RNA Editing Inhibitors and Their Uses

Abstract

The present invention provides oligonucleotides targeting the core editing site complementary sequence (ECS) of the AZIN1 gene, wherein the core ESC of the AZIN1 gene comprises the sequence 5'-GCTTTTCC-3', and wherein the oligonucleotide comprises one or more nucleotides with sugar modifications and one or more modified internucleotide linkages. On the other hand, a pharmaceutical composition comprising the oligonucleotides disclosed herein is provided. On the other hand, a method of inhibiting AZIN1 pre-mRNA editing in cells is provided, wherein the AZIN1 pre-mRNA editing is mediated by the adenosine deaminase acting on RNA-1 (ADAR-1), and a method of treating cancers associated with AZIN1 pre-mRNA editing, including liver cancer, using the oligonucleotide is provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to Singapore Application No. 10201906239R, filed on Jul. 4, 2019, the content of which is incorporated herein by reference in its entirety for all purposes. Technical field

[0003] The present invention generally relates to the fields of molecular biology, cell biology, and biotechnology. In particular, the present invention relates to oligonucleotides for inhibiting RNA editing, compositions comprising the oligonucleotides, and uses of the oligonucleotides and compositions. Background art

[0004] Cancer generally refers to a group of diseases involving abnormal cell growth that have the potential to invade or spread to other parts of the body. Cancer has a high prevalence worldwide, estimated to be as high as 90.5 million in 2015. The Centre for Disease Control (CDC) projects that between 2010 and 2020, the number of new cancer cases in the United States may increase by approximately 24% in men, exceeding 1 million cases per year, and by approximately 21% in women, exceeding 900,000 cases per year. The cancer types expected to increase the most are: melanoma in white men and women, prostate, kidney, liver, and bladder cancers in men, and lung, breast, uterine, and thyroid cancers in women.

[0005] RNA editing is a prevalent process that introduces changes in the RNA sequences encoded by the genome, resulting in “RNA mutations”. Over the past decade, abnormal RNA editing of specific genes and its association with cancer progression have been found in many cancer types, including but not limited to hepatocellular carcinoma (HCC), esophageal squamous cell carcinoma (ESCC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC).

[0006] Adenosine deaminase acting on RNA (ADAR) is an enzyme encoded by the ADAR gene in the human body. ADAR is an RNA-binding protein that plays a role in RNA editing by post-transcriptionally modifying mRNA transcripts by altering the nucleotide content of RNA. ADAR is responsible for binding to double-stranded RNA (dsRNA) and converting adenosine (A) to inosine (I) through deamination. Inosine is structurally similar to guanine (G), causing I to pair with cytosine (C). Inosine typically mimics guanosine during translation. Thus, the conversion from A to I in RNA disrupts normal A:U pairing and makes the RNA unstable. Codon changes may also be caused by editing that can lead to changes in the protein-coding sequence and its function. ADAR also affects the transcriptome in an editing-independent manner, possibly by interfering with other RNA-binding proteins.

[0007] In mammals, there are three types of ADAR, namely ADAR1, ADAR2, and ADAR3. ADAR1 and ADAR2 are present in many tissues in the body, while ADAR3 is only present in the brain. Studies have shown that ADAR1 and ADAR2 are often dysregulated in cancer. It is thought that ADAR1 is responsible for the disruption of the A-to-I editing pattern observed in various cancers. Dysregulation of ADAR1 expression may alter the frequency of A-to-I conversion in the protein-coding regions of oncogenes or tumor suppressor genes, leading to mutations in oncogene or tumor suppressor gene products and thus promoting the development of cancer.

[0008] Since the ADAR protein has thousands of editing substrates, simply regulating ADAR expression can lead to significant off-target effects. Therefore, ADAR inhibitors that specifically inhibit the RNA editing of oncogenes or tumor suppressor genes targeted by ADAR are needed. Summary of the Invention

[0010] In one aspect of the present invention, oligonucleotides targeting the core editing-site complementary sequence (ECS) of the AZIN1 gene are provided, wherein the core ECS of the AZIN1 gene comprises the sequence 5'-GCTTTTCC-3', and wherein the oligonucleotides comprise one or more nucleotides with sugar modifications and one or more modified internucleotide linkages. In another aspect, a pharmaceutical composition comprising the oligonucleotides disclosed herein is provided. In another aspect, a method of inhibiting AZIN1 pre-mRNA editing in cells is provided, the method comprising contacting the cells with the oligonucleotides disclosed herein or the pharmaceutical composition disclosed herein. In another aspect, a method of treating cancer in an individual in need thereof is provided, comprising administering to the individual a therapeutically effective amount of the oligonucleotides disclosed herein or the pharmaceutical composition disclosed herein, wherein the cancer is associated with AZIN1 pre-mRNA editing.

[0011] Brief Description of the Drawings

[0012] The present invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the drawings, in which:

[0013] Figure 1 It shows that the 3'-terminal sequence of exon 12 is required for AZIN1 editing. Figure 1 A is a schematic diagram of AZIN1 minigene constructs generated by inserting five different fragments (FA, FB, FC, FD or FE) covering the edited exon 11 and the flanking exons (exons 10 and 12) and introns (introns 9, 10, 11 and 12) into the pRK7 or pcDNA3.1 vector. The arrows indicate the relative positions of the editing sites. Figure 1 B and 1C are sequencing chromatograms illustrating the endogenous AZIN1 ( Figure 1 B, left panel) and exogenous HTR2C ( Figure 1 B, right panel) or AZIN1 ( Figure 1 C) transcripts edited in HEK293T cells co-transfected with the indicated pRK7 minigene and an empty vector (EV) or an ADAR1 expression construct (ADAR1). Figure 1 D shows a sequencing chromatogram illustrating the editing of endogenous and exogenous AZIN1 transcripts in HEK293T cells co-transfected with a pcDNA3.1-based minigene and EV or ADAR1. In Figure 1 B-1D, the editing percentage was calculated as the area of the "G" peak divided by the total area of the "A" and "G" peaks. * indicates no editing detected. The black arrows indicate the positions of the editing sites.Figure 1 Panel E shows the RNA secondary structure of AZIN1 predicted by RNAfold. The 8-nt sequence indicated by the small arrow on the right is the potential core ECS. The editing site is shown by the solid arrow on the left. The minimum free energy (MFE) structure depicting the coding base pair probabilities is shown. The base pair probabilities are shown as a chromatogram. In summary, Figure 1 it was shown that among all AZIN1 minigenes (using either the pRK7- or pcDNA3.1-based minigene systems), only the AZIN1 transcripts transcribed from the minigenes containing fragment A (FA), which lacks a 90-bp sequence at the 3′ end of exon 12, could not be edited. This indicates that the ECS of AZIN1 is located at the 3′ end of exon 12.

[0014] Figure 2 It was shown that the 8-nt sequence at the 3′ end of exon 12 is the core ECS and is essential for AZIN1 editing. Figure 2 Panel A is a schematic diagram of the FE-1, 2, and 3 minigene constructs. The small arrow at the bottom indicates the mutation introduced into the FE-3 minigene. The large arrow at the top indicates the relative position of the editing site. Figure 2 Panel B shows the RNA secondary structure of the AZIN1 transcript transcribed from the indicated minigene predicted by RNAfold. The black arrow indicates the editing site. The MFE structure depicting the coding base pair probabilities is shown. The base pair probabilities are shown as a chromatogram. Figure 2 Panel C shows the sequencing chromatograms illustrating the editing of endogenous and exogenous AZIN1 transcripts in HEK293T cells co-transfected with the pRK7-based minigene and either EV or ADAR1. Figure 2 Panel D shows the results of in vitro RNA editing analysis of the AZIN1 transcript. Figure 2 Left panel of D: In vitro transcribed HTR2C or AZIN1 transcripts from the indicated minigene constructs were incubated with purified ADAR1 protein and then subjected to RNA editing analysis using Sanger sequencing. In vitro transcribed HTR2C was used as a positive control. Figure 2 Right panel of D: The data are represented as the mean ± s.d. of three technical replicates of a representative experiment in a bar graph. n.d., not detected. In Figure 2 Panels C and 2D, the percentage of editing was calculated as the area of the “G” peak divided by the total area of the “A” and “G” peaks. *, editing not detected. The black arrow indicates the position of the editing site. In summary, Figure 2We showed that transcripts from the FE-1 (missing the 29-bp sequence at the 3' end of exon 12), FE-2 (missing the 8-bp sequence near the 3' end of exon 12), and FE-3 (point mutation near the 3' end of exon 12) minigenes could not be edited when ADAR1 was overexpressed, suggesting that the 8-nt sequence at the 3' end of exon 12 (5'-GCUUUUCC-3') is the core ECS edited by AZIN1.

[0015] Figure 3 Screening for effective antisense oligonucleotides (ASOs) that can bind to AZIN1 duplexes and inhibit AZIN1 editing in vitro is shown. Figure 3 A shows a schematic representation of ASO design. ASOs targeting the editing region or ECS region were designed using short RNA duplexes containing a partial exon 11 with adenosine undergoing deamination (editing site, solid underline) and a partial exon 12 sequence containing an ECS with a core 8-nt ECS (ECS region, dotted underline). ASP1, DSP1, and DSP2 are peptide nucleic acids (PNA), while ASOs 1-7 are ASOs using canonical bases modified with 2'-O-Me. Table 3 lists the sequence of each oligonucleotide and its characteristics. Figure 3 B shows the results of REMSA performed to examine the binding of each ASO (2.5 μM) to 32P-labeled AZIN1 RNA duplex (86-nt). Figure 9 A provides the sequence and predicted structure of the duplex probe. Vehicle control (VC) means no ASO was added. Figure 3 C shows the binding of ASO1, 3, 5 or 7 to 32P-labeled AZIN1 RNA duplex at different concentrations as shown detected by REMSA. Figure 3 D shows in vitro RNA editing analysis of AZIN1 transcripts transcribed from the FE minigene after incubation with purified ADAR1 protein and 200 nM of the indicated ASOs. Figure 3 D Top panel: Sequencing chromatograms illustrating editing of in vitro transcribed AZIN1 transcripts in the indicated samples. Percent editing was calculated as the area of ​​the "G" peak divided by the total area of ​​the "A" and "G" peaks. Arrows indicate the locations of editing sites. *, no editing detected. Figure 3 D Bottom panel: Data are presented in bar graphs as mean ± SD of three independent experiments. The value shown at the top of each bar is the mean. nd, not detectable. Figure 3It was shown that ASO1, ASO3, ASO5, and ASO7 could bind to AZIN1 dsRNA in a dose-dependent manner, and ASO1 and ASO3 could completely inhibit AZIN1 editing in vitro, while ASO5 could substantially inhibit AZIN1 editing in vitro.

[0016] Figure 4 It was shown that ECS-targeting ASOs eliminated or inhibited AZIN1 editing in cancer cells. Figure 4 A shows a schematic illustration of ASO chemical modification. The fully 2'-O-Me modified ASO1 and ASO3 were further fully or partially modified with phosphorothioate (PS) bonds indicated by asterisks (see also Table 3). Figure 4 B shows the results of semi-quantitative PCR analysis of AZIN1 transcripts in KYSE510 and H358 cells treated with 100 nM of each of the indicated ASOs. Agarose gel electrophoresis of the PCR amplicons showed two isoforms of AZIN1. The fast-moving band indicates the exon 11-skipping isoform of AZIN1. Figure 10 B shows the Sanger sequencing chromatogram data of the junction between exon 10 and exon 12. Figure 4 The results in B indicated that 7 ASOs (ASO1, 1.1, 1.2, 1.3, 5, 6, and 7) targeting the editing region led to exon 11 skipping. Figure 4 C shows the results of in silico prediction of splicing factor binding sites on the editing region of AZIN1 pre-mRNA by SpliceAid231. It was predicted that SRSF3, SRSF6, and SRSF1 would bind to the editing region. The editing sites are underlined. Figure 4 D shows the results of QPCR analysis of AZIN1 expression in KYSE510 cells treated with 100 nM of each of the indicated ASOs. The data were represented as the mean ± sd of three technical replicates of a representative experiment. Figure 4 E shows the results of Western blot analysis of AZIN1 and ADAR1 protein expression in KYSE510 cells treated with 100 nM of each of the indicated ASOs. Approximately 20 μg of protein lysates extracted from HEK293T cells transfected with the AZIN1 expression construct were included as a positive control for AZIN1 protein. GAPDH was used as a loading control. Figure 4 F and 4G are sequencing chromatograms showing the editing of AZIN1 transcripts in KYSE510 cells treated with 100 nM of each of the indicated ASOs. The editing percentage was calculated as the area of the "G" peak divided by the total area of the "A" and "G" peaks. The arrow indicates the position of the editing site. *, no editing was detected. The data (G) were represented as the mean ± sd of three technical replicates of a representative experiment. The value shown at the top of each bar is the mean. n.d., not detected. In summary, Figure 4D-4G indicated that among the three ECS-targeting ASOs, ASO3.1 and ASO3.2 completely abolished AZIN1 editing, while ASO3.3 significantly inhibited editing but did not affect the splicing and expression of AZIN1 at the mRNA and protein levels.

[0017] Figure 5 Shown that ASO3.2 specifically inhibits the G1 / S transition and cancer cell viability. Figure 5 A shows the cell viability of KYSE510 (K510), H358 or KYSE180 (K180) cells measured by (CTG) assay after treatment with different concentrations (1, 10, 25, 50, 100, 150, 200 and 250 nM) of ASO3.1, ASO3.2 or ASO-ctl for 48 h. The corresponding half-maximal inhibitory concentration (IC50) values for each cell line are shown. Data are represented as the mean ± sd of four replicates of a representative experiment. The results in A indicated that both ASO3.1 and ASO3.2 significantly inhibited the cell viability of KYSE510 and H358 with low IC50 values, while their inhibitory effects on the cell viability of KYSE180 were much smaller. Figure 5 B shows the cell viability of each of the three cancer cell lines and normal hepatocytes measured by CTG assay after treatment with 50 nM ASO3.2 or ASO-ctl for 48 h. ASO1 and ASO3, which were unable to inhibit AZIN1 editing, served as two additional negative controls. Figure 5 C shows the foci formation assay in each of the three cell lines after treatment with the indicated concentrations of ASO3.2 or ASO-ctl for 48 h. Cells were stained with crystal violet. Figure 5 The results in B and 5C indicated that ASO3.2 could specifically inhibit the cell viability of cancer cells expressing edited AZIN1 Figure 5 S367G of. The results in D left panel: Cells were treated with 50 nM ASO3, ASO3.2 or ASO-ctl for 48 h, then stained with PI and subjected to cell cycle analysis by flow cytometry. The raw FACS data were analyzed using BDFACSDiva software, which plotted cell counts versus DNA content. Figure 5 D right panel: Bar graphs show the percentages of cells in the sub-G1, G1, S and G2 / M phases of a representative experiment. Figure 5 The results in D indicated that compared with cells treated with ASO-ctl or ASO3, after ASO3.2 treatment, KYSE510 and H358 cells showed a marked attenuation of G1 / S transition and a significant increase in the percentage of sub-G1 phase (apoptotic cells). Figure 5 ​Figure 5 E shows Figure 5 The results of Western blot analysis of CCND1 and ODC protein expression in KYSE510 cells described in D. GAPDH was used as a loading control. Figure 5 The results in E showed that a significant decrease in CCND1 and ODC protein expression was observed in cells treated with ASO3.2, thus supporting Figure 5 the G1 / S arrest induced by ASO3.2 shown in D.

[0018] Figure 6 It shows that ASO3.2 specifically inhibits tumorigenesis and growth in vivo. Figure 6 A shows the cumulative tumor incidence curves of NOD scidγ (NOD scid gamma, NSG) mice subcutaneously injected with KYSE510 cells, and the KYSE510 cells were pretreated with 100 nM ASO3.2 or ASO-ctl for 48 hours. The cells pretreated with ASO3.2 or ASO-ctl were injected into the right or left dorsal side of the mice, respectively. Figure 6 The results in A showed that the tumor incidence in the ASO3.2 pretreatment group was significantly lower than that in the ASO-ctl pretreatment group. Figure 6 B shows representative tumors derived from the above-pretreated KYSE510 cells 6 weeks after subcutaneous injection (n = 6 mice per group), and the growth curves of tumors derived from the pretreated cells of each group within 6 weeks. Data are presented as mean ± sd. **P < 0.01, ***P < 0.001, determined by unpaired two-tailed Student's t-test. Figure 6 The results in B showed that within the 6-week observation period, the growth of tumors derived from ASO-ctl-pretreated cells was significantly faster than that of tumors derived from ASO3.2-pretreated cells. Figure 6 C shows representative fluorescence microscopy images of KYSE510 cells treated with ASO3.2 loaded into CFSE-labeled RBCEV. DAPI staining indicates the cell nuclei. Scale bar, 500 μm. Figure 6 The results in C showed that most ASO3.2-RBCEV could enter the cells. Figure 6 D shows representative tumors (n = 6 mice per group) derived from KYSE510 cells after intratumoral (i.t.) injection of ASO3.2-RBCEV or ASO-ctl-RBCEV every 4 days. For each injection, a total of 1 μg ASO was loaded into 50 μg RBCEV and resuspended in 20 μL PBS. The growth curves of tumors in each group within 7 weeks are shown. Data are presented as mean ± sd. *P < 0.05, **P < 0.01, determined by unpaired two-tailed Student's t-test. Black arrows indicate each injection.Figure 6 The results in D showed that intratumoral injection of ASO3.2-RBCEV significantly inhibited tumor growth. Figure 6 E shows representative tumors after multiple i.t. injections of naked ASO3.2 or ASO-ctl. The same experimental procedures as Figure 6 described in D were performed. Figure 6 The results in E showed that no significant difference in tumor growth was observed between mice treated with naked ASO-ctl and ASO3.2. Figure 6 The sequencing chromatograms shown in F illustrate the editing of the AZIN1 transcript in the indicated PDX lines. The editing percentage was calculated as the area of the "G" peak divided by the total area of the "A" and "G" peaks. The black arrow indicates the position of the editing site. *, no editing was detected. Figure 6 The results in F showed that four PDX cells (PDX-1; and PDX-22-T1, T4, and T5 from different sectors of PDX-22) had more than 20% edited AZIN1 transcripts. Figure 6 G shows the cell viability of PDX1 (top panel) or PDX22-T3 (bottom panel) measured by the CTG assay after treatment with the indicated concentrations of ASO3.2 or ASO-ctl (delivered by Lipofectamine). Data are represented as the mean ± sd of four replicates of a representative experiment. *P < 0.05, **P < 0.01, ***P < 0.001, determined by unpaired two-tailed Student's t-test. In summary, Figure 6 The results in F and 6G showed that ASO3.2 treatment could significantly reduce the cell viability of AZIN1 editing-positive cell lines, but not that of non-AZIN1 editing cell lines.

[0019] Figure 7 The results of quantitative real-time PCR (QPCR) analysis of ADAR1 expression in HEK293T cells co-transfected with the indicated pRK7 minigene and empty vector (EV) or ADAR1 expression construct (ADAR1) are shown. The results showed that ADAR1 was successfully overexpressed in all samples co-transfected with the ADAR1 expression construct.

[0020] Figure 8 The results of quantitative real-time PCR (QPCR) analysis of ADAR1 expression in HEK293T cells co-transfected with the indicated pRK7 minigene and empty vector (EV) or ADAR1 expression construct (ADAR1) are shown. The results showed that ADAR1 was successfully overexpressed in all samples co-transfected with the ADAR1 expression construct.

[0021] Figure 9Panel A shows the dsRNA secondary structure of the 86-nt AZIN1 duplex probe for REMSAs predicted by RNAFold. Figure 9 The REMSAs data shown in Panel B demonstrate the binding of ASP1, DSP1, or DSP2 to the truncated AZIN1 duplex probe. The truncated RNA duplex was 0.25 μM. The ASP1 concentrations were 0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.4, 0.7, 1, 1.5, and 2 μM from left to right. ASP1 showed no binding up to 2 μM. For DSP1 and DSP2, the truncated RNA duplex was 1 μM. The DSP1 and DSP2 concentrations were 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.4, 1, 2, 4, 10, and 20 μM from left to right. Both DSP1 and DSP2 showed binding to the RNA duplex at μM concentrations. Figure 9 Panel B also shows the complete sequences of the ASOs and PNAs and their positions on the short duplexes in the editing regions and ECS regions on exons 11 and 12 of AZIN1. Figure 9 The results in Panel B indicate that ASP1 cannot bind to the shortened AZIN1 RNA duplex, while DSP1 and DSP2 can bind with moderate binding affinity through PNA-dsRNA triplex formation. Figure 9 Panel C shows the sequencing chromatograms of in vitro RNA editing analysis of AZIN1 transcripts transcribed from the FE mini-gene after incubation with purified ADAR1 protein and 10 μM (left) and 200 nM (right) of DSP1 or DSP2. The editing percentage was calculated as the area of the "G" peak divided by the total area of the "A" and "G" peaks. Arrows indicate the positions of the editing sites. *, no editing detected. Figure 9 The results in Panel C indicate that DSP1 and DSP2 are able to abolish AZIN1 editing at a concentration of 10 μM, but their editing inhibitory effects are significantly attenuated at 200 nM.

[0022] Figure 10 The sequencing chromatograms shown in Panel A illustrate the editing of AZIN1 transcripts in the indicated HCC, ESCC, and NSCLC cell lines. The editing percentage was calculated as the area of the "G" peak divided by the total area of the "A" and "G" peaks. Arrows indicate the positions of the editing sites. *, no editing detected. Figure 10 The results in Panel A indicate that AZIN1 editing was detected only in the ESCC line KYSE510 and the NSCLC line H358 among the screened HCC, ESCC, and NSCLC cell lines. Figure 10 The sequencing chromatograms shown in Panel B illustrate exon 11 skipping of AZIN1 transcripts detected in KYSE510 cells treated with 100 nM ASO1.1 for 48 hours.

[0023] Definition

[0024] As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of linked nucleotides. As used herein, the term "oligomeric compound" refers to a polymeric structure comprising two or more substructures and capable of hybridizing to a region of a nucleic acid molecule. In some instances, the oligonucleotide may be introduced in single-stranded, double-stranded, circular, branched, or hairpin form, and may contain structural elements such as internal or terminal bulges or loops. A double-stranded oligonucleotide may be formed from two oligonucleotide strands hybridized together or a single oligonucleotide strand having sufficient self-complementarity to permit hybridization and formation of a fully or partially double-stranded compound.

[0025] As used herein, the term "nucleoside" refers to a glycosylamine comprising a nucleobase and a sugar. Nucleosides include, but are not limited to, natural nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and / or sugars. As used herein, the term "natural nucleoside" or "unmodified nucleoside" refers to a nucleoside comprising a natural nucleobase and a natural sugar. Natural nucleosides include RNA and DNA nucleosides. As used herein, the term "nucleobase" refers to the base portion of a nucleoside or nucleotide. A nucleobase may comprise any atom or group of atoms capable of forming a hydrogen bond with the base of another nucleic acid. As used herein, the term "natural nucleobase" refers to a nucleobase that has not been modified from its naturally occurring form in RNA or DNA. Examples of "natural nucleobases" include the purine nucleobases adenine (A) and guanine (G) and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U). In addition to "natural nucleobases", many modified nucleobases or nucleobase mimetics known to those of ordinary skill in the art are suitable for use in the compounds described herein. The terms "modified nucleobase" and "nucleobase mimetic" may overlap, but generally "modified nucleobase" refers to a nucleobase that is structurally very similar to the parent nucleobase, such as 7-deazapurine, 5-methylcytosine, or G-clamp, while "nucleobase mimetic" would include more complex structures such as tricyclic phenoxazine nucleobase mimetics.

[0026] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group covalently linked to the sugar. Nucleotides may be modified with any of a variety of substituents.

[0027] As used herein, the terms "targeting" or "targeted to" refer to the association of a compound with a specific target nucleic acid molecule or a specific nucleotide region within a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to permit hybridization under physiological conditions.

[0028] As used herein, the term "RNA editing" refers to a co-transcriptional or post-transcriptional modification process that introduces changes into the RNA sequence encoded by the genome, resulting in RNA mutations. Adenosine editing to inosine (A-to-I) in double-stranded RNA (dsRNA), catalyzed by adenosine deaminases acting on RNA (ADAR) enzyme family, is a common type of RNA editing in mammals. In vertebrates, the family of three ADAR proteins, ADAR1, ADAR2, and ADAR3, has been previously characterized. ADAR1 and ADAR2 (ADAR) catalyze all currently known A-to-I editing sites. ADAR3 has no known deaminase activity. Inosine (I) mimics guanosine (G), so ADAR proteins introduce a virtual A-to-G substitution in the transcript. Such changes can lead to specific amino acid substitutions, alternative splicing, microRNA-mediated gene silencing, or changes in transcript localization and stability.

[0029] As used herein, the term "AZIN1 gene" refers to the gene encoding the antizyme inhibitor 1 protein. Antizyme inhibitor 1 belongs to the antizyme inhibitor family and plays a role in cell growth and proliferation by maintaining intracellular polyamine homeostasis. Antizyme inhibitors are homologs of ornithine decarboxylase (ODC, a key enzyme in polyamine biosynthesis) that have lost the ability to decarboxylate ornithine but retain the ability to bind to antizyme. Antizyme negatively regulates intracellular polyamine levels by binding to ODC and targeting it for degradation, as well as by inhibiting polyamine uptake. Antizyme inhibitors act as positive regulators of polyamine levels by chelating antizyme and neutralizing its action. Antizyme inhibitor 1 is widely expressed and localized in the nucleus and cytoplasm of cells. Overexpression of the AZIN1 gene is associated with increased proliferation, cell transformation, and tumorigenesis. In one specific example, the sequence of the AZIN1 gene is SEQ ID NO:3, encoding the protein of SEQ ID NO:4.

[0030] An "ADAR enzyme" is a double-stranded RNA-specific adenosine deaminase capable of modifying a polynucleotide at a specific nucleic acid (e.g., mRNA). In some examples, the ADAR enzyme post-transcriptionally modifies or "edits" the mRNA sequence, for example, by converting adenosine to inosine. Since inosine mimics the activity of guanosine (e.g., pairing with cytosine), this can effectively result in the formation of single nucleotide polymorphisms in the transcribed mRNA sequence. In some examples, the editing can lead to the formation of "cryptic" splicing sites, recombination motifs, or other nucleic acid elements.

[0031] As used herein, the term "editing site complementary sequence" or the abbreviation "ECS" refers to a sequence that can form a double-stranded RNA structure and cover the adenosine-to-inosine editing site and its surrounding region in the untranslated region (UTR), exon, or intron of the gene to be edited. In some examples, the ECS is located in the intron of the gene to be edited. In some examples, the ECS can form an imperfect folded-back double-stranded RNA structure, while the exon sequence surrounds the adenosine-to-inosine editing side. In some examples, the ECS for ADAR1-mediated pre-mRNA editing of AZIN1 comprises the 29-nucleotide sequence 5'-AAGAAGACAGCUUUUCCGCUGAAGCUUAA-3' (SEQ ID NO:1) located near the 3' end of exon 12 of AZIN1 or consists thereof. As used herein, the term "core ECS" in the context of "core ECS for ADAR1-mediated pre-mRNA editing of AZIN1" refers to a specific portion of the ECS that the inventors of the present application have found to be crucial for ADAR1-mediated pre-mRNA editing of AZIN1 (i.e., deletion of the core ECS results in inhibition of ADAR1-mediated pre-mRNA editing of AZIN1). In some examples, the core ECS for ADAR1-mediated pre-mRNA editing of AZIN1 comprises the 8-nucleotide sequence 5'-GCTTTTCC-3' located near the 3' end of exon 12 of AZIN1 or consists thereof.

[0032] As used herein, the term "editing region" refers to a sequence in a gene, such as the AZIN1 gene, that is recognized and / or targeted by ADAR-1 for editing. In some examples, the editing region for ADAR1-mediated pre-mRNA editing of AZIN1 comprises the sequence 5’-UGAGCUUGAUCAAAUUGUGGAA A GCUGUCUUCUUCCUGAGCU-3’ (SEQID NO:2) (underlined "A" is the adenosine-to-inosine editing site) or consists thereof. In some examples, the sequence 5'-GGAA A GC-3' is considered to be a "sequence or region containing the editing site". It is generally believed that ADAR1-mediated pre-mRNA editing does not occur in the absence of the adenosine-to-inosine editing site.

[0033] As used interchangeably herein, the terms "sugar modification" or "modified sugar" refer to a sugar moiety that is not the ribofuranosyl found in natural RNA or the deoxyribofuranosyl found in natural DNA. Modified sugar moieties can be used to alter, and generally increase, the affinity of an antisense compound for its target and / or increase nuclease resistance. "Modified sugars" include, but are not limited to, substituted sugars, bicyclic or tricyclic sugars, or sugar surrogates. As used herein, a "substituted sugar moiety" refers to a furanosyl that contains at least one substituent that is different from the naturally occurring sugar moiety. Substituted sugars include, but are not limited to, furanosyls that contain substituents at the 2'-position, 3'-position, 5'-position, and / or 4'-position. As used herein, a "2'-substituted sugar" refers to a furanosyl that contains a substituent other than H or OH at the 2'-position. Unless otherwise specified, a 2'-substituted sugar is not a bicyclic sugar (i.e., the 2'-substituent of the 2'-substituted sugar moiety does not form a bridge with another atom of the furanosyl ring). Examples of sugar substituents suitable for the 2'-position include, but are not limited to: 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro. In some instances, the sugar substituent at the 2'-position is selected from allyl, amino, azido, thio, O-allyl, O-C 1 -C 10 alkyl, O-C 1 -C 10 substituted alkyl; O-C1-C 10 alkoxy; O-C 1 -C 10 substituted alkoxy, OCF3, O(CH 2 ) 2 SCH 3 、O(CH 2 ) 2 —O—N(Rm)(Rn) and O—CH 2 —C(═O)—N(Rm)(Rn), where each Rm and Rn is independently H or substituted or unsubstituted C 1 -C 10 alkyl.

[0034] As used herein, a "bicyclic sugar" refers to a modified sugar that contains a 4- to 7-membered ring (including, but not limited to, a furanosyl), the 4- to 7-membered ring containing a bridge that connects two atoms of the 4- to 7-membered ring to form a second ring, resulting in a bicyclic structure. In some instances, the 4- to 7-membered ring is a sugar ring. In some instances, the 4- to 7-membered ring is a furanosyl. In some such instances, the bridge connects the 2'-carbon and the 4'-carbon of the furanosyl.

[0035] As used herein, the term "bicyclic nucleoside" or "BNA" refers to a nucleoside in which the furanosyl moiety of the nucleoside includes a bridge connecting two atoms on the furanose ring, thereby forming a bicyclic system. BNA includes, but is not limited to, α-L-LNA, β-D-LNA, ENA, oxa-amino BNA (2'-O-N(CH 3 )-CH 2 -4') and amino-oxy BNA (2'-N(CH 3 )-O-CH 2 -4').

[0036] Representative structures of BNA include, but are not limited to:

[0037]

[0038] As used herein, the term "4'-to-2' bicyclic nucleoside" refers to a BNA in which the bridge connecting two atoms of the furanose ring bridges the 4'-carbon atom and the 2'-carbon atom of the furanose ring, thereby forming a bicyclic ring system.

[0039] As used herein, "locked nucleic acid" or "LNA" refers to a nucleotide that is modified such that the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4'-carbon atom of the sugar ring by a methylene group, thereby forming a 2'-C,4'-C-methylene linkage. LNA includes, but is not limited to, α-L-LNA and β-D-LNA.

[0040] As used herein, the term "sugar surrogate" refers to a structure that does not contain a furanosyl group and is capable of replacing the naturally occurring sugar of a nucleoside such that the resulting nucleoside is capable of (1) incorporation into an oligonucleotide and (2) hybridization with a complementary nucleoside. Such structures include rings containing a different number of atoms than a furanosyl group (e.g., 4-, 6-, or 7-membered rings); replacement of the oxygen of a furanosyl group with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and replacement of oxygen. Such structures may also include substitutions corresponding to those described for the substituted sugar moiety (e.g., a 6-membered carbon ring bicyclic sugar surrogate optionally containing additional substituents). Sugar surrogates also include more complex sugar surrogates (e.g., the acyclic system of peptide nucleic acid). Sugar surrogates include, but are not limited to, morpholine, modified morpholine, cyclohexenyl, and cyclohexanol.

[0041] As used herein, the term "peptide nucleic acid" or the abbreviation "PNA" refers to a synthetic polymer that is structurally similar to DNA or RNA. In some instances, PNA is resistant to cleavage by RNAi or RNase H, and / or resistant to degradation by nucleases and proteases. Compared to comparable oligonucleotides, PNA can also have increased stability and a longer half-life. In some instances, PNA has a high binding affinity for DNA and RNA. In some instances, PNA contains a backbone of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. Various purine and pyrimidine bases are attached to the backbone by methylene bridges (-CH 2 -) and carbonyl groups (-(C=O)-). PNA is typically described as a peptide with the N-terminus in the first (left) position and the C-terminus in the last (right) position. In some instances, PNA has a primary amide at the C-terminus to form a primary amide bond. In some instances, the N-terminus of PNA contains a lysine amino acid. In some cases, PNA has two C-termini or two N-termini. In some instances, the backbone of PNA does not contain charged phosphate groups.

[0042] As used herein, "internucleotide bond" refers to a covalent bond between adjacent nucleotides.

[0043] As used herein, "native internucleotide bond" refers to a 3'-to-5' phosphodiester bond.

[0044] As used herein, the term "modified internucleotide bond" refers to any bond between nucleotides other than the native internucleotide bond. Compared to the native phosphodiester bond, modified internucleotide bonds can be used to alter, typically increase, the nuclease resistance of an antisense compound.

[0045] As used herein, the term "antisense compound" refers to an oligomeric compound that is at least partially complementary to the target nucleic acid molecule to which it hybridizes. In some instances, the antisense compound modulates (increases or decreases) the expression of the target nucleic acid. Antisense compounds include, but are not limited to, compounds that are oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations thereof. Thus, while all antisense compounds are oligomeric compounds, not all oligomeric compounds are antisense compounds.

[0046] As used herein, the term "antisense oligonucleotide" refers to an antisense compound that is an oligonucleotide.

[0047] As used herein, the term "complementary" refers to the ability of an oligomeric compound to hybridize to another oligomeric compound or nucleic acid through nucleobase complementarity. In some instances, an antisense compound and its target are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases that can bond to one another to permit stable binding between the antisense compound and the target. Those skilled in the art will recognize that it is possible to include mismatches without eliminating the ability of the oligomeric compound to remain bound. Thus, the antisense compounds disclosed herein may contain up to about 20% mismatched nucleotides (i.e., nucleobases that are not complementary to the corresponding nucleotides of the target). Preferably, the antisense compound contains no more than about 15%, more preferably no more than about 10%, and most preferably no more than 5% mismatches or no mismatches. The remaining nucleotides are nucleobase complementary or do not disrupt hybridization (e.g., universal bases). One of ordinary skill in the art will recognize that the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleobase complementary to the target nucleic acid.

[0048] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common pairing mechanism involves hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonding. For example, the natural base adenine is a nucleobase that is complementary to the natural nucleobases thymidine and uracil, which pair by forming hydrogen bonds. The natural base guanine is a nucleobase that is complementary to the natural bases cytosine and 5-methylcytosine. Hybridization can occur under different circumstances.

[0049] Two sequences can be complementary and hybridize to each other under moderately stringent or preferably stringent conditions. Hybridization to a desired sequence can be carried out under moderately stringent conditions or under stringent conditions by methods known in the art. Hybridization conditions can also be varied according to known methods, depending on the sequences of interest.

[0050] As used herein, the term "percent complementarity" refers to the number of nucleobases of an oligomeric compound that have nucleobase complementarity to the corresponding nucleobases of another oligomeric compound or nucleic acid divided by the total length (number of nucleobases) of the oligomeric compound.

[0051] As used herein, the term "percent identity" refers to a value determined by dividing the number of identical nucleotides or amino acids that match by the length of the sequence for which the percent identity is reported. The percent amino acid sequence similarity can be determined by the same calculation used to determine the percent amino acid sequence identity, but can include, for example, conservative amino acid substitutions in addition to identical amino acids in the calculation. Oligonucleotide alignment algorithms, such as BLAST (GenBank; using default parameters), can be used to calculate the percent sequence identity.

[0052] As used herein, the term "pharmaceutically acceptable salt" refers to a salt of an active compound that retains the desired biological activity and does not impart undesired toxicological effects. Sodium salts of antisense oligonucleotides are useful and are widely accepted for therapeutic administration to humans.

[0053] As used herein, the term "prodrug" refers to a therapeutic agent prepared in an inactive or less active form that is converted in vivo or within its cells to an active form (i.e., the drug) by the action of endogenous enzymes, chemicals, and / or conditions. In particular, prodrug forms of oligonucleotides can be prepared as SATE ((S-acetyl-2-thioethyl) phosphate) derivatives according to the methods disclosed in WO 93 / 24510 or WO 94 / 26764. Prodrugs can also include antisense compounds in which one or both termini contain nucleobases that are cleaved (e.g., by incorporation of a phosphodiester backbone bond at the terminus) to produce the active compound.

[0054] As used herein, the term "treatment" refers to the administration of a composition of the present invention to effect a change or improvement in a disease or medical condition. Prevention, amelioration, and / or treatment may require the administration of multiple doses, either regularly or before the onset of a disease or medical condition, to alter the course of the disease or medical condition. In addition, for each prevention, amelioration, and treatment of a disorder or medical condition, a single agent can be used sequentially or simultaneously in a single individual.

[0055] As used herein, the term "agent" refers to a substance that provides a therapeutic benefit when administered to an individual.

[0056] As used herein, the term "therapeutically effective amount" refers to the amount of an agent that provides a therapeutic benefit to an animal.

[0057] As used herein, "administering" refers to providing an agent to an animal, including but not limited to administration by a medical technician and self-administration.

[0058] As used herein, the term "pharmaceutical composition" refers to a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition can contain an oligonucleotide and a sterile aqueous solution.

[0059] As used herein, the term "animal" refers to a human or non-human animal, including but not limited to mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including but not limited to monkeys and chimpanzees. Detailed Description of the Invention

[0061] The inventors of the present application have found that the 3'-terminal sequence of exon 12 is a complementary sequence (ECS) of the editing site of AZIN1 that forms double-stranded RNA (dsRNA) with the sequence edited at exon 11 of AZIN1. Surprisingly, it has been found that compounds targeting this ECS, especially oligonucleotides, can inhibit ADAR1-mediated AZIN1 pre-mRNA editing. Inhibiting AZIN1 pre-mRNA editing can effectively reduce the viability of cancer cells associated with AZIN1 pre-mRNA editing in vitro and inhibit the occurrence and growth of tumors / cancers associated with AZIN1 pre-mRNA editing in vivo. Therefore, the compounds targeting ECS identified by the inventors of the present application can serve as promising therapeutic candidates for tumors / cancers associated with AZIN1 pre-mRNA editing.

[0062] Accordingly, in one aspect, there is provided an oligonucleotide targeting the core editing site complementary sequence (ECS) of the AZIN1 gene, wherein the core ECS of the AZIN1 gene comprises the sequence 5'-GCTTTTCC-3', and wherein the oligonucleotide comprises one or more nucleotides with sugar modifications and one or more modified internucleotide linkages. The oligonucleotide can inhibit ADAR1-mediated AZIN1 pre-mRNA editing.

[0063] As shown in the following working examples, in some instances, the AZIN1 pre-mRNA comprises an editing region (e.g., 5’-UGAGCUUGAUCAAAUUGUGGAA A GCUGUCUUCUUCCUGAGCU-3’ (SEQ ID NO:2), with the underlined "A" being the adenosine-to-inosine editing site), which is recognized and / or targeted by ADAR-1 for editing. In these instances, ADAR-1 edits the sequence 5’-GGAA A GC-3’ to 5’-GGAA I GC-3’, resulting in a mutation in the translated AZIN1 protein.

[0064] In some instances, the antisense oligonucleotides disclosed herein prevent the recognition and / or binding of ADAR-1, thereby inhibiting or blocking the activity of ADAR-1. This can be achieved, for example, by preventing the formation of a dsRNA structure within the AZIN1 pre-mRNA strand or preventing ADAR-1 from recognizing the dsRNA structure.

[0065] The oligonucleotide can comprise ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In one specific instance, the oligonucleotide is an RNA oligonucleotide. In some instances, the oligonucleotide is not a peptide nucleic acid (PNA).

[0066] In some instances, the oligonucleotides disclosed herein have a length of at least about 8 nucleotides, such as, but not limited to, about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides. Additionally, the length of the oligonucleotide can be defined by any two values provided above or a range of any two values therebetween. In some specific instances, the oligonucleotide has a length of about 20 - 30 nucleotides. In one specific instance, the oligonucleotide has a length of at least about 20 nucleotides. In one specific instance, the oligonucleotide has a length of about 20 nucleotides.

[0067] In some instances, at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100% of the nucleotides in the oligonucleotide are modified with a sugar modification. Additionally, the percentage of nucleotides modified with a sugar modification can be defined by any two values provided above or a range of any two values therebetween. In one specific instance, at least about 50% of the nucleotides in the oligonucleotide are modified with a sugar modification. In another specific instance, at least about 70% of the nucleotides in the oligonucleotide are modified with a sugar modification. In yet another specific instance, all of the nucleotides in the oligonucleotide are modified with a sugar modification.

[0068] In some instances, the nucleotides modified with a sugar modification are located at or near the 5' end of the oligonucleotide. In some other instances, the nucleotides modified with a sugar modification are located at or near the 3' end of the oligonucleotide. In some instances, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides at or near the 5' end of the oligonucleotide are modified with a sugar modification. In some other instances, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides at or near the 3' end of the oligonucleotide are modified with a sugar modification.

[0069] In some instances, the nucleotide with a sugar modification is a 2'-O-methyl-modified nucleotide, a 2'-O-methoxyethyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2',4'-bridged nucleic acid-modified nucleotide, a locked nucleic acid (LNA)-modified nucleotide, or a morpholino-ring-modified nucleotide. In a specific instance, the nucleotide with a sugar modification is a 2'-O-methyl-modified nucleotide. In a specific instance, all nucleotides in the oligonucleotide are modified with 2'-O-methyl sugar modification.

[0070] When there is more than one nucleotide with a sugar modification in the oligonucleotide, these nucleotides can be modified with the same sugar modification or different sugar modifications.

[0071] In some instances, the oligonucleotides disclosed herein are antisense oligonucleotides. In some instances, the antisense oligonucleotides are non-degradable antisense oligonucleotides, i.e., the antisense oligonucleotides cannot effect target degradation through the RNase H or RNA interference (RNAi) mechanism. In some instances, the non-degradable antisense oligonucleotides bind to their target RNA and sterically prevent other molecules from accessing to base pair with the RNA. In some specific instances, such steric-blocking antisense oligonucleotides are fully modified at the 2'-sugar position such that the RNase H cannot degrade the target RNA.

[0072] In some instances, the oligonucleotides disclosed herein comprise one or more modified internucleotide linkages. Examples of modified internucleotide linkages include but are not limited to phosphorus-containing internucleoside linkages such as phosphotriesters, methylphosphonates, aminophosphates, diamino-phosphates, and phosphorothioates. In some specific instances, the oligonucleotides disclosed herein comprise one or more phosphorothioate, aminophosphate, or diamino-phosphate linkages.

[0073] In some instances, up to about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the internucleotide linkages in the oligonucleotide are modified internucleotide linkages. In some other instances, at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% of the internucleotide linkages in the oligonucleotide are modified internucleotide linkages. Additionally, the percentage of modified internucleotide linkages in the oligonucleotide can be defined by any two values provided above or by a range of any two values therebetween. In one specific instance, at least about 10% of the internucleotide linkages in the oligonucleotide are modified internucleotide linkages. In one specific instance, about 25% of the internucleotide linkages in the oligonucleotide are modified internucleotide linkages. In another specific instance, all of the internucleotide linkages in the oligonucleotide are modified internucleotide linkages. In yet another specific instance, at least about 10% of the internucleotide linkages in the oligonucleotide are phosphorothioate linkages. In yet another specific instance, about 25% of the internucleotide linkages in the oligonucleotide are phosphorothioate linkages. In yet another specific instance, all of the internucleotide linkages in the oligonucleotide are phosphorothioate linkages.

[0074] In some instances, the modified internucleotide linkages are located at or near the 5'-end of the oligonucleotide. In some other instances, the modified internucleotide linkages are located at or near the 3'-end of the oligonucleotide. In some instances, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the internucleotide linkages at or near the 5'-end of the oligonucleotide are modified internucleotide linkages. In some other instances, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the internucleotide linkages at or near the 3'-end of the oligonucleotide are modified internucleotide linkages.

[0075] In one specific instance, when the nucleotide with a sugar modification is a morpholino-modified nucleotide and the modified internucleotide linkage connecting the morpholino-modified nucleotide to an adjacent nucleotide is a phosphorodiamidate internucleotide linkage, a phosphorodiamidate morpholino oligomer (PMO) is formed.

[0076] In some instances, the oligonucleotide comprises or consists of an antisense sequence complementary to the core ECS of the AZIN1 gene, such that the oligonucleotide can effectively target the ECS of the AZIN1 gene. Since the core ECS of the AZIN1 gene contains the sequence 5'-GCTTTTCC-3', the antisense sequence that is fully complementary to the core ECS of the AZIN1 gene is 5'-GGAAAAGC-3'. Those skilled in the art should recognize that it is possible to include mismatches without eliminating the complementary activity of the antisense sequence. Thus, in some instances, the antisense sequence complementary to the core ECS of the AZIN1 gene can contain up to 1, 2, or 3 nucleotides that do not base pair with the core ECS of the AZIN1 gene.

[0077] In some instances, the antisense sequence complementary to the core ECS of the AZIN1 gene is located at or near the 3'-end of the oligonucleotide that targets the ECS of the AZIN1 gene. For example, the 3'-end of the antisense sequence complementary to the core ECS of the AZIN1 gene can be up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides away from the 3'-end of the oligonucleotide.

[0078] The antisense sequence complementary to the core ECS of the AZIN1 gene is crucial for the oligonucleotide containing the antisense sequence to effectively target the ECS of the AZIN1 gene. Thus, in some instances, at least some of the nucleotides in the antisense sequence complementary to the core ECS of the AZIN1 gene are modified with a sugar modification. This can increase the affinity of the antisense sequence for the ECS of the AZIN1 gene or increase the nuclease resistance of the antisense sequence. In some instances, at least 5, 6, 7, or 8 nucleotides in the antisense sequence complementary to the core ECS of the AZIN1 gene are modified with a sugar modification. In some specific instances, at least 5, 6, 7, or 8 nucleotides in the antisense sequence complementary to the core ECS of the AZIN1 gene are 2'-O-methyl-modified nucleotides, 2'-O-methoxyethyl-modified nucleotides, 2'-fluoro-modified nucleotides, 2',4'-bridged nucleic acid-modified nucleotides, locked nucleic acid (LNA)-modified nucleotides, or morpholino-ring-modified nucleotides or a combination thereof. In one specific instance, at least 5, 6, 7, or 8 nucleotides in the antisense sequence complementary to the core ECS of the AZIN1 gene are 2'-O-methyl-modified nucleotides. In another specific instance, all of the nucleotides in the antisense sequence complementary to the core ECS of the AZIN1 gene are 2'-O-methyl-modified nucleotides.

[0079] In some other instances, at least some of the internucleotide linkages in the antisense sequence complementary to the core ECS of the AZIN1 gene are modified internucleotide linkages. This can increase the nuclease resistance of the antisense sequence. In some instances, at least 3, 4, 5, 6, or 7 of the internucleotide linkages in the antisense sequence complementary to the core ECS of the AZIN1 gene are modified internucleotide linkages. In some specific instances, at least 3, 4, 5, 6, or 7 of the internucleotide linkages in the antisense sequence complementary to the core ECS of the AZIN1 gene are phosphorothioate, phosphoroamidate, or diaminophosphate linkages or combinations thereof. In one specific instance, at least 3, 4, 5, 6, or 7 of the internucleotide linkages in the antisense sequence complementary to the core ECS of the AZIN1 gene are phosphorothioate linkages. In another specific instance, at least 5 of the internucleotide linkages in the antisense sequence complementary to the core ECS of the AZIN1 gene are phosphorothioate linkages. In yet another specific instance, all of the internucleotide linkages in the antisense sequence complementary to the core ECS of the AZIN1 gene are phosphorothioate linkages.

[0080] In some instances, none of the internucleotide linkages in the antisense sequence complementary to the core ECS of the AZIN1 gene are modified internucleotide linkages, i.e., all of the internucleotide linkages in the antisense sequence complementary to the core ECS of the AZIN1 gene are natural 3' to 5' phosphodiester linkages. In such instances, other portions of the oligonucleotide (i.e., portions not complementary to the core ECS of the AZIN1 gene) can contain modified internucleotide linkages to increase the nuclease resistance of the oligonucleotide.

[0081] In some instances, the oligonucleotide is completely modified with sugar modifications and modified internucleotide linkages, i.e., each nucleotide in the oligonucleotide is modified with a sugar modification and is linked to an adjacent nucleotide by a modified internucleotide linkage. In some specific instances, each nucleotide in the oligonucleotide is modified with a 2'-O-methyl sugar modification and is linked to an adjacent nucleotide by a phosphorothioate linkage.

[0082] In some instances, the oligonucleotides disclosed herein have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to the sequence 5'-UUAAGCUUCAGCGGAAAAGC-3' (SEQ ID No: 5). In some instances, the oligonucleotide comprises or consists of the sequence 5'-UUAAGCUUCAGCGGAAAAGC-3' (SEQ ID No: 5). The sequence 5'-UUAAGCUUCAGCGGAAAAGC-3' (SEQ ID No: 5) contains one or more nucleotides having the sugar modifications described herein and optionally one or more of the modified internucleotide linkages described herein.

[0083] In some instances, the oligonucleotide has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to one or more of the following sequences: 5'-mUmUmAmAmGmCmUmUmCmAmGmCmGmGmAmAmAmAmGmC-3' (SEQ ID No:6), 5'-mU*mU*mA*mA*mG*mC*mU*mU*mC*mA*mG*mC*mG*mG*mA*mA*mA*mA*mG*mC-3' (SEQ ID No:7), 5'-mU*mU*mA*mA*mG*mCmUmUmCmAmGmCmGmGmAmAmAmAmGmC-3' (SEQ ID No:8) and 5'-mUmUmAmAmGmCmUmUmCmAmGmCmGmGmA*mA*mA*mA*mG*mC-3' (SEQ ID No:9), where m represents a 2'-O-Me sugar modification and * represents a phosphorothioate bond. In some instances, the oligonucleotide comprises or consists of one of the following sequences: 5'-mUmUmAmAmGmCmUmUmCmAmGmCmGmGmAmAmAmAmGmC-3' (SEQ ID No:6), 5'-mU*mU*mA*mA*mG*mC*mU*mU*mC*mA*mG*mC*mG*mG*mA*mA*mA*mA*mG*mC-3' (SEQ ID No:7), 5'-mU*mU*mA*mA*mG*mCmUmUmCmAmGmCmGmGmAmAmAmAmGmC-3' (SEQ ID No:8) and 5'-mUmUmAmAmGmCmUmUmCmAmGmCmGmGmA*mA*mA*mA*mG*mC-3' (SEQ ID No:9), where m represents a 2'-O-Me sugar modification and * represents a phosphorothioate bond.

[0084] In some instances, the oligonucleotides disclosed herein can be labeled with suitable moieties known in the art, such as but not limited to one or more fluorophores, radiolabels, chemical substituents, enzymes, antibodies, etc., to facilitate identification in hybridization assays and other assays or tests.

[0085] The oligonucleotides provided herein can be used in pharmaceutical compositions, for example, by adding an effective amount of the oligonucleotide to a suitable pharmaceutically acceptable diluent or carrier. Thus, in one aspect, a pharmaceutical composition comprising the oligonucleotides disclosed herein is provided.

[0086] Acceptable carriers and diluents are well known to those skilled in the art. The choice of diluent or carrier is based on many factors, including but not limited to the solubility of the oligonucleotide and the route of administration. These considerations are well understood by those skilled in the art.

[0087] The oligonucleotides provided herein include any pharmaceutically acceptable salt, ester, or salt of such an ester, or any other functional chemical equivalent that is capable of (directly or indirectly) providing a bioactive metabolite or a residue thereof when administered to an animal, including a human. Thus, for example, the present disclosure also provides prodrugs of the oligonucleotides and pharmaceutically acceptable salts of the oligonucleotides, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0088] The oligonucleotides disclosed herein can also be mixed, encapsulated, conjugated, or otherwise combined with other molecules, molecular structures, or mixtures of compounds.

[0089] The pharmaceutical compositions can be administered in a variety of ways, depending on whether local or systemic treatment is desired and the area to be treated.

[0090] The pharmaceutical formulations described herein, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing the active ingredient into association with a pharmaceutical carrier or excipient. Generally, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product (e.g., forming a specific particle size for delivery).

[0091] A "pharmaceutical carrier" can be a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal and is known in the art. When combined with the nucleic acids and other components of a given pharmaceutical composition, the carrier can be liquid or solid and is selected, taking into account the planned mode of administration, to provide the desired volume, consistency, etc. Liquid carriers can be aqueous carriers, non-aqueous carriers, or both, and include, but are not limited to, aqueous suspensions, oil emulsions, water-in-oil emulsions, water-in-oil-in-water emulsions, site-specific emulsions, long-residence emulsions, viscous emulsions, microemulsions, and nanoemulsions. Solid carriers can be biological carriers, chemical carriers, or both, and include, but are not limited to, viral vector systems, particles, microparticles, nanoparticles, microspheres, nanospheres, micropumps, bacterial cell wall extracts, and biodegradable or non-biodegradable natural or synthetic polymers that permit sustained release of the oligonucleotide composition.

[0092] Preferred aqueous carriers include, but are not limited to, water, saline, and pharmaceutically acceptable buffers. Preferred non-aqueous carriers include, but are not limited to, mineral oil or neutral oil, including, but not limited to, diglycerides, triglycerides, phospholipids, lipids, oils, and mixtures thereof, where the oil contains an appropriate mixture of polyunsaturated and saturated fatty acids. Examples include, but are not limited to, squalene, soybean oil, rapeseed oil, palm oil, olive oil, and myglyol, where the fatty acids can be saturated or unsaturated. Optionally, regardless of the pharmaceutically acceptable carrier, excipients may be included. These excipients include, but are not limited to, antioxidants, buffers, and bacteriostatic agents, and may include suspending and thickening agents.

[0093] In embodiments, the compositions of the present invention can be prepared by conventional pharmaceutical techniques in combination with, for example, one or more pharmaceutically acceptable carriers or excipients. Such techniques include the step of bringing into association a composition containing the active ingredient and a pharmaceutical carrier or excipient. Generally, the formulations are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier.

[0094] The composition, shape, and type of dosage form of the pharmaceutical compositions disclosed herein will generally vary depending on the intended use. For example, a dosage form for the acute treatment of the disease or a related disease may contain a greater amount of one or more of the active compounds it contains compared to a dosage form for the long-term treatment of the same disease. Similarly, a parenteral dosage form may contain a lesser amount of one or more of the active compounds it contains compared to an oral dosage form for the treatment of the same disease or condition. These and other ways in which the specific dosage forms encompassed by the present invention differ from one another should be apparent to those skilled in the art. Examples of dosage forms include, but are not limited to: tablets; cachets; capsules, such as soft elastic gelatin capsules; sachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms; pastes; powders; poultices; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions or water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms particularly suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide a liquid dosage form suitable for parenteral administration to a patient. Thus, in one example, the pharmaceutical compositions disclosed herein can be provided in a form selected from, but not limited to, the following: tablets, cachets, capsules, hard capsules, soft capsules, soft elastic gelatin capsules, hard gelatin capsules, sachets, troches, lozenges, dispersions, suppositories, ointments, cataplasms, pastes, powders, poultices, creams, plasters, solutions, injections, patches, aerosols, nasal sprays, inhalers, gels, suspensions, aqueous liquid suspensions, non-aqueous liquid suspensions, oil-in-water emulsions, water-in-oil liquid emulsions, solutions, sterile solids, crystalline solids, amorphous solids, solids for reconstitution, or combinations thereof.

[0095] In one aspect, a method of inhibiting AZIN1 pre-mRNA editing in a cell is provided, the method comprising contacting the cell with an oligonucleotide disclosed herein or a pharmaceutical composition disclosed herein. Such methods can be in vivo, ex vivo, or in vitro. In particular, the AZIN1 pre-mRNA editing inhibited by the oligonucleotides or pharmaceutical compositions disclosed herein is mediated by the adenosine deaminase acting on RNA-1 (ADAR-1).

[0096] Methods of contacting a body fluid, organ, or tissue with an effective amount of one or more of the oligonucleotides or pharmaceutical compositions provided herein are also contemplated. A body fluid, organ, or tissue can be contacted with one or more oligonucleotides to modulate AZIN1 pre-mRNA editing in the cells of the body fluid, organ, or tissue. The effective amount can be determined by monitoring the modulation of AZIN1 pre-mRNA editing by the oligonucleotide or pharmaceutical composition by methods routine to those skilled in the art.

[0097] Pre-mRNA editing of the AZIN1 gene is associated with increased proliferation, cell transformation, and tumorigenesis. Thus, the oligonucleotides capable of inhibiting ADAR-1-mediated AZIN1 pre-mRNA editing disclosed herein can be effective in treating cancers associated with ADAR-1-mediated AZIN1 pre-mRNA editing. Accordingly, in one aspect, provided are methods of treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the oligonucleotides disclosed herein or the pharmaceutical compositions disclosed herein, wherein the cancer is associated with AZIN1 pre-mRNA editing.

[0098] RNA editing of the protein recoding type can promote tumorigenesis by enhancing the activity of oncogenes or reducing the activity of tumor suppressor genes. In some instances, the AZIN1 pre-mRNA can be edited by the ADAR1 protein, resulting in a serine (S) to glycine (G) substitution at residue 367. In some instances, AZIN1 S367G is more stable than wild-type AZIN1 and has a stronger affinity for antizyme. Antizyme regulates growth by binding to and degrading proteins associated with cell growth and proliferation, such as ornithine decarboxylase (ODC) and cyclin D1 (CCND1). AZIN1 S367G can inhibit antizyme-mediated degradation of ODC and CCND1 by competing with wild-type AZIN1 for binding to antizyme, thereby promoting entry into the cell cycle and having a stronger tumorigenic ability than wild-type AZIN1.

[0099] Examples of cancers associated with AZIN1 pre-mRNA editing, particularly ADAR-1-mediated AZIN1 pre-mRNA editing, include but are not limited to liver cancer, esophageal cancer, lung cancer, and colorectal cancer. Specific cancer types include but are not limited to hepatocellular carcinoma (HCC), esophageal squamous cell carcinoma (ESCC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC). In some instances, elevated levels of AZIN1 RNA editing are prognostic factors for overall survival and disease-free survival and independent risk factors for lymph node and distant metastasis.

[0100] In some instances, the methods for treating cancer described herein include administering multiple therapeutic agents. In some instances, any of the oligonucleotides or pharmaceutical compositions described herein is a first therapeutic agent, and the method further includes administering a second therapeutic agent. The second therapeutic agent can be administered before, simultaneously with, or after the first therapeutic agent. In some instances, the second therapeutic agent is an RNA-based therapeutic agent or a small molecule drug.

[0101] It should be understood that small molecule drugs can refer to drugs known in the art for targeting cancer. Suitable drugs include: sorafenib, gefitinib, osimertinib, crizotinib, pemetrexed (Alimta), paclitaxel, carboplatin, gemcitabine, capecitabine, eribulin, 5-FU (5-fluorouracil), etc. Some drugs can be used in combination with the oligonucleotides described herein to treat specific diseases or disease conditions. For example, when treating non-small cell lung cancer (NSCLC), the oligonucleotides described herein can be combined with gefitinib, osimertinib (for EGFR mutants), crizotinib (for ALK mutants), or a combination thereof. In some instances, the oligonucleotides disclosed herein can be combined with chemotherapy drugs such as pemetrexed (Alimta), which is used when the tumor is unresponsive to targeted drugs. In the case of targeting breast cancer, the oligonucleotides described herein can be combined with paclitaxel, carboplatin, gemcitabine, capecitabine, eribulin, or a combination thereof. In the case of targeting colon cancer, the oligonucleotides described herein can be combined with 5-FU (5-fluorouracil) or capecitabine.

[0102] Also provided herein is the use of the oligonucleotides disclosed herein or the pharmaceutical compositions disclosed herein in the manufacture of a drug for treating cancer, wherein the cancer is associated with AZIN1 pre-mRNA editing. Also provided herein is the use of the oligonucleotides disclosed herein or the pharmaceutical compositions disclosed herein for treating, particularly for treating cancer, wherein the cancer is associated with AZIN1 pre-mRNA editing.

[0103] In some instances, the oligonucleotides disclosed herein inhibit the growth, cell viability, and / or proliferation of cells, particularly cancer cells, by reducing the G1 / S cell cycle transition. Cancer cells can be understood as any of the cancer cells described herein, or any cancer cells that produce mutant antizyme inhibitors. In some instances, reducing the G1 / S cell cycle transition includes reducing the amount of mutant antizyme inhibitor translated from the edited AZIN1 RNA transcript. Blocking ADAR-1-mediated dsRNA formation and / or abnormal AZIN1 RNA transcript editing results in a decrease in the production of mutant antizyme inhibitor.

[0104] In some instances, to determine whether a patient's cancer is associated with AZIN1 pre-mRNA editing and thus should be treated with the oligonucleotides or pharmaceutical compositions disclosed herein, a sample is obtained from the patient to measure the level of edited AZIN1 pre-mRNA. Accordingly, in some instances, the treatment methods disclosed herein further include measuring the level of edited AZIN1 pre-mRNA in a sample obtained from an individual prior to administering a therapeutically effective amount of the oligonucleotides disclosed herein or the pharmaceutical compositions disclosed herein. In some instances, measuring the level of edited AZIN1 pre-mRNA includes isolation and sequencing of the RNA transcript of AZIN1.

[0105] In some instances, for cancer in a patient to be considered associated with AZIN1 pre-mRNA editing, the level of edited AZIN1 pre-mRNA in a sample obtained from the patient is at least 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% higher than the level of edited AZIN1 in a sample obtained from a healthy individual. In the provided instances, the level of edited AZIN1 pre-mRNA is calculated as the area of the "G" (representing editing by ADAR-1) peak divided by the total area of the "A" and "G" peaks.

[0106] In some instances, the level of edited AZIN1 pre-mRNA is also determined during treatment to indicate the efficacy of the treatment, and / or examined after treatment to determine whether the treatment is effective.

[0107] As used herein, the term "sample" refers to a biological sample, or a sample that contains at least some biological material such as cells, DNA or RNA. Examples of biological samples include but are not limited to solid tissue samples such as bone marrow, and liquid samples such as whole blood, serum, plasma, cerebrospinal fluid, central spinal fluid, lymph fluid, cyst fluid, sputum, feces, pleural effusion, mucus, hydrothorax, ascites, amniotic fluid, peritoneal fluid, saliva, bronchial washings and urine. In some instances, the biological sample is a blood sample. In some other instances, the biological sample is a tumor sample obtained from a tumor biopsy or surgical resection.

[0108] The biological samples of the present disclosure can be obtained from any organism, including mammals such as humans, primates (e.g., monkeys, chimpanzees, orangutans and gorillas), cats, dogs, rabbits, farm animals (e.g., cows, horses, goats, sheep, pigs) and rodents (e.g., mice, rats, hamsters and guinea pigs).

[0109] It should be understood that, according to any method related to testing, profiling or screening described herein, the method may further include other tests or screenings for one or more other gene mutations, blood tests, blood enzyme tests, counseling, providing support resources or administering other agents based on the results of such tests and / or screenings. Similarly, it is also contemplated that the method may be carried out after one or more steps, such as but not limited to selecting an individual having cancer or considered at risk of developing cancer, or selecting a pre-cancerous or suspected pre-cancerous individual at risk.

[0110] Oligonucleotides can be delivered by what is understood as "naked delivery" directly to the body and taken up by the recipient into cells. Oligonucleotides can also be conjugated to ligands such as cell-penetrating peptides, neamine, N-acetylgalactosamine (GalNAc). Oligonucleotides can also be delivered by suitable carriers such as nanoparticles. Oligonucleotides can also be transfected, lipofected or electroporated into cells.

[0111] Methods of administering the oligonucleotides or pharmaceutical compositions disclosed herein include but are not limited to the following: oral (e.g., buccal or sublingual), anal, rectal, as a suppository, intracolonic, topical, parenteral, nasal, aerosol, inhalation, intrathecal, intraperitoneal, intravenous, intraarterial, transdermal, intradermal, subdermal, subcutaneous, intramuscular, intralymphatic, intrauterine, intravesicular, vaginal, visceral, into body cavities, surgical administration at the site of inflamed tissue such as adipose tissue, into the lumen or parenchyma of an organ, into the bone marrow and into any mucosal surface of the gastrointestinal, reproductive, urinary and urogenital systems. It should be understood that the choice of the route of administration should be made by a person of ordinary skill in the art of treatment so as to achieve inhibition or reduction of the RNA editing level.

[0112] It should be noted that, as used herein, the terms "organism", "individual", "subject" or "patient" are used as synonyms and are interchangeable.

[0113] The dosing depends on the severity and responsiveness of the disease state to be treated, and the course of treatment lasts from several days to several months, or until cure or alleviation of the disease state is achieved. The optimal dosing regimen can be calculated from measurements of the drug accumulation or its metabolites in the patient. The dosing physician can readily determine the optimal dose, dosing method and repetition rate. The optimal dose can vary according to the relative potency of the composition and can generally be estimated based on an arithmetic mean, such as based on the EC found to be effective in in vitro and in vivo animal models 50a value, or based on the embodiments described herein. Generally, the dosage of the pharmaceutical compositions of the present disclosure is from about 0.01 μg to 100 g / kg body weight, and may be administered once or more daily, weekly, monthly, or annually. The treating physician can estimate the repetition rate of the quantitative administration based on the measured residence time and the concentration of the drug in body fluids or tissues. After successful treatment, it may be necessary to subject the individual to maintenance therapy to prevent recurrence of the disease state, wherein the composition is administered at a maintenance dose of 0.01 μg to 100 g / kg body weight, once or more daily to once every 2 years.

[0114] As described above, those skilled in the art should be able to determine the required dosage and dosage regimen required to achieve the desired clinical effect based on, for example, the severity of the disease. The following are illustrative examples of intravenous injection, which can be modified according to the needs of other modes of administration. In one example, the methods disclosed herein administer at least one injection to an individual. In one example, more than a single injection may be administered to a patient at any given time. In yet another example, the methods disclosed herein may require multiple administrations of a single injection to a patient within a specified treatment time frame or regimen. In yet another example, the methods disclosed herein may require multiple administrations of more than two or more injections to a patient within a specified treatment time frame or regimen. This means that, depending on the clinical requirements, an individual may be given an initial treatment in the form of an injection, whereby further treatment may be carried out at intervals of, for example, 3 days, 7 days, weekly, 2 weeks, bi-weekly, 1 month, monthly, quarterly, semi-annually, annually, or longer, depending on the treatment designed for the individual. If desired, the methods disclosed herein can also be used as combination therapy with other drugs or pharmaceutical compositions.

[0115] Also described herein are methods for identifying RNA therapeutics that modulate pre-mRNA editing. The methods for targeting AZIN1 pre-mRNA editing described herein may not be limited to AZIN1, but may be used to target genes other than AZIN1. Such methods may include determining the editing region and ECS of the desired RNA transcript, determining the dsRNA structure of the desired transcript, and designing RNA therapeutics that target and disrupt dsRNA assembly or bind to the dsRNA structure. Antisense oligonucleotides are designed to disrupt pre-mRNA editing by targeting dsRNA structure, such as by targeting the editing region or ECS. Such antisense oligonucleotides may include any chemical modification described herein. Any of the steps described may be implemented using the methods described herein. For example, the minigene assay described herein can be used to determine the dsRNA structure of the desired target sequence. Minigenes are the smallest gene fragments that include exons and control regions required to express themselves in the same manner as wild-type gene fragments. This is a minigene in the most basic sense. More complex minigenes containing multiple exons and introns can be constructed. Minigenes provide researchers with valuable tools for evaluating splicing patterns in in vivo and in vitro biochemical evaluation experiments. Specifically, minigenes are used as splicing reporter vectors (also called exon capture vectors) and as probes to determine which factors are important for splicing outcomes. They are constructed to test how cis-regulatory elements (RNA effectors) and trans-regulatory elements (associated proteins / splicing factors) affect gene expression.

[0116] The invention illustratively described herein may be suitably practiced in the absence of any element, limitation or limitations not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", and the like are to be understood broadly and not restrictively. Furthermore, the terms and expressions used herein have been used as descriptive and not restrictive terms, and in the use of such terms and expressions, it is not intended to exclude any equivalents of the features shown and described, or portions thereof, but it is to be recognized that various modifications may be made within the scope of the claimed invention. Therefore, it should be understood that although the present invention has been specifically disclosed through preferred embodiments and optional features, those skilled in the art may resort to modifications and variations embodied in the invention disclosed herein, and it is considered that such modifications and variations are within the scope of the present invention.

[0117] It will be appreciated by those skilled in the art that various methods and techniques known in the art may be used to implement certain embodiments of the present invention. For example, various methods and techniques well known in the art may be used to complete the detection of mutations and other polymorphisms or mutations in any oncogene or tumor suppressor gene described herein.

[0118] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description of a range format is merely for convenience and brevity and should not be construed as inflexibly limiting the scope of the disclosed range. Thus, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual numerical values within that range. For example, a description of a range such as 1-6 should be considered to have specifically disclosed sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within the range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0119] The present invention has been described broadly and generically herein. Each narrower genus and sub-genus grouping falling within the generic disclosure also forms part of the present invention. This includes the generic description of the present invention with qualifying or negative limitations removing any subject matter from the generic concept, whether or not the excised material is specifically recited herein.

[0120] Other embodiments fall within the appended claims and non-limiting examples. Additionally, in instances where the present invention is described in terms of a Markush group, those skilled in the art will recognize that the present invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. Examples

[0121] The following examples illustrate aspects of how the present invention may be practiced or methods of preparing materials suitable for practicing certain embodiments of the present invention.

[0122] Materials and Methods

[0123] Cell line

[0124] All cell lines were maintained in Roswell Park Memorial Institute (RPMI-1640) medium (Biowest) supplemented with 10% FBS (Biowest). All cell lines used in this study were regularly verified by morphological observation and tested for mycoplasma contamination. Patient-derived xenograft (PDX) cell lines were cultured in DMEM / F12 (Biowest) supplemented with 1:50 B27 supplement without vitamin A (Thermo Fisher), 1:100 insulin-transferrin-selenium supplement (Gibco), 1.25 mM N-acetyl-L-cysteine (Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 10 nM recombinant human (Leu15)-gastrin I (Sigma-Aldrich), 25 ng / mL recombinant human HGF (Abcam), 50 ng / mL recombinant human EGF (Abcam), 50 ng / mL recombinant human bFGF (Abcam), 5 μg / mL heparin (Sigma-Aldrich), 10-ng / mL recombinant human FGF-10 (Abcam). All cancer cell lines and PDX cell lines were incubated at 37 °C in a humidified incubator containing 5% CO 2 2.

[0125] Normal human hepatocytes

[0126] After humanizing the liver with fresh or frozen human liver / hepatocytes from cadavers, human hepatocytes were prepared from mice (Mus musculus). HepaCur human hepatocytes were isolated from the perfused livers of humanized TM mice. It was ensured that freshly isolated hepatocytes were ≥95% human and had a viability ≥70%. The isolated hepatocytes were cultured in FRS (BioLife Solutions, catalog number: 101373), an optimized cryopreservation medium. Before plating, FRS was replaced with HMM (HepaCur Maintenance Medium, catalog number HMM500) according to the manufacturer's protocol. Subsequently, 1.0×10 TM cells were plated into each well of a 96-well plate and treated with ASO on the same day. At 37 °C, the cells were cultured in a humidified incubator containing 5% CO 4 2. 2 2.

[0127] RNA extraction, cDNA synthesis, quantitative PCR (qPCR), and Sanger sequencing

[0128] Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. cDNA synthesis was performed using the Advantage Reverse Transcription Kit (Clontech Laboratories) according to the manufacturer's protocol. Real-time quantitative PCR (qPCR) was performed using GoTaq DNA polymerase (Promega) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems). The relative expression of AZIN1 or ADAR1 (defined as "relative expression") was expressed as 2 -ΔCT (ΔC T = C T (AZIN1 / ADAR1) – C T (β-actin)), and normalized to the relative expression defined as 1.0 detected in the corresponding control cells. Semi-quantitative PCR was performed using the FastStart Taq kit (Roche) according to the manufacturer's protocol. The purified PCR amplicons were identified by Sanger sequencing. ImageJ was used to calculate the percentage of A-to-I (G) editing. The editing percentage was calculated as the area of the "G" peak divided by the total area of the "A" and "G" peaks. Table 1 lists the primer sequences.

[0129]

[0130]

[0131]

[0132] Table 1. Primer sequences used.

[0133] Generation of small gene constructs

[0134] To clone the AZIN1 sequence for pRK7 or pcDNA3.1 minigene construction, placental DNA (Sigma-Aldrich) was used for PCR using PrimeSTAR Max DNA polymerase (Takara) according to the manufacturer's protocol. Internal deletions or point mutations were introduced using the KAPA HiFi HotStart PCR Kit (KAPA Biosystems). Table 1 lists the primer sequences used for cloning.

[0135] In vitro RNA editing assay

[0136] First, FLAG-tagged ADAR1 protein was forcibly overexpressed by transfecting FLAG-ADAR1 plasmid into HEK293T cells. Cells were harvested 48 hours after transfection and lysed in lysis buffer containing 50 mM Tris-HCl pH 7.5 (Ambion), 150 mM NaCl (Ambion), 1 mM EDTA (Ambion), Triton-X100 (Sigma-Aldrich), and 1×cOmplete TM EDTA-free Protease Inhibitor Cocktail (1×cOmplete TM EDTA-free protease inhibitor mixture, Roche). M2 Magnetic Beads (anti- M2 magnetic beads, Sigma-Aldrich) were used for immunoprecipitation of cell lysates to obtain FLAG-ADAR1 protein. 100 μg / mL FLAG elution buffer was obtained by dissolving 3×FLAG peptide (Sigma) in Tris-buffered saline (TBS) containing 50 mM Tris-HCl pH 7.4 and 150 mM NaCl. The FLAG elution buffer was used to elute the protein from the magnetic beads. The eluate was stored at -80 °C until further use. In vitro transcription of the small gene construct was performed using RiboMAX TM Large Scale RNA Production System-SP6 (RiboMAX TM Large Scale RNA Production System-SP6, Promega) according to the manufacturer's protocol. Next, 5 μL of FLAG-ADAR1 protein and the purified RNA transcript transcribed from the AZIN1 FE small gene were incubated at 37 °C for 3 hours, then RNA purification was performed using the RNeasy Mini Kit (QIAGEN), and cDNA synthesis was carried out. To test the editing inhibition of ASO in vitro, the ASO was incubated with the in vitro transcribed RNA transcript before adding the purified ADAR1 protein. Then PCR amplification was performed, and the purified PCR product was sent for Sanger sequencing. Table 2 lists the primer sequences used for in vitro editing analysis.

[0137] RNA electrophoretic mobility shift assay (REMSA) - Binding of each oligonucleotide to the AZIN1 RNA duplex probe

[0138] First, RiboMAX TM Large Scale RNA Production System-T7 (RiboMAX TMA large-scale RNA production system - T7, Promega) was used to transcribe the 86-nt AZIN1 RNA duplex probe in vitro according to the manufacturer's protocol. Next, 50 pmol of the RNA probe was incubated with rSAP (NEB) at 37 °C for 30 min (minutes) to dephosphorylate the RNA. EDTA (0.8 μL, 250 mM) was added and the mixture was incubated at 65 °C for 20 min to heat-inactivate rSAP. Then the mixture was incubated with 1 μL of 100 mM MgCl containing T4 PNK (NEB) and ATP, [γ-32P] (PerkinElmer) 2 at 37 °C for 30 min. Then the mixture was heated to 95 °C to denature the duplex and slowly annealed to room temperature. After that, 80 μL of distilled water was added and the mixture was transferred to an Illustra MicroSpin G-25 column (GE Healthcare) for purification. Using LightShift TM Chemiluminescent RNA EMSA Kit (Thermo Scientific) 10×REMSA Binding Buffer (100 mM HEPES pH 7.3, 200 mM KCl, 10 mM MgCl 2 , 10 mM DTT) to incubate the samples. The samples were mixed with 1 μL of RNA duplex (final concentration 25 nM) and the corresponding oligonucleotides and incubated for 30 min. After that, FLAG-ADAR1 protein was added and further incubated for 30 min if needed. The TBE gel was pre-run before adding the samples. The gel was dried and then exposed to BioMax Light Film (Carestream, Sigma-Aldrich) and developed; or a BAS Storage Phosphor Screen (GE Healthcare), imaged with a Typhoon Trio Variable Mode Imager (GE Healthcare), and analyzed with ImageQuant TL (GE Healthcare).

[0139] RNA electrophoretic mobility shift assay (REMSA) - Binding of each PNA to the truncated AZIN1 RNA duplex probe

[0140] Two strands (ECS-s and ES-s) were added together and slowly cooled from 95 °C to room temperature to form a truncated RNA duplex, and then PNA was annealed at 40 °C for 10 min. Both steps were carried out in an incubation buffer of 200 mM NaCl, 0.5 mM EDTA and 20 mM HEPES (pH 7.5). After annealing of PNA, the sample was allowed to cool to room temperature and then incubated overnight at 4 °C. The gel was run at a constant voltage of 250 V for 5 h in a running buffer of 1× TBE, pH 8.3. The gel was then stained with ethidium bromide for 30 min and then imaged using a Typhoon Trio Variable Mode Imager. Table 2 lists the sequences of the probes.

[0141]

[0142] Table 2. Probe sequences used.

[0143] ASO treatment

[0144] All ASOs were purchased from Integrated DNA Technologies (IDT). PNAs (ASP1, DSP1 and DSP2) were synthesized and purified according to a previously reported protocol (Toh, D.K., Patil, K.M. & Chen, G. Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids. J Vis Exp, doi:10.3791 / 56221 (2017)). Cells were seeded one day prior to treatment to reach 80% confluence on the day of treatment. Cells were then treated (transfected) with ASO diluted to the desired concentration in Opti-MEM by Lipofectamine 2000 (lipid transfection amine 2000). Subsequent analyses were performed 48 h after ASO treatment. Three independent experiments were performed, with three technical replicates for each experiment.

[0145] Cell viability assay

[0146] After treating the cells with ASO, with Cell viability was measured by Luminescent Cell Viability (CTG) Assay (Promega). Cells were seeded in 96-well clear flat-bottom plates (Corning) and treated for 2 days before adding the CTG assay reagent. A total of 100 μL of cell lysate was transferred to 96-well white flat-bottom plates (Corning). The luminescence intensity was read using a Discover Microplate Reader (Promega).

[0147] Western blot analysis

[0148] Protein lysates were prepared using RIPA buffer (Sigma) supplemented with 1× cOmplete EDTA-free protease inhibitor mixture (Roche) and quantified using the Bradford assay (Bio-Rad). The protein lysates were then separated by 8 - 10% SDS-PAGE, followed by overnight incubation with primary antibodies (1:1000 dilution) at 4°C and incubation with secondary antibodies (1:10,000 dilution) for 1 hour at room temperature. The primary antibodies used were anti-ADAR1 (Abcam, ab88574), anti-AZIN1 (Proteintech, 11548-1-AP), anti-GAPDH (Santa Cruz Biotechnology, sc-59540), anti-ODC (Abcam, ab66067), and anti-CCND1 (Cell signalling technology, 2978).

[0149] Focus formation assay

[0150] For the colony formation assay, cells were seeded to obtain 80% confluence before ASO treatment. Cells were stained with crystal violet (Sigma-Aldrich) 48 hours after treatment.

[0151] Cell cycle analysis by PI staining and FACS

[0152] Before cell cycle analysis, cells were treated with ASO for 48 hours. After treatment, cells were fixed overnight at -20 °C with 70% ethanol. After washing with phosphate-buffered saline (PBS; 10 mM phosphate, 137 mM NaCl, and 2.7 mM KCl), cells were resuspended in 1 mL of staining solution containing 200 μL of 1 mg / mL PI (Invitrogen) and 20 μL of 10 mg / mL RNase A (Thermo Scientific) and incubated at 37 °C for 1 hour. Stained cells were analyzed on an LSRII (BD Biosciences), and results were analyzed on FACSDiva Software (BD Biosciences).

[0153] Loading of ASO into RBCEV

[0154] Blood samples were obtained from healthy donors by the Hong Kong Red Cross, China, and EVs were generated from RBCs according to an established protocol (Usman, W.M. et al. Efficient RNA drug delivery using red blood cell extracellular vesicles. Nat Commun 9, 2359, doi:10.1038 / s41467-018-04791-8 (2018)). According to the manufacturer's protocol, ASO was loaded into extracellular vesicles (EVs) derived from red blood cells (RBCEVs) at a ratio of 1:50 using ExoFect transfection reagent (SystemBioSciences). RBCEVs were washed twice with PBS at 21,000 x g for 30 minutes at 4 °C to remove free ASO and transfection reagent.

[0155] Labeling of RBCEV with CFSE

[0156] A total of 200 μg of ASO-loaded RBCEVs were incubated with 400 μL of 10 μM CFSE at 37 °C for 2 hours. A total of 0.5 ml of CFSE-labeled RBCEVs were loaded onto a pre-packed qEV original size exclusion chromatography column (Izon Science, New Zealand) and eluted with PBS into 40 fractions (0.5 ml / fraction). Fractions 7 - 11 were pooled and centrifuged at 21,000 × g for 30 min at 4 °C. The supernatant was removed, and the RBCEV pellet was washed twice with PBS, resuspended, and quantified using a Nanodrop spectrophotometer (ThermoFisher).

[0157] Fluorescence imaging

[0158] The cells were cultured on cover slips for 24 hours and treated with CFSE-labeled ASO3.2-RBCEV. At 48 hours after treatment, the cells were washed with PBS and then fixed with methanol for 10 min at room temperature. The fixed cells were washed three times with PBS for 5 min each time. The cover slips were mounted on glass slides using SlowFade Gold antifade mounting medium with DAPI (Thermo Fisher Scientific) and observed under a Zeiss Axio Imager M2 microscope.

[0159] In vivo tumorigenicity assay

[0160] - Pre-treatment model

[0161] KYSE510 cells were pre-treated with 100 nM ASO3.2 and ASO-ctl for 48 hours using Lipofectamine 2000 (Invitrogen), and then 4×10 6 The pre-treated cells were subcutaneously injected into the left and right dorsum of 4- to 6-week-old NOD scidγ (NSG) mice (n = 6 mice per group). Tumor growth was monitored by measuring the tumor length (L) and width (W) at the specified time points. The tumor volume was calculated by the formula V = 0.5×L×W2. All animal experiments were approved by the Institutional Animal Care and Use Committees of National University of Singapore (NUS, Singapore) and were conducted in accordance with it.

[0162] - Intratumoral injection model

[0163] A total of 2×10 6KYSE510 cells were subcutaneously injected into the right and left flanks of 4-6-week-old NSG mice for tumor development. When the tumors were visible (approximately 1 mm in diameter), the mice were divided into 2 groups (6 mice per group) for multiple intratumoral (i.t.) injections of ASO-loaded RBCEVs (Group 1: RBCEV-based delivery) or naked ASO (Group 2: naked ASO), once every 4 days for 7 weeks. For each tumor, each injection of ASO-loaded RECEV, a total of 1 μg ASO was loaded into 50 μg RBCEV and resuspended in 20 μL PBS. For each tumor, each injection of naked ASO, a total of 13.5 μg ASO (ASO-ctl or ASO3.2) was dissolved in 20 μL PBS. Tumor growth was monitored by measuring tumor length (L) and width (W) at the specified time points. Tumor volume was calculated by the formula V = 0.5 × L × W2. All animal experiments were approved by the Institutional Animal Care and Use Committee of the National University of Singapore (NUS, Singapore) and were conducted in accordance with it.

[0164] Statistical analysis

[0165] Unpaired two-tailed Student's t-tests were used to statistically analyze the changes in cell viability and tumor growth rate between the control group and the treatment group. For all numbers: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

[0166] Results

[0167] The 8-nt sequence at the 3' end of exon 12 is the core ECS and is essential for AZIN1 editing

[0168] Revealing the ECS of the AZIN1 transcript will help decipher the precise dsRNA structure crucial for AZIN1 editing. To this end, AZIN1 minigene constructs were generated by inserting different-length fragments into the pRK7 or pcDNA3.1 vectors, which covered the edited exon 11 and the flanking exons and introns ( Figure 1 A). Each AZIN1 minigene construct was co-transfected with the ADAR1 expression construct or the empty vector into HEK293T cells, and then editing analysis was performed on endogenous AZIN1 and exogenous transcripts transcribed from the AZIN1 minigene. First, it was examined whether ADAR1 could act effectively on exogenous transcripts transcribed from the pRK7-based minigene system. The HTR2C minigene was generated using HTR2C as a positive control, which is a well-characterized editing target and whose dsRNA structure has been well depicted in many studies. After co-transfection of the HTR2C minigene and ADAR1, three known A-to-I editing sites were detected in the exogenous HRT2C transcript ( Figure 1B), which supports the feasibility of using the pRK7 minigene system in this study. Additionally, approximately 75.8% of endogenous AZIN1 was edited, indicating successful overexpression of ADAR1( Figure 1 Band Figure 7 ). Among all the AZIN1 minigenes, only the AZIN1 transcript transcribed from the minigene containing fragment A (FA) could not be edited, and this fragment A lacked a 90-bp sequence at the 3'-end of exon 12( Figure 1 A, 1C). This observation could be reproduced using the pcDNA3.1-based minigene, thus excluding the possibility of artifacts in the pRK7 minigene system( Figure 1 D). All these findings suggest that the ECS of AZIN1 is most likely located at the 3'-end of exon 12.

[0169] To precisely map the ECS, secondary structure prediction was performed on the RNA sequence corresponding to fragment E (FE) by RNAFold30. As expected, the 3'-end of exon 12 formed dsRNA with the edited sequence( Figure 1 E). By introducing a 29-bp deletion at the 3'-end of the 29-bp sequence of exon 12 (FE-1), an 8-bp internal deletion (FE-2), or point mutations (FE-3) in the sequence directly opposite to the editing region( Figure 2 A), three additional minigenes were generated using the FE minigene. Secondary structure prediction indicated that both the deletion and mutations could significantly alter the secondary structure( Figure 2 B). Using the same strategy, it was observed that the transcripts transcribed from the FE-1, 2, and 3 minigenes could not be edited upon overexpression of ADAR1( Figure 2 Cand Figure 8 ). Additionally, in vitro RNA editing assays were performed and it was found that in the presence of purified ADAR1 protein, the in vitro transcribed AZIN1 transcripts from the FB or FE minigenes but not from the FA, FE-2, and FE-3 minigenes were edited at the expected editing sites( Figure 2 D). All these data strongly suggest that the 8-nt sequence (5'-GCUUUUCC-3') at the 3'-end of exon 12 is the core ECS and is essential for dsRNA formation and AZIN1 editing.

[0170] Identification of ASOs with significant in vitro editing inhibitory effects

[0171] Based on the elucidation of the AZIN1 dsRNA structure, seven fully 2'-O-Me modified ASOs (ASO1 - ASO7) and three PNAs (including 1 antisense PNA (ASP1) and 2 dsRNA-binding PNAs (DSP1 and DSP2)) that can form triplexes with AZIN1 dsRNA were designed and synthesized, and their binding abilities to AZIN1 dsRNA were evaluated (Table 3 and Figure 3 A). RNA electrophoretic mobility shift assay (REMSA) was performed on each oligonucleotide to examine their binding abilities to the 32P-labeled AZIN1 RNA duplex probe ( Figure 9 A). Strong band shifts were observed in the presence of ASO1, ASO3, ASO5, and ASO7, while very weak or no band shifts were detected after the addition of ASO2, ASO4, ASO6, and all three PNAs ( Figure 3 B). Notably, with the increasing amount of ASO1, ASO3, ASO5, or ASO7 added, a dose-dependent increase in AZIN1 duplex binding was detected, further confirming the binding ability of these ASOs to AZIN1 (with submicromolar affinities) ( Figure 3 C). Since PNA is shorter than ASO, further tests using a shortened AZIN1 duplex confirmed that 12-mer ASP1 could not bind to the shortened AZIN1 RNA duplex, while DSP1 and DSP2 could bind with moderate binding affinities (micromolar; Figure 9 B).

[0172]

[0173]

[0174]

[0175] m, 2'-O-Me modified; *, PS modified Table 3. Characteristics of ASOs and PNAs used

[0176] Next, to examine whether the binding of each oligonucleotide to the AZIN1 duplex was sufficient to inhibit AZIN1 editing, in vitro RNA editing assays were performed on ASO1, 3, 5, and 7. With the addition of ASO3, AZIN1 editing was completely abolished. Compared with ASO3, the efficiency of ASO1 was slightly lower but significantly inhibited editing from 87.6% to 2.8%; while for ASO5 or ASO7, they showed low or no inhibition of AZIN1 editing, respectively ( Figure 3D). Notably, ASO5 is a 25-mer ASO with 5 nucleotides (GCUUU) added to the 5'-end of ASO1 ( Figure 3 A and Table 3). Although ASO5 can target the editing site due to this extension, it fails to improve or maintain the editing inhibition of ASO1, which is consistent with the fact that ASO5 binds slightly weaker compared to ASO1 ( Figure 3 C), probably because the base pairs of the sequence involved in editing (AAAGC) (possibly 3 A-U and 2 G-C pairs) are relatively more stable and difficult to be invaded by ASO ( Figure 3 A). This is also supported by the following observation: ASO6, which shares the same sequence with ASO5 except being 5-nt shorter than ASO5 at the 3'-end ( Figure 3 A and Table 3), largely fails to bind to the AZIN1 duplex ( Figure 3 C). In addition, DSP1 and DSP2 can abolish AZIN1 editing at a concentration of 10 μM, but their editing inhibition significantly decays at 200 nM ( Figure 9 C), indicating that for editing inhibition, the formation of PNA-dsRNA triplex may not be as effective as traditional Watson-Crick base pairing. All these data suggest that ASO1 targeting the editing region flanking the editing site and ASO3 targeting the ECS can bind to the AZIN1 transcript and abolish or significantly inhibit AZIN1 editing in vitro at nanomolar concentrations.

[0177] ECS-targeting ASO significantly inhibits AZIN1 editing in cancer cells

[0178] Currently, the most widely used chemistries for pre-mRNA binding and splicing regulation are PS backbones that are fully modified with 2'-O-Me / 2'-O-MOE / LNA or PMO throughout the oligonucleotide length. Their stability, nuclease resistance, target affinity, and inability to trigger RNase H / RNAi responses make them ideal tools for pre-mRNA binding, splicing, and potentially RNA editing. Therefore, the 2'-O-Me modified ASO1 and ASO3 were further fully or partially modified with PS (ASO1.1, 1.2, 1.3 and ASO3.1, 3.2 and 3.3; Figure 4 A and Table 3) and included in this study. The basal editing levels of AZIN1 were screened in 9 HCC, 3 ESCC, and 3 NSCLC cell lines, and AZIN1 editing was detected only in the ESCC line KYSE510 and the NSCLC line H358 ( Figure 10A). Next, KYSE510 and H358 cells were treated with each chemically modified ASO. Unexpectedly, all 7 ASOs (ASO1, 1.1, 1.2, 1.3, 5, 6, and 7) targeting the editing region resulted in exon 11 skipping ( Figure 4 B and Figure 10 B), which might be due to the presence of splice factor binding sites in the editing region predicted by SpliceAid231 ( Figure 4 C). Notably, among the 3 ECS-targeting ASOs, ASO3.1 and ASO3.2 completely abolished AZIN1 editing, and ASO3.3 significantly inhibited editing without affecting the splicing and expression of AZIN1 at the mRNA and protein levels ( Figure 4 D-G). Other PS modifications in 2'-O-Me modified ASO3 might increase chemical stability, thus enhancing the editing inhibition effects of ASO3.1, ASO3.2, and ASO3.3 in cells. The above findings indicate that the editing region at exon 11 of the AZIN1 transcript is not targetable, and only ASOs targeting ECS can effectively inhibit AZIN1 editing in cancer cells.

[0179] ASO3.2 specifically inhibits G1 / S transition and cancer cell viability

[0180] Next, it was investigated whether the most effective ASOs, namely ASO3.1 and ASO3.2, specifically inhibited cancer cell viability by suppressing AZIN1 editing. For this purpose, in addition to KYSE510 and H358, the AZIN1 editing null ESCC cell line KYSE180 was also included in the study. The three cell lines were treated with increasing concentrations of ASO3.1, ASO3.2, or ASO-ctl by Lipofectamine transfection, and then cell viability analysis was performed. It was observed that both ASO3.1 and ASO3.2 significantly inhibited the cell viability of KYSE510 and H358 with lower IC 50 values (ASO3.1: K510: 45.8 nM and H358: 37.6 nM; ASO3.2: K510: 62.3 nM and H358: 51.0 nM); while their inhibitory effects on KYSE180 cell viability were much smaller (ASO3.1: 271 nM; ASO3.2: 699 nM) ( Figure 5 A). Notably, the IC50 sensitivity of KYSE180 to ASO3.2 was approximately 2.6-fold lower than that to ASO3.1, which means that ASO3.2 is most likely to confer higher specificity in inhibiting editing and cancer cell viability than ASO3.1. Figure 5A). To further confirm the specific inhibitory effect of ASO3.2, after treatment with low-dose ASO3.2, cell viability and focus formation assays were performed on three cell lines. As a result, ASO3.2 only inhibited the cell viability of KYSE510 and H358, but not KYSE180( Figure 5 B, C). In addition to cancer cells, normal human hepatocytes isolated from the perfused livers of humanized mice were also treated with ASO3.2 or ASO-ctl Figure 5 B). It was found that normal hepatocytes were insensitive to ASO3.2 treatment. All these data support that ASO3.2 can specifically inhibit the cell viability of cancer cells expressing edited AZIN1 S367G . Cell cycle analysis showed that compared with cells treated with ASO-ctl or ASO3, both KYSE510 and H358 cells after ASO3.2 treatment exhibited a significant attenuation of G1 / S transition and a significant increase in the percentage of sub-G1 phase (apoptotic cells)( Figure 5 D). In addition, the decrease in the expression of CCND1 and ODC proteins supported the G1 / S arrest induced by ASO3.2 Figure 5 E). In summary, ASO3.2 can specifically inhibit AZIN1 editing in cancer cells, resulting in a decrease in CCND1 expression and the consequent G1 / S arrest and reduced cancer cell viability.

[0181] ASO3.2 effectively inhibits tumorigenesis and growth in vivo

[0182] The role of ASO3.2 in tumorigenesis and growth was investigated using two xenograft tumor models. Using lipofectamine transfection, KYSE510 cells were pretreated with ASO-ctl or ASO3.2 and then subcutaneously injected into the two dorsal sides of mice to compare their tumor incidence and growth rate. The tumor incidence in the ASO3.2 pretreatment group was significantly lower than that in the ASO-ctl pretreatment group Figure 6 A). In addition, during the 6-week observation period, the growth rate of tumors derived from ASO-ctl-pretreated cells was significantly faster than that of tumors derived from ASO3.2-pretreated cells Figure 6B). In addition to the pre-treatment model, the effect of ASO3.2 on tumor growth was also studied by intratumoral injection. Extracellular vesicles (EVs) are small membrane vesicles released from different types of cells and are increasingly recognized as natural RNA carriers and novel drug delivery carriers. After entering the cell, the cargo is released from the EVs and the ASO is transported to the nucleus. To deliver ASO into tumor cells, ASO3.2 or ASO-ctl was loaded into EVs derived from human red blood cells (RBCEVs), which are an ideal source of EVs with promising properties for RNA drug delivery. To test the cellular uptake of ASO3.2-RBCEVs, ASO3.2-RBCEVs were labeled with carboxyfluorescein succinimidyl ester (CFSE), and CFSE fluoresces only in the presence of esterase when it is loaded into RBCEVs or internalized into cells. It was found that most ASO3.2-RBCEVs could enter cells ( Figure 6 C). Next, KYSE510 cells were subcutaneously injected into the two dorsa of mice to promote tumor development. When the tumors were visible (about 1 mm in diameter), ASO-ctl-RBCEVs or ASO3.2-RBCEVs were injected intratumorally every 4 days. This experiment also included naked (unloaded) ASO-ctl or naked ASO3.2 to examine whether RBCEV-based delivery enhanced the uptake of ASO into tumor cells. As expected, intratumoral injection of ASO3.2-RBCEVs significantly inhibited tumor growth ( Figure 6 D); while no obvious difference in tumor growth was observed between mice treated with naked ASO-ctl and naked ASO3.2 ( Figure 6 E). In summary, ASO3.2 can effectively inhibit the occurrence and growth of tumors in vivo.

[0183] ASO3.2 specifically inhibits the cell viability of cells derived from HCC PDX

[0184] To evaluate whether ASO3.2 could be a promising RNA therapeutic agent for cancer treatment, the effect of ASO3.2 in Derived from cells of HCC PDX was examined. Since HCC is a highly heterogeneous cancer, this study also included cells derived from PDXs to investigate whether ASO3.2 specifically targets the tumor cell population expressing edited AZIN1, and PDXs were generated from different regions of the same primary HCC tumor (such as PDX22-T1 and PDX22-T2). First, the editing level of AZIN1 was examined in all PDX cells. Four PDX cells (PDX-1; and PDX-22-T1, T4, and T5 from different parts of PDX-22) had more than 20% of edited AZIN1 transcripts ( Figure 6 F). By Figure 6As can be seen from Figure G, PDX1, which is an AZIN1 editing-positive PDX line, showed significantly reduced cell viability after treatment with ASO3.2; while no obvious change in cell viability was observed in the non-AZIN1 editing-positive PDX line PDX22-T3. These data indicate that ASO3.2 has a specific inhibitory effect on isolated cell viability.

[0185] Discussion

[0186] Dysregulated A-to-I RNA editing is associated with a variety of human diseases, including cancer. Dysregulated A-to-I editing is a key driver of the pathogenesis of various cancers, such as breast cancer, glioma, multiple myeloma (MM), chronic myeloid leukemia, HCC, CRC, gastric cancer, and ESCC. Transcripts abnormally edited by ADAR in cancer tissues, such as AZIN1, Gli1 (glioma-associated oncogene 1), and DHFR (dihydrofolate reductase), significantly promote cancer progression and metastasis. Different from DNA editing, the genetic information manipulated by RNA editing is reversible and adjustable. Since ADAR1 has multiple functions crucial for normal development, such as hematopoiesis and organ development, simply regulating the expression of ADAR can lead to considerable off-target effects. Another strategy is to disrupt the ADAR enzyme at specific editing sites of target transcripts.

[0187] This study found that the 3'-terminal sequence of exon 12 of AZIN1 forms dsRNA with the editing region. Therefore, a variety of 2'-O-Me / PS-modified ASOs and PNAs targeting the editing region or ECS were designed and synthesized. It was observed that 1) ASO1 or ASO3 could sufficiently inhibit or completely eliminate AZIN1 editing in vitro, 2) ASO2, 4, and 6 could not bind to the AZIN1 duplex, and 3) although ASO7 could bind to AZIN1, it still could not inhibit editing in vitro. All these observations indicate that the 5' of the editing site position and the 3' of ECS are beneficial for the binding of ASO to AZIN1 and the inhibition of the ADAR1 reaction, which can provide a useful model for understanding ADAR1 substrate binding and deamination in the future.

[0188] In addition, it has been reported that PNAs incorporating modified nucleobases such as thiomorpholine pseudoisocytosine (L) and guanidine-modified 5-methylcytosine (Q) can selectively bind dsRNA in a sequence-specific manner relative to ssRNA and dsDNA. In addition, PNA has a neutral peptide-like backbone, is chemically stable, resistant to nucleases, and provides enhanced specificity for RNA sequence and structure recognition. This study also tested the inhibitory effect of PNA on AZIN1 editing. Since ASO4 (20-mer) cannot bind to the AZIN1 duplex, it is not surprising that the antisense PNA ASP1 (12-mer) also cannot bind to AZIN1. Although the dsRNA-binding PNAs DSP1 and DSP2 can bind to AZIN1 with moderate affinity, they cannot inhibit editing at nanomolar concentrations, which may be attributed to the insufficient blocking of the ADAR1-AZIN1 dsRNA interaction by DSP1 (10-mer) or DSP2 (8-mer) due to their relatively short lengths. All these observations suggest that relatively long and chemically stable ASOs (e.g., a combination of 2'-O-Me sugar ring modification and PS backbone modification) may be the optimal chemicals for inhibiting RNA editing.

[0189] Although ASO1 showed very promising editing inhibition in vitro, it was found that ASO1 and other ASOs targeting the 42-nt editing region led to a large amount of exon 11 skipping, which may be due to the blockade of splicing regulators (such as SRSF1, SRSF3, SRSF6) in the editing region, indicating that the editing region is not targetable. In addition, even though the ECS-targeted, 2'-O-Me-modified ASO3 can completely eliminate AZIN1 editing in vitro, only ASO3.1 and ASO3.2 (which have the same sequence as ASO3 and have complete or partial PS modifications, respectively) can effectively eliminate AZIN1 editing in cancer cells. This may be attributed to the advantages of PS modification, such as strong resistance to endonuclease and exonuclease digestion, increased serum stability, and reduced renal clearance. Notably, compared with ASO3.1, ASO3.2 showed higher specificity for inhibiting cancer cell viability by inhibiting AZIN1 editing, which may be because of the adverse consequences of non-specific binding to proteins and other nucleotide sequences caused by PS modification. This finding is further supported by the following observation, that is, ASO3.2 was observed to inhibit the S367G cell viability of cancer cells expressing AZIN1 and cells derived from HCC PDX, but not AZIN1 S367G- Cell viability of null cancer cells, PDX cell lines, and normal hepatocytes. Additionally, in a pre - treated xenograft tumor model, ASO3.2 significantly inhibited tumorigenesis and growth. This observation was also supported by the intratumoral injection model, in which ASO3.2 was delivered into tumor cells using an RBCEV - based delivery method, indicating that intratumoral injection of ASO3.2 loaded into RBCEV rather than naked (unloaded) ASO3.2 significantly inhibited tumor growth.

[0190] In summary, the data indicate that ASO - mediated inhibition of AZIN1 editing effectively inhibits tumorigenesis and growth, supporting the possibility that a large number of cancer patients, especially HCC patients showing high levels of AZIN1 editing, could benefit from AZIN1 - targeted, ASO - based therapeutic strategies. Due to the liver structure and rapid endocytosis, hepatocytes have a high receptivity to ASO uptake. Conjugation of the tris N - galactosamine (GalNAc) targeting domain to ASO results in a 10 - fold increase in ASO activity, which significantly improves the chemical properties of second - generation ASOs and increases the potential of ASO therapeutics for treating liver diseases, including HCC. In this study, the discovery of this ASO - based RNA editing inhibitor provides an attractive approach for targeting cancer - related RNA editing substrates. Sequence Listing <110> National University of Singapore Nanyang Technological University <120> RNA Editing Inhibitor and Its Use <130> 10104SG1273 <160> 52 <170> PatentIn version 3.5 <210> 1 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 1 aagaagacag cuuuuccgcu gaagcuuaa 29 <210> 2 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 2 ugagcuugau caaauugugg aaagcugucu ucuuccugag cu 42 <210> 3 <211> 37899 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 3 ctaatataaa tactggcgtc gctggcgccg ccttctcaca ctttcaggct ctgatcgcgg 60 ccgcagtttt tccttttttc ttctgccgtc gccttctctg cctcttctca tcctttctcg 120 ctctgctgct ctgcagtgtg acgagtccga atcctcttcc cacccagccc gcgcctttct 180 tcttttgcct gcgctgttct atttctcctt cggccgccgc cgccactgct gcacacagct 240 ggtgtcggtg ccgcgctttt acccccaagt cgttcccgca gcctatggcc caggccgcct 300 tgggtatttc tgctcaaggt aaccacatcc ctctttaaaa attccgccga aaaagagaag 360 acgctttacc cgactctttg ggccgttatc tcacgtgagt accgagccga ggagcgcggg 420 aggggccagg acgcggggct ggccttgggg ggcggctggg gggctccggc tgggccccgc 480 cgcccttaag acgtccccgc agcgccccgc ggccggccgc ctcgaacatg gcggcggcgg 540 ctgtctgtgc ccccggcggc ggcggcggcg gcggggctgg gcggcgggcc gcgcctcagg 600 ctgtctgtgc ccccggcggc ggcggcggcg gcggggctgg gcggcgggcc gcgcctcagg 600 accttcgggc ggctgctgga cgcttggcgg agccggagcc gcagcagttg gggcgagggg 660 accttcgggc ggctgctgga cgcttggcgg agccggagcc gcagcagttg gggcgagggg 660 gcgtgtgcca gtgggggagc gcgcgggggt ctcgccgccg ggggtcgccg tcccctcctg 720 gcgtgtgcca gtgggggagc gcgcgggggt ctcgccgccg ggggtcgccg tcccctcctg 720 gcggcgtgcg gcccccaccc ctctcccgtg gtgcattgct gtttccgggg agggggcccg 780 gcggcgtgcg gcccccaccc ctctcccgtg gtgcattgct gtttccgggg agggggcccg 780 gccgcagccc tgaggggccg gggtcccgca gcccctactc cgcggtgcgc cgccgccacc 840 gccgcagccc tgaggggccg gggtcccgca gcccctactc cgcggtgcgc cgccgccacc 840 tttgcctccc gaagcccggc gggcgggcgg cgctgtgggc tgcaacggct ggggtcctgg 900 tttgcctccc gaagcccggc gggcgggcgg cgctgtgggc tgcaacggct ggggtcctgg 900 ttatgaatgg agcggggcgg gccccgcgcc ggcctctggt gacagctgcg gaggaagtcg 960 ttatgaatgg agcggggcgg gccccgcgcc ggcctctggt gacagctgcg gaggaagtcg 960 gattcctggg gggcgcgggg agcgcggtcc ccctctggtg gaggaaagtt gacgccactt 1020 gattcctggg gggcgcgggg agcgcggtcc ccctctggtg gaggaaagtt gacgccactt 1020 ggggcctggc ggcggggaag ccccggtgtc aggtagccag cgagaggctt ttggcccctt 1080 ggggcctggc ggcggggaag ccccggtgtc aggtagccag cgagaggctt ttggcccctt 1080 ctggaagcct cgaggctgct gctccgcccc tgcggcgctc ctgattgggt caagtcgcct 1140 ctggaagcct cgaggctgct gctccgcccc tgcggcgctc ctgattgggt caagtcgcct 1140 tcactcaccc cagtcacctc ctggagcctg aggattagtc accatggctt cgtttttccg 1200 tcactcaccc cagtcacctc ctggagcctg aggattagtc accatggctt cgtttttccg 1200 tccgcgccat ggttcctagg cagaaagagg gagattggta gacgggtggc ttatcaacta 1260 tccgcgccat ggttcctagg cagaaagagg gagattggta gacgggtggc ttatcaacta 1260 ctgtcttgac tgccaacagt tggtggtggt aacatgttct aacccttaca cggtatcctg 1320 ctgtcttgac tgccaacagt tggtggtggt aacatgttct aacccttaca cggtatcctg 1320 aatgtgtgtt tcgtagacaa agctgctggt tcagaaaccc gaaacagtcg gggacacagt 1380 aatgtgtgtt tcgtagacaa agctgctggt tcagaaaccc gaaacagtcg gggacacagt 1380 ttaccgcctc atgtttcagc acatttaact gaattttgtg gtggcatctt ggttatatta 1440 ttaccgcctc atgtttcagc acatttaact gaattttgtg gtggcatctt ggttatatta 1440 gttctgtgtc ccgtcgtgtt tctatcgtga attcccagac tgggtgggaa tctaaggact 1500 gttctgtgtc ccgtcgtgtt tctatcgtga attcccagac tgggtgggaa tctaaggact 1500 caaaggaggg ggaattgcct tgtgctcagc gaaggttgat gtagaggtgc atcaaagcac 1560 caaaggaggg ggaattgcct tgtgctcagc gaaggttgat gtagaggtgc atcaaagcac 1560 aaagtagaag tacatcaaag cacatcaaat acagcaaggg ttggcagaaa catggctcat 1620 aaagtagaag tacatcaaag cacatcaaat acagcaaggg ttggcagaaa catggctcat 1620 gattgtctag aaaggccctc ataattcatt aatttctttt acatatacga gagcgtgcag 1680 gattgtctag aaaggccctc ataattcatt aatttctttt acatatacga gagcgtgcag 1680 atgctgatca tgattagaaa taagccccaa ggagtttagg aaagactcca aaagaaaatg 1740 atgctgatca tgattagaaa taagccccaa ggagtttagg aaagactcca aaagaaaatg 1740 attatcggtg gtgataagtg aggtcgagta gtcagtgcct ttctccccca aggttttatc 1800 attatcggtg gtgataagtg aggtcgagta gtcagtgcct ttctccccca aggttttatc 1800 acatacgttt tcaaacagaa ggtgaaaaag agttatactt ccacctagat tctgtaatta 1860 acatacgttt tcaaacagaa ggtgaaaaag agttatactt ccacctagat tctgtaatta 1860 acattttacc ttatttgtcg atgcatttca aagtaaagtt ggagacccct ccaactttag 1920 acattttacc ttatttgtcg atgcatttca aagtaaagtt ggagacccct ccaactttag 1920 tatgtgcata ttaaatagag ctcagtaccg cttactattt tttaaatctt agagggtaaa 1980 tatgtgcata ttaaatagag ctcagtaccg cttactattt tttaaatctt agagggtaaa 1980 attcacatca gtataaaccc acaagtctta agtatgcaat ttgatgaacg ttgacaaaaa 2040 tacagctgtg taacctaaac cttttgtcaa gatgtagaac atcgccatca ccctcaaaag 2100 ttttttcttg tcctttcaaa tcgtcttcca gtcaccacag agccaaccac gatcttggtt 2160 tttttttttt ggagtctagc tctgtcgccc aggctggagt gcagtggcac aatctcggct 2220 tactgcaacc tccgcttccc aggttcaagc gattctcctt cctcagcctc ccgagcagct 2280 gggattacag gcgcccacca cgcccagcta atttttgtat ttttagtaga aaaggggttt 2340 cactgttggt caggctggtc tggaactcct gacctcatga tccgcccatc tcggcctccc 2400 agagtgctgg gattacaagc gtgagccact gcgcccggct gatcttgatt ttttttttta 2460 aacccataga ttattttcgt ctattccaga ctttccacat gaatggaatc atacagtatg 2520 cattctttgg tataaaaggc ttcttttaac tcagcacatt tttaatattc atccctgttg 2580 tctttatctg tagattatca gtagattatt tgtgttgctg agtagtagcc cattaggtga 2640 ctgtcccttg gttgtttttt ttaaaaataa actttattag gccgggcgcg gtggctcaag 2700 cctgtaatcc cagcactttg ggaggctgag gcgggtggat tgcctgaggt caggagacca 2760 gcctggctaa catgctgaaa ctccatctct actaaaaata caaaaattag ccgggcgtgg 2820 tggctggctc ctgtaatccc agctcctcgg gaggctgagt caggagaatc gcttgaactg 2880 gggaggcaga ggttgcagtg agccgagatc tcaccagggc actccagcct gggggacaag 2940 agcagcggga cttcatctca aaaaataata ataataatga aaaataaact ttattttgga 3000 gtatcttagt tgcgcagcaa agtcacaaag atacagtttc ttcatacttc ccacccagtt 3060 attagtaaca tcttacgtta ttacagtaca tttgttaaaa ctaagaaact gactttggca 3120 ctatgaaacg ttcgagactt ttatttggat ttcgccaatt tttctttaat gtctttttct 3180 gttccagaat ccaagttacc acattgtatt tagttgtcat gtcttcccag ttttctttgg 3240 tttgtgacag tttctcagtc ttcctttgtt tttcattacc atcacagtct agaaaagtgc 3300 tgccagatat ctggtagaat actcccctcc cctaaacttg gggtttacct gatgtttttc 3360 tcatgattag actggggttt ggagttttta caaagaatac cacagaacga ttttaaaaat 3420 cattggtaaa ggcattgtgt tttcagagac gtactgataa ttttgagata ctaagtttgt 3480 aatagttact gtcaaagtga tagaaagttt ttgtagttgg gcacggtggt ttgcacctgt 3540 aatcccagca cttcggaagg ctgatgcagg cggatcactt gagctcagga attcaaggcc 3600 agcttgaatt cctattttat ttttaagaaa ataaaatagg ccaggcgtgg tggctcatgc 3660 ctgtaatccc agcactttgg gaggtcgagg tgggtggatc gcaaggtcag gagttcgaga 3720 ccagcctggc caatatgatg aaaccccatc tctactaaaa atacaaaaaa attagccggg 3780 catggtggca catgcctgta atccgagcta ctcggaaggc tgaggcagga gaattgcttg 3840 aatctgagag gcagaggttg cagtgagcca agatcgcgcc actgcactcc agcctgggtg 3900 acagagcgag actccgtctc aaaaaaataa aataaaataa attacataaa atgcttcttg 3960 tttcttactg accaaccgag ccctctctgt gctattctct aaggtgttag agaatttgca 4020 gtgtttacat acaccattct aagtgaaaat aatttatttt tttccatact gccttttctc 4080 cgcatttcca tttaacaagt ccttcttcat gcttttttat cctaactgtc cggataaaca 4140 ttctatctat cctgtatgta agtgcttata agaagtgctt atctatcctg tatgtgttta 4200 taagaagtgc ttatcctatg caccttttgg gtcttgctgt gttgcccagg ctggactcaa 4260 attcctgggc tcaagcagtc ctcctgcctt aacctcctca gtagttgaga ctataggtgc 4320 atactgtcac atctggctca ttttttcttt tcttttcttt ttttggagac aggttctccc 4380 tctgttgcct gggctggagt acagtggcac aatctcggct cactgcaacc tccgcctctt 4440 gagttcaagc aattcttgtg cctcaggctc ctgagtagct gggattacag gtgtgcaccg 4500 ccatgcccag ctgatttttg tatttttagt agaagatggg atttcactgt gttgaccaga 4560 ctgatcttga actcctggcc tcaagtgatc cactggctca gagccactgc gcctagccct 4620 attttttgta tttaaataat gcctttcaaa ttctagagcc ttcattccct actagtatct 4680 ttttgttaaa tggaaatgtt gtagggttct cactgaggaa cattctggaa tgtctgacgt 4740 ttaatgtcca taatgtttca catcattgct cttgggttgc attaagcagc aaaatagctg 4800 tggatatatg tattgagaaa aagatgaata atgtcttacc tagggtatct gatcctattc 4860 cttaggataa ttgagaatga tctgtcccca gcatgactca actttttatt tatttagaaa 4920 attacgttgg tgttgaaaga atcagaatgt tatggagtag tacattattt ggaatctaat 4980 gtaagattat tatcatcatt gcatgtttgt aggagttctt tcctcatcat ccataaatgt 5040 cattcattga gcatgtgatc agcttctcct agatagatga aacattttgc ctccaccagg 5100 agtgccttga tcctggtgag atacacttta ggtatgccta cagtgcttct gaaatccctc 5160 caactcttga ctttataatt tctggttttc tggcttcctt cttaccaccc ctttctagca 5220 aacactgtca gattatttta cttgaaaaca gtaaccactg aattatacat agaaacatag 5280 gtatatcatt ttagcttaga agtgatgagt ggtaacaatg tctaaaactc acctaccatg 5340 ttgtatagtg tgctgtgcac ttatcacatt tatttgttcc tggtgataat agaatgaatt 5400 atgtccaaat aggttagata tgagaagata aatcttaaga gattagttgc ttaatattat 5460 aaggaagttc tcaagttatt ttatgttttt caaaagttat gttagataca tatataatgc 5520 attagctaag tcataaaatc ttttggtatt gtggtggatt tttacattta aaatgtaaaa 5580 gacaaatatt tgaggagtaa aagagtgttc tgggtggttc caaactattg ggtcatgcta 5640 gtacattatt catataaagt cttgtatata cactaggttg gtgagggtac ttggaatttt 5700 ttaaattctg gaccacttaa aaatgtggaa ttttagagga caaataaaag caaaaatgaa 5760 gttttcatct tcaaaaatga aggctggtaa ctcttgagta ggcagataaa acgacttggt 5820 aagatggcta tgacatcaag ttctgtgaat tttttatagc agcttggaaa tgtttactgc 5880 ataaagtact tggtactgtg gtaaaccctt tgaaaatatc acttgtgtaa tgtgacttta 5940 atgaaacatt gttataaaat gaacacgtat tctacagttc atgtttaagt cagcctaaac 6000 atttttttaa cataggacta tcttctgcta cttttatttc cttcttttag ggcgaacttt 6060 ctgaccaagt atacaactac ccagagggcc taggagaagt gctgtataga gagcagttcg 6120 acttcaacgc tgagccacct tgggaaccta gctgatgata ggggggttcc atctcccaac 6180 ttgtccatgt aagtatttgc acatttgttg gtggggagga ggtctttaaa atcgaaggaa 6240 gagtgctgcc ttgcttagta tttttcagaa tttgaaattg catttacctc ttaaagtgca 6300 gtgactttta aatttctgaa atagagataa gacagttact ggttacagac ttgactgatt 6360 tagttgtgtg cattgtttac caatgactgg atcatttaaa actatcttgt atgcataact 6420 tagaaaatat acgttgaaga gggcttttgg gaaataaggt atagtttatc acatcattca 6480 accatttaac ttagtgaaat gatttttgct actcagctgg taggaacaac taactgattg 6540 tttttagagg acttgctgaa agatgataga gatagataga gatacatata tatatctcaa 6600 tttatatatg tatctaaata tgtatatggt attaaaatat atgtatggta ttaaaatata 6660 gatatttaac agattttcaa ggatagtttt tgactatact tggttttcta tgtgtgaatt 6720 ttactttaga attgagcccc tgtgcaaaat gtaattccac agtaaactga atttgggcta 6780 ctagatttgt gtgggttcat ttaaaaattt tgtggtatat agacttattg ttacggaaat 6840 tcagattttg ttattaaaat ttaagtgttt ttaagtttgg tcagagactt agaggtaagt 6900 tttcaggaaa gctgagggta tcagttttag tgtttcttta atctgatctt gttaaacaga 6960 tccctattcg ataaaagtgt ttgctctaag caaacatttg atgttaatac aaattagaat 7020 tttagactca ggtttttaag gctaaagaga aggaaacaca gtttaaatag taagatttct 7080 tgaaaccttt gacttagaat gttggctaga gggtttccta tactggcttt ctcctacctg 7140 tgtaaatttg agcaaatgtg aatcccagct ataacataat gttggactgg aacagttctt 7200 tttgtgttaa actggcataa tttctttttt tgagtgtatt actgtccttg gttatgcagt 7260 gtagtcttcg gacgaattgc attagcatcc tcacctagaa cagaaataca gaatctcagc 7320 ctcacccaaa cctgaatcag agtctatatc caggtggctt gtgtgcacat taaagagaag 7380 cagggtcatt gctgtttcaa tactaagtca gttgcctaat ttttgttagt ccatctgttc 7440 tatttgcaac atacctgatg tttttctccc aagttgactt ggagatataa aacctttttg 7500 ggtggtgcaa tctgttaaag ttaacatcat agttgtgaat ccattctaag gcagaaattt 7560 aagaacaagc ttcgtctttt gaggaagacc gtaataaaag ataatccttc ttttaattcg 7620 tgtctcttgc tttaaagaaa agttaccttg cattatagtt aatatttcta ctggtgtttc 7680 ctcttagaaa cttgagagac tattgtcagt gagctgaatg attctgcaaa atcagggaaa 7740 agtagtcttt agattaataa tgtaggattc agaaagtcta tagtgtctcc ctctttgtgg 7800 gtaggaggga atgggagcag atagaagcta aagataattt attctgcagg ttctggcagt 7860 cagaataact ggcttttaac tttattatgc ccgggtacaa tgcatattat gtcaaacaaa 7920 tataacttct ttaaatattc acatgtaatc ttctcatttt gcatgtctag tatttgatgc 7980 tttgctataa agctacaggt ccacaatccc tttttcatag aattctgaaa tccaaaaagt 8040 ctttaaaaaa atttttttat agcttccttg atgtgacctt aactgatttg ggaaaaaaaa 8100 aaaaaaacaa aaaaaacttg acctggatct gtgctgattt gaggctattt ttgtctttac 8160 ttatcccatt tagtgttaat attcctaaat tttgcttcag aaatattgtg gttgcagggt 8220 gctgactcat tctcctgaag aatattacat aacatatgta aagtatgtgt actacattat 8280 ggttcaaaat ctggagaatt ctgaattcac aaagatacct agtcccaaag gtttcttgac 8340 tagaatttgt aaaattggca agagaaaatt ccgcattcca gaaatgtgtt atccttacaa 8400 atcaataaga gtttgctcta ttggacaccg aaatcctggg gttaattttc tgtagatcaa 8460 caatattatt tgaaaaccat taaaaggagt taaaacagct tcatggaaat tatttggccc 8520 atagcattgc aaagcatgta gctatatgaa ctctgcttga actggtccac taggattccc 8580 gtgcaggtag tctgagatcc agaattggct tcagagttag gttttaaatc aaccacctga 8640 attttgatgg aataaactaa gtatctggaa attttggaag ttgcttacat aatccttttc 8700 tttcataatt tagaatgtgt gtatgatgta gtacgcaaac actgtaaaaa tgtcttaaca 8760 aaatatttta aggtcaatat caaagattaa tgggccccta attgtgaaaa agtttttgtg 8820 agccttttga gaaatcccct aggccaggtg aggtggctca cacctgtaat cccagcactt 8880 tgggaggcca aggcgggcgg attacatgag gtcaggagtt caagaccagc ttggccaaca 8940 tggtgaaacc ctgtctctac taaaaaaaaa tataaaatta gtcgagcgtc gtggcaggtg 9000 cctgtaatgc caactactcg tgggaggctg aggcaggaga atcacttgaa tctgggaggc 9060 ggaggctgca gtgagccgag atcgtgccat tgcacttcag cctaggctac aacagcaaat 9120 ctccgtctca aaaaaaaaga aatcccctag gtcttgatgc tggagtaaag gttttccagt 9180 gggctagttt tctatttatt ttggttgtag gtttaattat aattaatgaa ttagcctaat 9240 ggaaaatatt ttaaaactta aaaagctaag tttgcttctc atagtggatc cagtgatgtg 9300 gtcagattca ttatcacata ccctttaatg ttgtgcagta tggaaaaggg attcagttaa 9360 ttgatttgct taaatgggct cttaacaagt ttgctttaaa aagttttttt ttttacctgg 9420 ttgtcattag tcggctctat atattcatgt aggaacttca gaagaacatc agcattttaa 9480 atattcacat tttgtctcct aatctttagt atttacacat gccttagtag gagctcccta 9540 cctaattagg attattttaa ctagggaaaa aaaatatata tatatatata tatttttttt 9600 ttttttgaca gagtctcctc ctttcgccca ggctggagtg cagtggcaca atcttggctc 9660 actgcaacct ccgcctccct ggttcaactg attcttccgc ctcagcctcc caagtagttg 9720 ggattacagg cacccaccat tatgcccggc taaattttgt atttttgtgg agatgggttt 9780 caccatgtgg ccaggctggt cttgaactcc agacctcagg tgatccgccc cccttgcctc 9840 ccaaaatgct gggattatag gcgtgagcca ccgcgcctgg ctggaaagat gttctttacg 9900 aatttgctag tactccttcc tcatgtttca ctgtaagtta tcagtaggaa agggaggttg 9960 tccgctgggt gcggtgcctc acacctataa tcccagcact ttgggaggct gaggccggca 10020 gatcatctga agtcaggagt ttgagaccag cctggccaat atggtgaaac cttgtctcta 10080 ctaaaaatac aaaaataaat tagttgggca tggtggtgca cgcatgtaat cccagctact 10140 agggaggctg aggcctgctt gaacccggga ggcggaggtt gcagtgagcc aagatggtgc 10200 caccgcactg tagactagga gacagtgcaa gactgtctca agggaaaggg aggttgttga 10260 ccctgactgc agagaaaggg gaaaatttca gttttgatat ttggtacttt tttttctttt 10320 ctccccccac cccccgattt aatttctcac agatgatatt tggtacttta aggatactgt 10380 gatatgagca aatgtaacag aacattcatt aatggcggtt gatactcttt gaccaaaaag 10440 atgagtaaaa ccactatcgt tgaaatacac agtctgtata tccccttggc atacataaag 10500 tagagtcaat ggtgtgtcag tgcttgtgta aagtagcaga aaaatattct gtaaaagcac 10560 aatgtgaata ctagcatatt aatggataaa caaggtttta gagacatggt atttcttgct 10620 ttgagcaaat ttagcatttt ttttaggaag gtcaaaaaga ttggaaagca acgtagacca 10680 ttgagcaaat ttagcatttt ttttaggaag gtcaaaaaga ttggaaagca acgtagacca 10680 gttttttctt tcttttttga gacgaagttt cgctcttgtt gcccaggcta gagtgcagtg 10740 gttttttctt tcttttttga gacgaagttt cgctcttgtt gcccaggcta gagtgcagtg 10740 gcgcgatttc agctcactgc aaactccacc tcccgggttc catcaattct cctgcctcag 10800 gcgcgatttc agctcactgc aaactccacc tcccgggttc catcaattct cctgcctcag 10800 cgtcctgagt ggctggtatt ataggcatgc accaccatgc ccgactaatt ttgtatttct 10860 cgtcctgagt ggctggtatt ataggcatgc accaccatgc ccgactaatt ttgtatttct 10860 agtagagaca gggtttcacc atattggtca gacttgtctc gaactcctga ccccatgatc 10920 agtagagaca gggtttcacc atattggtca gacttgtctc gaactcctga ccccatgatc 10920 cgccttcctt ggcctcccaa agttctagga ttacaggcat gcgccactgc gcccgaccat 10980 cgccttcctt ggcctcccaa agttctagga ttacaggcat gcgccactgc gcccgaccat 10980 agaccagttt ttaaacggga taaatggctt ttgcatttat tacagaggtt agtttagctg 11040 agaccagttt ttaaacggga taaatggctt ttgcatttat tacagaggtt agtttagctg 11040 agaaaattaa tactgactac caatctgtgt gttccatcac agtcttcacc caacctgatc 11100 agaaaattaa tactgactac caatctgtgt gttccatcac agtcttcacc caacctgatc 11100 ttgtttcaga gttttccagt ttgggtctcc ccatacccca ccccaggtaa accaatccct 11160 ttgtttcaga gttttccagt ttgggtctcc ccatacccca ccccaggtaa accaatccct 11160 tttgcgatat ttaacttcca ttagaataaa tgagatattg catctatgaa aaaggcttgt 11220 tttgcgatat ttaacttcca ttagaataaa tgagatattg catctatgaa aaaggcttgt 11220 aaaccttaaa atctagtata tggcaggcta ctgttctaaa ctggccattg ttcagataag 11280 aaaccttaaa atctagtata tggcaggcta ctgttctaaa ctggccattg ttcagataag 11280 cctaaggaga ctcttccaag ttgaagtgct tacatactgg tgaggcccca gggagcacag 11340 atattttgtc cttcctccat tccagggaca tccatgccca gtcaaatcaa gcaatttgtc 11400 acaagttaac taaagcagat atttagctct ccttttcgtt aatccactgt tttttacatt 11460 atgcatgagt agaaaatact gtgtagcata ctgggttacg gggattagga tagtcaggtt 11520 caacctgccc aggccatagc cagccatcat gaggtccagc cacgttgtgc atgccaccac 11580 gccctgctaa tttttgtatt aaaaaatttt ttttttacta ttattttttt gagatggagt 11640 ttcacctttg ttgcccaggc tggagtgcag tggttcagtc tcactgcaac ttctgcctcc 11700 ttagttcaag caattctcct gcctcagcct cccaagtagc tggaattaca ggcacacacc 11760 accatgccca gctaattttg tatttttagt agagacaagg tttcgccatg ttggtcaggc 11820 tggtctcgaa ctcctgacct caggtgatct gccctcctca gcctcccaaa gtgctgggat 11880 tacaggcgtg agccaccatg cttggcagta tgagctttat gttgacttca gattactttt 11940 tatctgcttc ttgctcttta ctgcaaggat cctctgatgc ccagtttgca aagatctcct 12000 aaacaagtat ggtatcttct tagctcatgt aggttcagca tgaacatgtt atatttggca 12060 tgaccaaagg taggatgggg ttatcctggt atgttcaaca tggaaattct gacaaaacaa 12120 tcaccccaaa aataatactc ctaatgagag gtttgctatc ctaaattcat gaagagctcc 12180 ttcagataaa taaaaggcaa aaacaagaaa ctgagcaaaa gacttgaaaa ctgtttgcca 12240 ctaacaatct agcagttcca gtcccaggta tagactgtag tccctagatc agtgctactt 12300 aaactgccag ccagtggaat aaggactcta gtactagaat gtaaagtacc acattgccca 12360 acactgtttt agggtcacat agaagaataa aaccattaag taacaaagga atgatgatta 12420 gagaaggtct aaacaaattc taagggagag gtacaggggc ctctgagata ttacatctta 12480 tttctttctt tttttttgtt ttctgagaca gtcttgctct gtcacccagg gcagtggcgt 12540 gatcttggcc cactgcaacc tccacctccc aggttcaagc gattctcctg cctcagcctc 12600 ccaagtagct gggattacag gtgcccacca ccacgcccag ctaatttttg tatttttagt 12660 agagacaggg tttcaccatc ttggccaggc tggtctggaa ctcctgatct catgatccac 12720 ctgcgtcagc ctcccaaagt gctgggatta caggcgtgag ccaccatgtc cggccagcat 12780 ctgatttctt aataacctgc ttaacttggt cttggttgca attactgtat tgcacatgta 12840 ttctatatgt tttccttggg tgagtagtgg acaggcaggc aatgagctct gccttaaggt 12900 aaccaaaata gtgtgtctct ttaaaggatt attccctatg cttaattgta atcaggaaag 12960 ctagaaaact tgaaattaat atgattgtgt acttcatgtt gtattgtaat agttatcttt 13020 aatgttatca catcttaatt gccaaaaggg atataaatat ttcacttgag tgcaggtaca 13080 cacacacttt agtacacaca cattttctcc agaaaaaaaa tttagtcttc acattgaata 13140 gtcaagacat ttttaaggga gtctaattgt gttaattcca cgtgcaaaaa taatattgct 13200 gtgagaattg tgtattttgt tttctcatta agctacattt taatatttat ggtgaggcag 13260 ttcattgggc agtctttaca caggcacttc aaacttagcc tgttgggctt ataacttttg 13320 ggattttttg cccccttata tatttcctat tcattcttgt tcttctcttt taaaataaat 13380 ctttagcagt cacctttagt cttcttcctc taaggttact cagttttttc aacctcatca 13440 cctgaaaaat aagtttaggg gtatgcattg tgattactgg ctttcttatt gaagcaagcc 13500 ttgtcctgat ttatcctgag tactcaatgt aggctgttgt tattgtctat cacttatttt 13560 ggggtccagg gtggcagaag tttacctgat cttatgatca ctaaatttat aggatttcct 13620 accagtttca gtatatagct tcctggaatt cttttgtagt gtttcagacc tactcaaggt 13680 ctcacatcta tggtagatgt cattctttag aagcttctag tgctcttctg gtcaattact 13740 tttaaaggta ttctatgtgg taagtaacat tttctgttgt aggtagaatg cagaggggac 13800 ttaagcaacg ttgtatattt ttaaagctgt gagctgggtg ctaagtcatc aaatgcctat 13860 gtaaaaataa aagtagaaat gcacctaggc atcttcagag taacagtaat agtgtaaatt 13920 tcagaatatc ttttctcatt tttaatgagt aaaaatcctt ttagaaaaat ggttgatgta 13980 ttaaggttgg gtttcctgct taggtatctc tgaaacagta ttctcaacct ttcagctctc 14040 cctgttctac ttccagggag gtcttcactt cagaaatcca agactcatat tcatccagct 14100 tggtgtcaag tgggctgttg ctgccagaat tatcttgtga ttatttgaga gatgtatcag 14160 tttcttctga agtacaatca actgtagaag cctttgtagc aggtgtgtaa caaggcaagt 14220 gcccagctcc tgcagccact actcttggag ctaccctgtc gatgaacatg aggactcagc 14280 ggcctcatga aaggctccag ggagcagtgt gtggttcttt ggagcctttt gtttcttttg 14340 gcagctcttc atggaaaaat gtattaaaaa aacacccata tgaaggtgtg aataagggat 14400 gttacatatt ttttcttctc agttatgagt ctaagaggaa attattaata aaagtagtat 14460 tgtggttata cgctactgta ttagtgtaaa ataacctgag ctttctcaac cgtatgttaa 14520 taaggtggtt gaaaatagct taaaagtgct tgtgagtaga gaatactttc tagtgtctat 14580 tgataatact agacatcaga gctagatttg ctgttcagtt ctaatgaaga gaatctaaga 14640 tttacatgga gtagtttttt tttcaagttt aatataatta gttcaaggtc caatagcacc 14700 agaatgtgtt gcagccagta aatcagtcct gtttctgatc tgtaagccag aagaagtggt 14760 acagtttctt ttgggaatta tttccactca gaattgcaag gagatacttg atatatcacc 14820 ttggtttgag tctgcattct tagtggaggg tatggagacg tcctggaaag agtccctgtg 14880 cgggggtttt tgtttcttgt ttttgttttg agatggagtc ttgctctgtc accaggctgg 14940 agtgctgtgg cacaatctca gctcactgta acctccgcct cctgggttca agtgattctc 15000 ctgcctcatc ctcctgagta cctgggacta caggtgtgca ccaccacacc cagctaattt 15060 ttatattttt agtagagaca agtttcacca tgttggccag gatggtctcg atctcaacct 15120 cgtgatctgc ctgcctcggc ctcccagagt gctgggatta cactgtgccc agccccctgt 15180 ggtgttgaag attgaggtag attaacttag tgaatctgga gtaaggtata aactgctcac 15240 ccccgaaaca tgcacactta ttttcatgct tgctccctag aacatgctgt gtcttaagac 15300 ttttggtttg acatattgtc gccaattaac tttaatacct cctgaaaacg ttttggcaaa 15360 atatttctta gtgctttcag taaggcataa atgtcacaac ataaaccttt ctttttttta 15420 acaatggaac tgtaaactat ttttgctaat atattaagaa aactgcaagc agtgatccca 15480 gaggtatgat ttgagctgct tttttaggac tctttcagtg agattggggc acaacatatg 15540 aaagactcaa gcaacctacc tcacccacct acttgtccct tttcagttgg aaatactggg 15600 tttccaagtg gcatttcatt tgaacaaaga gtttgttgcc tcaagatata ttagaaaatt 15660 tgaccaggca tagtggctca cactggtaat atcagcactt aagagaggca gagacaggag 15720 gatagcttga gcccaagaat tagagactag cctggccaat atagtgagac cccattctcc 15780 ataaagagaa aaacaaaaaa agaaaacttg ttgctgcaga tgtttgtgtt ttacagattc 15840 ttttgaggaa ttggcttttt tttttttttt tttttttggc cttttagtct ttaacagtag 15900 ctgatagttg agctttgcat tcacaggcat gggtagctta atgacagaga tagattctga 15960 gaaatgtgtc gttaggtgac attgtggttg tttgaacatc atagtgtaca tacagaaacc 16020 tagatagtac agcctacgac acacccaagc tgtatggtgt agcttactgc tcctaggcta 16080 ccatcctgta taccgagtta ctgtactgaa tactgttgac agttgtaaca cagtggtaag 16140 tatctgtgta tctaaacata gaaaaggtac agtaaaaata tggtattatg agaccacctt 16200 catatatttg atctgtcctt attaaaaatg ttgttatgca acgcatgact atacatctgg 16260 attgcttgca tatcaaagtt acgggcttct tttaagaatg tgaaagagct ggacagagtg 16320 gctcatgccg gtaattgcaa cactttggga ggccatggtg ggaggattgc ttgaggccag 16380 gagcttgaga ccagcctggg gcaacatagt gagacactat gaacaaagag ttttctacaa 16440 aaaataaaaa attagctgga catgatggca tgtgcctgta gtctcagcta cagggactga 16500 ctgagtggag gtgggactga aggctgtggt gggaggatcg cctgagtggt tgaggcttcg 16560 gtgggctgtg atgccactgc actccagtct gggtgacaag agtgagacct tttctcaaaa 16620 aaaaaaaaaa tgggaaaaat tgagctactt tttcaataca cttccaggga gttcattacc 16680 accaccccta agtttaagaa ctccccactg tttgttgtga agtcttttgt ccatttgttt 16740 ctgagtacct attctgggct aggaatttac agcaatctca aagccttcaa gagagcatca 16800 aagaaaataa acctccatta gagctttatt gttttttttt tttttttttt aagagatggg 16860 ttctccctat attgctcagg ctgatctcaa acttctggcc tcacaaaatc ttgcctcagc 16920 ttcccaaagt attgggttca atcaactact ttattgtagt tctctgtata atgaaagaaa 16980 acaaatgtta atgtactaca tctgtggatt caggtgatcc ttaatccatt ttgtcaagta 17040 ctgccaatat agcaaatgaa ttttatttca tttttgcaac agaatgattt tgtcactcag 17100 ctgggacgtg gttaacaact ctgctgttac ttctgaagta acagttccat ttcaaagatg 17160 aatagtcaac tccacaaaca ttttatcttt cctttagaag aggtcatatt ttttcactat 17220 ttttatttat attgataatg acactatatg tcagctgttt tggtttagcc ttcagttgat 17280 taaaattatt tagcttctat aaaagtctta atttatagtg acaagaagct ggtcagtggt 17340 tgcctggggt ggagttgggg atttgctgca aaagggacat catatgaaga aaggctgtga 17400 gtaggcgtga taatgttttt ataccagtgt gaataaactt agcatttttc aacaagtatg 17460 agtgttcata tgtctgaaag gggttagttg attatgtgga tatgataaag ttcttttaaa 17520 gagactgaga actgtgttgg atttcccaaa aaaaaaagga gtcctgtgtt ttatagggat 17580 ggggtgctgg ggaagcaagg ttttcatatt gttttaaatg tagtttgatc attagtatct 17640 ctagagctaa cctacttaat tattctattc ctcagtatat caatggagat aataccgtca 17700 ttgtggtttt aagggttaag agaattaata ttgaatagat gaaacactta ggctgggaga 17760 ctgctataaa catgtttact gttagattta agctagctta gcaggcagtc agagttgaac 17820 ctatgaaatt atatggtttg aaaatatttg ggaaaaaata tctttatcca tatcatcata 17880 ctactgtagc agaagctctg aagaatgtag gtagtcagcc agtaagactt gatccagtgg 17940 gcctgatgtt tttataagta ctgaggatcc tgactgttaa aggaaaatta cccttcctaa 18000 gaacactatg gttgtaggaa atggaggttt atcttatcct gacttaaaag ctagaccttc 18060 tgtatttggc agcagttgtg tgttgtcgtt tataatcaat aaccaaaggt tttcaactaa 18120 gtactaaata ctacatatat attagactat gcccagagac ttagcaacag caactgagat 18180 acaattaagt cctcattgaa catcatcagt aggttcttgg aaactgcgac tttaagcaaa 18240 aagacataga cagcaagccc tcaaataaca tttcattata atgttaagaa aaaaaatggt 18300 ttcgttttac ctcttactta aaagacacag tttgcaagaa gctattgaca taaagtgagg 18360 acacaattta cttatttttt tttccatttc aaagtatttt atttaggcca aaagtcttga 18420 tttcgtattt tttcatttat taatagtaag ataaatgaaa actggctgct atgccaaaaa 18480 ggttagtgat gttaaactta acaggaagtt tctaccttat tttcttatat ttatcttttt 18540 aaatgagtga cctttatttt tttaattgtt cttagtgctc agttgaatat ccccccaaaa 18600 ttggtgcttt aaattgctta aaataaacat gtattcttag cctaaaatgt ctatattttc 18660 tgggcacaga tttacagcag tcctgttata acagtcttta gagaatgtgc aggctgtgat 18720 ttgacgtcac tgcacaggta ttaggtctat ttagataggc ctcaattaac ttggtaaaat 18780 aaaaacatcc taaacttttc agtctgtgct atatggcatg aaagatctga tgagttgaac 18840 tgaatgtagt tggcttcttg gaaatgaagc tgttagtgtt tgaaaagtta aaactgagta 18900 tttgtatttc atgaaatttg actaatacag cttattaagg actgttttgg tgcagccaaa 18960 atgctgacac ttttaagaac catcgaaatg gagtatactt tcaggacagt ggccatcacc 19020 caagggtgaa ctctagtttg gggcattagg gagggtggag gggggtggtg ggaggaatca 19080 aggacacagg aaaggatgtg caggttttaa aacaggctgc ccagacagct ggtcaatggc 19140 cattattaat gtggcaacca gaaattagta ccagatattg agtagtatcc tgtctggtgg 19200 acctgtttta tctcatttat gtcgaagtta tttcctaaag taacctaagg accagtgaag 19260 ttcaacaaag aagataaacc tttatgtcag tatagtatgt gctgccttag gacaatgagt 19320 gctccggggg agtgggcagg tttatttcat tgatttgagt ggagagggat aggatttagc 19380 ctaaagtctt tttaaaaatc agattactta gtaactggct caggatgtac agatacctaa 19440 tttacaaatg tagtttctaa ttgtgttatt tttgcatttc tggtaattga caattttagc 19500 tctattctag ccagcactga aacagatgtt tttaatttat atgctctatg attttttttt 19560 ttttttacaa caacctgttg aagaaataat taaggtagtt aactgaggtt tagagaaatt 19620 gatgtagatt ctcctaaatg atatagccag gacgaggtct tctgactcct agtccagtga 19680 tttcaccaaa gcaactcacc tcataacatt gctaacttga tacagctagt actattttag 19740 cttgaggtaa ggcttaggca gtctgtatca aagcttcaga ttgttccctc aagccaccct 19800 ccactctcac ccatttttat tactttcacc tatttgggaa tttttttttt ttttttagga 19860 gagacggagt cttgctatgt tgtccaggcc ggtctcaaac tcctaggctc atgcaatcta 19920 ccggcctggg cctctcaaag tgctgggatt acaggcatgg gccaccatgc ccggccagga 19980 cttttattta taagacattt atctgcaatt acctggcagt gctaatacgt ggctgacagt 20040 atattggaag ttgagactgg tattttgatt tcttggatgc agatacttat ttcactagct 20100 ttctttactc ttaaaatggg taaaattatg tgtgttatag gaacgcaaaa caagataata 20160 atacagattg ggtctgcaac tctgtaagta aggtgaccat ataggcttgt aatacattgt 20220 gtatatatgt gagttacccc ttcaaattat attgaccttt ttgggtaaat gtattttaat 20280 tcatgtattt gcttttttaa atgggcgtta aaggaaatta ataatctgat gtttctgaaa 20340 tcatgtattt gcttttttaa atgggcgtta aaggaaatta ataatctgat gtttctgaaa 20340 ttgtaaaatt gcgtaaattc ctcactaaat taggcactta gcacactttc attgactcgt 20400 ttgtaaaatt gcgtaaattc ctcactaaat taggcactta gcacactttc attgactcgt 20400 acattatgtc tattgaaata atacagactt ttatataact tatattttga cagtttgttg 20460 acattatgtc tattgaaata atacagactt ttatataact tatattttga cagtttgttg 20460 catattctaa ggacccagac ataggcttgg tggcccgtct cttgtctttc ctggtttatg 20520 catattctaa ggacccagac ataggcttgg tggcccgtct cttgtctttc ctggtttatg 20520 actttcggct ttgtggaata cggctgagat gaaaggattt attgatgatg caaactactc 20580 actttcggct ttgtggaata cggctgagat gaaaggattt attgatgatg caaactactc 20580 cgttggcctg ttggatgaag gaacaaacct tggaaatgtt attgataact atgtttatga 20640 cgttggcctg ttggatgaag gaacaaacct tggaaatgtt attgataact atgtttatga 20640 acataccctg gtaagtgcca tttctcaaat acagtgtttg tcattttcca agctctgaaa 20700 acataccctg gtaagtgcca tttctcaaat acagtgtttg tcattttcca agctctgaaa 20700 cgagtgcttt ttaatttcca agatggtaat ctgatcagaa gtacttggtt catatgctat 20760 cgagtgcttt ttaatttcca agatggtaat ctgatcagaa gtacttggtt catatgctat 20760 ctgatttaac ttgaccttcc tcccccacac ccactcccct tgagtagtga tccatttttc 20820 ctgatttaac ttgaccttcc tcccccacac ccactcccct tgagtagtga tccatttttc 20820 tttccaatcc acattatttt aaaatcctcc ctatggccct ttaccttcta tgccttttag 20880 tttccaatcc acattatttt aaaatcctcc ctatggccct ttaccttcta tgccttttag 20880 catctggcac ccacatatgt tctgtttatt ctgttgatgt gatgccagcc aaccttctaa 20940 catctggcac ccacatatgt tctgtttatt ctgttgatgt gatgccagcc aaccttctaa 20940 tcagtacatg gcttcttact catttttctc aattttcttt tttttttttt tttttggaga 21000 tcagtacatg gcttcttact catttttctc aattttcttt tttttttttt tttttggaga 21000 cgagtctcac gctgtcgccc aggctggagt gcagtggcgt gatcttggct cacgtgcaac 21060 cgagtctcac gctgtcgccc aggctggagt gcagtggcgt gatcttggct cacgtgcaac 21060 cctcgcctcc tgggctcaag cagttctctc tcagcctcct gaattagcta gaattacagg 21120 cctcgcctcc tgggctcaag cagttctctc tcagcctcct gaattagcta gaattacagg 21120 cgcacaccac cacacccggc taatttttgt attttagtag agacgaggtt tcgccatgct 21180 cgcacaccac cacacccggc taatttttgt attttagtag agacgaggtt tcgccatgct 21180 ggtcttgaac tcctgacctc aggtgatcta cccacctcag cctcctgaag tgctgggatt 21240 ggtcttgaac tcctgacctc aggtgatcta cccacctcag cctcctgaag tgctgggatt 21240 acaggcacga gccaccgcgc ccagcctcaa ttcttttttt ttgcttgctt gtttgagatg 21300 acaggcacga gccaccgcgc ccagcctcaa ttcttttttt ttgcttgctt gtttgagatg 21300 gattcttaca ctgttgccca ggctggagtt cagtggcacg atctcagctc actgtgacct 21360 gattcttaca ctgttgccca ggctggagtt cagtggcacg atctcagctc actgtgacct 21360 ctgcctcctg cgttcaagca attgatttag gatccttatg cctcagcctc ccgagtagct 21420 ctgcctcctg cgttcaagca attgatttag gatccttatg cctcagcctc ccgagtagct 21420 gggattacag gtctgtgcca ccatgcccag ctaagtggtt ttttttttgt tttttgtttt 21480 gggattacag gtctgtgcca ccatgcccag ctaagtggtt ttttttttgt tttttgtttt 21480 ttgttttttt ttttgagatg gagtctccct ctgtatccca ggctggaggg caatggcagt 21540 ttgttttttt ttttgagatg gagtctccct ctgtatccca ggctggaggg caatggcagt 21540 atctcggctc actgcaacct ccgcctccca ggttcaagcg attctcctgc ctcagcctcc 21600 atctcggctc actgcaacct ccgcctccca ggttcaagcg attctcctgc ctcagcctcc 21600 caagtagctg ggattacagg cgcccgccac catgcccagc taattttttg tatttttagt 21660 caagtagctg ggattacagg cgcccgccac catgcccagc taattttttg tatttttagt 21660 agaggtgggg tttcaccatg ttggccaggc tggtctcgaa ctcctgacct gaggtgatcc 21720 gcccaccttg gcctcccaaa gggctgggat tacaggcgtg agccactgcg cctggcctaa 21780 gtttttgtat ttttagtaga tggggttttg ccatgttggc caggctggta ttgaacgcct 21840 gaccaacatg tgatctgccc gcctcagcct cccagagtgt tggattaaca ggcgtgagcc 21900 actgtgcctg gtctcagttg tattcttcag tggatattaa tttactttca gataattttg 21960 gttgtgtatg ttcattgaga atttgataat ttatcctgag tacttactct ctgctgctct 22020 aggagtcaac ttctacagaa tcatatactc actgctactt aaggtgattt tctttttttt 22080 tttctctttg agacgaagtt tcactcttgt tgcccaggct ggagcgcagt ggtgcgatct 22140 caactcacta caacctcggc ctccctggtt caagcgattc tcctgcctta gcctcccaag 22200 tagctgggat tacaggcgcc cagcaccagg cccagctgat ttttgtattt ttagtagaga 22260 cagggtttca ccgtgttgac cagtctggtc tcaaactcct gacctcaggt gatccaccca 22320 ccttggcctc ccaaagtgct gggattacag gcgcgagcca ccgcgcccag cctggtcttt 22380 tttttttttt tttttatata tatatatatt agacttaact gatacaatct ttctttattg 22440 gaggaaattt cctgtattca accaaacttg ttagataatt gttttcatgt taccggtgtt 22500 cagtaattga atatccaaaa ttttctcctt ctcttgacct taagtctgca gacgtgttca 22560 tcttcccctc atcctagcaa aaatcccttt gtcttcctct gccattcaac ttgttaaaac 22620 ttgacatatt ggtcatgaaa cttgtcatat tggtcaacca atttctgcca agccattctt 22680 cagttgccac tgggaagctc ctagtcaaat tctgatgatc atttttatca cttaacttta 22740 ttcttctgac tttttgcaac acttttcggg ggtcagatat aatctttgaa atattccttc 22800 catcccacat ctaaattaac ccacgtttag tagcattcaa tgaggattac tcctcaaccc 22860 cattcttttc tggctctggg gtgacacttc tgtgttctct aagtcattca gtccaccttt 22920 tgtttgtcct ctcttattta accttggtct tgccttcata acatcctggt attcatctcc 22980 tccattccca ctatctatcc gtctttgcac atgtcattat agtgacctcc taacttattt 23040 cccttttcat cgtcctattg tttgcccaag cctgcttaat tttgttgaat attcattaaa 23100 tacaaggctt ccctgctccc taaatttgtt tttcgttgcc tactgagaat tttccagact 23160 cacatcatgc ctaagaaagg aagctgaata ccttgttgtt tcatcccatt tctcttgtgg 23220 ttcccgtaaa taccttattt tttcccaaat taaatataaa ctttgtgtct ccggaatacc 23280 atagatatca tatcttggga ggatggataa cttaatacaa ctgttaaaat tctgctgcac 23340 ataatgtgcg ttttgcattt atatcagttg ggcactatat tctaatgaca ttttcaaata 23400 ctgtgaatta aagagttgaa aacagtaaca ttgtcttcat ggattctcaa acagtatttg 23460 gatgttgtac gtcctaaata agattttttt gcctgctatc taacactgag taatatatgt 23520 tctgtccaac tgcataagtc atctctctag tcctagaata gccacaactt taaggaagat 23580 tattcttatt caggcttatc tgatatttgg tagacttatg tacttttaaa atctaatttt 23640 gacatctcca tgtgcagact aacaaccttg tctttttaaa aatggtttcc tggttggtca 23700 cggtggctca cacctgtaat cccagcactt tgggaggcct aacatgggag gatagcttga 23760 gctccaggag ttggaggctg tagtaagcta tgattgcacc actgccctcc agtctgttgc 23820 tacagagcaa gacctcaact cttgaaaaag taataaaata agaggtttcc cagtgaagat 23880 ggtctcttat tttaccatgt tgtcttaagg tgcagtttta atggcgatat atagcttgtt 23940 ttatttagca tttctgattc tacttactgt attaggaaat tatatcctga actgccaaag 24000 tattttaaac agtgggggaa gcatgggaga aaataggttc ccttccttca actgccaaaa 24060 agtaaaatca aggcagtttc attctgactt ttgtcactgt aattgataat agttttttta 24120 tctcaaaggg gagaatattt tatatgctag tgtagcacag caaaagtaat caaatataag 24180 aagaaagaac aaatgtattc agagtctctt ttaatgttta ctaaatctag ttaacaagct 24240 ttaaatgtat tactgtatgt aaatatatgt cttgacccat atatttgtgg aggaaaagag 24300 gtgttctttc ctgttttgat acttgatttt caatggtttt ttgttcatat ttgtcttttt 24360 ttttataacc ataatagaca gggaaaaatg cattttttgt gggagatctt ggaaagattg 24420 tgaagaaaca cagtcaatgg cagaatgtag tggctcagat aaagccattc tacacagtga 24480 tgaagaaaca cagtcaatgg cagaatgtag tggctcagat aaagccattc tacacagtga 24480 agtgcaactc tgctccagct gtacttgaga ttttggcagc tcttggaacc ggatttgctt 24540 agtgcaactc tgctccagct gtacttgaga ttttggcagc tcttggaacc ggatttgctt 24540 gttccagtaa agtaagtatt tttcattttt taactaaact gaaacattga ataatttaac 24600 gttccagtaa agtaagtatt tttcattttt taactaaact gaaacattga ataatttaac 24600 ttaatgtagt tacagcggtt agagaacaaa tgcaaggaag tttgactttt ggatggagtt 24660 ttaatgtagt tacagcggtt agagaacaaa tgcaaggaag tttgactttt ggatggagtt 24660 tcaaaataat tgtttaaatc tccagcctca tgttggaaaa ttacaagctt ttcaagtgca 24720 tcaaaataat tgtttaaatc tccagcctca tgttggaaaa ttacaagctt ttcaagtgca 24720 ccaatgaaag ggaagtttca gtgtaatgag cctttgaaac gttttatctt tattaattga 24780 ccaatgaaag ggaagtttca gtgtaatgag cctttgaaac gttttatctt tattaattga 24780 aaactttaat ttgttcctca ttcttaggct agagctactt ctagaccaaa ctacagcaga 24840 aaactttaat ttgttcctca ttcttaggct agagctactt ctagaccaaa ctacagcaga 24840 tatctaaact ctgagatttc tttggtgctt ctttttagta atgtttaggc caggagcgat 24900 tatctaaact ctgagatttc tttggtgctt ctttttagta atgtttaggc caggagcgat 24900 ggctaacacc tgtaatccca ccactttgag aggctgaggt gagagttgct tgagccagga 24960 ggctaacacc tgtaatccca ccactttgag aggctgaggt gagagttgct tgagccagga 24960 gtttaggatc agcctgggca acatagtgag acctcgtctc actttttatt tatttatgtt 25020 gtttaggatc agcctgggca acatagtgag acctcgtctc actttttatt tatttatgtt 25020 tttaaataaa aatattggtt tggctaggca cagtgactca tacctgtact cgtagcactt 25080 tttaaataaa aatattggtt tggctaggca cagtgactca tacctgtact cgtagcactt 25080 tgggaggctg aggcaggagg atcacttgag cagccccaga gatggaggct gcattgagct 25140 atgattgtgc tattgcactt cagcctggaa tacaaagagt gagaccctgg ttttaaaagg 25200 ggtgggggta atagtattag aggaagcatt accagaatat gaactgtgac attatgtatt 25260 ctcactttaa cctcattttc acagaatgaa atggctttag tgcaagagtt gggtgtacct 25320 ccagaaaaca ttatttacat aagtccttgc aagcaagtgt ctcagataaa gtatgcagca 25380 aaagttggag tgaatatcct gacatgtgac aatgaaattg aattgaagaa aattgcacgt 25440 aatcacccaa atgccaagta agtataaaat taagtacttg aaaatattat ttagcactta 25500 gagggttggg tttgtctttt gtctgggaag aaaggagcat ttgtggtagg gcaatgatgt 25560 gtatcataat tataacttat ttaaagggcc tctgtgggaa acactagtaa cattttattg 25620 gctctctacc tctgatcctt catgtggaat tggaatttaa aaaaataaga gcagtttggg 25680 gatatgatct aataatcttc aaggaatcct tgatagttgg tgtgataatc aaattagggc 25740 catggctgta ttttcattat ttctataact ttgtttgatg tttgataacg ttaggaacag 25800 gataaacttt ggtcagcatt ttttcctgtt tagctgagga tttaatctac cctaggccat 25860 gat aa act tt ggt cag cat ttt tcc tgt t agc tga gga ttt aat cta ccc tag gcc at 25860 agttcttgaa tggtcatctt tactgacagt gtcttcgaag cctagaccat agatttgggg 25920 agt tct tga a tgg tca tct t tac tga cag t gt ctt cga agc cta gac cat aga ttt gggg 25920 ggttttgttt ttaaggctaa tacttttatt tttgaacatg aagtttaatt ttttacttgc 25980 ggt ttt gtt tta agg cta a tac ttt tat ttt tga aca tg aag ttt aat ttt tta ctt gc 25980 atcttcatgt atccagagaa tttttttttt ttcttgatta gaaagtttac tttttaaagg 26040 atc ttc atg tat cca gag aaa ttt ttt ttt ttc ttg att aga aag ttt act ttt taa agg 26040 ctgggcgcag tggctcacac ctgtaatccc agcactttag gagactgagg ctggtggatc 26100 ctg ggc gca gtg gct cac acc tgt aat ccc agc act ttt agg aga ctg agg ctg gtg gat c 26100 atttaaggtc aaggtggcct gcgcctgtaa tcccagctac ttgggaagct gaggcaagag 26160 att taa ggt c aa ggt ggc ctg cgc ctg taa tcc cag cta ctt ggg aag ctg agg caa gag 26160 aatcccttga acccaggagc cggaggctgc agtgagccaa gatcgagcca ctgcactcca 26220 aaa tcc ctt ga acc cag gag ccg gag gct gc agt gag cca aga tcg agc ca ctg cac tcc a 26220 gcctgggcaa cagagcaaga ctccctcata aaaaggttta cttttttaaa agcaaaaaga 26280 gcc tgg gca a ca gag caa ga ctc cct cat aaa aag gtt ta ctt ttt taa aag caa aaa aga 26280 tttaaggcaa attttatgtt ttcacaaact ggtatatcga gagggcttaa gaatatgcta 26340 ttt aag gca a att tta tgt ttt cac aaa ctg gta tat cga gag ggc tta aaa tat gct a 26340 aaagcatatt gaattatgca gttgttggct tactttttaa tgagttatgt gtgtttccgg 26400 aaa gca tat tga att atg ca gtt gtt ggc tta ctt ttt taa tga gtt atg tgt gtt tcc gg 26400 aaatgaattc tatctgtatt ttctttgtat gtctataatt ttcatactga ctttaatggt 26460 aaa tga att c tat ctg tat ttt ctt tgt at gt cta tat att ttc ata ctg act tta atg gt 26460 gtacttaaga tatcctgaca gcattgatta ctctggtaaa tctttgtgtg gataaaagcc 26520 ttgttgggaa aattctaggg aaccacttgt ttttctaaaa aatattgttc tgtgatcctc 26580 tgaagacaag ctgtgatttg tgatgtttag tatgttagat tgtggactgg catcttcttg 26640 atggagctga ttctaacctt agaataattt ttgctttcat caatcttgtc ctctgattat 26700 caaattaggg ccatagctgt attttcatgg ccatattatt aaccttctta gtttatgtaa 26760 ttattacatc cataggaaaa cagttacaca aaaagaattt gtatattttc aacttctagc 26820 agtttgtaat tactcagctc ctgaaattaa agaaatttaa tcagttttag tcatcttgtc 26880 ttggttgcca tggtttggaa ggaaatacca aatagatttg aatcagtaga ctagaaggct 26940 gctgtttaaa cacatgaaat aattttttaa aaagctttct gggttgggcg cagtagctca 27000 tgcctgtaat cccagcactt ggggaggccg aggcgggtgg atcacttgaa gtcaggagtt 27060 tgagaccagc ctggccaaca tggtgaaacc ctgtctccac taaaaataaa aaaaattagc 27120 tgggcgtggt ggtgcaccct tgtatttcca gctactttgg gaggctgagg caggagaatt 27180 gcttgaactc aggaggcact ctcatgaggc ggaggttgca gtgagctgaa attgcgccac 27240 tgcactccag cctgggcgac agagtgaaac tccatctcag aaaaaaaaga aaaaaaaatt 27300 taaaaaatgc ttcctatttt tttccagtta gagacctgct gggatgtaac aatgttagta 27360 ggaagaatgt ttgcagtaac catggaggtt tagctagtta gcacaatcaa agaaatttga 27420 agacatttaa agccctgata tttttattga aagacaatga tggcattgct attcaattta 27480 gtcttcctag gtggatttat ggactgatgg acttcatttg aaagtaatgt cgtctttcat 27540 tacagagccc aaccaactct tgatgctaca acagagtctt aacttgctgc attttccaaa 27600 atcccatcct tgtaagcttc atgctttagg ccctaattgc tagttttttc tctatacagg 27660 attgtttttc attaggttca cttgattcat ccgtcgctgg atttgggagc actggcaaca 27720 taatcaacac acttcctaca atcttcaggc ttcacatgtg ctgatgatga tgtaaaccaa 27780 ctctgcccca atcatctccc cttctcttag ggtcttacta catattgcaa cagaagataa 27840 tattggaggt gaagagggta acatgaagtt tggcactacc ctgaagaact gtaggcatct 27900 cttggaatgt gctaaggaac ttgatgtcca aataattggg gttaagtgag tatttgtttt 27960 aaacttttaa agctgtgaac attttatggt ggtaacctgt ctttctggtt attgatttaa 28020 gactttattt gcatgaaatc taagatgcaa ttttttctag tcttttaaca cacatgtatg 28080 tgttttcttc ttagtttagg caaaacaact taaatatgcc ttgtcccttt tcatactgtt 28140 ttgtgactgt acctccttcc ccacaataaa aagaatagca aataatattt tgattcagtt 28200 agtgcagagt aacttaaatc gccgcctttt aaattggatg ggaggggatt tgctagaatt 28260 cttaatgaaa ataaaagtgt cagacttttt tgtcctttta acgattactc tgggggactt 28320 caataaatta gactttgctt tagcctttta attttatatg tgaggaaacc gagactccaa 28380 gaggttcaaa tacttgataa tagcctcaga actaattaca ggcatagtaa cactaagccc 28440 tagatgttat ttgccagccc attgctactt ttactaaagt aaaaaggtct tgctttttca 28500 aacactcaca aagatgtatt gtgtatcttt ctttaatttt attatttatt ttttgagata 28560 gtatctcatt ctgtctccca tgctggagtg cagtggcacg atcacggctc actgcagcct 28620 caaactcctg gctcaagcaa tcttcctgcc ttggcctccc aaagtggtgg gattacagat 28680 gtgagcctct gtgcctggcc ttattgtgta tctttctata ctactttgta cataaatagg 28740 tggtagtctt ttaaacaaag aatgggatca tgctgtgcct tacacaatag ataacattcc 28800 atgtcagtct agttttaagt tttaatttaa gtcaacataa aaattaatga agtactatct 28860 cgaatagatg aagggaaaag aagaatggac tttacgggta cagttctatg gattttagta 28920 cttgtgtgac cactgccaca atcaggaagt attctattct gaaactttat cactagtaat 28980 actagtgaat aatagtaatg ctgttactca gcttgattaa tgttcatttt tatttatttc 29040 acaacagaca catttttgta tttggttggg taaaattgaa tgttcagttt tatgctcaac 29100 tgaatgtgat acgatttggt agtgcttgtt acttcactat ataaacataa aagtgccaca 29160 tatacataaa agcacacctt attgtataac cgtggaacag tactgataat tgacttattg 29220 ttaagaattt ttgtttatag aattacagtt ttatcttggc attgcctttt ctataaagct 29280 ggtactgtaa cttttgggta attagaagct aatgcttcta atactttaaa tatttggtaa 29340 agttaatggg gatacttaat ggtatgaata tgatgagtga cagttgaaag aaattacagg 29400 aggtgtctct acaactttct gctcctttaa atttagtatt ctatgtgttt cagatttcat 29460 gtttcgagtg cttgcaaaga atctcaagta tatgtacatg ctctatctga tgctcgatgt 29520 gtgtttgaca tggctgtaag ttctttcttt taagttattt tgatatttta ttttagccag 29580 gatgttaagt taaagactaa gtattttctg cctgtgtgaa ttactctgac tacattcaac 29640 gtgacacaca cctcattttt attatataag cattcagagc tctgtacagt cacacctttt 29700 aagcaggtaa aggagtgaaa tagtaattta cttgcataag tctagaagag agtacaaata 29760 aaatcaaaga gacagcatac ctaacattga ataatctgtt tctatcagta aaaaaccaag 29820 aatttgctgt actaaagatc agaacagata tttacctccc taggaggggg tttttaaaat 29880 gttctcatat ccattacaat ttggaaaaac atttaaattt tttttttcca tctctaggga 29940 gaaattggct ttacgatgaa catgttagac attggtggag gattcacggg aactgaattt 30000 gaaattggct ttacgatgaa catgttagac attggtggag gattcacggg aactgaattt 30000 caattggaag aggtaaactt ctcagtagat gacattaaca gaataattgc tttctttgtt 30060 caattggaag aggtaaactt ctcagtagat gacattaaca gaataattgc tttctttgtt 30060 acttaagtgg tggatgtgta gagcaggatt tttacaatgg cagaaactaa accctataac 30120 acttaagtgg tggatgtgta gagcaggatt tttacaatgg cagaaactaa accctataac 30120 atctctttaa aattatgttc ttagaaaagg aataggcagt tagtagacct agctaacttt 30180 atctctttaa aattatgttc ttagaaaagg aataggcagt tagtagacct agctaacttt 30180 gaaaagtttg atagaattct ttgaacatta tcataaaaca ttttctattt cagttgtgtt 30240 gaaaagtttg atagaattct ttgaacatta tcataaaaca ttttctattt cagttgtgtt 30240 tctgtactta atggcacaaa tgatttaata cttagaaaac ttcactacta cctttttttt 30300 tctgtactta atggcacaaa tgatttaata cttagaaaac ttcactacta cctttttttt 30300 ttttttttta aagtcttcct gtgttgccca ggctggagtg gagtggcgca attatagctc 30360 ttttttttta aagtcttcct gtgttgccca ggctggagtg gagtggcgca attatagctc 30360 actatagctt ccaactcctg ggctcaagcg atcctcctgc ttcaacctcc caagtagctg 30420 actatagctt ccaactcctg ggctcaagcg atcctcctgc ttcaacctcc caagtagctg 30420 ggacttaaaa aaaaaaaaaa aaaaaaattg agtctttcta tattgcccag gctggtctcg 30480 ggacttaaaa aaaaaaaaaa aaaaaaattg agtctttcta tattgcccag gctggtctcg 30480 gaactcctgg cctcaaatga tcctcctgtc cccaagtgat cctcctgccc cagcctccca 30540 gaactcctgg cctcaaatga tcctcctgtc cccaagtgat cctcctgccc cagcctccca 30540 aagttttggg gttacaggtg tgagccacca ctcccagcca actttagcat ttaggccaga 30600 aagttttggg gttacaggtg tgagccacca ctcccagcca actttagcat ttaggccaga 30600 tacacgtcac acctgtttct caccttcctg ctctgccctc cttccttctg tgatatctag 30660 aaactatgat ttgaaaattt tacttctgtt tttagtgatc aagtatagaa aaatgactta 30720 aaaaaaaaca aaaacaaata ccagtgggta tttgttatta gagctatttt taaaattaat 30780 gccattctga tagttacaaa ataaactgac ttgaaagcat gcctaattaa ttgcttggag 30840 ataactacaa agttgctaag gttcatgatc tgtgattgga actgcttcat tttcctagaa 30900 aattttgtta atatcaatct ctgaatattg cgaattatct aatccaatat tgaaaccata 30960 ctgtggcatt taaaatccat aggttaatca tgttatcagc cctctgttgg atatctactt 31020 tcctgaagga tctggtgtta agataatttc agaacccgga agctactatg tgtcttctgc 31080 atttacactc gcagttaata tcatagcaaa gaaagttgtt gaaaatgata aatttccctc 31140 tggaggtaag ttataaagtt tatagtttta tttttgttgg tagataattg cagttagtct 31200 ggaaatgagc agatggtcat gaagttgtag gtggttttca gcttttaaaa atcttaaaac 31260 tggccgggcg tggtggctca cgcctataat cccagcactt tgggaggcag agccaggtgg 31320 atcacaaggt caggagttcg agaccagcct ggccaatatg gtggaaaccc catctctact 31380 aaaaatataa aacttagctg gtcatggtgg cgcacacctg taatcccggc tactcgggag 31440 gctgaggcac aaaaatcgct tgaatccggg aggcagaggt tgcagtgagc caagattgtg 31500 ccactggact ccagcctggg tgacaagagt gagactccgt ctcaaaaaaa aaaaaaatct 31560 taaagccaag gtcccattcc gtaagacttc caaatccaac tttcttggta ctgatgttta 31620 gatacgtgtg ttttagataa atgtctagca gatgattaat tctcatccat tattcagaac 31680 tacttttatt gtgatctttt ctgttctcct tacctcacat gggtcagagg gaggctctgt 31740 tttcacactt tctcacataa gggccacaaa gagtccctgt aatctaactc aagtcaaacc 31800 aacaaaaagg cactagacag taccatcttg ttggctaata ttgtaggaaa tactgtttta 31860 cattaagaag actttttttt ttggctgtgg actaatccac agcccatagg aaagttgtcc 31920 atttccccaa tattgtcttt tttttacagc tttgtttttc cttgcttaga atccagtcca 31980 ggaccacatt gaattcgtca tttctccttg aatctagata aagtttctca gctcattact 32040 ttgtattttt taactttgac atttttgaag agtagaaatt tcatagctga tccttagttt 32100 gggtttgttg gatgattcct catgattaat tttttaggct atgcatttgg ggcatgaagt 32160 ccactaaagt gttactctgt tcttctcact tcatcatgtt aggaggcaca tgatagtggc 32220 caattctaat attgatgatg ctaggtgttt tttcttgttg ttttgtcgcc caggctggag 32280 tacagtggct ccatcagggc tcattgcagt ctccacctcc cggcttaagc agtcctccta 32340 ccttcaacct cttgcatagc tgggactaca ggcatatgcc accatgcctg gctaggtttt 32400 tttctatttt tattttgtag agggtttcgt cgtgtttctc aggctggtct caaactcctg 32460 agctcaagtg atccacctgc ctcagcctcc caaagtgctg ggattacaaa cttgagtcaa 32520 tacacctggc cttatgattt tttttttttt tttttttttg agactgtttt gctcttgttg 32580 cccaagctgg aatgcagtgg catgatctca gctcactgca acctccacct cccaggttca 32640 agcaattctc ctgcctcagc ctcccaagta gctgggatta caggtgcgtg ccaccacacc 32700 cggctagatt tttttttttt tttttaattt aaatgctagt tttgagcact tgtttaaggt 32760 tgtgtcaggc aggtttcttt ccattgtgaa gtggcaagtt taaattagag actctgtaaa 32820 ctacctgttt ttaaccagat tttcacccca ttagttttag catctattgt tgaatttcta 32880 acttcattat tgtttatatt agttggcctt tacttaaaga atatcttttc cttccctcct 32940 gtttatgagt ttagactcat agatctttat tcaataggtt tatataatct gttactctta 33000 tttttccctt gacatgtctc cattattctc agagcaattc gttatttttt ggtatatcaa 33060 gatgttccag gctcatttta accttttaaa tgagcctcag tcccgtaatc agttgtttct 33120 ccagggagcc ttggtttgtt ttcgtggaga gtgatatttt aaaaacctga aaattagtat 33180 tggttgtgct aagagtagag tagctgtgag tagaatgggc gttttcaaag gtttcaaagt 33240 gaggtgagag gatctattga tccctgaagc tcgaggctgc ggtgacctgt gatcacgcta 33300 ctgcactgta gcctgagtaa cagtgaggcc ctgtctctac acacacacac agattcccac 33360 tggcagtgta tacatgtgag ctctggttgc tcctcctgct ctctgatgtt ttgtagtgtc 33420 agtctttaag ttgatccagt cagtaggttg gtggtaatgg tagtccattg tggttttaat 33480 ttgcatttac ctgatgacta acgatgtaga gcactgtttt ctttttcttt ttcttttctt 33540 tttttttttt gtttttgaga cagggttttt gctctgttat ccaggctgga atgagtggca 33600 caatctcaac ttttattttt aattctacat tcaaacacat atttaactcc ttgtgcatat 33660 attgccttcc ccttgctgat ttgaaaagaa actaaaaagt gtttttcctt gctgcttcat 33720 ttcctctttt tttgaaacag caactatcag aaagtcttct agaatcttaa tacttttttc 33780 tttttttttt tttttttttg agacagggtc tcattctgtc acccaggctg gagtgcagta 33840 gcacagcctt ggctcactgt ctcactgtct tactgtaacc tccgcctccc ggattcaagg 33900 gattctagtg cctcagcctc ccaagtagct gtgattacag gcatgcacca ccacacctgg 33960 ctaatttttg tatttttagt agagatggag ttttgccatg ttgttcaggc tgatcttgaa 34020 ctcctgacct caagtgatcc tcccacctca gcctcacaaa gtgctaggat tacaggtatg 34080 agccaccgca tccagcccta gagcactctt ttcttgtgct cattggttat cttcctttgt 34140 gaaatgcgtg ctctatgtgt ctttttaagt ttaaatagtt tgagaaatgt gaaaattaca 34200 ttaatttgtt tcatatgagc cattttatta aaaattcatt ttcccctttc ctgttttatt 34260 ccagtagaaa aaaccggaag tgatgaacca gccttcatgt attatatgaa tgatggtgtt 34320 tatggttctt ttgcaagtaa actgtctgag gacttaaata ccattccaga ggttcacaag 34380 gcaagtttta tcagaaatat caaaacctat ttggcatttt ataagctgag ctgttatttt 34440 taagatgctt cccagttttt tgaaaaacat cttagaatgg ggaaaaatgt cctttttgag 34500 gatttgcgtt cttctggtgg agtccctttt ttagtttgtt gcctttacgt ggagttaatg 34560 gtaccatcga ggcttaagta gcaccctgcc cttggctcta ggcctttttt ccatgagcac 34620 attggatctg agacagtgaa aatacttatt tactcacaaa ttcaatacct gcgtaagtat 34680 gagtaaaaat taaattgtaa aacttttcct acagaaatac aaggaagatg agcctctgtt 34740 tacaagcagc ctttggggtc catcctgtga tgagcttgat caaattgtgg aaagctgtct 34800 tcttcctgag ctgaatgtgg gagattggct tatctttgat aacatgggag cagattcttt 34860 ccatgaacca tctgctttta atgattttca gaggccagcc atttattaca tgatgtcatt 34920 cagtgattgg taaggtgatt ttattttaag gcagatggga tatttgaact gtccttttag 34980 acttgctaat aagttttaat tctgtaatga atgctaaact tagaagtaca gtcatgtatt 35040 gcctatacag tgtagttact taatgatggg aatttgttct gacaaataca acattaggcg 35100 attgtatgat ggcataacct actacataat ctaggccatg tggtatattg atcctaggct 35160 acaaacctgt acagcatgtt actctgttta atactgttgg cagttgtaat gcagtggcgt 35220 ttgtgtatct taagttactt aaacatagaa aggataatac attgtgctat gacatgattg 35280 ctgcgatgta acgaggtgat aggacttttt aaacttcact gtaattttac ggtataccac 35340 agtcatatgt gtagtctttg accaaaacat cactctgcag tgcatgacta tcatagaaaa 35400 atgatgatct tttaaaggtt tttggaagac tagttataat cctgttttat tatccatttg 35460 ttatgtgatt acgtggtctt tgggcttaaa ctgagaattt agctgatttt ttttctacgt 35520 aggtatgaga tgcaagatgc tggaattact tcagactcaa tgatgaagaa cttcttcttt 35580 gtgccttctt gcattcagct gagccaagaa gacagctttt ccgctgaagc ttaaacaggc 35640 attaacgctt ctttagatct gaagttgcag gttaagcttg tctggtcaac attccagtgt 35700 ggaaaaataa tttaaacaat cttattctct taattctttt ggcaacaaaa actattagta 35760 atagctattt gggaccagac aaaatcagct ttcatctata attcattggg gataatggga 35820 gatttagata atgtatccag atttaaacct accagtttgt cctacccctt aagcgtttaa 35880 aataaaatat gcaacaaaat ggatgactta gtggagatgg aagcccatta attgggttcc 35940 ccattaaatc gtttacatac aagaacacag tttttatact aaggatttgt gtttaaagtc 36000 ttgtaaagtt catgtctttc acccagatat atcaaatgtt agaagaccag tgtgacttca 36060 ttagataacg tttagtgtat ttagaatgtg taaatttgtg ctttgaactg tagtttaata 36120 aatgtaaaat tgcatcatag tatttgttga cctaatgtaa cccttgtatg attgcaataa 36180 aattttgtgt agattttact gttttttcag gctaaaactt tgggaaaggg gctagctagc 36240 aaaggtagtt ttgaaataga tgtgtatatg gactgttttg aagggttttt ttctttatag 36300 cccagttaag ttttgtttgg ctcggtgcat ttttcattta tttaattagt aatttaagta 36360 aagtgtttgg taaatcattg tgaagttcag attcattatg gagagttgat gtgcagtaag 36420 catgatgttt aacaatttta acaccaaaaa tgttaatcct gcataaatca actgtaataa 36480 taaataggtg tttctgtata gatagaatgc atagagtacc ttagtaaatc tttgaatcac 36540 aatcttttgg ctgaaatgga agattctgtt aaatactttg aataaacttg gggggaggga 36600 aataaaattg cagaaaactg cagagcacta aaacttaaag aagggctaca tctttatcca 36660 gaaacctgtt gctcttttgc acggaatgtt taaattcaga gttgggatgg gggttggggt 36720 gaagcacact tattatcttc agttgcagtg atttcaaatt taggattttt tgttgttggt 36780 ttgaactgtc cccttagttt cttgttattt ccaatttgtt ctgcttagtc attactttta 36840 attcttttct tactaaaatt ttatggtggt tgggggaagg gagttagcat cactaacctg 36900 acagttgttg ccaggaattt gctttgttta ctgctagtat attagaaatc ctagatctca 36960 gaatcacaat agtaataaac aacaggggtc attttttcct aacttactct gtgttcaggt 37020 gtggaatttc tgtctcccaa gaggaaatgt gacttcactt tggtgccaat ggacagaaaa 37080 ttctacctgt gctacatagg agaagtttgg aatgcactta atagctggtt tttacacctt 37140 gatttcgagg tggaaagaaa ttgatcatga atctctaata aatttaaatc tcttaaacca 37200 gtaggtgctt aatatttttt gatttgatta atgcccattt aaatctcatg ggttctatta 37260 aaaatatata tatatagggc cccaatccat tgccatcaaa ttgcccttgg acttttccaa 37320 ggtatattat ggggttttat gcaaaattcc aagctaccat gtaacttttt ttaaccattt 37380 aacaaggagg gggaactgtt tcctaccttc tttacatgtt gtgcattgtt gtggtccaga 37440 aatgccaaac ctttttaaag atggtgcaac tttgagtcct tggcttgact atacaggcct 37500 tgaacttcat ggcatatcaa ctttgccata tctgcaggag agctgttcta taagaaatag 37560 ctcagagttg caaatatcac atgtgaatga tacggtaact tttaagaaat gtctgtattg 37620 tatttgaaga ctgtttgcca taaatctgaa atttgaacct atgtatttca atttggtatg 37680 ctaaaaagtt ctgaattaat gtaaagtttt ttgttataat attgtaatct cagttcaaaa 37740 gttaactgca aatataaaac ccaatgattt ctatatagta aattgaactg taaaggtaac 37800 ttgtgtgtga ttctgaatac atagataaat gtttttattc ctcatgtttt actttggctt 37860 ctatctgaaa tagaggtaaa attttacata tcagcttta 37899 <210> 4 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 4 Met Lys Gly Phe Ile Asp Asp Ala Asn Tyr Ser Val Gly Leu Leu Asp 1 5 10 15 Glu Gly Thr Asn Leu Gly Asn Val Ile Asp Asn Tyr Val Tyr Glu His 20 25 30 Thr Leu Thr Gly Lys Asn Ala Phe Phe Val Gly Asp Leu Gly Lys Ile 35 40 45 Val Lys Lys His Ser Gln Trp Gln Asn Val Val Ala Gln Ile Lys Pro 50 55 60 Phe Tyr Thr Val Lys Cys Asn Ser Ala Pro Ala Val Leu Glu Ile Leu 65 70 75 80 Ala Ala Leu Gly Thr Gly Phe Ala Cys Ser Ser Lys Asn Glu Met Ala 85 90 95 Leu Val Gln Glu Leu Gly Val Pro Pro Glu Asn Ile Ile Tyr Ile Ser 100 105 110 Pro Cys Lys Gln Val Ser Gln Ile Lys Tyr Ala Ala Lys Val Gly Val 115 120 125 Asn Ile Leu Thr Cys Asp Asn Glu Ile Glu Leu Lys Lys Ile Ala Arg 130 135 140 Asn His Pro Asn Ala Lys Val Leu Leu His Ile Ala Thr Glu Asp Asn 145 150 155 160 Ile Gly Gly Glu Glu Gly Asn Met Lys Phe Gly Thr Thr Leu Lys Asn 165 170 175 Cys Arg His Leu Leu Glu Cys Ala Lys Glu Leu Asp Val Gln Ile Ile 180 185 190 Gly Val Lys Phe His Val Ser Ser Ala Cys Lys Glu Ser Gln Val Tyr 195 200 205 Val His Ala Leu Ser Asp Ala Arg Cys Val Phe Asp Met Ala Gly Glu 210 215 220 Ile Gly Phe Thr Met Asn Met Leu Asp Ile Gly Gly Gly Phe Thr Gly 225 230 235 240 Thr Glu Phe Gln Leu Glu Glu Val Asn His Val Ile Ser Pro Leu Leu 245 250 255 Asp Ile Tyr Phe Pro Glu Gly Ser Gly Val Lys Ile Ile Ser Glu Pro 260 265 270 Gly Ser Tyr Tyr Val Ser Ser Ala Phe Thr Leu Ala Val Asn Ile Ile 275 280 285 Ala Lys Lys Val Val Glu Asn Asp Lys Phe Pro Ser Gly Val Glu Lys 290 295 300 Thr Gly Ser Asp Glu Pro Ala Phe Met Tyr Tyr Met Asn Asp Gly Val 305 310 315 320 Tyr Gly Ser Phe Ala Ser Lys Leu Ser Glu Asp Leu Asn Thr Ile Pro 325 330 335 Glu Val His Lys Lys Tyr Lys Glu Asp Glu Pro Leu Phe Thr Ser Ser 340 345 350 Leu Trp Gly Pro Ser Cys Asp Glu Leu Asp Gln Ile Val Glu Ser Cys 355 360 365 Leu Leu Pro Glu Leu Asn Val Gly Asp Trp Leu Ile Phe Asp Asn Met 370 375 380 Gly Ala Asp Ser Phe His Glu Pro Ser Ala Phe Asn Asp Phe Gln Arg 385 390 395 400 Pro Ala Ile Tyr Tyr Met Met Ser Phe Ser Asp Trp Tyr Glu Met Gln 405 410 415 Asp Ala Gly Ile Thr Ser Asp Ser Met Met Lys Asn Phe Phe Phe Val 420 425 430 Pro Ser Cys Ile Gln Leu Ser Gln Glu Asp Ser Phe Ser Ala Glu Ala 435 440 445 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 5 uuaagcuuca gcggaaaagc 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <400> 6 uuaagcuuca gcggaaaagc 20 <210> 7 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <220> <221> misc_binding <222> (1)..(20) <223> Phosphorothioate bond <400> 7 uuaagcuuca gcggaaaagc 20 <210> 8 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <220> <221> misc_binding <222> (1)..(6) <223> Phosphorothioate bond <400> 8 uuaagcuuca gcggaaaagc 20 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <220> <221> misc_binding <222> (15)..(20) <223> phosphorothioate bond <400> 9 uuaagcuuca gcggaaaagc 20 <210> 10 <211> 37 <212> DNA <213> artificial sequence <220> <223> synthetic sequence <400> 10 cgggatccat tcattttccc ctttcctgtt ttattcc 37 <210> 11 <211> 31 <212> DNA <213> artificial sequence <220> <223> synthetic sequence <400> 11 cggaattctc cagcatcttg catctcatac c 31 <210> 12 <211> 32 <212> DNA <213> artificial sequence <220> <223> synthetic sequence <400> 12 cggaattcgc ttaaggggta ggacaaactg gt 32 <210> 13 <211> 44 <212> DNA <213> artificial sequence <220> <223> synthetic sequence <400> 13 cgggatccgc aagttttatc agaaatatca aaacctattt ggca 44 <210> 14 <211> 39 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 14 cgggatccaa atacaaggaa gatgagcctc tgtttacaa 39 <210> 15 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 15 cggaattctt aagcttcagc ggaaaagctg tc 32 <210> 16 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 16 ctgagccgaa ttcaatcgat ggccgccatg 30 <210> 17 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 17 gaattcggct cagctgaatg caagaaggca caaag 35 <210> 18 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 18 gaagacagct gaagcttaag aattcaatcg atggcc 36 <210> 19 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 19 ttcagctgtc ttcttggctc agctgaatgc aag 33 <210> 20 <211> 41 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 20 ctcattcagt gcagaagctt aagaattcaa tcgatggccg c 41 <210> 21 <211> 40 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 21 tgcactgaat gagtcttctt ggctcagctg aatgcaagaa 40 <210> 22 <211> 32 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 22 cgggatccat catgcacctc tgcgctatat cg 32 <210> 23 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 23 cggaattcag aacccgatca aacgcaaatg ttacc 35 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 24 gctgaagctg gaagcaagaa agtg 24 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 25 cagggccttc tttggacagg a 21 <210> 26 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 26 attgatgatg caaactactc cgttgg 26 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 27 ctggaggtac acccaactct tg 22 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 28 accctgaagt accccatcga 20 <210> 29 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 29 ctcaaacatg atctgggtca tct 23 <210> 30 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 30 gcatttacac tcgcagttaa tatcatagc 29 <210> 31 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 31 aatgcaagaa ggcacaaaga agaag 25 <210> 32 <211> 24 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 32 cggattcccc gtgccaagag tgac 24 <210> 33 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 33 ggccatggcg gccatcgatt 20 <210> 34 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 34 tacaaggaag atgagcctct gtttacaa 28 <210> 35 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 35 tccagcatct tgcatctcat acc 23 <210> 36 <211> 27 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 36 cctacagaaa tacaaggaag atgagcc 27 <210> 37 <211> 30 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 37 taaaatcacc ttaccaatca ctgaatgaca 30 <210> 38 <211> 86 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 38 ugagcuugau caaauugugg aaagcugucu ucuuccugag cuagcugagc caagaagaca 60 gcuuuuccgc ugaagcuuaa acaggc 86 <210> 39 <211> 21 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 39 caagaagaca gcuuuuccgc u 21 <210> 40 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <400> 40 uguggaaagc ugucuucuuc 20 <210> 41 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <400> 41 ccacaauuug aucaagcuca 20 <210> 42 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <220> <221> misc_binding <222> (1)..(20) <223> Phosphorothioate bond <400> 42 ccacaauuug aucaagcuca 20 <210> 43 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <220> <221> misc_binding <222> (15)..(20) <223> Phosphorothioate bond <400> 43 ccacaauuug aucaagcuca 20 <210> 44 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <220> <221> misc_binding <222> (1)..(6) <223> Phosphorothioate bond <400> 44 ccacaauuug aucaagcuca 20 <210> 45 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <400> 45 gcugucuucu uggcucagcu 20 <210> 46 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <400> 46 cuucagcgga aaagcugucu 20 <210> 47 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(25) <223> 2'-O-Me sugar modification <400> 47 gcuuuccaca auuugaucaa gcuca 25 <210> 48 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <400> 48 gcuuuccaca auuugaucaa 20 <210> 49 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <400> 49 cagcucagga agaagacagc 20 <210> 50 <211> 10 <212> DNA <213> Artificial sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (1)..(1) <223> NH2-Lys modified <220> <221> misc_feature <222> (3)..(3) <223> m represents thioguanosine <220> <221> misc_feature <222> (6)..(6) <223> m represents thioguanosine <220> <221> misc_feature <222> (8)..(8) <223> n represents guanidine-modified 5-methylcytosine <220> <221> misc_feature <222> (10)..(10) <223> m represents thioguanosine <220> <221> misc_feature <222> (10)..(10) <223> CONH2-modified <400> 50 ttmttmtntm 10 <210> 51 <211> 12 <212> DNA <213> artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (1)..(1) <223> NH2-Lys-modified <220> <221> misc_feature <222> (12)..(12) <223> CONH2-modified <400> 51 agcggaaaag ct 12 <210> 52 <211> 20 <212> DNA <213> artificial sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (1)..(20) <223> 2'-O-Me sugar modification <220> <221> misc_binding <222> (15)..(20) <223> phosphorothioate bond <400> 52 cguguguucu acgcucuggu 20

Claims

1. An oligonucleotide targeting the core editing complementary sequence (ECS) of the AZIN1 gene, wherein the core ECS of the AZIN1 gene comprises the sequence 5'-GCTTTTCC-3', and wherein the oligonucleotide comprises a sequence complementary to 5'-GCTTTTCC-3' at the 3'-end of the oligonucleotide, the oligonucleotide having the sequence 5'-UUAAGCUUCAGCGGAAAAGC-3' (SEQ ID NO:5), wherein the oligonucleotide comprises one or more nucleotides with sugar modifications, and optionally one or more modified internucleotide linkages.

2. The oligonucleotide according to claim 1, wherein the oligonucleotide is a ribonucleic acid (RNA) oligonucleotide.

3. The oligonucleotide according to claim 1, wherein the oligonucleotide is an antisense oligonucleotide.

4. The oligonucleotide according to claim 3, wherein the antisense oligonucleotide is a non-degradable antisense oligonucleotide.

5. The oligonucleotide according to claim 1, wherein the nucleotides with sugar modifications are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 2',4'-bridged nucleic acid modified nucleotides, locked nucleic acid (LNA) modified nucleotides, and morpholino ring modified nucleotides.

6. The oligonucleotide according to claim 1, wherein at least five nucleotides in the sequence complementary to 5'-GCTTTTCC-3' are modified by sugar modification.

7. The oligonucleotide according to claim 1, wherein each nucleotide in the oligonucleotide is modified by sugar modification.

8. The oligonucleotide according to claim 7, wherein the oligonucleotide comprises the sequence 5'-mUmUmAmAmGmCmUmUmCmAmGmCmGmGmAmAmAmAmGmC-3' (SEQ ID NO:6), wherein m represents a 2'-O-Me sugar modification.

9. The oligonucleotide according to claim 1, wherein the oligonucleotide comprises one or more nucleotides with sugar modifications, and one or more modified internucleotide linkages.

10. The oligonucleotide according to claim 9, wherein the nucleotides with sugar modifications are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-O-methoxyethyl modified nucleotides, 2'-fluoro modified nucleotides, 2',4'-bridged nucleic acid modified nucleotides, locked nucleic acid (LNA) modified nucleotides, and morpholino ring modified nucleotides.

11. The oligonucleotide according to claim 9, wherein the modified internucleotide linkages are selected from the group consisting of phosphorothioates, phosphoramidates, and diamidophosphates.

12. The oligonucleotide according to claim 11, wherein the oligonucleotide comprises a diamidophosphate morpholino oligomer (PMO).

13. The oligonucleotide according to claim 9, wherein at least five nucleotides in the sequence complementary to 5'-GCTTTTCC-3' are modified by sugar modification.

14. The oligonucleotide according to claim 9, wherein the oligonucleotide comprises at least three modified internucleotide linkages in the sequence complementary to 5'-GCTTTTCC-3'.

15. The oligonucleotide according to claim 9, wherein the oligonucleotide does not comprise any modified internucleotide linkages in the sequence complementary to 5'-GCTTTTCC-3'.

16. The oligonucleotide according to claim 9, wherein at least 50% of the nucleotides are modified by sugar modification.

17. The oligonucleotide according to claim 9, wherein at least 10% of the internucleotide linkages in the oligonucleotide are modified internucleotide linkages.

18. The oligonucleotide according to claim 9, wherein each nucleotide in the oligonucleotide is modified by sugar modification.

19. The oligonucleotide according to claim 9, wherein each nucleotide in the oligonucleotide is modified by sugar modification and is linked to an adjacent nucleotide by a modified internucleotide linkage.

20. The oligonucleotide according to claim 9, which comprises or consists of a sequence selected from the following: 5'-mU*mU*mA*mA*mG*mC*mU*mU*mC*mA*mG*mC*mG*mG*mA*mA*mA*mA*mG*mC-3' (SEQ ID NO:7), 5'-mU*mU*mA*mA*mG*mCmUmUmCmAmGmCmGmGmAmAmAmAmGmC-3' (SEQ ID NO:8), and 5'-mUmUmAmAmGmCmUmUmCmAmGmCmGmGmA*mA*mA*mA*mG*mC-3' (SEQ ID NO:9), wherein m represents a 2'-O-Me sugar modification and * represents a phosphorothioate bond.

21. A pharmaceutical composition comprising the oligonucleotide according to any one of claims 1-20.

22. A method for inhibiting AZIN1 pre-mRNA editing in a cell ex vivo or in vitro, the method comprising contacting the cell with the oligonucleotide according to any one of claims 1-20 or the pharmaceutical composition according to claim 21.

23. The method according to claim 22, wherein the AZIN1 pre-mRNA editing is mediated by an adenosine deaminase (ADAR-1) acting on RNA-1.

24. Use of the oligonucleotide according to any one of claims 1-20 or the pharmaceutical composition according to claim 21 in the preparation of a medicament for treating cancer in an individual in need thereof, wherein the cancer is associated with AZIN1 pre-mRNA editing.

25. The use according to claim 24, wherein the cancer is selected from hepatocellular carcinoma (HCC), esophageal squamous cell carcinoma (ESCC), non-small cell lung cancer (NSCLC), and colorectal cancer (CRC).

26. The use according to claim 25, wherein the cancer is liver cancer.

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