were fed with a methionine (0.1%) and choline (0%) deficient diet for 4 weeks. At the end of the treatment animals were sacrificed and liver were split into several pieces for subsequent analysis including: RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. Blood for serum analysis was collected once a week during the treatment.

Pre-clinical Study: NAFLD Animal Model With siRNA Therapy

[0615] C57BL/6J wild-type mice were fed with a methionine (0.1%) and choline (0%) deficient diet for 4 weeks. 2 weeks after the beginning of the diet mice were divided in two groups and subjected to an in vivo silencing CNNM4 or unrelated siRNA control, receiving either 200 .Math.ll of a 0.75 .Math.g/.Math.l solution of or CNNM4-specific in vivo siRNA (Custom Ambion, USA) or control siRNA (Sigma-Aldrich, USA) using Invivofectamine ®3.0 Reagent (Invitrogen, USA) following the manufacturer’s instructions. Tail vein injection was performed twice a week until the fourth week. At the end of the treatment animals were sacrificed and liver were split into several pieces for subsequent analysis including: RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. Blood for serum analysis was collected once a week during the treatment.

Cirrhosis Animal Model: Bile Duct Ligation (BDL)

[0616] Adult C57BL/6J wilt-type mice were subjected to BDL as described previously (Fernandez-Alvarez et al., 2015. Lab Invest. 95(2):223-36). Briefly, mice were anesthetized with 1.5% isofluorane in O2 and the abdomen was opened. The bile duct was separated from the portal vein and the hepatic artery, performing a suture around the bile duct and securing with a surgical know. Finally, the abdomen was closed and mice sacrificed at 24 h, 48 h, 72 h, 3 days and 21 days. Liver were split into several pieces for subsequent analysis including: RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. Blood for serum analysis was collected once a week during the treatment.

Hepatocellular Carcinoma Animal Model (HCC): GNMT-/- Mice

[0617] Adult GNMT-/- mice were grown from 7 to 9 months, when they are described to develop spontaneously HCC (Wagner et al., 2009. Toxicol Appl Pharmacol. 1;237(2):246; author reply 247). At that time animals were sacrificed and liver were split into several pieces for subsequent analysis including: RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. Blood for serum analysis was collected once a week during the treatment.

Drug Induced Liver Injury (DILI): Acetaminophen (APAP) Overdose

[0618] Adult C57BL/6J wilt-type mice were treated with 500 mg/kg of acetaminophen (APAP) to induce acute liver injury. After 48 h of treatment, mice were sacrificed and liver were split into several pieces for subsequent analysis including: RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. Blood for serum analysis was collected once a week during the treatment.

Isolation of Primary Hepatocytes, Culture and Treatments

[0619] Primary hepatocytes from 3-month old wild-type (C57BL/6J) mice were isolated by perfusion with collagenase Type I (Worthington, USA). Briefly, animals were anesthetized with isoflurane (1.5% isoflurane in O2). Then, the abdomen was opened and a catheter was inserted into the inferior vena cava. Liver was perfused with buffer A (1x PBS, 5 mM EGTA, 37° C. and oxygenated) and the portal vein was cut. Next, liver was perfused with buffer B (1x PBS, 1 mM CaCl2 37° C. and oxygenated) to remove EGTA, and finally perfused with buffer C (1x PBS, 2 mM CaCl2, 0.65 BSA, collagenase type I, 37° C. and oxygenated). After buffer C perfusion, liver was separated from the resto of the body and placed into a petri dish with MEM (Gibco, USA). Gall bladder was carefully removed and, then, liver was mechanically disaggregated with forceps. The digested liver diluted in MEM was filtered through a sterile gauze and filtered liver cells were collected and washed three times (1x4′ at 400 RPM and 2x5′ at 500 RPM) in 10% FBS (Gibco)/1% PSG (Gibco) supplemented MEM, conserving all supernadant Kupffer and Hepatic Stellate cells isolation. After the final wash, hepatocytes contained in the pellet were resuspended in 10% FBS 1% PSG MEM for subsequently culturing.

[0620] Primary hepatocytes were seeded over previously collagen-coated culture dishes at a density of 7600 cells/mm2 in 10% FBS/1% PSG supplemented MEM and placed in an incubator at 37° C., 5%CO2-95% air. After 6 hours of attachment, culture medium and unattached hepatocytes were removed with fresh 0% FBS/1% PSG MEM for the aimed treatment.

THLE2 Cells

[0621] THLE-2 cells were purchased from ATCC (ATCC® CRL-2706TM). They were maintained on Bronchial Epithelial Growth Medium (BEGMTM, Lonza) supplemented with BEGM Bullet KitTM (Lonza) and 10%FBS. They were split with 0.05% trypsin-EDTA and collected in BEGM. After centrifugation at 123 g during 5 minutes, supernatant was discarded and pellet resuspended.

Plasmid Transfection

[0622] Plasmids were transfected into primary mouse hepatocytes for overexpression using jetPRIME® (Polyplus, USA) transfection reagent following manufacturer’s protocol. In a 24-well plate, 0.5 .Math.g of plasmid were added to the jePRIME® buffer and vortexed 10s before adding 1 .Math.ljetPRIME® reagent. The mix was vortexed 10 s, spun down and incubated 10′ at RT). Transfections were performed in cell suspension medium and transfection mix was replaced for fresh medium 6 h after transfection unless indicated.

Gene Silencing by siRNA Delivery

[0623] Cells were transfected with specific siRNAs (Hs n7o for human CNNM4 and Mm n7o for mouse CNNM4) at a final concentration of 100 nM using DharmaFECT 1 reagent (Dharmacon) following manufacturer’s protocol. DharmaFECT 1 and siRNA were diluted separately in 0% FBS/1% PSG MEM for 5′ at RT. Dilutions were then mixed and incubated 20′ at RT. siRNA transfection mixes were then added to cell suspension medium and replaced for fresh medium after 6 h.

Gene Silencing by shRNA Delivery

[0624] Cells were transfected with a CNNM4 shRNA (SEQ ID NO: 454) by using lipofectamine® 3000 (Thermofisher) and following the protocol according manufacturer instructions. 7.5.Math.l lipofectamine and the shRNA were diluted separately in 0.2ml culture medium and incubated during 5′ at room temperature. After incubation they were mixed again and incubated for 30′ at room temperature before delivering to the cells.

RNA Isolation

[0625] Total RNA from whole liver or cultured cells was isolated using TRIzol reagent (Invitrogen) according to manufacturer’s instruction. In case of cell mRNA extraction, 5 .Math.g of Glycogen (Ambion, USA) were used in the RNA precipitation step to facilitate the visibility of the RNA pellet. RNA concentration was determined spectrophometrically using the Nanodrop ND-100 spectrophotometer (ThermoFisher Scientific, USA).

Retrotranscription

[0626] 1-2 .Math.g of isolated RNA were treated with DNAse I (Invitrogren) and used to synthesize cDNA by M-MLV reverse transcriptase in the presence of random primers and RNAseOUT (all from Invitrogen). Resulting cDNA was diluted ⅒ (1/20 if 2 .Math.g were used) in RNAse free water (Sigma-Aldrich).

Real Time Quantitative PCR (RT-qPCR)

[0627] qPCRs were performed using either the ViiA 7 or the QS6 Real time PCR System with SYBR Select Master Mix (Applied Biosystems, USA). 1.5 .Math.l of cDNA were used and including the specific primers for a total reaction volume of 6.5 .Math.l in a 384-well plate (Applied Biosystems). All reactions were performed in triplicate. PCR conditions for the primers were optimized and 40 cycles with a melting temperature of 60° C. and 30 s per step were used. Both Homo Sapiens and Mus musculus primers were designed using the Primer 3 software via the NCBI-Nucleotide webpage (www.ncbi.nlm.nih.gov/nucleotide) and synthesized by Sigma Aldrich. After checking the specificity of the PCR products with the melting curve, data were normalized to the expression of a housekeeping gene (GAPDH, ARP).

Protein Extraction and Analysis

[0628] Extraction of total protein was performed as indicated. Cells were washed with cold PBS buffer and resuspended in 200 .Math.l of RIPA lysis buffer (1.6 mM NaH2PO4, 8.4 mM Na2HPO4, 0.5% Azide, 0.1 M NaCl, 0.1% SDS, 0.1% Triton X-100, 5 mg/ml sodium deoxycholate). The lysis buffer was supplemented with protease and phosphatase inhibitor cocktails (Roche, Switzerland). They were centrifuged (13000 rpm, 20′ at 4° C.) and the supernatand (protein extract) was quantified for total protein content by the Bradford protein assay (Bio-Rad) and determined using a Spectramax M3 spectrophotometer (Molecular Devices, USA). In the case of frozen liver tissue, approximately 50 .Math.g of tissue was homogenized by using a Precellys 24 tissue homogenizer (Precellys, France) in 500 .Math.l of buffer. In all cases, the lysates were centrifuged (13000 rpm, 20 min, 4° C.) and the supernatant (protein extract) was quantified for total protein content by the Bradford protein assay or by BCA protein assay (Pierce, USA) depending on the type of lysis buffer used and determined using a Spectramax M3 spectrophotometer.

Western Blotting

[0629] Protein extracts were boiled at 95° C. for 5 min in SDS-PAGE sample buffer (250 mM Tris-HCl pH 6.8, 500 mM β-mercaptoethanol, 50% glycerol, 10% SDS and bromophenol blue). An appropriate amount of protein (between 5 and 50 .Math.g), depending on protein abundance and antibody sensitivity, were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in 3% to 15% acrylamide gels (depending on the molecular weight of the protein of interest), using a Mini-PROTEAN Electrophoresis System (Bio-Rad). Gels were transferred onto nitrocellulose membranes by electroblotting using a Mini Trans-Blot cell (Bio-Rad). Membranes were blocked with 5% nonfat milk in TBS pH 8 containing 0.1% Tween-20 (Sigma Aldrich) (TBST-0.1%), for 1 hour at RT, washed three times during 10′ with TBST-0.1% and incubated overnight at 4° C. with commercial primary antibodies. Primary antibodies and their optimal incubation conditions are detailed in Table 6. Membranes were then washed three times during 10′ with TBST-0.1% and incubated for 1 hour at RT in blocking solution containing secondary antibody conjugated to horseradish-peroxidase (HRP, Table 6). Immunoreactive proteins were detected by using Western Lightning Enhanced Chemiluminescence reagent (ECL, PerkinElmer, USA) and exposed to Super Rx-N X-ray films (Fuji, Japan) in a Curix 60 Developer (AGFA, Belgium).

Sudan Red for Lipid Staining

[0630] O.C.T-included frozen liver samples were cut into 10 .Math.m sections. Sections were washed in 60% isopropanol and then stained with fresh Sudan III (0.5% in isopropanol; Sigma Aldrich) solution for 30 min. Samples were then washed again in 60% isopropanol and then counterstained with hematoxylin and eosin. The sections were then washed with distilled water and mounted in DPX mounting medium. Images were taken with an upright light microscope (Zeiss).

ROS Determination by DHE

[0631] O.C.T-embedded 8 .Math.m sections were incubated with MnTBAP 150 .Math.M 1h at RT. The samples were then incubated with dihydroethidium (DHE) 5 .Math.M for 30 min at 37° C. and sections were mounted with Fluoromount-G (Southern Biotech, USA) containing 0.7 mg/l of DAPI to counterstain nuclei. Images were taken using an Axioimager D1 (Zeiss).

Immunohistochemistry for CNNM4 Determination

[0632] Paraffin-embedded sections (5 .Math.m thick) were unmasked according to the primary antibody to be used and subjected to peroxide blocking (3% H2O2 in PBS, 10′, RT). For stainings with mouse-hosted antibodies in mouse tissues, samples were blocked with goat anti-mouse Fab fragment (Jackson Immunoresearch, USA) (1:10, 1 h, RT) and the blocked with 5% goat serum (30′, RT). Then, sections were incubated in a humid chamber with the CNNM4 primary antibody (Ab191207, Abcam) in DAKO antibody diluent (DAKO) at 1:100 followed by Envision anti rabbit (DAKO) HRP-conjugated secondary antibody incubation (30′, RT). Colorimetric detection was confirmed with Vector VIP chromogen (Vector) and sections were counterstained with hematoxylin. Samples were mounted using DPX mounting medium. Images were taken with an upright light microscope (Zeiss).

Immunofluorescence for αSMA Determination

[0633] For α-SMA staining, O.C.T-embedded 10 .Math.m sections were incubated with a 1/200 dilution in 2%BSA in 0.01%PBS-azide of the primary antibody conjugated to Cy3 (C6198, Sigma Aldrich) and mounted with Fluoromount-G (Southern Biotech) containing 0.7 mg/l of DAPI to counterstain nuclei. Images were taken using an Axioimager D1 (Zeiss).

BODIPY for Lipid Quantification in Primary Hepatocytes

[0634] Primary hepatocytes cultured in high lipid content medium (OA) or methionine/choline deficient medium (MDMC) were fixed in 4% paraformaldehyde (10′, RT) in PBS and incubated with BODIPY 493/503 (Molecular Probes, Invitrogen) at 1 mg/ml (1h, RT). BODIPY immunocytofluorescence images were taken using an Axioimager D1 (Zeiss) microscope. Quantification of lipid bodies was performed using Frida Software and represented as mean area per total number of cells.

Data Analysis

[0635] The average sum of intensities or stained area percentage of each sample were calculated using the FRIDA software (http://bui3.win.ad.jhu.edu/frida/, John Hopkins University).

Liver Lipid Quantification

[0636] 30 mg of frozen liver were homogenized with 10 volumes of ice-cold PBS in a potter homogenizer. Fatty acids were measured in the homogenates using the Wako Chemicals kit (Richmond, VA) and lipids were quantified as described (Folch et al., 1957. J Biol Chem. 226(1):497-509). Briefly, lipids were extracted from 1.5 mg of protein from liver homogenates. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), fatty acids (FAs) and cholesterol (Ch) were separated by thin layer chromatography (TLC) and quantified as described (Ruiz and Ochoa, 1997). Triglycerides (TGs) were measured in the lipid extract with the A. Menarini Diagnostics (Italy) kit.

Intracellular Magnesium Levels

[0637] Primary hepatocytes grown in glass coverslips were loaded with 2 .Math.M Mag-S-Tz or 1 .Math.M Mag-S-Tz-AM diluted REF (Gruskos et al., 2016. J. Am. Chem. Soc. 138 (44), pp 14639-14649) in 0% FBS/1% PSG medium and incubated at 37° C. and 5% CO2 during 30′ or 1 h respectively. After removing the dye-containing medium, a 30′ incubation in 0% FBS/1% PSG was performed. Coverslips were then washed in a 20 mM Tris-HCl, 2.4 mM CaCl2, 10 mM glucose, pH 7.4 buffer and mounted on a perfusion chamber with thermostat on a Eclipse TE 300-based microspectrofluorometer (Nikon, USA) and visualized with a 40x oil-immersion fluorescence. Intracellular Mg2+ levels were determines using the method described by Grynkiewicz (Grynkiewiz et al., 1985. J Biol Chem. 260(6):3440-50). The 340/380 nm excited light ratio was determined with a Delta system (Photon Technologies International, Princeton) and converted into Mg.sup.2+ concentration from the standard equation:

[00001]$\left[M{g}^{2+}\right]i=\frac{\left(R-R_{\min }\right)}{\left(R_{max}-R\right)}\times K_d\times Q$

Where Kd is the Mg.sup.2+ dissociation constant of Mag-S-Tz (3.2 mM) and Mag-S-Tz-AM (8.9 mM) and Q is the ratio of the minimum/maximum fluorescence intensity at 380 nm.

Extracellular Magnesium Levels

[0638] Extracellular magnesium was quantified using the QuantiCromTM Magnesium Assay Kit (BioAssay Systems, USA). Briefly, 5 .Math.l of serum or culture media were mixed with 200 .Math.l of a 1:1 mix of Reagent A and Reagent B. After 2′ incubation at RT, OD was determined at 500 nm length using a Spectramax M3 spectrophotometer (Molecular Devices, USA). Then, 10 .Math.l of EDTA were added and OD500 was determined again. Magnesium concentrations were finally calculated by comparing to the OD500 from a standard concentration (2 mg/ml).

Mitochondrial ROS Determination

[0639] Mitochondrial ROS was measured using MitoSOXTM Red reagent (Life Technologies), following manufacturer’s instructions. Briefly, primary hepatocytes and hepatoma cells were incubated with MitoSOX reagent (2.5 .Math.M, 10′, 37° C. in a CO2 incubator) in normal culture medium. Then, cells were washed twice with PBS and the fluorescence was measured at an excitation of 510 nm and emission of 595 nm using a spectrophotometer. Final values were normalized to total protein concentration.

Cell Death Determination by TUNEL

[0640] Cell Death was analysed by using the In situ Cell Death detection Kit (Roche) following the manufacturer’s instructions as above indicated. Cells were subjected peroxide block (3% H2O2 in methanol) for 3 minutes before incubation with TUNEL diluent buffer containing FITC-conjugated primary antibody (dilution 1/50) for 1 hour at 37° C. Sections were mounted in Dako fluorescence mounting medium (Dako). Images were taken using an Axioimager D1 (Zeiss) microscope and cell viability was calculated by determining the % of TUNEL positive cells.

Results

CNNM4 Overexpression in Liver Pathologies

[0641] Chronic liver disease includes a group of different pathologies. A method to detect CNNM4 expression by immunohistochemistry (IHC) in human liver biopsies and livers from mouse animal models has been developed. Herein CNNM4 expression has been characterized in DILI and all the stages from chronic liver disease, both in human biopsies and animal models, observing an overexpression of the protein in all the pathologies (FIG. 1). These results were confirmed by a method to detect CNNM4 expression by qPCR of CNNM4 mRNA levels in human liver samples from healthy subjects compared with samples from steatotic and NASH patients (FIG. 2A). CNNM4 expression could also be determined by qPCR of CNNM4 mRNA levels in an in vivo NASH mouse model (FIG. 2B), as well as in an in vitro NASH mouse cell model (FIG. 2C).

CNNM4 a New Target for Treating Liver Disease

[0642] The overexpression observed in CNNM4 determination by IHC suggests CNNM4 as a potential target for treating liver disease, both for DILI and chronic disease. An in vitro study was performed inducing NASH in primary hepatocytes and treating them with a siRNA CNNM4 therapy. In case of NASH model-primary hepatocytes the lipid content and inflammation by reactive oxygen species (ROS) were measured, observing that both get restored in NASH hepatocytes treated with the siRNA therapy (FIGS. 3A and 3B). A further in vitro study was performed on DILI induced by acetaminophen overdose. The expected cell death in the DILI model-hepatocytes was observed, as well as restoration upon siRNA treatment (FIG. 3C). A further in vitro study was performed in which NASH was induced in human cells and these cells were then treated with a siRNA CNNM4 therapy or with a shRNA CNNM4 therapy. Relative lipid accumulation restoration was observed both in NASH-induced human cells (THLE2 cells) treated with the siRNA therapy (FIG. 4A) and in NASH-induced human cells treated with the shRNA therapy (FIG. 4B).

Specificity and Need of Targeting CNNM4

[0643] Having observed the protective effect of siRNA CNNM4-therapies from NASH and DILI, the effect of targeted silencing the other proteins of CNNM family (CNNM1, CNNM2 and CNNM3) was determined. NASH was induced in primary hepatocytes and they were treated with siRNAs against CNNM1, CNNM2 and CNNM3. Differently than with siRNA CNNM4 therapy, silencing the other proteins of the CNNM family had no effect, indicating the specificity of a possible the treatment based only on CNNM4 and not on other CNNM family proteins (FIG. 5A). Furthermore, experiments were conducted which prove the need of a CNNM4-based treatment. In a first experiment, lipid accumulation was induced in primary hepatocytes by CNNM4 overexpression to mimic the situation observed in NASH patients and animal models. Magnesium supplementation had no effect on the level of lipid accumulation in those hepatocytes (FIG. 5B). In a second experiment, lipid accumulation was induced by magnesium deprivation in primary hepatocytes, which is leads to a similar physiological condition as CNNM4 overexpression. In this case, siRNA CNNM4 therapy reduced lipid accumulation (FIG. 5C). These last two results indicate that magnesium supplementation is not sufficient for treating NASH and that there is a need for a CNNM4-based therapy.

Preclinical Study of a siRNA CNNM4-based Therapy

[0644] A preclinical study in NAFLD, the first stage of chronic liver disease, was developed to test the efficacy of CNNM4 modulation not only in cells but also in animals. Mice were fed a 0.1% methionine and choline-deficient diet (0.1%MCDD) during two weeks in order for them to develop NAFLD. A subgroup was then treated with a siRNA CNNM4 therapy and another with a siCtrl RNA. Animals were sacrificed and different biomarkers were measured in order to analyse NAFLD progression. Sudan red staining was used to measure lipid accumulation (FIG. 6A). Transaminase levels in serum, which are indicative of liver damage, were also measured (FIG. 6B). DHE staining was used to quantify inflammation caused by ROS (FIG. 6C) and α-smooth-muscle actin (αSMA) levels were measured to indicate the progression of fibrosis (FIG. 6D). These results clearly indicate that siRNA CNNM4 therapy reduces NAFLD.

Pharmacological Inhibition of CNNM4

[0645] In addition to siRNA therapy, CNNM4 activity can be also modulated pharmacologically. An in vitro assay was performed inducing NASH in primary hepatocytes and treating them with the compound 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one. It can be observed that this pharmacological inhibition of CNNM4 has the same effect as siRNA therapy in NASH-induced hepatocytes, it decreases its lipid content (FIG. 7A) and leads them to accumulate magnesium (FIG. 7B). It is therefore expected that a combination therapy of a CNNM4 siRNA and 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one or one of its derivatives with similar function could lead to a potent treatment of NASH.

CNNM4 Over-Expression in Pathologies of Different Organs

[0646] As shown in FIG. 1, CNNM4 is overexpressed in different liver pathologies, both in animal models and in human samples. However, fibrosis development occurs not only in liver but also in other organs such as the kidneys. There may be similarities in the development of fibrosis in both tissues and CNNM4 may also be deregulated in this organ. This is indeed the case. We observed a CNNM4 overexpression in a renal fibrosis mouse model, indicating that a therapy that reduces CNNM4 expression may also be effective in the treatment of kidney fibrosis (FIG. 8). In addition, an analysis of TCGA (The Cancer Genome Atlas) data show that CNNM4 is overexpressed in primary tumour samples of Liver Hepatocellular Carcinoma (HHC) and in primary tumour samples of lung adenocarcinoma (LUAD) compared to normal tissue (FIGS. 9A and 9B). Overexpression of CNNM4 has also been detected in cholangiocarcinoma (bile duct cancer). restore Mg.sup.2+ homeostasis

[0647] In summary, the presented results prove that CNNM4 is a suitable target for preventing NAFLD progression and indicates that inhibiting it could also ameliorate other liver pathologies (DILI, cirrhosis and HCC), renal fibrosis and lung cancer. CNNM4 is overexpressed in all these diseases. In addition, for DILI, the in vitro studies show a protective effect of siRNA therapy from APAP overdose. Inhibiting or silencing CNNM4, especially by siRNA therapy, is a suitable method for treating liver disease, renal fibrosis and lung cancer.

Example 2 - Synthesis of Building Blocks

[0648] The synthesis route for DMT-Serinol(GaINAc)-CEP and CPG as described below is outlined in FIG. 10. 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.

DMT-Serinol(GalNAc) (3)

[0649] 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 (2x) 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 (2x) to yield to yield 10.6 g (51%) of DMT-Serinol(GalNAc) (3) as an off-white foam.

DMT-Serinol(GaINAc)-CEP (5)

[0650] 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 (2x) to yield 13.5 g (77%) of the colorless DMT-Serinol(GaINAc)-CEP (5) foam.

DMT-Serinol(GalNAc)-succinate (6)

[0651] 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 (3x) to yield 5.9 g (70%) DMT-Serinol(GalNAc)-succinate (6).

DMT-Serinol(GalNAc)-succinyl-lcaa-CPG (7)

[0652] 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-lcaa-CPG (500 A, 88 .Math.mol/g, 1 eq.). The suspension was gently shaken at room temperature on a wrist-action shaker for 16 h, then filtered and washed with acetonitrile. The solid support was dried under reduced pressure for 2 h. The unreacted amines on the support were capped by stirring with Ac.sub.2O/2,6-lutidine/NMl at room temperature (2x15 min). The washing of the support was repeated as above. The solid was dried under vacuum to yield DMT-Serinol(GalNAc)-succinyl-lcaa-CPG (7) (loading: 34 .Math.mol/g, determined by detritylation assay).

Example 3 - Oligonucleotide Synthesis

[0653] 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.

[0654] Downstream cleavage, deprotection and purification followed standard procedures that are known in the art.

[0655] 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 2.

[0656] 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 (0.3 M 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: 0.05 M 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.

[0657] Attachment of the Serinol(GalNAc) moiety was achieved by use of either base-loaded (S)-DMT-Serinol(GalNAc)-succinyl-lcaa-CPG (7) or a (S)-DMT-Serinol(GalNAc)-CEP (5). Tri-antennary 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.

[0658] 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 .Math.L TMSBr and 177 .Math.L pyridine in CH.sub.2Cl.sub.2 (0.5 ml/.Math.mol solid support bound oligonucleotide) at room temperature and the reactor was sealed with a Luer cap. The reaction vessels were slightly agitated over a period of 2x15 min, the excess reagent discarded, and the residual CPG washed 2x with 10 ml acetonitrile. Further downstream processing did not alter from any other example compound.

[0659] 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 lyophilised until further use.

[0660] 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 4 - Double-Strand Formation

[0661] 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 5

[0662] Reduction of human CNNM4 mRNA level in human HepG2 cells by transfection of CNNM4 siRNAs.

[0663] In vitro test shows reduction of CNNM4 mRNA levels in human HepG2 cells by transfection of CNNM4 siRNA molecules.

[0664] HepG2 cells were seeded in 96 well plates at a density of 40 000 cells per well with 10 nM siRNA and 0.3 .Math.l RNAiFect added to the culture medium. The following day, cells were lysed for RNA extraction and CNNM4 and HPRT1 mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene HRPT1 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 FIGS. 11A and 11B.

Example 6

[0665] Reduction of CNNM4 mRNA level in murine Hepa 1-6 cells by transfection of CNNM4 siRNAs.

[0666] In vitro test shows reduction of CNNM4 mRNA levels in murine Hepa 1-6 cells by transfection of different CNNM4 siRNA molecules at a concentration of 10 nM.

[0667] Hepa 1-6 cells were seeded in 96 well plates at a density of 12 500 cells per well in the presence of 10 nM siRNA and 0.6 .Math.l RNAiFect added to the culture medium. The following day, cells were lysed for RNA extraction and CNNM4 and ApoB mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping 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 duplexes used in this study are listed in Table 2. Results are shown in FIGS. 12A and 12B.

Example 7

[0668] Dose-dependent reduction of CNNM4 mRNA level in murine Hepa 1-6 cells by transfection of CNNM4 siRNAs.

[0669] In vitro test shows reduction of CNNM4 siRNAs mRNA levels in murine Hepa 1-6 cells by transfection of different CNNM4 siRNA molecules in a dose range of 4 nM to 0.0001 nM.

[0670] Hepa 1-6 cells were seeded in 96 well plates at a density of 12 500 cells per well in the presence of 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.006 nM, 0.001 nM, 0.0003 nM or 0.0001 nM siRNA and 0.6 .Math.l RNAiFect added to the culture medium. The following day, cells were lysed for RNA extraction and CNNM4 and ApoB mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping 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 duplexes used in this study are listed in Table 2. Results are shown in FIGS. 13A to 13D.

Example 8

[0671] Inhibition of human CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

[0672] The example shows dose-dependent reduction of human CNNM4 mRNA levels by EU401 to EU414 in primary human hepatocytes.

[0673] 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 incubated with CNNM4 siRNA conjugates EU401 to EU414, at concentrations of 100 nM, 10 nM and 1 nM as shown in FIG. 14, or they were incubated with non-targeting control conjugates (Ctr) at 100 nM (Ctr is EU400). The following day, cells were lysed for RNA extraction and CNNM4 and HPRT1 mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene HRPT1 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 FIG. 14.

Example 9

[0674] Inhibition of mouse CNNM4 gene expression in primary mouse hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

[0675] The example shows dose-dependent reduction of mouse CNNM4 mRNA levels by EU401 to 408 and by EU410 to EU414 in primary mouse hepatocytes.

[0676] Primary mouse hepatocytes were seeded in a 96 well plate at a density of 25 000 cells per well in plating medium and incubated with CNNM4 siRNA conjugates EU401 to 408 and EU410 to EU414, at concentrations of 100 nM, 10 nM and 1 nM as shown in FIG. 15, or they were incubated with non-targeting control conjugates (Ctr) at 100 nM (Ctr is EU400). The following day, cells were lysed for RNA extraction and CNNM4 and ApoB mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping 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 FIG. 15.

Example 10

[0677] Inhibition of human CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

[0678] The example shows dose-dependent reduction of human CNNM4 mRNA levels by EU415 to EU422 in primary human hepatocytes.

[0679] 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 incubated with CNNM4 siRNA conjugates EU415 to EU422 in concentrations of 100 nM, 10 nM and 1 nM as shown in FIG. 16, or they were incubated with a non-targeting control conjugates (Ctr) at 100 nM (Ctr is EU423). The following day, cells were lysed for RNA extraction and CNNM4 and HPRT1 mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene HRPT1 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 FIG. 16.

Example 11

[0680] Inhibition of CNNM4 gene expression in primary murine hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

[0681] The example shows dose-dependent reduction of mouse CNNM4 mRNA levels by EU415 to EU424 in primary mouse hepatocytes.

[0682] Primary mouse hepatocytes were seeded in a 96-well plate at a density of 25 000 cells per well in plating medium and incubated with CNNM4 siRNA conjugates EU415 to EU422, in concentrations of 100 nM, 10 nM and 1 nM as shown in FIG. 17, or they were incubated with non-targeting control conjugates (Ctr) at 100 nM (Ctr is EU423). The following day, cells were lysed for RNA extraction and CNNM4 and ApoB mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping 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 FIG. 17.

Example 12

[0683] Dose-dependent inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

[0684] The example shows reduction of CNNM4 mRNA levels in the liver of wild-type mice two weeks after single dosing of EU403, EU404, EU408, EU412 and EU114 by subcutaneous injection.

[0685] Five- to seven-week old male C57BL/6 mice were treated with a single dose of 1 or 5 mg siRNA conjugate per kg body weight by subcutaneous injection. Control groups received a subcutaneous injection with the vehicle PBS. Two weeks after the treatment, liver samples were collected from all mice and snap frozen. RNA was extracted from liver samples and CNNM4 and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene Actin and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean value from 6 animals +/-SD.

[0686] siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 mRNA in mouse liver after treatment with siRNA conjugates is shown in FIG. 18.

Example 13

[0687] Dose-dependent inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

[0688] The example shows reduction of CNNM4 mRNA levels in the liver of wild-type mice two weeks after single dosing of EU418, EU420 and EU422 by subcutaneous injection.

[0689] Five- to seven-week old male C57BL/6 mice were treated with a single dose of 0.3 or 1 mg siRNA conjugate per kg body weight by subcutaneous injection. Control groups received a subcutaneous injection with the vehicle PBS. Two weeks after the treatment, liver samples were collected from all mice and snap frozen. RNA was extracted from liver samples and CNNM4 and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene Actin and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean value from 6 animals +/-SD.

[0690] siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 mRNA in mouse liver after treatment with siRNA conjugates is shown in FIG. 19.

Example 14

[0691] Long-lasting inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

[0692] The example shows reduction of CNNM4 mRNA levels in the liver of wild-type mice five weeks after a single dosing of EU418, EU420 and EU422 by subcutaneous injection.

[0693] Five- to seven-week old male C57BL/6 mice were treated with a single dose of 1 mg siRNA conjugate per kg body weight by subcutaneous injection. Control groups received a subcutaneous injection with the vehicle PBS. Five weeks after the treatment, liver samples were collected from all mice and snap frozen. RNA was extracted from liver samples and CNNM4 and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene Actin and related to mean of PBS treated cohort (PBS) set at 1-fold target gene expression. Each bar represents mean value from 6 animals +/-SD.

[0694] siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 mRNA in mouse liver after treatment with siRNA conjugates is shown in FIG. 20.

Example 15

[0695] Inhibition of CNNM4 expression in rodent a NASH model treated with CNNM4 siRNA conjugates.

[0696] The example shows reduction of CNNM4 mRNA levels in mice with NASH after treatment with a CNNM4 siRNA conjugate. The NASH phenotype was induced by feeding the mice with a diet devoid of choline and with 0.1% methionine for six weeks.

[0697] Three-month old male C57BL/6 mice were maintained on a diet deficient in choline with 0.1% methionine (0.1%MCDD) (A02082006i, Research Diets, Inc., New Jersey, USA) for six weeks. After three weeks of 0.1% MCDD, mice were treated with 1 mg or 5 mg siRNA per kg body weight of CNNM4 siRNA conjugate (EU414). Control groups received 1 mg/kg non-targeting siRNA conjugate (EU400). Mice were then maintained on 0.1% MCDD for another three weeks and liver samples were subsequently collected from all mice and snap frozen. RNA was extracted from liver samples and CNNM4 and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene Actin and related to mean of EU400 treated cohort set at 1-fold target gene expression. Each bar represents mean value from 6-8 animals +/-SD.

[0698] siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 mRNA in the liver of murine NASH models three weeks after treatment with CNNM4 siRNA conjugate is shown in FIG. 21.

Example 16

[0699] Treatment with CNNM4 siRNA conjugates reduces lipid accumulation in hepatocytes.

[0700] The example shows that lipid accumulation in hepatocytes is induced by oleic acid supplementation or by culturing the cells in methionine and choline deficient medium. Lipid accumulation is attenuated by treatment with CNNM4 siRNA conjugates.

[0701] Freshly isolated murine hepatocytes were seeded on coated cover slides in multi-well plates. Upon attachment, cells were incubated for 6 hours with 10 or 100 nM of EU403, EU404, EU408, EU412 or EU414. On the following day, the untreated cells (ut) were maintained in control medium (MEM/Gibco) for another 24 hours. The treated cells were cultured in the presence of 400 .Math.M oleic acid (Sigma) or incubated with methionine- and choline- deficient DMEM/F12 medium (custom-made, Gibco) for 24 hours. Cells were then fixed in 4% paraformaldehyde solution. Lipid accumulation in hepatocytes was determined by staining of lipid bodies using boron-dipyromethene (BODIPY 493/503, Molecular Probes, Thermo Fisher Scientific). Images were acquired by fluorescence microscopy and BODIPY staining was quantified by ImageJ software.

[0702] Two independent experiments were carried out, each in quadruplicates. Values were normalized to mean values of untreated samples set at 1-fold. Each bar represents mean +/-SD. siRNA conjugates used in this study are listed in Table 2. The effect of CNNM4 siRNA treatment on lipid accumulation in methionine- and choline-deficient medium is shown in FIG. 22A. The effect of CNNM4 siRNA treatment on lipid accumulation induced by oleic acid is shown in FIG. 22B. Statistics: 2-way ANOVA with Dunnett’s post-hoc test against respective oleic acid and MCD control group on log-transformed values. * p≤ 0.05; **p ≤ 0.01;***p ≤ 001; ****p ≤ 0.0001. There are no significant inter-experimental differences.

Example 17

[0703] Treatment with CNNM4 siRNA conjugates reduces mitochondrial reactive oxygen species (ROS) production in hepatocytes.

[0704] The example shows that mitochondrial ROS induced by oleic acid supplementation or by culturing cells in methionine- and choline-deficient medium is reduced by treatment with CNNM4 siRNA molecules.

[0705] Freshly prepared murine hepatocytes were seeded in multi-well plates. Upon attachment, cells were incubated for 6 hours with 1 nM, 10 or 100 nM of EU404 or EU414. On the following day, the untreated cells (ut) were maintained in control medium (MEM/Gibco) for another 24 hours. The treated cells were cultured in the presence of 400 .Math.M oleic acid (Sigma) or incubated with methionine- and choline-deficient DMEM/F12 medium (custom-made, Gibco BRL) for 24 hours. Mitochondrial ROS production in hepatocytes was assessed using MitoSOX Red mitochondrial superoxide indicator (Invitrogen, USA). The cells were loaded with 2 .Math.M MitoSOX Red for 10 min at 37° C. in a CO.sub.2 incubator. The cells were then washed three times with PBS. Fluorescence was measured at 510 nm (excitation) and 595 (emission) using a plate reader SpectraMax M2 (bioNova, USA).

[0706] Two independent experiments were each carried out in quadruplicates. Values were normalized to mean values of untreated samples set at 1-fold. Each bar represents mean +/-SD. siRNA conjugates used in this study are listed in Table 2. The effect of CNNM4 siRNA treatment on mitochondrial ROS production in methionine- and choline-deficient medium is shown in FIG. 23A. The effect of CNNM4 siRNA treatment on ROS production induced by oleic acid is shown in FIG. 23B. Statistics: 2-way ANOVA with Dunnett’s post-hoc test against respective oleic acid and MCD control group on log-transformed values. * p ≤ 0.05; **p ≤ 0.01;***p ≤ 001; ***p ≤ 0.0001; # indicates significant inter-experimental differences (p≤0.0001).

Example 18

[0707] Treatment of rodent NASH model with CNNM4 siRNA conjugates reduces development of NASH.

[0708] The example shows that treatment with a CNNM4 siRNA conjugate reduces lipid accumulation, reactive oxygen species (ROS) and fibrosis in a rodent NASH model. The NASH phenotype was induced by feeding the mice with a diet devoid of choline and with 0.1% methionine for six weeks.

[0709] Three-month old male C57BL/6 mice were maintained on a diet deficient in choline with 0.1% methionine (0.1%MCDD) (A02082006i, Research Diets, Inc., New Jersey, USA) for six weeks or fed a standard chow (SC). After three weeks of 0.1% MCDD, mice were treated with 1 mg or 5 mg CNNM4 siRNA conjugate (EU414) per body weight. Control groups fed with 0.1% MCD received 1 mg/kg non-targeting siRNA conjugate (EU400). Control mice fed with normal chow (SC) received the vehicle PBS at the same time point (three weeks). Mice were subsequently maintained on 0.1% MCDD or standard chow (SC) for another three weeks as indicated in FIG. 24. Liver samples were collected from all mice at the end of the study and cryopreserved by embedding in optical coherence tomography cryocompound (OCT) or fixed with formalin and embedded in paraffin for preparation of sections. Lipid bodies and reactive oxygen species (ROS) were detected in cryosections by staining with Sudan red and Dihydroxyetidium (DHE), respectively. Liver fibrosis was assessed in paraffine sections by staining of the smooth muscle cell marker alpha smooth muscle actin (αSMA) by immunohistochemistry, as well as by detection of collagen fibers by Sirius red staining. Macrophages were detected by immunohistochemistry, by F4/80 staining of paraffin-embedded liver sections. Blood samples were collected from all animals at the six-week time point for serum preparation. Serum Mg.sup.2+ levels were then determined using the QuantiCrom™Magnesium Assay Kit (BioAssay Systems, USA).

[0710] siRNA conjugates used in this study are listed in Table 2. The reduction of liver steatosis by CNNM4 siRNA treatment in the 0.1% MCDD NASH model is shown in FIG. 24A. FIG. 24B shows reduction of reactive oxygen species by CNNM4 siRNA treatment. FIG. 24C shows the reduction of macrophage infiltration (F4/80 positive cells) in the liver by CNNM4 siRNA treatment. FIGS. 24D and 24E show the reduction in liver fibrosis by CNNM4 siRNA treatment in the same NASH model. These figures show quantifications of stained area percentages from each individual staining calculated using FIJI (https://imagej.net/Fiji.) in box and whisker plots with 5-95% percentile. n=5-7. (a.u. = arbitrary units). FIG. 24F shows reduction of serum Mg.sup.2+ levels in 0.1 %MCDD NASH model by treatment with EU414.

Example 19

[0711] CNNM4 siRNA conjugates protect hepatocytes from apoptosis and cell death induced by acetaminophen (APAP).

[0712] The example shows that inhibition of CNNM4 mRNA levels in primary hepatocytes with protects these cells from cell death induced by acetaminophen challenge.

[0713] Freshly prepared murine hepatocytes were seeded in multi-well plates. Upon attachment, cells were incubated for 6 hours with 1 nM and 10 nM of EU404 or EU414 or left untreated. Thereafter, cells were cultured in serum-free culture medium (MEM) for 13 to 14 hours. Subsequently, they remained untreated (Ut) or 10 mM acetaminophen was added to the medium (APAP). 6 hours later, cells were stained by Tunel assay or Trypan Blue staining to determine the proportion of cells that were necrotic (Tunel positive) or had undergone cell death (Trypan Blue positive).

[0714] siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 expression by EU414 and EU404 is shown in FIG. 25A. The reduction of Tunel positive cells (necrosis marker) and Tripan Blue positive cells (cell death marker) by CNNM4 siRNA treatment after exposure to Acetaminophen is shown in FIGS. 25A and 25B, respectively.

Example 20

[0715] Reduction of CNNM4 mRNA level in human Huh-7 cells by transfection of CNNM4 siRNAs.

[0716] In vitro test shows reduction of CNNM4 mRNA levels in human Huh-7 cells by transfection of CNNM4 siRNA molecules.

[0717] Huh7 cells were seeded in 96 well plates at a density of 20 000 cells per well and transfected with 0.5 nM siRNA and RNAiMAX at a final concentration of 1 .Math.l/ml culture medium. The following day, cells were lysed for RNA extraction and CNNM4 and HPRT1 mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the house keeping gene HRPT1 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 FIG. 26.

Example 21

[0718] Dose-dependent reduction of CNNM4 mRNA level in human Huh-7 cells by transfection of CNNM4 siRNAs.

[0719] In vitro test shows reduction of CNNM4 mRNA levels in human Huh-7 cells by transfection of different CNNM4 siRNA molecules in a dose range of 10 nM to 0.01 nM.

[0720] Huh-7 cells were seeded in the 96 well plate at a density of 20 000 cells per well and transfected with 10 nM, 1 nM, 0.1 nM, 0.01 nM siRNA and RNAiMAX at a final concentration of 1 .Math.l/ml culture medium. The following day cells were lysed for RNA extraction and CNNM4 and HPRT1 mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the house keeping gene HPRT1 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 FIGS. 27A and B.

Example 22

[0721] Inhibition of CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake.

[0722] The example shows dose-dependent reduction of human CNNM4 mRNA levels by EU424 to EU433 in primary human hepatocytes by receptor mediated uptake.

[0723] 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 CNNM4 siRNA conjugates EU424 to EU433 in concentrations of 100 nM, 10 nM, 1 nM, 0.1 nM or 0.01 nM or non-targeting control siRNA, EU423 (depicted as Ctr) as shown in FIG. 28. Values obtained for CNNM4 mRNA were normalized to values generated for the house keeping gene HPRT1 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 with EU424 to EU433 are shown in FIG. 28.

Example 23

[0724] Inhibition of CNNM4 gene expression in primary cynomolgus hepatocytes by receptor mediated uptake.

[0725] The example shows dose-dependent reduction of CNNM4 mRNA levels by EU429 to EU433 in primary cynomolgus hepatocytes by receptor mediated uptake.

[0726] Primary hepatocytes were seeded in a 96 well plate at a density of 45 000 cells per well in plating medium and were subsequently incubated with CNNM4 siRNA conjugates EU429 to EU433 in concentrations of 100 nM, 10 nM, 1 nM, 0.1 or 0.01 nM or 100 nM non-targeting control siRNA, EU423 (depicted as Ctr) as shown in FIG. 29. Values obtained for CNNM4 mRNA were normalized to values generated for the house keeping gene PPIB 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 with EU429 to 433 are shown in FIG. 29.

Example 24

[0727] Inhibition of CNNM4 gene expression in rodent model for NASH.

[0728] The example shows dose dependent reduction of CNNM4 mRNA levels by EU422 in a murine model for NASH.

[0729] 5-week old C57BL/6 male mice were maintained on a DIO-NASH diet for about 32 weeks and randomized based on pre-liver biopsy fibrosis and steatosis score. Only animals with fibrosis score of ≥ 1 and steatosis score of ≥ 2 were included in the study. Animals were then maintained on DIO-NASH diet for another 16 weeks. At the 4 week, 8 week, and 12 week time points after the pre liver biopsy mice were treated with 1 mg/kg or 5 mg/kg EU422, respectively or with the vehicle PBS by subcutaneous injection. 4 weeks after the last treatment, liver samples were collected from all mice and snap frozen. RNA was extracted from liver samples and CNNM4 and HPRT mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the house keeping gene HPRT and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean value from 15 animals +/-SD.

[0730] siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 mRNA in mouse liver after treatment with siRNA conjugates is shown in FIG. 30.

Summary Tables

[0731] TABLE-US-00002 Summary duplex table Duplex Single Strands Duplex Single Strands Duplex Single Strands EU200 EU200A EU259 EU259A EU414 EU414A EU200B EU259B EU414B EU201 EU201A EU260 EU260A Mm n1 Mm n1 A EU201B EU260B Mm n1 B EU202 EU202A EU261 EU261A Mn n2 Mm n2 A EU202B EU261B Mm n2 B EU203 EU203A EU262 EU262A Mm n3 Mm n3 A EU203B EU262B Mm n3 B EU204 EU204A EU263 EU263A Mm n4 Mm n4 A EU204B EU263B Mm n4 B EU205 EU205A EU264 EU264A Mm n5 Mm n5 A EU205B EU264B Mm n5 B EU206 EU206A EU265 EU265A Mm n6 Mm n6 A EU206B EU265B Mn n6 B EU207 EU207A EU266 EU266A Hs n1 Hs n1 A EU207B EU266B Hs n1 B EU208 EU208A EU267 EU267A Hs n2 Hs n2 A EU208B EU267B Hs n2 B EU209 EU209A EU268 EU268A Hs n3 Hs n3 A EU209B EU268B Hs n3 B EU210 EU210A EU269 EU269A Hs n4 Hs n4 A EU210B EU269B Hs n4 B EU211 EU211A EU270 EU270A Hs n5 Hs n5 A EU211B EU270B Hs n5 B EU212 EU212A EU271 EU271A Hs n6 Hs n6 A EU212B EU271B Hs n6 B EU213 EU213A EU272 EU272A Hs n7 Hs n7 A EU213B EU272B Hs n7 B EU214 EU214A EU273 EU273A Mm n7 Mm n7 A EU214B EU273B Mm n7 B EU215 EU215A EU274 EU274A Hs n7o Hs n7 Ao EU215B EU274B Hs n7 Bo EU216 EU216A EU275 EU275A Mm n7o Mm n7 Ao EU216B EU275B Mm n7 Bo EU217 EU217A EU276 EU276A EU415 EU404A EU217B EU276B EU415B EU218 EU218A EU277 EU277A EU416 EU416A EU218B EU277B EU415B EU219 EU219A EU278 EU278A EU417 EU302A EU219B EU278B EU415B EU220 EU220A EU279 EU279A EU418 EU418A EU220B EU279B EU418B EU221 EU221A EU280 EU280A EU419 EU418A EU221B EU280B EU419B EU222 EU222A EU281 EU281A EU420 EU414A EU222B EU281B EU420B EU223 EU223A EU282 EU282A EU421 EU277A EU223B EU282B EU420B EU224 EU224A EU283 EU283A EU422 EU422A EU224B EU283B EU422B EU225 EU225A EU284 EU284A EU423 EU423A EU225B EU284B EU423B EU226 EU226A EU285 EU285A EU304 EU304A EU226B EU285B EU304B EU227 EU227A EU286 EU286A EU305 EU305A EU227B EU286B EU305B EU228 EU228A EU287 EU287A EU306 EU306A EU228B EU287B EU306B EU229 EU229A EU288 EU288A EU307 EU307A EU229B EU288B EU307B EU230 EU230A EU289 EU289A EU308 EU308A EU230B EU289B EU308B EU231 EU231A EU290 EU290A EU309 EU309A EU231B EU290B EU309B EU232 EU232A EU291 EU291A EU310 EU310A EU232B EU291B EU310B EU233 EU233A EU292 EU292A EU311 EU311A EU233B EU292B EU311B EU234 EU234A EU293 EU293A EU312 EU312A EU234B EU293B EU312B EU235 EU235A EU294 EU294A EU313 EU313A EU235B EU294B EU313B EU236 EU236A EU295 EU295A EU314 EU314A EU236B EU295B EU314B EU237 EU237A EU296 EU296A EU315 EU315A EU237B EU296B EU315B EU238 EU238A EU297 EU297A EU316 EU316A EU238B EU297B EU316B EU239 EU239A EU298 EU298A EU317 EU317A EU239B EU298B EU317B EU240 EU240A EU299 EU299A EU318 EU318A EU240B EU299B EU318B EU241 EU241A EU300 EU300A EU319 EU319A EU241B EU300B EU319B EU242 EU242A EU301 EU301A EU320 EU320A EU242B EU301B EU320B EU243 EU243A EU302 EU302A EU321 EU321A EU243B EU302B EU321B EU244 EU244A EU303 EU303A EU322 EU322A EU244B EU303B EU322B EU245 EU245A EU400 EU400A EU323 EU323A EU245B EU400B EU323B EU246 EU246A EU401 EU401A EU324 EU324A EU246B EU401B EU324B EU247 EU247A EU402 EU402A EU325 EU325A EU247B EU402B EU325B EU248 EU248A EU403 EU403A EU424 EU424A EU248B EU403B EU424B EU249 EU249A EU404 EU404A EU425 EU425A EU249B EU404B EU425B EU250 EU250A EU405 EU405A EU426 EU426A EU250B EU405B EU426B EU251 EU251A EU406 EU406A EU427 EU427A EU251B EU406B EU427B EU252 EU252A EU407 EU407A EU428 EU428A EU252B EU407B EU428B EU253 EU253A EU408 EU408A EU429 EU429A EU253B EU408B EU424B EU254 EU254A EU409 EU409A EU430 EU430A EU254B EU409B EU425B EU255 EU255A EU410 EU410A EU431 EU431A EU255B EU410B EU426B EU256 EU256A EU411 EU411A EU432 EU432A EU256B EU411B EU427B EU257 EU257A EU412 EU412A EU433 EU433A EU257B EU412B EU428B EU258 EU258A EU413 EU413A EU258B EU413B

TABLE-US-00003 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 [00067] (vp)-mU-phos [00068] ivA, ivC, ivU, ivG inverted RNA (3′-3′) nucleotides ST23 [00069] ST23-phos [00070] ST43 (or C6XLT) [00071] ST43-phos (or C6XLT-phos) [00072] Ser(GN) (when at the end of a chain, one of the O—— is OH) [00073] [ST23(ps)]3 ST43(ps) [00074] [ST23]3 ST43 [00075] [ST23(ps)]3 ST41 (ps) [00076] [ST23]3 ST41 [00077]

[0732] 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-00004 Summary sequence table SEQ ID NO: Name (A=1.sup.st strand; B=2.sup.nd strand) Sequence 5′-3′ Unmodified sequence 5′-3′ counterpart 1 EU200A mU (ps) fc (ps) mGfAmAfGmUfAmUfUmCfCmGfCmGfUmLA (ps) fC (ps) mG UCGAAGUAUUCCGCGUACG 2 EU200B mC (ps) mG (ps) mUmAmCmGfCfGfGmAmAmUmAmCmUmUmC (ps) mG (ps) mA CGUACGCGGAAUACUUCGA 3 EU201A mC (ps) fC (ps) mUfAmGfGmAfUmCfAmCfGmCfUmCfUmG (ps) fC (ps) mU CCUAGGAUCACGCUCUGCU 4 EU201B mA (ps) mG (ps) mCmAmGmAfGfCfGmUmGmAmUmCmCmUmA (ps) mG (ps) mG AGCAGAGCGUGAUCCUAGG 5 EU202A mG (ps) fA (ps) mCfUmUfGmUfUmGfCmAfGmCfUmCfGmC (ps) fC (ps) mA GACUUGUUGCAGCUCGCCA 6 EU202B mU (ps) mG (ps) mGmCmGmAfGfCfUmGmCmAmAmCmAmAmG (ps) mU (ps) mC UGGCGAGCUGCAACAAGUC 7 EU203A mC (ps) fA (ps) mCfGmAfCmUfUmGfUmUfGmCfAmGfCmU (ps) fC (ps) mG CACGACUUGUUGCAGCUCG 8 EU203B mC (ps) mG (ps) mAmGmCmUfGfCfAmAmCmAmAmGmUmCmG (ps) mU (ps) mG CGAGCUGCAACAAGUCGUG 9 EU204A mG (ps) fA (ps) mCfAmCfGmAfAmGfAmUfGmAfUmGfCmC (ps) fA (ps) mU GACACGAAGAUGAUGCCAU 10 EU204B mA (ps) mU (ps) mGmGmCmAfUfCfAmUmCmUmUmCmGmUmG (ps) mU (ps) mC AUGGCAUCAUCUUCGUGUC 11 EU205A mC (ps) fG (ps) mGfUmGfAmAfGmGfAmGfAmUfCmAfGmG (ps) fU (ps) mU CGGUGAAGGAGAUCAGGUU 12 EU205B mA (ps) mA (ps) mCmCmUmGfAfUfCmUmCmCmUmUmCmAmC (ps) mC (ps) mG AACCUGAUCUCCUUCACCG 13 EU206A mC (ps) fU (ps) mCfAmCfGmUfUmGfAmCfCmAfGmCfUmG (ps) fC (ps) mU CUCACGUUGACCAGCUGCU 14 EU206B mA (ps) mG (ps) mCmAmGmCfUfGfGmUmCmAmAmCmGmUmG (ps) mA (ps) mG AGCAGCUGGUCAACGUGAG 15 EU207A mG (ps) fG (ps) mCfUmCfCmUfCmCfArnCfGmAfUmGfAmA (ps) fG (ps) mA GGCUCCUCCACCAUGAAGA 16 EU207B mU (ps) mC (ps) mUmUmCmAfUfGfGmUmGmGmAmGmGmAmG (ps) mC (ps) mC UCUUCAUGGUGGAGGAGCC 17 EU208A mG (ps) fA (ps) mAfAmAfUmAfUmGfCmCfCmGfAmCfAmG (ps) fC (ps)mA GAAAAUAUGCCCGACAGCA 18 EU208B mU (ps) mG (ps) mCmUmGmUfCfGfGmGmCmAmUmAmUmUmU (ps) mU (ps) mC UGCUGUCGGGCAUAUUUUC 19 EU209A mG (ps) fA (ps) mCfCmCfGmAfUmGfAmGfGmUfUmGfUmC (ps) fU (ps) mA GACCCGAUGAGGUUGUCUA 20 EU209B mU (ps) mA (ps) mGmAmCmAfAfCfCmUmCmAmUmCmGmGmG (ps) mU (ps) mC UAGACAACCUCAUCGGGUC 21 EU210A mG (ps) fG (ps) mAfCmCfCmGfAmUfGmAfGmGfUmUfGmU (ps) fC (ps) mU GGACCCGAUGAGGUUGUCU 22 EU210B mA (ps) mG (ps) mAmCmAmAfCfCfUmCmAmUmCmGmGmGmU (ps) mC (ps) mC AGACAACCUCAUCGGGUCC 23 EU211A mG (ps) fU (ps) mAfAmAfCmAfGmUfGmCfGmAfAmUfCmU (ps) fC (ps) mC GUAAACAGUGCGAAUCUCC 24 EU211B mG (ps) mG (ps) mAmGmAmUfUfCfGmCmAmCmUmGmUmUmU (ps) mA (ps) mC GGAGAUUCGCACUGUUUAC 25 EU212A mG (ps) fU (ps) mUfGmUfAmAfAmCfAmGfUmGfCmGfAmA (ps) fU (ps) mC GUUGUAAACAGUGCGAAUC 26 EU212B mG (ps) mA (ps) mUmUmCmGfCfAfCmUmGmUmUmUmAmCmA (ps) mA (ps) mC GAUUCGCACUGUUUACAAC 27 EU213A mC (ps) fA (ps) mUfUmAfUmAfGmGfGmCfUmCfUmGfUmC (ps) fA (ps) mC CAUUAUAGGGCUCUGUCAC 28 EU213B mG (ps) mU (ps) mGmAmCmAfGfAfGmCmCmCmUmAmUmAmA (ps) mU (ps) mG GUGACAGAGCCCUAUAAUG 29 EU214A mG (ps) fU (ps) mCfAmUfUmAfUmAfGmGfGmCfUmCfCmG (ps) fU (ps) mC GUCAUUAUAGGGCUCCGUC 30 EU214B mG (ps) mA (ps) mCmGmGmAfGfCfCmCmUmAmUmAmAmUmG (ps) mA (ps) mC GACGGAGCCCUAUAAUGAC 31 EU215A mG (ps) fA (ps) mUfCmAfUmAfUmUfGmAfGmCfUmCfCmU (ps) fC (ps) mU GAUCAUAUUGAGCUCCUCU 32 EU215B mA (ps) mG (ps) mAmGmGmAfGfCfUmCmAmAmUmAmUmGmA (ps) mU (ps) mC AGAGGAGCUCAAUAUGAUC 33 EU216A mG (ps) fC (ps)mUfGmGfGmUfCmAfUmGfAmUfAmUfCmC (ps) fU (ps) mC GCUGGGUCAUGAUAUCCUC 34 EU216B mG (ps) mA (ps) mGmGmAmUfAfUfCmAmUmGmAmCmCmCmA (ps) mG (ps) mC GAGGAUAUCAUGACCCAGC 35 EU217A mC (ps) fU (ps) mGfCmGfGmAfUmCfAmUfGmAfAmGfCmA (ps) fG (ps) mU CUGCGGAUCAUGAAGCAGU 36 EU217B mA (ps) mC (ps) mUmGmCmUfUfCfAmUmGmAmUmCmCmGmC (ps) mA (ps) mG ACUGCUUCAUGAUCCGCAG 37 EU218A mG (ps) fG (ps) mAfGmAfUmUfUmUfCmAfCmUfUmUfGmA (ps) fG (ps) mC GGAGAUUUUCACUUUGAGC 38 EU218B mG (ps) mC (ps) mUmCmAmAfAfGfUmGmAmAmAmAmUmCmU (ps) mC (ps) mC GCUCAAAGUGAAAAUCUCC 39 EU219A mC (ps) fU (ps) mUfAmUfUmUfCmGf GmGfUmGfUmAfCmA (ps) fG (ps) mG CUUAUUUCGGGUGUACAGG 40 EU219B mC (ps) mC (ps) mUmGmUmAfCfAfCmCmCmGmAmAmAmUmA (ps) mA (ps) mG CCUGUACACCCGAAAUAAG 41 EU220A mG (ps) fC (ps) mGfCmCfCmGfUmCfUmCfAmAfAmCfUmU (ps) fC (ps) mA GCGCCCGUCUCAAACUUCA 42 EU220B mU (ps) mG (ps) mAmAmGmUfUfUfGmAmGmAmCmGmGmGmC (ps) mG (ps) mC UGAAGUUUGAGACGGGCGC 43 EU221A mC (ps) fU (ps) mUfAmAfCmCfCmUfGmUfCmUfUmCfCmC (ps) fU (ps) mU CUUAACCCUGUCUUCCCUU 44 EU221B mA (ps) mA (ps) mGmGmGmAfAfGfAmCmAmGmGmGmUmUmA (ps) mA (ps) mG AAGGGAAGACAGGGUUAAG 45 EU222A mC (ps) fA (ps) mGfUmUfCmCfAmGfGmGfUmAfUmGfGmC (ps) fU (ps) mC CAGUUCCAGGGUAUGGCUC 46 EU222B mG (ps) mA (ps) mGmCmCmAfUfAfCmCmCmUmGmGmAmAmC (ps) mU (ps) mG GAGCCAUACCCUGGAACUG 47 EU223A mC (ps) fC (ps) mGfAmGfAmGfUmCfAmAfAmCfUmCfAmU (ps) fA (ps) mA CCGAGAGUCAAACUCAUAA 48 EU223B mU (ps) mU (ps) mAmUmGmAfGfUfUmUmGmAmCmUmCmUmC (ps) mG (ps) mG UUAUGAGUUUGACUCUCGG 49 EU224A mG (ps) fU (ps) mCfAmUfAmAfCmAfAmCfAmAfAmAfCmU (ps) fC (ps) mC GUCAUAACAACAAAACUCC 50 EU224B mG (ps) mG (ps) mAmGmUmUfUfUfGmUmUmGmUmUmAmUmG (ps) mA (ps) mC GGAGUUUUGUUGUUAUGAC 51 EU225A mC (ps) fC (ps) mAfUmAfUmAfUmCfGmUfUmUfAmGfCmA (ps) fA (ps) mA CCAUAUAUCGUUUAGCAAA 52 EU225B mU (ps) mU (ps) mUmGmCmUfAfAfAmCmGmAmUmAmUmAmU (ps) mG (ps) mG UUUGCUAAACGAUAUAUGG 53 EU226A mA (ps) fG (ps) mAfUmGfAmUfGmCfCmAfUmCfCmGfGmG (ps) fU (ps) mU AGAUGAUGCCAUCCGGGUU 54 EU226B mA (ps) mA (ps) mCmCmCmGfGfAfUmGmGmCmAmUmCmAmU (ps) mC (ps) mU AACCCGGAUGGCAUCAUCU 55 EU227A mA (ps) fC (ps) mGfAmAfGmAfUmGfAmUfGmCfCmAfUmC (ps) fC (ps) mG ACGAAGAUGAUGCCAUCCG 56 EU227B mC (ps) mG (ps) mGmAmUmGfGfCfAmUmCmAmUmCmUmUmC (ps) mG (ps) mU CGGAUGGCAUCAUCUUCGU 57 EU228A mU (ps) fC (ps) mGfGmAfCmAfCmGfAmAfGmAfUmGfAmU (ps) fG (ps) mC UCGGACACGAAGAUGAUGC 58 EU228B mG (ps) mC (ps) mAmUmCmAfUfCfUmUmCmGmUmGmUmCmC (ps) mG (ps) mA GCAUCAUCUUCGUGUCCGA 59 EU229A mA (ps) fA (ps) mGfUmUfCmAfCmCfGmUfGmCfUmGfCmC (ps) fC (ps) mU AAGUUCACCGUGCUGCCCU 60 EU229B mA (ps) mG (ps) mGmGmCmAfGfCfAmCmGmGmUmGmAmAmC (ps) mU (ps) mU AGGGCAGCACGGUGAACUU 61 EU230A mU (ps) fC (ps) mGfGmUfGmAfAmGfGmAfGmAfUmCfAmG (ps) fG (ps) mU UCGGUGAAGGAGAUCAGGU 62 EU230B mA (ps) mC (ps) mCmUmGmAfUfCfUmCmCmUmUmCmAmCmC (ps) mG (ps) mA ACCUGAUCUCCUUCACCGA 63 EU231A mA (ps) fC (ps) mCfUmCfGmGfUmGfAmAfGmGfAmGfAmU (ps) fC (ps) mA 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mUmCmAmAfCfAfCmCmUmCmCmCmUmCmA (ps) mC (ps) mA GGUCAACACCUCCCUCACA 75 EU237A mA (ps) fU (ps) mUfGmUfGmAfGmGfGmAfGmGfUmGfUmU (ps) fG (ps) mA AUUGUGAGGGAGGUGUUGA 76 EU237B mU (ps) mC (ps) mAmAmCmAfCfCfUmCmCmCmUmCmAmCmA (ps) mA (ps) mU UCAACACCUCCCUCACAAU 77 EU238A mU (ps) fC (ps) mUfAmGfAmAfGmGfAmUfUmGfUmGfAmG (ps) fG (ps) mG UCUAGAAGGAUUGUGAGGG 78 EU238B mC (ps) mC (ps) mCmUmCmAfCfAfAmUmCmCmUmUmCmUmA (ps) mG (ps) mA CCCUCACAAUCCUUCUAGA 79 EU239A mU (ps) fU (ps) mGfUmCfUmAfGmAfAmGfGmAfUmUfGmU (ps) fG (ps) mA UUGUCUAGAAGGAUUGUGA 80 EU239B mU (ps) mC (ps) mAmCmAmAfUfCfCmUmUmCmUmAmGmAmC (ps) mA (ps) mA UCACAAUCCUUCUAGACAA 81 EU240A mU (ps) fG (ps) mAfGmGfUmUfGmUfCmUfAmGfAmAfGmG (ps) fA (ps) mU UGAGGUUGUCUAGAAGGAU 82 EU240B mA (ps) mU (ps) mCmCmUmUfCfUfAmGmAmCmAmAmCmCmU (ps) mC (ps) mA AUCCUUCUAGACAACCUCA 83 EU241A mA (ps) fU (ps) mCfUmCfCmCfCmAfAmAfGmAfUmGfAmC (ps) fA (ps) mA AUCUCCCCAAAGAUGACAA 84 EU241B mU (ps) mU (ps) mGmUmCmAfUfCfUmUmUmGmGmGmGmAmG (ps) mA (ps) mU UUGUCAUCUUUGGGGAGAU 85 EU242A mA (ps) fG (ps) mAfAmCfAmAfUmAfGmUfGmUfUmGfGmC (ps) fA (ps) mC AGAACAAUAGUGUUGGCAC 86 EU242B mG(ps) mU (ps) mGmCmCmLAfAfCfAmCmUmAmUmUmGmUmU (ps) mC (ps) mU GUGCCAACACUAUUGUUCU 87 EU243A mA (ps) fG (ps) mCfAmUfAmAfAmGfAmAfUmUfUmGfGmU (ps) fG(ps) mA AGCAUAAAGAAUUUGGUGA 88 EU243B mU (ps) mC (ps) mAmCmCmAfAfAfUmUmCmUmUmUmAmUmG (ps) mC (ps) mU UCACCAAAUUCUUUAUGCU 89 EU244A mU (ps) fU (ps) mGfUmAfAmAfCmAfGmUfGmCfGmAfAmU (ps) fC (ps) mU UUGUAAACAGUGCGAAUCU 90 EU244B mA (ps) mG (ps) mAmUmUmCfGfCfAmCmUmGmUmUmUmAmC (ps) mA (ps) mA AGAUUCGCACUGUUUACAA 91 EU245A mA (ps) fC(ps) mAfGmUfUmUfUmGfGmUfCmCfUmUfAmG (ps) fU (ps) mU ACAGUUUUGGUCCUUAGUU 92 EU245B mA (ps) mA (ps) mCmUmAmAfGfGfAmCmCmAmAmAmAmCmU (ps) mG (ps) mU AACUAAGGACCAAAACUGU 93 EU246A mA (ps) fU (ps) mAfUmCfGmUfGmUfAmCfAmGfUmUfUmU (ps) fG (ps) mG AUAUCCUCUACAGUUUUGG 94 EU246B mC (ps) mC (ps) mAmAmAmAfCfUfGmUmAmGmAmGmGmAmU (ps) mA (ps) mU CCAAAACUGUAGAGGAUAU 95 EU247A mA (ps) fU (ps) mAfUmCfUmAfCmAfAmUfAmUfUmGfGmA (ps) fC (ps) mU AUAUCUACAAUAUUGGACU 96 EU247B mA (ps) mG (ps) mUmCmCmAfAfUfAmUmUmGmUmAmGmAmU (ps) mA (ps) mU AGUCCAAUAUUGUAGAUAU 97 EU248A mA (ps) fA (ps) mUfAmUfGmUfAmCfAmAfUmAfUmUfGmG (ps) fA (ps) mC AAUAUCUACAAUAUUGGAC 98 EU248B mG (ps) mU (ps) mCmCmAmAfUfAfUmUmGmUmAmGmAmUmA (ps) mU (ps) mU GUCCAAUAUUGUAGAUAUU 99 EU249A mU (ps) fU (ps) mGfAmCfGmUfAmGfAmGfAmAfUmAfUmC (ps) fU (ps) mA UUGACGUAGAGAAUAUCUA 100 EU249B mU (ps) mA (ps) mGmAmUmAfUfUfCmUmCmUmAmCmGmUmC (ps) mA (ps) mA UAGAUAUUCUCUACGUCAA 101 EU250A mA (ps) fA (ps) mGfUmCfUmUfUmGfAmCfGmUfAmGfAmG (ps) fA (ps) mA AAGUCUUUGACGUAGAGAA 102 EU250B mU (ps) mU (ps) mCmUmCmUfAfCfGmUmCmAmAmAmGmAmC (ps) mU (ps) mU UUCUCUACGUCAAAGACUU 103 EU251A mU (ps) fA (ps) mGfAmAfGmCfGmAfGmUfGmAfUmAfGmU (ps) fC (ps) mU UAGAAGCGAGUGAUAGUCU 104 EU251B mA (ps) mG (ps) mAmCmUmAfUfCfAmCmUmCmGmCmUmUmC (ps) mU (ps) mA AGACUAUCACUCGCUUCUA 105 EU252A mU (ps) fU (ps) mGfAmAfGmGfCmAfGmAfGmAfAmGfUmC (ps) fA (ps) mC UUGAAGGCAGAGAAGUCAC 106 EU252B mG (ps) mU (ps) mGmAmCmUfUfCfUmCmUmGmCmCmUmUmC (ps) mA (ps) mA GUGACUUCUCUGCCUUCAA 107 EU253A mU (ps) fU (ps) mUfCmAfCmUfUmUfGmAfmGmCfmUmCfAmU (ps) fU (ps) mG UUUCACUUUGAGCUCAUUG 108 EU253B mC (ps) mA (ps) mAmUmGmAfGfCfUmCmAmAmAmGmUmGmA (ps) mA (ps) mA CAAUGAGCUCAAAGUGAAA 109 EU254A mA (ps) fA (ps) mCfUmGfGmUfUmGfCmUfGmCfUmGfCmC (ps) fU (ps) mG AACUGGUUGCUGCUGCCUG 110 EU254B mC (ps) mA (ps) mGmGmCmAfGfCfAmGmCmAmAmCmCmAmG (ps) mU (ps) mU CAGGCAGCAGCAACCAGUU 111 EU255A mU (ps) fU (ps) mGfAmGfAmAfGmAfGmUfUmGfUmGfGmU (ps) fC (ps) mU UUGAGAAGAGUUGUGGUCU 112 EU255B mA (ps) mG (ps) mAmCmCmAfCfAfAmCmUmCmUmUmCmUmC (ps) mA (ps) mA AGACCACAACUCUUCUCAA 113 EU256A mU (ps) fU (ps) mCfAmUfGmUfGmGfGmCfCmCfAmCfUmG (ps) fG (ps) mG UUCAUGUGGGCCCACUGGG 114 EU256B mC (ps) mC (ps) mCmAmGmUfGfGfGmCmCmCmAmCmAmUmG (ps) mA (ps) mA CCCAGUGGGCCCACAUGAA 115 EU257A mU (ps) fC (ps) mUfUmCfAmUfGmUfGmGfGmCfCmCfAmC (ps) fU (ps) mG UCUUCAUGUGGGCCCACUG 116 EU257B mC (ps) mA (ps) mGmUmGmGfGfCfCmCmAmCmAmUmGmAmA (ps) mG (ps) mA CAGUGGGCCCACAUGAAGA 117 EU258A mU (ps) fG (ps) mGfAmCfUmAfAmCfAmGfGmUfUmCfCmC (ps) fU (ps) mC UGAACUAACAGGUUCCCUC 118 EU258B mG (ps) mA (ps) mGmGmGmAfAfCfCmUmGmUmUmAmGmUmC (ps) mC (ps) mA GAGGGAACCUGUUAGUCCA 119 EU259A mU (ps) fU (ps) mCfCmUfUmAfAmCfCmCfUmGfUmCfUmU (ps) fC (ps) mC UUCCUUAACCCUGUCUUCC 120 EU259B mG (ps) mG (ps) mAmAmGmAfCfAfGmGmGmUmUmAmAmGmG (ps) mA (ps) mA GGAAGACAGGGUUAAGGAA 121 EU260A mA (ps) fG (ps) mUfUmCfCmUfUmAfAmCfCmCfUmGfUmC (ps) fU (ps) mU AGUUCCUUAACCCUGUCUU 122 EU260B mA (ps) mA (ps) mGmAmCmAfGfGfGmUmUmAmAmGmGmAmA (ps) mC (ps) mU AAGACAGGGUUAAGGAACU 123 EU261A mA (ps) fU (ps) mAfAmAfGmUfUmCfCmUfUmAfAmCfCmC (ps) fU (ps) mG AUAAAGUUCCUUAACCCUG 124 EU261B mC (ps) mA (ps) mGmGmGmUfUfAfAmGmGmAmAmCmUmUmU (ps) mA (ps) mU CAGGGUUAAGGAACUUUAU 125 EU262A mA (ps) fA (ps) mAfUmAfAmAfGmUfUmCfCmUfUmAfAmC (ps) fC (ps) mC AAAUAAAGUUCCUUAACCC 126 EU262B mG (ps) mG (ps) mGmUmUmAfAfGfGmAmAmCmUmUmUmAmU (ps) mU (ps) mU GGGUUAAGGAACUUUAUUU 127 EU263A mU (ps) fA (ps) mCfAmUfAmAfAmGfUmUfCmCfUmUfAmA (ps) fC (ps) mC UACAUAAAGUUCCUUAACC 128 EU263B mG (ps) mG (ps) mUmUmAmAfGfGfAmAmCmUmUmUmAmUmG (ps) mU (ps) mA GGUUAAGGAACUUUAUGUA 129 EU264A mA (ps) fU (ps) mUfAmUfCmAfUmGfUmCfCmCfUmGfAmU (ps) fC (ps) mA AUUAUCAUGUCCCUGAUCA 130 EU264B mU (ps) mG (ps) mAmUmCmAfGfGfGmAmCmAmUmGmAmUmA (ps) mA (ps) mU UGAUCAGGGACAUGAUAAU 131 EU265A mA (ps) fU (ps) mAfAmAfGmAfAmCfCmUfGmCfUmUfCmC (ps) fG (ps) mU AUAAAGAACCUGCUUCCGU 132 EU265B mA (ps) mC (ps) mGmGmAmAfGfCfAmGmCmUmUmCmUmUmU (ps) mA (ps) mU ACGGAAGCAGGUUCUUUAU 133 EU266A mA (ps) fC (ps) mUfAmAfCmUfUmCfAmGfCmAfAmGfGmG (ps) fC (ps) mA ACUAACUUCAGCAAGGGCA 134 EU266B mU (ps) mG (ps) mCmCmCmUfUfGfCmUmGmAmAmGmUmUmA (ps) mG (ps) mU UGCCCUUGCUGAAGUUAGU 135 EU267A mU (ps) fG (ps) mGfUmGfCmAfAmGfAmCfAmAfUmCfCmC (ps) fU (ps) mG UGGUGCAAGACAAUCCCUG 136 EU267B mC (ps) mA (ps) mGmGmGmAfUfUfGmUmCmUmUmGmCmAmC (ps) mC (ps) mA CAGGGAUUGUCUUGCACCA 137 EU268A mA (ps) fU (ps) mAfAmAfUmAfGmGfAmGfUmCfAmUfCmU (ps) fG (ps) mC AUAAAUAGGAGUCAUCUGC 138 EU268B mG (ps) mC (ps) mAmGmAmUfGfAfCmUmCmCmUmAmUmUmU (ps) mA (ps) mU GCAGAUGACUCCUAUUUAU 139 EU269A mU (ps) fU (ps) mGfGmAfAmAfUmAfAmUfGmAfUmGfAmC (ps) fC (ps) mC UUGGAAAUAAUGAUGACCC 140 EU269B mG (ps) mG (ps) mGmUmCmAfUfCfAmUmUmAmUmUmUmCmC (ps) mA (ps) mA GGGUCAUCAUUAUUUCCAA 141 EU270A mA (ps) fA (ps) mAfCmUfCmAfUmAfAmCfAmAfUmGfCmC (ps) fA (ps) mA AAACUCAUAACAAUGCCAA 142 EU270B mU (ps) mU (ps) mGmGmCmAfUfUfGmUmUmAmUmGmAmGmU (ps) mU (ps) mU UUGGCAUUGUUAUGAGUUU 143 EU271A mA (ps) fU (ps) mUfAmUfCmAfUmUfGmUfUmUfUmGfUmG (ps) fA (ps) mC AUUAUCAUUGUUUUGUGAC 144 EU271B mG (ps) mU (ps) mCmAmCmAfAfAfAmCmAmAmUmGmAmUmA (ps) mA (ps) mU GUCACAAAACAAUGAUAAU 145 EU272A mU (ps) fU (ps) mGfUmUfUmAfGmCfAmAfAmUfAmUfUmA (ps) fU (ps) mC UUGUUUAGCAAAUAUUAUC 146 EU272B mG (ps) mA (ps) mUmAmAmUfAfUfUmUmGmCmUmAmAmAmC (ps) mA (ps) mA GAUAAUAUUUGCUAAACAA 147 EU273A mU (ps) fC (ps) mCfCmCfGmAfGmAfGmUfCmAfAmAfCmU (ps) fC (ps) mA UCCCCGAGAGUCAAACUCA 148 EU273B mU (ps) mG (ps) mAmGmUmUfUfGfAmCmUmCmUmCmGmGmG (ps) mG (ps) mA UGAGUUUGACUCUCGGGGA 149 EU274A mA (ps) fA (ps) mCfUmCfCmCfCmGfAmGfAmGfUmCfAmA (ps) fA (ps) mC AACUCCCCGAGAGUCAAAC 150 EU274B mG (ps) mU (ps) mUmUmGmAfCfUfCmUmCmGmGmGmGmAmG (ps) mU (ps) mU GUUUGACUCUCGGGGAGUU 151 EU275A mA (ps) fA (ps) mCfAmAfCmAfAmAfAmCfUmCfCmCfCmG (ps) fA (ps) mG AACAACAAAACUCCCCGAG 152 EU275B mC (ps) mU (ps) mCmGmGmGfGfAfGmUmUmUmUmGmUmUmG (ps) mU (ps) mU CUCGGGGAGUUUUGUUGUU 153 EU276A mA (ps) fU (ps) mAfAmCfAmAfCmAfAmAfAmCfUmCfCmC (ps) fC (ps) mG AUAACAACAAAACUCCCCG 154 EU276B mC (ps) mG (ps) mGmGmGmAfGfUfUmUmUmGmUmUmGmUmU (ps) mA (ps) mU CGGGGAGUUUUGUUGUUAU 155 EU277A mU (ps) fG (ps) mAfCmAfAmAfAmGfAmCfAmCfAmAfGmA (ps) fG (ps) mU UGACAAAAGACACAAGAGU 156 EU277B mA (ps) mC (ps) mUmCmUmUfGfUfGmUmCmUmUmUmUmGmU (ps) mC (ps) mA ACUCUUGUGUCUUUUGUCA 157 EU278A mU (ps) fU (ps) mGfUmUfUmUfGmUfGmAfCmAfAmAfAmG (ps) fA (ps) mC UUGUUUUGUGACAAAAGAC 158 EU278B mG (ps) mU (ps) mCmUmUmUfUfGfUmCmAmCmAmAmAmAmC (ps) mA (ps) mA GUCUUUUGUCACAAAACAA 159 EU279A mU (ps) fC (ps) mUfAmGfGmAfUmCfAmCfGmCfUmCfUmG (ps) fC (ps) mU UCUAGGAUCACGCUCUGCU 160 EU279B mA (ps) mG (ps) mCmAmGmAfGfCfGmUmGmAmUmCmCmUmA (ps) mG (ps) mA AGCAGAGCGUGAUCCUAGA 161 EU280A mU (ps) fA (ps) mCfUmUfGmUfUmGfCmAfGmCfUmCfGmC (ps) fC (ps) mA UACUUGUUGCAGCUCGCCA 162 EU280B mU (ps) mG (ps) mGmCmGmAfGfCfUmGmCmAmAmCmAmAmG (ps) mU (ps) mA UGGCGAGCUGCAACAAGUA 163 EU281A mU (ps) fA (ps) mCfGmAfCmUfUmGfUmUf GmCfAmGfCmU (ps) fC (ps) mG UACGACUUGUUGCAGCUCG 164 EU281B mC (ps) mG (ps) mAmGmCmUfGfCfAmAmCmAmAmGmUmCmG (ps) mU (ps) mA CGAGCUGCAACAAGUCGUA 165 EU282A mU (ps) fA (ps) mCfAmCfGmAfAmGfAmUfGmAfUmGfCmC (ps) fA (ps) mU UACACGAAGAUGAUGCCAU 166 EU282B mA (ps) mU (ps) mGmGmCmAfUfCfAmUmCmUmUmCmGmUmG (ps) mU (ps) mA AUGGCAUCAUCUUCGUGUA 167 EU283A mU (ps) fG (ps) mGfUmGfAmAfGmGfAmGfAmUfCmAfGmG (ps) fU (ps) mU UGGUGAAGGAGAUCAGGUU 168 EU283B mA (ps) mA (ps) mCmCmUmGfAfUfCmUmCmCmUmUmCmAmC (ps) mC (ps) mA AACCUGAUCUCCUUCACCA 169 EU284A mU (ps) fU (ps) mCfAmCfGmUfUmGfAmCfCmAfGmCfUmG (ps) fC (ps) mU UUCACGUUGACCAGCUGCU 170 EU284B mA (ps) mG (ps) mCmAmGmCfUfGfGmUmCmAmAmCmGmUmG (ps) mA (ps) mA AGCAGCUGGUCAACGUGAA 171 EU285A mU (ps) fG (ps) mCfUmCfCmUfCmCfAmCfCmAfUmGfAmA (ps) fG (ps) mA UGCUCCUCCACCAUGAAGA 172 EU285B mU (ps) mC (ps) mUmUmCmAfUfGfGmUmGmGmAmGmGmAmG (ps) mC (ps) mA UCUUCAUGGUGGAGGAGCA 173 EU286A mU (ps) fA (ps) mAfAmAfUmAfUmGfCmCfCmGfAmCfAmG (ps) fC (ps) mA UAAAAUAUGCCCGACAGCA 174 EU286B mU (ps) mG (ps) mCmUmGmUfCfGfGmGmCmAmUmAmUmUmU (ps) mU (ps) mA UGCUGUCGGGCAUAUUUUA 175 EU287A mU (ps) fA (ps) mCfCmCfGmAfUmGfAmGfGmUfUmGfUmC (ps) fU (ps) mA UACCCGAUGAGGUUGUCUA 176 EU287B mU (ps) mA (ps) mGmAmCmAfAfCfCmUmCmAmUmCmGmGmG (ps) mU (ps) mA UAGACAACCUCAUCGGGUA 177 EU288A mU (ps) fG (ps) mAfCmCfCmGfAmUfGmAfGmGfUmUfGmU (ps) fC (ps) mU UGACCCGAUGAGGUUGUCU 178 EU288B mA (ps) mG (ps) mAmCmAmAfCfCfUmCmAmUmCmGmGmGmU (ps) mC (ps) mA AGACAACCUCAUCGGGUCA 179 EU289A mU (ps) fU (ps) mAfAmAfCmAfGmUfGmCfGmAfAmUfCmU (ps) fC (ps) mC UUAAACAGUGCGAAUCUCC 180 EU289B mG (ps) mG (ps) mAmGmAmUfUfCfGmCmAmCmUmGmUmUmU (ps) mA (ps) mA GGAGAUUCGCACUGUUUAA 181 EU290A mU (ps) fU (ps) mUfGmUfAmAfAmCfAmGfUmGfCmGfAmA (ps) fU (ps) mC UUUGUAAACAGUGCGAAUC 182 EU290B mG (ps) mA (ps) mUmUmCmGfCfAfCmUmGmUmUmUmAmCmA (ps) mA (ps) mA GAUUCGCACUGUUUACAAA 183 EU291A mU (ps) fA (ps) mUfUmAfUmAfGmGfGmCfUmCfUmGfUmC (ps) fA (ps) mC UAUUAUAGGGCUCUGUCAC 184 EU291B mG (ps) mU (ps) mGmAmCmAfGfAfGmCmCmCmUmAmUmAmA (ps) mU (ps) mA GUGACAGAGCCCUAUAAUA 185 EU292A mU (ps) fU (ps) mCfAmUfUmAfUmAfGmGfGmCfUmCfCmG (ps) fU (ps) mC UUCAUUAUAGGGCUCCGUC 186 EU292B mG (ps) mA (ps) mCmGmGmAfGfCfCmCmUmAmUmAmAmUmG (ps) mA (ps) mA GACGGAGCCCUAUAAUGAA 187 EU293A mU (ps) fA (ps) mUfCmAfUmAfUmUfGmAfGmCfUmCfCmU (ps) fC (ps) mU UAUCAUAUUGAGCUCCUCU 188 EU293B mA (ps) mG (ps) mAmGmGmAfGfCfUmCmAmAmUmAmUmGmA (ps) mU (ps) mA AGAGGAGCUCAAUAUGAUA 189 EU294A mU (ps) fC (ps) mUfGmGfGmUfCmAfUmGfAmUfAmUfCmC (ps) fU (ps) mC UCUGGGUCAUGAUAUCCUC 190 EU294B mG (ps) mA (ps) mGmGmAmUfAfUfCmAmUmGmAmCmCmCmA (ps) mG (ps) mA GAGGAUAUCAUGACCCAGA 191 EU295A mU (ps) fU (ps) mGfCmGfGmAfUmCfAmUfGmAfAmGfCmA (ps) fG (ps) mU UUGCGGAUCAUGAAGCAGU 192 EU295B mA (ps) mC (ps) mUmGmCmUfUfCfAmUmGmAmUmCmCmGmC (ps) mA (ps) mA ACUGCUUCAUGAUCCGCAA 193 EU296A mU (ps) fG (ps) mAfGmAfUmUfUmUfCmAfCmUfUmUfGmA (ps) fG (ps) mC UGAGAUUUUCACUUUGAGC 194 EU296B mG (ps) mC (ps) mUmCmAmAfAfGfUmGmAmAmAmAmUmCmU (ps) mC (ps) mA GCUCAAAGUGAAAAUCUCA 195 EU297A mU (ps) fU (ps) mUfAmUfUmUfCmGfGmGfUmGfUmAfCmA (ps) fG (ps) mG UUUAUUUCGGGUGUACAGG 196 EU297B mC (ps) mC (ps) mUmGmUmAfCfAfCmCmCmGmAmAmAmUmA (ps) mA (ps) mA CCUGUACACCCGAAAUAAA 197 EU298A mU (ps) fC (ps) mGfCmCfCmGfUmCfUmCfAmAfAmCfUmU (ps) fC (ps) mA UCGCCCGUCUCAAACUUCA 198 EU298B mU (ps) mG (ps) mAmAmGmUfUfUfGmAmGmAmCmGmGmGmC (ps) mG (ps) mA UGAAGUUUGAGACGGGCGA 199 EU299A mU (ps) fU (ps) mUfAmAfCmCfCmUfGmUfCmUfUmCfCmC (ps) fU (ps) mU UUUAACCCUGUCUUCCCUU 200 EU299B mA (ps) mA (ps) mGmGmGmAfAfGfAmCmAmGmGmGmUmUmA (ps) mA (ps) mA AAGGGAAGACAGGGUUAAA 201 EU300A mU (ps) fA (ps) mGfUmUfCmCfAmGfGmGfUmAfUmGfGmC (ps) fU (ps) mC UAGUUCCAGGGUAUGGCUC 202 EU300B mG (ps) mA (ps) mGmCmCmAfUfAfCmCmCmUmGmGmAmAmC (ps) mU (ps) mA GAGCCAUACCCUGGAACUA 203 EU301A mU (ps) fC (ps) mGfAmGfAmGfUmCfAmAfAmCfUmCfAmU (ps) fA (ps) mA UCGAGAGUCAAACUCAUAA 204 EU301B mU (ps) mU (ps) mAmUmGmAfGfUfUmUmGmAmCmUmCmUmC (ps) mG (ps) mA UUAUGAGUUUGACUCUCGA 205 EU302A mU (ps) fU (ps) mCfAmUfAmAfCmAfAmCfAmAfAmAfCmU (ps) fC (ps) mC UUCAUAACAACAAAACUCC 206 EU302B mG (ps) mG (ps) mAmGmUmUfUfUfGmUmUmGmUmUmAmUmG (ps) mA (ps) mA GGAGUUUUGUUGUUAUGAA 207 EU303A mU (ps) fC (ps) mAfUmAfUmAfUmCfGmUfUmUfAmGfCmA (ps) fA (ps) mA UCAUAUAUCGUUUAGCAAA 208 EU303B mU (ps) mU (ps) mUmGmCmUfAfAfAmCmGmAmUmAmUmAmU (ps) mG (ps mA UUUGCUAAACGAUAUAUGA 209 EU400A mU (ps) fC (ps) mGfAmAfGmUfAmUfUmCfCmGfCmGfUmA (ps) fC (ps) mG UCGAAGUAUUCCGCGUACG 210 EU400B [ST23 (ps) ] 3ST43 (ps) mCmGmUmAmCmGfCfGfGmAmAmUmAmCmUmUmC (ps) mG (ps) mA CGUACGCGGAAUACUUCGA 211 EU401A (vp) mUfAmCfGmAfCmUfUmGfUmUfGmCfAmGfCmU (ps) fC (ps) mG UACGACUUGUUGCAGCUCG 212 EU401B [ST23 (ps) ] 3ST43 (ps) mCmGmAmGmCmUfGfCfAmAmCmAmAmGmUmCmG (ps) mU (ps) mG CGAGCUGCAACAAGUCGUG 213 EU402A (vp)mUfGmAfGmAfUmUfUmUfCmAfCmUfUmUfGmA(ps)fG(ps)mC UGAGAUUUUCACUUUGAGC 214 EU402B [ST23 (ps) ] 3ST43 (ps) mGmCmUmCmAmAfAfGfUmGmAmAmAmAmUmCmU (ps) mC (ps) mC GCUCAAAGUGAAAAUCUCC 215 EU403A (vp) mUfCmGfAmGfAmGfUmCfAmAfAmCfUmCfAmU (ps) fA (ps) mA UCGAGAGUCAAACUCAUAA 216 EU403B [ST23(ps) ] 3ST43 (ps) mUmUmAmUmGmAfGfUfUmUmGmAmCmUmCmUmC (ps) mG (ps) mG UUAUGAGUUUGACUCUCGG 217 EU404A (vp) mUfUmCfAmUfAmAfCmAfAmCfAmAfAmAfCmU (ps) fC (ps) mC UUCAUAACAACAAAACUCC 218 EU404B [ST23(ps) ] 3ST43 (ps) mGmGmAmGmUmUfUfUfGmUmUmGmUmUmAmUmG (ps) mA (ps) mC GGAGUUUUGUUGUUAUGAC 219 EU405A (vp) mUfCmGfGmAfCmAfCmGfAmAfGmAfUmGfAmU (ps) fG (ps) mC UCGGACACGAAGAUGAUGC 220 EU405B [ST23 (ps) ] 3ST43 (ps) mGmCmAmUmCmAfUfCfUmUmCmGmUmGmUmCmC (ps) mG (ps) mA GCAUCAUCUUCGUGUCCGA 221 EU406A (vp) mUfUmAfUmCfUmAfCmAfAmUfAmUfUmGfGmA (ps) fC (ps) mU UUAUCUACAAUAUUGGACU 222 EU406B [ST23 (ps) ] 3ST43 (ps) mAmGmUmCmCmAfAfUfAmUmUmGmUmAmGmAmU (ps) mA (ps) mU AGUCCAAUAUUGUAGAUAU 223 EU407A (vp) mUfAmUfAmUfCmUfAmCfAmAfUmAfUmUfGmG (ps) fA (ps) mC UAUAUCUACAAUAUUGGAC 224 EU407B [ST23 (ps) ] 3ST43 (ps) mGmUmCmCmAmAfUfAfUmUmGmUmAmGmAmUmA (ps) mU (ps) mU GUCCAAUAUUGUAGAUAUU 225 EU408A (vp) mUfAmGfAmAfGmCfGmAfGmUfGmAfUmAfGmU (ps) fC (ps) mU UAGAAGCGAGUGAUAGUCU 226 EU408B [ST23 (ps) ] 3ST43 (ps) mAmGmAmCmUmAfUfCfAmCmUmCmGmCmUmUmC (ps) mU (ps) mA AGACUAUCACUCGCUUCUA 227 EU409A (vp) mUfGmGfAmCfUmAfAmCfAmGfGmUfUmCfCmC (ps) fU (ps) mC UGGACUAACAGGUUCCCUC 228 EU409B [ST23 (ps) ] 3ST43 (ps) mGmAmGmGmGmAfAfCfCmUmGmUmUmAmGmUmC (ps) mC (ps) mA GAGGGAACCUGUUAGUCCA 229 EU410A (vp) mUfUmAfAmAfGmUfUmCfCmUfUmAfAmCfCmC (ps) fU (ps) mG UUAAAGUUCCUUAACCCUG 230 EU410B [ST23 (ps) ] 3ST43 (ps) mCmAmGmGmGmUfUfAfAmGmGmAmAmCmUmUmU (ps) mA (ps) mU CAGGGUUAAGGAACUUUAU 231 EU411A (vp) mUfAmCfAmUfAmAfAmGfUmUfCmCfUmUfAmA (ps) fC (ps) mC UACAUAAAGUUCCUUAACC 232 EU411B [ST23 (ps) ] 3ST43 (ps) mGmGmUmUmAmAfGfGfAmAmCmUmUmUmAmUmG (ps) mU (ps) mA GGUUAAGGAACUUUAUGUA 233 EU412A (vp) mUfUmUfAmUfCmAfUmUfGmUfUmUfUmGfUmG (ps) fA (ps) mC UUUAUCAUUGUUUUGUGAC 234 EU412B [ST23 (ps) ] 3ST43 (ps) mGmUmCmAmCmAfAfAfAmCmAmAmUmGmAmUmA (ps) mA (ps) mU GUCACAAAACAAUGAUAAU 235 EU413A (vp) mUfUmAfAmCfAmAfCmAfAmAfAmCfUmCfCmC (ps) fC (ps) mG UUAACAACAAAACUCCCCG 236 EU413B [ST23 (ps) ] 3ST43 (ps) mCmGmGmGmGmAfGfUfUmUmUmGmUmUmGmUmU (ps) mA (ps) mU CGGGGAGUUUUGUUGUUAU 237 EU414A (vp) mUfGmAfCmAfAmAfAmGfAmCfAmCfAmAfGmA (ps) fG (ps) mU UGACAAAAGACACAAGAGU 238 EU414B [ST23 (ps) ] 3ST43 (ps) mAmCmUmCmUmUfGfUfGmUmCmUmUmUmUmGmU (ps) mC (ps) mA ACUCUUGUGUCUUUUGUCA 239 EU201Aun CCUAGGAUCACGCUCUGCU CCUAGGAUCACGCUCUGCU 240 EU201Bun AGCAGAGCGUGAUCCUAGG AGCAGAGCGUGAUCCUAGG 241 EU202Aun GACUUGUUGCAGCUCGCCA GACUUGUUGCAGCUCGCCA 242 EU202Bun UGGCGAGCUGCAACAAGUC UGGCGAGCUGCAACAAGUC 243 EU203Aun CACGACUUGUUGCAGCUCG CACGACUUGUUGCAGCUCG 244 EU203Bun CGAGCUGCAACAAGUCGUG CGAGCUGCAACAAGUCGUG 245 EU204Aun GACACGAAGAUGAUGCCAU GACACGAAGAUGAUGCCAU 246 EU204Bun AUGGCAUCAUCUUCGUGUC AUGGCAUCAUCUUCGUGUC 247 EU205Aun CGGUGAAGGAGAUCAGGUU CGGUGAAGGAGAUCAGGUU 248 EU205Bun AACCUGAUCUCCUUCACCG AACCUGAUCUCCUUCACCG 249 EU209Aun GACCCGAUGAGGUUGUCUA GACCCGAUGAGGUUGUCUA 250 EU209Bun UAGACAACCUCAUCGGGUC UAGACAACCUCAUCGGGUC 251 EU210Aun GGACCCGAUGAGGUUGUCU GGACCCGAUGAGGUUGUCU 252 EU210Bun AGACAACCUCAUCGGGUCC AGACAACCUCAUCGGGUCC 253 EU211Aun GUAAACAGUGCGAAUCUCC GUAAACAGUGCGAAUCUCC 254 EU211Bun GGAGAUUCGCACUGUUUAC GGAGAUUCGCACUGUUUAC 255 EU212Aun GUUGUAAACAGUGCGAAUC GUUGUAAACAGUGCGAAUC 256 EU212Bun GAUUCGCACUGUUUACAAC GAUUCGCACUGUUUACAAC 257 EU213Aun CAUUAUAGGGCUCUGUCAC CAUUAUAGGGCUCUGUCAC 258 EU213Bun GUGACAGAGCCCUAUAAUG GUGACAGAGCCCUAUAAUG 259 EU214Aun GUCAUUAUAGGGCUCCGUC GUCAUUAUAGGGCUCCGUC 260 EU214Bun GACGGAGCCCUAUAAUGAC GACGGAGCCCUAUAAUGAC 261 EU215Aun GAUCAUAUUGAGCUCCUCU GAUCAUAUUGAGCUCCUCU 262 EU215Bun AGAGGAGCUCAAUAUGAUC AGAGGAGCUCAAUAUGAUC 263 EU216Aun GCUGGGUCAUGAUAUCCUC GCUGGGUCAUGAUAUCCUC 264 EU216Bun GAGGAUAUCAUGACCCAGC GAGGAUAUCAUGACCCAGC 265 EU217Aun CUGCGGAUCAUGAAGCAGU CUGCGGAUCAUGAAGCAGU 266 EU217Bun ACUGCUUCAUGAUCCGCAG ACUGCUUCAUGAUCCGCAG 267 EU218Aun GGAGAUUUUCACUUUGAGC GGAGAUUUUCACUUUGAGC 268 EU218Bun GCUCAAAGUGAAAAUCUCC GCUCAAAGUGAAAAUCUCC 269 EU219Aun CUUAUUUCGGGUGUACAGG CUUAUUUCGGGUGUACAGG 270 EU219Bun CCUGUACACCCGAAAUAAG CCUGUACACCCGAAAUAAG 271 EU220Aun GCGCCCGUCUCAAACUUCA GCGCCCGUCUCAAACUUCA 272 EU220Bun UGAAGUUUGAGACGGGCGC UGAAGUUUGAGACGGGCGC 273 EU221Aun CUUAACCCUGUCUUCCCUU CUUAACCCUGUCUUCCCUU 274 EU221Bun AAGGGAAGACAGGGUUAAG AAGGGAAGACAGGGUUAAG 275 EU222Aun CAGUUCCAGGGUAUGGCUC CAGUUCCAGGGUAUGGCUC 276 EU222Bun GAGCCAUACCCUGGAACUG GAGCCAUACCCUGGAACUG 277 EU223Aun CCGAGAGUCAAACUCAUAA CCGAGAGUCAAACUCAUAA 278 EU223Bun UUAUGAGUUUGACUCUCGG UUAUGAGUUUGACUCUCGG 279 EU224Aun GUCAUAACAACAAAACUCC GUCAUAACAACAAAACUCC 280 EU224Bun GGAGUUUUGUUGUUAUGAC GGAGUUUUGUUGUUAUGAC 281 EU225Aun CCAUAUAUCGUUUAGCAAA CCAUAUAUCGUUUAGCAAA 282 EU225Bun UUUGCUAAACGAUAUAUGG UUUGCUAAACGAUAUAUGG 283 EU226Aun AGAUGAUGCCAUCCGGGUU AGAUGAUGCCAUCCGGGUU 284 EU226Bun AACCCGGAUGGCAUCAUCU AACCCGGAUGGCAUCAUCU 285 EU227Aun ACGAAGAUGAUGCCAUCCG ACGAAGAUGAUGCCAUCCG 286 EU227Bun CGGAUGGCAUCAUCUUCGU CGGAUGGCAUCAUCUUCGU 287 EU228Aun UCGGACACGAAGAUGAUGC UCGGACACGAAGAUGAUGC 288 EU228Bun GCAUCAUCUUCGUGUCCGA GCAUCAUCUUCGUGUCCGA 289 EU230Aun UCGGUGAAGGAGAUCAGGU UCGGUGAAGGAGAUCAGGU 290 EU230Bun ACCUGAUCUCCUUCACCGA ACCUGAUCUCCUUCACCGA 291 EU231Aun ACCUCGGUGAAGGAGAUCA ACCUCGGUGAAGGAGAUCA 292 EU231Bun UGAUCUCCUUCACCGAGGU UGAUCUCCUUCACCGAGGU 293 EU233Aun UACAGCUUCAUGCUCUCGC UACAGCUUCAUGCUCUCGC 294 EU233Bun GCGAGAGCAUGAAGCUGUA GCGAGAGCAUGAAGCUGUA 295 EU234Aun AUGAAGAGCAGUGAGUCCU AUGAAGAGCAGUGAGUCCU 296 EU234Bun AGGACUCACUGCUCUUCAU AGGACUCACUGCUCUUCAU 297 EU235Aun AGGUAGUUGCCCUUGCGCC AGGUAGUUGCCCUUGCGCC 298 EU235Bun GGCGCAAGGGCAACUACCU GGCGCAAGGGCAACUACCU 299 EU236Aun UGUGAGGGAGGUGUUGACC UGUGAGGGAGGUGUUGACC 300 EU236Bun GGUCAACACCUCCCUCACA GGUCAACACCUCCCUCACA 301 EU237Aun AUUGUGAGGGAGGUGUUGA AUUGUGAGGGAGGUGUUGA 302 EU237Bun UCAACACCUCCCUCACAAU UCAACACCUCCCUCACAAU 303 EU238Aun UCUAGAAGGAUUGUGAGGG UCUAGAAGGAUUGUGAGGG 304 EU238Bun CCCUCACAAUCCUUCUAGA CCCUCACAAUCCUUCUAGA 305 EU239Aun UUGUCUAGAAGGAUUGUGA UUGUCUAGAAGGAUUGUGA 306 EU239Bun UCACAAUCCUUCUAGACAA UCACAAUCCUUCUAGACAA 307 EU240Aun UGAGGUUGUCUAGAAGGAU UGAGGUUGUCUAGAAGGAU 308 EU240Bun AUCCUUCUAGACAACCUCA AUCCUUCUAGACAACCUCA 309 EU241Aun AUCUCCCCAAAGAUGACAA AUCUCCCCAAAGAUGACAA 310 EU241Bun UUGUCAUCUUUGGGGAGAU UUGUCAUCUUUGGGGAGAU 311 EU243Aun AGCAUAAAGAAUUUGGUGA AGCAUAAAGAAUUUGGUGA 312 EU243Bun UCACCAAAUUCUUUAUGCU UCACCAAAUUCUUUAUGCU 313 EU245Aun ACAGUUUUGGUCCUUAGUU ACAGUUUUGGUCCUUAGUU 314 EU245Bun AACUAAGGACCAAAACUGU AACUAAGGACCAAAACUGU 315 EU246Aun AUAUCCUCUACAGUUUUGG AUAUCCUCUACAGUUUUGG 316 EU246Bun CCAAAACUGUAGAGGAUAU CCAAAACUGUAGAGGAUAU 317 EU247Aun AUAUCUACAAUAUUGGACU AUAUCUACAAUAUUGGACU 318 EU247Bun AGUCCAAUAUUGUAGAUAU AGUCCAAUAUUGUAGAUAU 319 EU248Aun AAUAUCUACAAUAUUGGAC AAUAUCUACAAUAUUGGAC 320 EU248Bun GUCCAAUAUUGUAGAUAUU GUCCAAUAUUGUAGAUAUU 321 EU249Aun UUGACGUAGAGAAUAUCUA UUGACGUAGAGAAUAUCUA 322 EU249Bun UAGAUAUUCUCUACGUCAA UAGAUAUUCUCUACGUCAA 323 EU250Aun AAGUCUUUGACGUAGAGAA AAGUCUUUGACGUAGAGAA 324 EU250Bun UUCUCUACGUCAAAGACUU UUCUCUACGUCAAAGACUU 325 EU251Aun UAGAAGCGAGUGAUAGUCU UAGAAGCGAGUGAUAGUCU 326 EU251Bun AGACUAUCACUCGCUUCUA AGACUAUCACUCGCUUCUA 327 EU252Aun UUGAAGGCAGAGAAGUCAC UUGAAGGCAGAGAAGUCAC 328 EU252Bun GUGACUUCUCUGCCUUCAA GUGACUUCUCUGCCUUCAA 329 EU253Aun UUUCACUUUGAGCUCAUUG UUUCACUUUGAGCUCAUUG 330 EU253Bun CAAUGAGCUCAAAGUGAAA CAAUGAGCUCAAAGUGAAA 331 EU254Aun AACUGGUUGCUGCUGCCUG AACUGGUUGCUGCUGCCUG 332 EU254Bun CAGGCAGCAGCAACCAGUU CAGGCAGCAGCAACCAGUU 333 EU255Aun UUGAGAAGAGUUGUGGUCU UUGAGAAGAGUUGUGGUCU 334 EU255Bun AGACCACAACUCUUCUCAA AGACCACAACUCUUCUCAA 335 EU256Aun UUCAUGUGGGCCCACUGGG UUCAUGUGGGCCCACUGGG 336 EU256Bun CCCAGUGGGCCCACAUGAA CCCAGUGGGCCCACAUGAA 337 EU257Aun UCUUCAUGUGGGCCCACUG UCUUCAUGUGGGCCCACUG 338 EU257Bun CAGUGGGCCCACAUGAAGA CAGUGGGCCCACAUGAAGA 339 EU258Aun UGGACUAACAGGUUCCCUC UGGACUAACAGGUUCCCUC 340 EU258Bun GAGGGAACCUGUUAGUCCA GAGGGAACCUGUUAGUCCA 341 EU259Aun UUCCUUAACCCUGUCUUCC UUCCUUAACCCUGUCUUCC 342 EU259Bun GGAAGACAGGGUUAAGGAA GGAAGACAGGGUUAAGGAA 343 EU260Aun AGUUCCUUAACCCUGUCUU AGUUCCUUAACCCUGUCUU 344 EU260Bun AAGACAGGGUUAAGGAACU AAGACAGGGUUAAGGAACU 345 EU261Aun AUAAAGUUCCUUAACCCUG AUAAAGUUCCUUAACCCUG 346 EU261Bun CAGGGUUAAGGAACUUUAU CAGGGUUAAGGAACUUUAU 347 EU262Aun AAAUAAAGUUCCUUAACCC AAAUAAAGUUCCUUAACCC 348 EU262Bun GGGUUAAGGAACUUUAUUU GGGUUAAGGAACUUUAUUU 349 EU263Aun UACAUAAAGUUCCUUAACC UACAUAAAGUUCCUUAACC 350 EU263Bun GGUUAAGGAACUUUAUGUA GGUUAAGGAACUUUAUGUA 351 EU264Aun AUUAUCAUGUCCCUGAUCA AUUAUCAUGUCCCUGAUCA 352 EU264Bun UGAUCAGGGACAUGAUAAU UGAUCAGGGACAUGAUAAU 353 EU265Aun AUAAAGAACCUGCUUCCGU AUAAAGAACCUGCUUCCGU 354 EU265Bun ACGGAAGCAGGUUCUUUAU ACGGAAGCAGGUUCUUUAU 355 EU266Aun ACUAACUUCAGCAAGGGCA ACUAACUUCAGCAAGGGCA 356 EU266Bun UGCCCUUGCUGAAGUUAGU UGCCCUUGCUGAAGUUAGU 357 EU267Aun UGGUGCAAGACAAUCCCUG UGGUGCAAGACAAUCCCUG 358 EU267Bun CAGGGAUUGUCUUGCACCA CAGGGAUUGUCUUGCACCA 359 EU269Aun UUGGAAAUAAUGAUGACCC UUGGAAAUAAUGAUGACCC 360 EU269Bun GGGUCAUCAUUAUUUCCAA GGGUCAUCAUUAUUUCCAA 361 EU271Aun AUUAUCAUUGUUUUGUGAC AUUAUCAUUGUUUUGUGAC 362 EU271Bun GUCACAAAACAAUGAUAAU GUCACAAAACAAUGAUAAU 363 EU273Aun UCCCCGAGAGUCAAACUCA UCCCCGAGAGUCAAACUCA 364 EU273Bun UGAGUUUGACUCUCGGGGA UGAGUUUGACUCUCGGGGA 365 EU274Aun AACUCCCCGAGAGUCAAAC AACUCCCCGAGAGUCAAAC 366 EU274Bun GUUUGACUCUCGGGGAGUU GUUUGACUCUCGGGGAGUU 367 EU275Aun AACAACAAAACUCCCCGAG AACAACAAAACUCCCCGAG 368 EU275Bun CUCGGGGAGUUUUGUUGUU CUCGGGGAGUUUUGUUGUU 369 EU276Aun AUAACAACAAAACUCCCCG AUAACAACAAAACUCCCCG 370 EU276Bun CGGGGAGUUUUGUUGUUAU CGGGGAGUUUUGUUGUUAU 371 EU277Aun UGACAAAAGACACAAGAGU UGACAAAAGACACAAGAGU 372 EU277Bun ACUCUUGUGUCUUUUGUCA ACUCUUGUGUCUUUUGUCA 373 EU279Aun UCUAGGAUCACGCUCUGCU UCUAGGAUCACGCUCUGCU 374 EU279Bun AGCAGAGCGUGAUCCUAGA AGCAGAGCGUGAUCCUAGA 375 EU280Aun UACUUGUUGCAGCUCGCCA UACUUGUUGCAGCUCGCCA 376 EU280Bun UGGCGAGCUGCAACAAGUA UGGCGAGCUGCAACAAGUA 377 EU281Aun UACGACUUGUUGCAGCUCG UACGACUUGUUGCAGCUCG 378 EU281Bun CGAGCUGCAACAAGUCGUA CGAGCUGCAACAAGUCGUA 379 EU282Aun UACACGAAGAUGAUGCCAU UACACGAAGAUGAUGCCAU 380 EU282Bun AUGGCAUCAUCUUCGUGUA AUGGCAUCAUCUUCGUGUA 381 EU283Aun UGGUGAAGGAGAUCAGGUU UGGUGAAGGAGAUCAGGUU 382 EU283Bun AACCUGAUCUCCUUCACCA AACCUGAUCUCCUUCACCA 383 EU284Aun UUCACGUUGACCAGCUGCU UUCACGUUGACCAGCUGCU 384 EU284Bun AGCAGCUGGUCAACGUGAA AGCAGCUGGUCAACGUGAA 385 EU287Aun UACCCGAUGAGGUUGUCUA UACCCGAUGAGGUUGUCUA 386 EU287Bun UAGACAACCUCAUCGGGUA UAGACAACCUCAUCGGGUA 387 EU289Aun UUAAACAGUGCGAAUCUCC UUAAACAGUGCGAAUCUCC 388 EU289Bun GGAGAUUCGCACUGUUUAA GGAGAUUCGCACUGUUUAA 389 EU290Aun UUUGUAAACAGUGCGAAUC UUUGUAAACAGUGCGAAUC 390 EU290Bun GAUUCGCACUGUUUACAAA GAUUCGCACUGUUUACAAA 391 EU291Aun UAUUAUAGGGCUCUGUCAC UAUUAUAGGGCUCUGUCAC 392 EU291Bun GUGACAGAGCCCUAUAAUA GUGACAGAGCCCUAUAAUA 393 EU292Aun UUCAUUAUAGGGCUCCGUC UUCAUUAUAGGGCUCCGUC 394 EU292Bun GACGGAGCCCUAUAAUGAA GACGGAGCCCUAUAAUGAA 395 EU293Aun UAUCAUAUUGAGCUCCUCU UAUCAUAUUGAGCUCCUCU 396 EU293Bun AGAGGAGCUCAAUAUGAUA AGAGGAGCUCAAUAUGAUA 397 EU294Aun UCUGGGUCAUGAUAUCCUC UCUGGGUCAUGAUAUCCUC 398 EU294Bun GAGGAUAUCAUGACCCAGA GAGGAUAUCAUGACCCAGA 399 EU295Aun UUGCGGAUCAUGAAGCAGU UUGCGGAUCAUGAAGCAGU 400 EU295Bun ACUGCUUCAUGAUCCGCAA ACUGCUUCAUGAUCCGCAA 401 EU296Aun UGAGAUUUUCACUUUGAGC UGAGAUUUUCACUUUGAGC 402 EU296Bun GCUCAAAGUGAAAAUCUCA GCUCAAAGUGAAAAUCUCA 403 EU297Aun UUUAUUUCGGGUGUACAGG UUUAUUUCGGGUGUACAGG 404 EU297Bun CCUGUACACCCGAAAUAAA CCUGUACACCCGAAAUAAA 405 EU298Aun UCGCCCGUCUCAAACUUCA UCGCCCGUCUCAAACUUCA 406 EU298Bun UGAAGUUUGAGACGGGCGA UGAAGUUUGAGACGGGCGA 407 EU299Aun UUUAACCCUGUCUUCCCUU UUUAACCCUGUCUUCCCUU 408 EU299Bun AAGGGAAGACAGGGUUAAA AAGGGAAGACAGGGUUAAA 409 EU300Aun UAGUUCCAGGGUAUGGCUC UAGUUCCAGGGUAUGGCUC 410 EU300Bun GAGCCAUACCCUGGAACUA GAGCCAUACCCUGGAACUA 411 EU301Aun UCGAGAGUCAAACUCAUAA UCGAGAGUCAAACUCAUAA 412 EU301Bun UUAUGAGUUUGACUCUCGA UUAUGAGUUUGACUCUCGA 413 EU302Aun UUCAUAACAACAAAACUCC UUCAUAACAACAAAACUCC 414 EU302Bun GGAGUUUUGUUGUUAUGAA GGAGUUUUGUUGUUAUGAA 415 EU303Aun UCAUAUAUCGUUUAGCAAA UCAUAUAUCGUUUAGCAAA 416 EU303Bun UUUGCUAAACGAUAUAUGA UUUGCUAAACGAUAUAUGA 417 EU406Aun UUAUCUACAAUAUUGGACU UUAUCUACAAUAUUGGACU 418 EU407Aun UAUAUCUACAAUAUUGGAC UAUAUCUACAAUAUUGGAC 419 EU410Aun UUAAAGUUCCUUAACCCUG UUAAAGUUCCUUAACCCUG 420 EU412Aun UUUAUCAUUGUUUUGUGAC UUUAUCAUUGUUUUGUGAC 421 EU413Aun UUAACAACAAAACUCCCCG UUAACAACAAAACUCCCCG 422 Mm n1 A GAACUGAGAAGGAGAGAAA GAACUGAGAAGGAGAGAAA 423 Mm n1 B UUUCUCUCCUUCUCAGUUC UUUCUCUCCUUCUCAGUUC 424 Mm n2 A GGGAGAAGCUGAUGGAGAU GGGAGAAGCUGAUGGAGAU 425 Mm n2 B AUCUCCAUCAGCUUCUCCC AUCUCCAUCAGCUUCUCCC 426 Mm n3 A CAAUGAACUCAAAGUGAAA CAAUGAACUCAAAGUGAAA 427 Mm n3 B UUUCACUUUGAGUUCAUUG UUUCACUUUGAGUUCAUUG 428 Mm n4 A CGGGAGAAGCUGAUGGAGA CGGGAGAAGCUGAUGGAGA 429 Mm n4 B UCUCCAUCAGCUUCUCCCG UCUCCAUCAGCUUCUCCCG 430 Mm n5 A UGGUGAAGGAGGAGUUAAA UGGUGAAGGAGGAGUUAAA 431 Mm n5 B UUUAACUCCUCCUUCACCA UUUAACUCCUCCUUCACCA 432 Mm n6 A GUGAAGGAGGAGUUAAAUA GUGAAGGAGGAGUUAAAUA 433 Mn n6 B UAUUUAACUCCUCCUUCAC UAUUUAACUCCUCCUUCAC 434 Hs n1 A GAUUGUAGCUGUUAAGAAA GAUUGUAGCUGUUAAGAAA 435 Hs n1 B UUUCUUAACAGCUACAAUC UUUCUUAACAGCUACAAUC 436 Hs n2 A AGGCAGAGCUCAAGGGAGA AGGCAGAGCUCAAGGGAGA 437 Hs n2 B UCUCCCUUGAGCUCUGCCU UCUCCCUUGAGCUCUGCCU 438 Hs n3 A GAUUGUAGCUGUUAAGAAA GAUUGUAGCUGUUAAGAAA 439 Hs n3 B UUUCUUAACAGCUACAAUC UUUCUUAACAGCUACAAUC 440 Hs n4 A CGAUGGAGAUUUAGAGUAU CGAUGGAGAUUUAGAGUAU 441 Hs n4 B AUACUCUAAAUCUCCAUCG AUACUCUAAAUCUCCAUCG 442 Hs n5 A GCUGAUGAGUGCAAAGAAA GCUGAUGAGUGCAAAGAAA 443 Hs n5 B UUUCUUUGCACUCAUCAGC UUUCUUUGCACUCAUCAGC 444 Hs n6 A CCUCAAAGCUCAAGGCACA CCUCAAAGCUCAAGGCACA 445 Hs n6 B UGUGCCUUGAGCUUUGAGG UGUGCCUUGAGCUUUGAGG 446 Hs n7 A UCCAUUAUCUCCGACAUGG UCCAUUAUCUCCGACAUGG 447 Hs n7 B CCAUGUCGGAGAUAAUGGA CCAUGUCGGAGAUAAUGGA 448 Hs n7 Ao UCCAUUAUCUCCGACAUGGTG UCCAUUAUCUCCGACAUGG TG 449 Hs n7 Bo CCAUGUCGGAGAUAAUGGATT CCAUGUCGGAGAUAAUGGA TT 450 Mm n7 A UUUGGUGAGAACAAUAGUG UUUGGUGAGAACAAUAGUG 451 Mm n7 B CACUAUUGUUCUCACCAAA CACUAUUGUUCUCACCAAA 452 Mm n7 Ao UUUGGUGAGAACAAUAGUG UUUGGUGAGAACAAUAGUG TT 453 Mm n7 Bo CACUAUUGUUCUCACCAAA CACUAUUGUUCUCACCAAA TT 454 CNNM4 shRNA UCUCUGCCUUCAAGGAUGCGGACAAUGAG UCUCUGCCUUCAAGGAUGC GGACAAUGAG 455 EU415B [ST23 (ps) ] 3ST41 (ps) mGmGmAmGmUmUfUfUfGmUmUmGmUmUmAmUmG (ps) mA (ps) mC GGAGUUUUGUUGUUAUGAC 456 EU416A mG (ps) fU (ps) mCfAmUfAmLAfCmLAfAmCfAmLAfAmAfCmU (ps) fC (ps) mC GUCAUAACAACAAAACUCC 457 EU418A (vp) mUfUmCfAmUfAmAfCmAfAmCfAmAfAmAfCmUfC (ps2) mC UUCAUAACAACAAAACUCC 458 EU418B [ST23 ] 3ST41mG (ps2) mGmAmGmUmUfUfUfGmUmUmGmUmUmAmUmGmA (ps2) mC GGAGUUUUGUUGUUAUGAC 459 EU419B [ST23 ] 3ST41mG (ps2) mGmAmGmUmUfUfUfGmUmUmGmUmUmAmUmGmA (ps2) mA GGAGUUUUGUUGUUAUGAA 460 EU420B [ST23 (ps) ] 3ST41 (ps) mAmCmUmCmUmUfGfUfGmUmCmUmUmUmUmGmU (ps) mC (ps) mA ACUCUUGUGUCUUUUGUCA 461 EU422A (vp) mUfGmAfCmAfAmAfAmGfAmCfAmCfAmAfGmA fG (ps2) mU UGACAAAAGACACAAGAGU 462 EU422B [ST23 ] 3ST41mA (ps2) mCmUmCmUmUfGfUfGmUmCmUmUmUmUmGmUmC (ps2) mA ACUCUUGUGUCUUUUGUCA 463 EU423A mU (ps) fC (ps) mGfAmAfGmUfAmUfUmCfCmGfCmGfUmA (ps) fC (ps) mG UCGAAGUAUUCCGCGUACG 464 EU423B [ST23 (ps) ] 3ST41 (ps) fCmGfUmAfCmGfCmGfGmAfAmUfAmCfUmUfC (ps) mG(ps) fA CGUACGCGGAAUACUUCGA 465 EU304A mU (ps) fC (ps) mCfUmUfGmUfCmCfGmUfCmCfAmCfUmU (ps) fC (ps) mA UCCUUGUCCGUCCACUUCA 466 EU304B mU (ps) mG (ps) mAmAmGmUfGfGfAmCmGmGmAmCmAmAmG (ps) mG (ps) mA UGAAGUGGACGGACAAGGA 467 EU305A mC (ps) fA (ps) mAfUmCfUmUfGmCfGmGfGmCfAmUfArnG (ps) fC (ps) mG CAAUCUUGCGGGCAUAGCG 468 EU305B mC (ps) mG (ps) mCmUmAmUfGfCfCmCmGmCmAmAmGmAmU (ps) mU (ps) mG CGCUAUGCCCGCAAGAUUG 469 EU306A mU (ps) fA (ps) mAfAmCfAmGfUmGfCmGfAmAfUmCfUmC (ps) fC (ps) mU UAAACAGUGCGAAUCUCCU 470 EU306B mA (ps) mG (ps) mGmAmGmAfUfUfCmGmCmAmCmUmGmUmU (ps) mU (ps) mA AGGAGAUUCGCACUGUUUA 471 EU307A mA (ps) fU (ps) mCfAmUfAmUfUmGfAmGfCmUfCmCfUmC (ps) fU (ps) mU AUCAUAUUGAGCUCCUCUU 472 EU307B mA (ps) mA (ps) mGmAmGmGfAfGfCmUmCmAmAmUmAmUmG (ps) mA (ps) mU AAGAGGAGCUCAAUAUGAU 473 EU308A mU (ps) fU (ps) mGfUmUfCmUfUmCfUmCfAmGfAmCfArnC (ps) fC (ps) mC UUGUUCUUCUCAGACACCC 474 EU308B mG (ps) mG (ps) mGmUmGmUfCfUfGmAmGmAmAmGmAmAmC (ps) mA (ps) mA GGGUGUCUGAGAAGAACAA 475 EU309A mG (ps) fA (ps) mGfAmUfUmUfUmCfAmCfUmUfUmGfAmG (ps) fC (ps) mU GAGAUUUUCACUUUGAGCU 476 EU309B mA (ps) mG (ps) mCmUmCmAfAfAfGmUmGmAmAmAmAmUmC (ps) mU (ps) mC AGCUCAAAGUGAAAAUCUC 477 EU310A mA (ps) fA (ps) mUfGmAfCmAfUmCfUmGfGmGfUmAfCmU (ps) fU (ps) mG AAUGACAUCUGGGUACUUG 478 EU310B mC (ps) mA (ps) mAmGmUmAfCfCfCmAmGmAmUmGmUmCmA (ps) mU (ps) mU CAAGUACCCAGAUGUCAUU 479 EU311A mU (ps) fU (ps) mAfUmUfUmCfGmGfGmUfGmUfAmCfAmG (ps) fG (ps) mU UUAUUUCGGGUGUACAGGU 480 EU311B mA (ps) mC (ps) mCmUmGmUfAfCfAmCmCmCmGmAmAmAmU (ps) mA (ps) mA ACCUGUACACCCGAAAUAA 481 EU312A mU (ps) fU (ps) mGfAmUfGmUfAmCfUmGfCmAfAmGfUmC (ps) fC (ps) mA UUGAUGUACUGCAAGUCCA 482 EU312B mU (ps) mG (ps) mGmAmCmUfUfGfCmAmGmUmAmCmAmUmC (ps) mA (ps) mA UGGACUUGCAGUACAUCAA 483 EU313A mG (ps) fA (ps) mCfUmUfCmUfCmGfGmCfCmAfAmGfUmU (ps) fC (ps) mU GACUUCUCGGCCAAGUUCU 484 EU313B mA (ps) mG (ps) mAmAmCmUfUfGfGmCmCmGmAmGmAmAmG (ps) mU (ps) mC AGAACUUGGCCGAGAAGUC 485 EU314A mA (ps) fA (ps) mCfAmGfGmUfUmCfCmCfUmCfUmCfUmU (ps) fC (ps) mA AACAGGUUCCCUCUCUUCA 486 EU314B mU (ps) mG (ps) mAmAmGmAfGfAfGmGmGmAmAmCmCmUmG (ps) mU (ps) mU UGAAGAGAGGGAACCUGUU 487 EU315A mA (ps) fC (ps) mAfUmAfAmAfGmAfAmCfCmUfGmCfUmU (ps) fC (ps) mC ACAUAAAGAACCUGCUUCC 488 EU315B mG (ps) mG (ps) mAmAmGmCfAfGfGmUmUmCmUmUmUmAmU (ps) mG (ps) mU GGAAGCAGGUUCUUUAUGU 489 EU316A mU(ps)fA (ps) mCfUmAfCmUfAmAfCmUfUmCfAmGfCmA (ps) fA (ps) mG UACUACUAACUUCAGCAAG 490 EU316B mC(ps)mU(ps)mUmGmCmUfGfAfAmGmUmUmAmGmUmAmG(ps)mU(ps)mA CUUGCUGAAGUUAGUAGUA 491 EU317A mA (ps) fG (ps) mUfAmCfUmAfCmUfAmAfCmUfUmCfAmG (ps) fC (ps) mA AGUACUACUAACUUCAGCA 492 EU317B mU(ps)mG(ps)mCmUmGmAfAfGfUmUmAmGmUmAmGmUmA(ps)mC(ps)mU UGCUGAAGUUAGUAGUACU 493 EU318A mA(ps)fG(ps)mUfUmCfUmAfUmGfGmCfUmAfGmGfAmG(ps)fA(ps)mC AGUUCUAUGGCUAGGAGAC 494 EU318B mG(ps)mU(ps)mCmUmCmCfUfAfGmCmCmAmUmAmGmAmA(ps)mC(ps)mU GUCUCCUAGCCAUAGAACU 495 EU319A mA(ps)fA(ps)mCfAmAfAmAfGmUfCmUfGmGfUmGfUmC(ps)fU(ps)mU AACAAAAGUCUGGUGUCUU 496 EU319B mA(ps)mA(ps)mGmAmCmAfCfCfAmGmAmCmUmUmUmUmG(ps)mU(ps)mU AAGACACCAGACUUUUGUU 497 EU320A mG(ps)fA(ps)mAfAmUfAmAfUmGfAmUfGmAfCmCfCmU(ps)fC(ps)mU GAAAUAAUGAUGACCCUCU 498 EU320B mA(ps)mG(ps)mAmGmGmGfUfCfAmUmCmAmUmUmAmUmU(ps)mU(ps)mC AGAGGGUCAUCAUUAUUUC 499 EU321A mA(ps)fA(ps)mAfAmUfAmAfAmUfAmCfGmGfCmGfGmC(ps)fU(ps)mG AAAAUAAAUACGGCGGCUG 500 EU321B mC(ps)mA(ps)mGmCmCmGfCfCfGmUmAmUmUmUmAmUmU(ps)mU(ps)mU CAGCCGCCGUAUUUAUUUU 501 EU322A mA (ps) fU (ps) mGfCmAfAmAfAmUfAmAfAmUfAmCfGmG (ps) fC (ps) mG AUGCAAAAUAAAUACGGCG 502 EU322B mC(ps)mG(ps)mCmCmGmUfAfUfUmUmAmUmUmUmUmGmC(ps)mA(ps)mU CGCCGUAUUUAUUUUGCAU 503 EU323A mA(ps)fG(ps)mUfCmAfAmAfCmUfCmAfUmAfAmCfGmC(ps)fC(ps)mA AGUCAAACUCAUAACGCCA 504 EU323B mU(ps)mG(ps)mGmCmGmUfUfAfUmGmAmGmUmUmUmGmA(ps)mC(ps)mU UGGCGUUAUGAGUUUGACU 505 EU324A mC (ps) fA (ps) mUfAmAfCmAfAmCfAmAfAmAfCmUfCmC (ps) fC (ps) mC CAUAACAACAAAACUCCCC 506 EU324B mG(ps)mG(ps)mGmGmAmGfUfUfUmUmGmUmUmGmUmUmA(ps)mU(ps)mG GGGGAGUUUUGUUGUUAUG 507 EU325A mA(ps)fG(ps)mUfCmAfUmAfAmCfAmAfCmAfAmAfAmC(ps)fU(ps)mC AGUCAUAACAACAAAACUC 508 EU325B mG(ps)mA(ps)mGmUmUmUfUfGfUmUmGmUmUmAmUmGmA(ps)mC(ps)mU GAGUUUUGUUGUUAUGACU 509 EU424A mA(ps)fA(ps)mUfGmAfCmAfUmCfUmGfGmGfUmAfCmU(ps)fU(ps)mG AAUGACAUCUGGGUACUUG 510 EU424B [ST23(ps)]3ST41(ps)mCmAmAmGmUmAfCfCfCmAmGmAmUmGmUmCmA(ps)mU(ps)mU CAAGUACCCAGAUGUCAUU 511 EU425A mA(ps)fA(ps)mCfAmGfGmUfUmCfCmCfUmCfUmCfUmU(ps)fC(ps)mA AACAGGUUCCCUCUCUUCA 512 EU425B [ST23(ps)]3ST41(ps)mUmGmAmAmGmAfGfAfGmGmGmAmAmCmCmUmG(ps)mU(ps)mU UGAAGAGAGGGAACCUGUU 513 EU426A mA (ps) fA (ps) mAfAmUfAmAfAmUfAmCfGmGfCmGfGmC (ps) fU (ps) mG AAAAUAAAUACGGCGGCUG 514 EU426B [ST23(ps)]3ST41(ps)mCmAmGmCmCmGfCfCfGmUmAmUmUmUmAmUmU(ps)mU(ps)mU CAGCCGCCGUAUUUAUUUU 515 EU427A mA (ps) fU (ps) mGfCmAfAmAfAmUfAmAfAmUfAmCfGmG (ps) fC (ps) mG AUGCAAAAUAAAUACGGCG 516 EU427B [ST23 (ps) ] 3ST41 (ps) mCmGmCmCmGmUfAfUfUmUmAmUmUmUmUmGmC (ps) mA (ps) mU CGCCGUAUUUAUUUUGCAU 517 EU428A mA (ps) fG (ps) mUfCmAfUmAfAmCfAmAfCmAfAmAfAmC (ps) fU (ps) mC AGUCAUAACAACAAAACUC 518 EU428B [ST23(ps)]3ST41(ps)mGmAmGmUmUmUfUfGfUmUmGmUmUmAmUmGmA(ps)mC(ps)mU GAGUUUUGUUGUUAUGACU 519 EU429A (vp)mUfAmUfGmAfCmAfUmCfUmGfGmGfUmAfCmU(ps)fU(ps)mG UAUGACAUCUGGGUACUUG 520 EU430A (vp) mUfAmCfAmGfGmUfUmCfCmCfUmCfUmCfUmU (ps) fC (ps) mA UACAGGUUCCCUCUCUUCA 521 EU431A (vp) mUfAmAfAmUfAmAfAmUfAmCfGmGfCmGfGmC (ps) fU (ps) mG UAAAUAAAUACGGCGGCUG 522 EU432A (vp)mUfUmGfCmAfAmAfAmUfAmAfAmUfAmCfGmG(ps)fC(ps)mG UUGCAAAAUAAAUACGGCG 523 EU433A (vp) mUfGmUfCmAfUmAfAmCfAmAfCmAfAmAfAmC (ps) fU (ps) mC UGUCAUAACAACAAAACUC 524 EU304Aun UCCUUGUCCGUCCACUUCA UCCUUGUCCGUCCACUUCA 525 EU304Bun UGAAGUGGACGGACAAGGA UGAAGUGGACGGACAAGGA 526 EU307Aun AUCAUAUUGAGCUCCUCUU AUCAUAUUGAGCUCCUCUU 527 EU307Bun AAGAGGAGCUCAAUAUGAU AAGAGGAGCUCAAUAUGAU 528 EU310Aun AAUGACAUCUGGGUACUUG AAUGACAUCUGGGUACUUG 529 EU310Bun CAAGUACCCAGAUGUCAUU CAAGUACCCAGAUGUCAUU 530 EU312Aun UUGAUGUACUGCAAGUCCA UUGAUGUACUGCAAGUCCA 531 EU312Bun UGGACUUGCAGUACAUCAA UGGACUUGCAGUACAUCAA 532 EU314Aun AACAGGUUCCCUCUCUUCA AACAGGUUCCCUCUCUUCA 533 EU314Bun UGAAGAGAGGGAACCUGUU UGAAGAGAGGGAACCUGUU 534 EU316Aun UACUACUAACUUCAGCAAG UACUACUAACUUCAGCAAG 535 EU316Bun CUUGCUGAAGUUAGUAGUA CUUGCUGAAGUUAGUAGUA 536 EU317Aun AGUACUACUAACUUCAGCA AGUACUACUAACUUCAGCA 537 EU317Bun UGCUGAAGUUAGUAGUACU UGCUGAAGUUAGUAGUACU 538 EU319Aun AACAAAAGUCUGGUGUCUU AACAAAAGUCUGGUGUCUU 539 EU319Bun AAGACACCAGACUUUUGUU AAGACACCAGACUUUUGUU 540 EU321Aun AAAAUAAAUACGGCGGCUG AAAAUAAAUACGGCGGCUG 541 EU321Bun CAGCCGCCGUAUUUAUUUU CAGCCGCCGUAUUUAUUUU 542 EU322Aun AUGCAAAAUAAAUACGGCG AUGCAAAAUAAAUACGGCG 543 EU322Bun CGCCGUAUUUAUUUUGCAU CGCCGUAUUUAUUUUGCAU 544 EU323Aun AGUCAAACUCAUAACGCCA AGUCAAACUCAUAACGCCA 545 EU323Bun UGGCGUUAUGAGUUUGACU UGGCGUUAUGAGUUUGACU 546 EU325Aun AGUCAUAACAACAAAACUC AGUCAUAACAACAAAACUC 547 EU325Bun GAGUUUUGUUGUUAUGACU GAGUUUUGUUGUUAUGACU 548 EU429Aun UAUGACAUCUGGGUACUUG UAUGACAUCUGGGUACUUG 549 EU430Aun 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