Enhancer oligonucleotides for modulating FUBP1 expression

Abstract

This invention relates to enhancing antisense oligonucleotides that are complementary to distal upstream element-binding protein 1 (FUBP1) and capable of reducing FUBP1 target nucleic acids, such as FUBP1 mRNA. This invention relates to enhancing antisense oligonucleotides or conjugates thereof targeting FUBP1 for the treatment and / or prevention of hepatitis B virus (HBV) infection, particularly chronic HBV infection. This invention particularly relates to the use of said enhancing antisense oligonucleotides or conjugates thereof targeting FUBP1 for destabilizing cccDNA, such as HBV cccDNA. This invention further relates to enhancing antisense oligonucleotides or conjugates thereof targeting FUBP1 for the treatment of cancer. This invention also includes a pharmaceutical composition and its use in the treatment and / or prevention of HBV infection, or its use in the treatment of cancer.

Description

Technical Field

[0001] This invention relates to enhancing antisense oligonucleotides that are complementary to distal upstream element-binding protein 1 (FUBP1) and capable of reducing FUBP1 target nucleic acids, such as FUBP1 mRNA. This invention relates to enhancing antisense oligonucleotides or conjugates thereof targeting FUBP1 for the treatment and / or prevention of hepatitis B virus (HBV) infection, particularly chronic HBV infection. This invention particularly relates to the use of said enhancing antisense oligonucleotides or conjugates thereof targeting FUBP1 for destabilizing cccDNA, such as HBV cccDNA. This invention further relates to enhancing antisense oligonucleotides or conjugates thereof targeting FUBP1 for the treatment of cancer. This invention also includes a pharmaceutical composition and its use in the treatment and / or prevention of HBV infection, or its use in the treatment of cancer. Background Technology

[0002] Distal upstream element-binding protein 1 (FUBP1 or FBP1) is a single-stranded DNA-binding protein that binds to multiple DNA elements. This protein is also thought to bind RNA and possesses 3'-5' helicase activity, exhibiting in vitro activity on both DNA-DNA and RNA-RNA duplexes. FUBP1 is known to activate the transcription of the proto-oncogene c-myc by binding to the distal upstream element (FUSE) located upstream of c-myc in undifferentiated cells. This protein is primarily located in the cell nucleus. Upregulation of FUBP1 has been observed in many types of cancer. Furthermore, FUBP1 can bind to the RNA of hepatitis C virus and enteroviruses and mediate their replication (Zhang and Chen 2013 Oncogene vol 32p.2907-2916).

[0003] FUBP1 has also been identified in hepatocellular carcinoma (HCC), where it is believed to be involved in HCC tumorigenesis (Ramdzan et al. 2008 Proteomics Vol 8p.5086-5096), and as demonstrated by the use of shRNA expressed by lentiviruses targeting FUBP1, FUBP1 is essential for HCC tumor growth (Rabenhorst et al. 2009 Hepatology vol 50p1121-1129).

[0004] It has been demonstrated that knocking down FUBP1 carrying lentivirally expressed shRNA can enhance the therapeutic response to ovarian cancer (Zhang et al., 2017, Oncology Letters, Vol. 14, pp. 5819-5824).

[0005] WO 2004 / 027061 discloses a screening method comprising the steps of analyzing whether a test substance inhibits FBP (FBP is now called FUBP) and a pharmaceutical composition for treating proliferative diseases, which contains a substance that inhibits FBP as an active ingredient.

[0006] Poly(U)-binding splicing factor 60 (PUF60) is a potential regulator of transcriptional and post-transcriptional steps in HBV pregenome expression. PUF60 is known to form a complex with FUBP1, which is associated with c-myc repression. However, FUBP1 is not involved in PUF60-dependent regulation of HBV pregenome expression (Sun et al., 2017 Scientific Reports 7:12874).

[0007] HBV infection remains a major health problem worldwide, affecting approximately 350 million chronic carriers. About 25% of carriers eventually die from chronic hepatitis, cirrhosis, or liver cancer. Hepatitis B virus is the second leading cause of cancer after tobacco, causing 60% to 80% of all primary liver cancers. HBV is 100 times more infectious than HIV.

[0008] Hepatitis B virus (HBV) is an enveloped, partially double-stranded DNA virus. The compact 3.2kb HBV genome consists of four overlapping open reading frames (ORFs), encoding the core, polymerase (Pol), envelope, and X protein, respectively. The Pol ORF is the longest and contains the envelope ORF, while the X and core ORFs overlap with the Pol ORF. The HBV genome replication cycle has two main events: 1) the generation of closed circular DNA (cccDNA) from relaxed circular (RC DNA), and 2) the reverse transcription of pregenomic RNA (pgRNA) to produce RC DNA. RC DNA may originate from the infecting viral particle or serve as an intracellular replication intermediate.

[0009] HBsAg quantification is an important biomarker for the prognosis and treatment response of chronic hepatitis B, and the disappearance of circulating HBsAg in chronically infected patients is considered a key event for achieving cure. However, the disappearance of HBsAg and seroconversion (functional cure) are rarely observed in chronically infected patients. Hepatitis B e antigen (also known as HBV envelope antigen or HBeAg) is a viral protein secreted by hepatitis B-infected cells. HBeAg is associated with chronic hepatitis B infection and is used as a marker of active viral disease and the degree of infectivity in patients.

[0010] Therefore, compared with simply suppressing HBsAg secretion, reducing both HBeAg and HBsAg secretion will result in improved inhibition of the development of chronic HBV infection.

[0011] Current therapies, such as nucleoside (acid) analogs, are molecules that inhibit HBV DNA synthesis but do not target the reduction of HBsAg levels. Most therapies currently under development aim to achieve a functional cure, defined as persistent HBsAg disappearance, with or without anti-HBs seroconversion, undetectable serum DNA, and cccDNA in a transcriptionally inactive state, but they do not address the persistent presence of cccDNA. Conversely, complete cure of HBV infection is defined as the combination of cccDNA disappearance and persistent HBV DNA and HBsAg disappearance. The persistent presence of cccDNA in infected hepatocytes is a major obstacle to eradicating the virus in patients with chronic hepatitis B virus (CHB), and there is an urgent need to develop new therapies that eliminate cccDNA for a complete HBV cure.

[0012] In WO 2019 / 193165, it was demonstrated that inhibiting FUBP1 function using small molecules, siRNA, or LNA antisense oligonucleotides leads to a reduction in HBV cccDNA. In the examples section of WO 2019 / 193165, single-stranded LNA gapmer oligonucleotides capable of inhibiting FUBP1 expression were analyzed.

[0013] There is a need for therapeutic agents that can specifically inhibit FUBP1. This invention has screened over 2000 antisense oligonucleotides targeting human FUBP1 and identified sequences and compounds that are particularly effective at specifically targeting human FUBP1. Specifically, nine alternating flanking gapmers were identified, which conferred significant downregulation of human FUBP1 in vitro. Eight compounds target a region within exon 14 of human FUBP1, and one compound targets a region within exon 20 (CMP ID 18_1).

[0014] Purpose of the invention

[0015] This invention provides antisense oligonucleotides and their conjugates for regulating FUBP1 expression. The invention identifies specific target sequences present in exon 14 or exon 20 of human FUBP1 precursor mRNA, which can be targeted by antisense oligonucleotides or their conjugates to deliver effective FUBP1 inhibition. In particular, targeting positions 16184-16205 of SEQ ID NO:1 are advantageous for reducing FUBP1.

[0016] Furthermore, this invention identifies specific target sequences present in exon 20 of human FUBP1 precursor mRNA, which can be targeted by antisense oligonucleotides or their conjugates to provide effective FUBP1 inhibition. In particular, the target positions 30536-30553 of SEQ ID NO:1 are advantageous for reducing FUBP1.

[0017] Therefore, one object of the present invention is to provide an enhanced antisense oligonucleotide targeting FUBP1 or its conjugates, wherein the antisense oligonucleotide or its conjugates are capable of inhibiting FUBP1 expression in vitro and in vivo, thereby reducing cccDNA in HBV-infected cells. The enhanced antisense oligonucleotide targeting FUBP1 or its conjugates may be used for the treatment and / or prevention of HBV infection, or for the treatment of cancer. Summary of the Invention

[0018] This invention relates to antisense oligonucleotides or conjugates thereof that target FUBP1 (distal upstream element binding protein 1) nucleic acids, such as mammalian FUBP1 nucleic acids, and are capable of inhibiting the expression of said nucleic acids in cells expressing said nucleic acids, and their use in medicine. The antisense oligonucleotides are complementary to mammalian FUBP1 nucleic acids (such as human FUBP1).

[0019] The present invention provides an antisense oligonucleotide comprising a continuous nucleotide sequence that is regionally complementary (e.g., fully complementary) to nucleotides 16184 to 16205 of human FUBP1 precursor mRNA (as shown in SEQ ID NO:1).

[0020] The present invention also provides an antisense oligonucleotide comprising a continuous nucleotide sequence that is complementary (e.g., fully complementary) to the regions of nucleotides 30536 to 30553 of human FUBP1 precursor mRNA (as shown in SEQ ID NO:1).

[0021] In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence is complementary to regions 16184 to 16200 of SEQ ID NO:1, such as being fully complementary.

[0022] In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence is complementary to, for example, a region of nucleotides 16186 to 16203 of SEQ ID NO:1, such as being completely complementary.

[0023] In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence is complementary to regions 30536 to 30553 of SEQ ID NO:1, such as being fully complementary.

[0024] In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence is complementary to regions 16188 to 16205 of SEQ ID NO:1, such as being completely complementary. In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence is complementary to regions 16189 to 16205 of SEQ ID NO:1, such as being completely complementary.

[0025] The antisense oligonucleotides of the present invention are typically 12 to 30 nucleotides in length, such as 12 to 22 nucleotides, such as 16 to 20 nucleotides, and comprise a continuous nucleotide sequence of at least 12 nucleotides (such as 13, 14, 15, 16, 17, or 18 nucleotides) that is complementary (such as fully complementary) to a region of human FUBP1 precursor mRNA (as shown in SEQ ID NO:1), the region being selected from nucleotides 16184 to 16205, 16184 to 16200, 16186 to 16203, 16188 to 16205, 16189 to 16205, and 30536 to 30553 of SEQ ID NO:1.

[0026] The present invention provides antisense oligonucleotides of 12 to 22 nucleotides in length, wherein the antisense oligonucleotides comprise a continuous nucleotide sequence of 12 to 22 nucleotides in length, wherein the continuous nucleotide sequence is complementary to SEQ ID NO 10, such as being completely complementary.

[0027] The present invention provides antisense oligonucleotides of 12 to 20 nucleotides (such as 15, 16, 17 or 18 nucleotides), wherein the antisense oligonucleotides comprise a continuous nucleotide sequence of 12 to 18 nucleotides (such as 15, 16, 17 or 18 nucleotides), wherein the continuous nucleotide sequence is complementary to SEQ ID NO 11, such as being completely complementary.

[0028] The present invention provides antisense oligonucleotides of 12 to 20 nucleotides (such as 15, 16, 17 or 18 nucleotides), wherein the antisense oligonucleotides comprise a continuous nucleotide sequence of 12 to 18 nucleotides (such as 15, 16, 17 or 18 nucleotides), wherein the continuous nucleotide sequence is complementary to SEQ ID NO 19, such as being completely complementary.

[0029] The present invention provides antisense oligonucleotides of 10 to 30 nucleotides in length, comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, wherein the continuous nucleotide sequence is 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 6, 7, 8, 9 and 18; or at least 14 continuous nucleotides thereof.

[0030] The present invention provides antisense oligonucleotides of 10 to 30 nucleotides in length, comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, wherein the continuous nucleotide sequence is 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 6, 7, 8, 9 and 18; or at least 15 continuous nucleotides thereof.

[0031] The present invention provides antisense oligonucleotides of 10 to 30 nucleotides in length, comprising a continuous nucleotide sequence of 10 to 30 nucleotides in length, wherein the continuous nucleotide sequence is 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 6, 7, 8, 9 and 18; or at least 16 continuous nucleotides thereof.

[0032] The present invention provides an antisense oligonucleotide comprising a continuous nucleotide sequence that is 100% identical to the sequence selected from the group consisting of: SEQ ID NO: 6, 7, 8, 9 and 18, or at least 14, 15, 16 or 17 continuous nucleotides thereof.

[0033] The present invention provides an antisense oligonucleotide comprising or consisting of a continuous nucleotide sequence selected from the group consisting of: SEQ ID NO: 6, 7, 8, 9 and 18.

[0034] The present invention provides an antisense oligonucleotide comprising a continuous nucleotide sequence that is 100% identical to the following: SEQ ID NO:6 (CTTATGCTTTTTATGGT) or 14, 15 or 16 of the following continuous nucleotides.

[0035] The present invention provides an antisense oligonucleotide comprising a continuous nucleotide sequence that is 100% identical to the following: SEQ ID NO:7 (CTTATGCTTTTTATGGTT) or 14, 15, 16 or 17 of the following continuous nucleotides.

[0036] The present invention provides an antisense oligonucleotide comprising a continuous nucleotide sequence that is 100% identical to the following: SEQ ID NO:8 (GCTTTTTATGGTTTCAC) or 14, 15 or 16 of the following continuous nucleotides.

[0037] The present invention provides an antisense oligonucleotide comprising a continuous nucleotide sequence that is 100% identical to the following: SEQ ID NO:9 (TATGCTTTTTATGGTTTC) or 14, 15, 16 or 17 of the following continuous nucleotides.

[0038] The present invention provides an antisense oligonucleotide comprising a continuous nucleotide sequence that is 100% identical to the following: SEQ ID NO:18 (ACCAATTTTCATTTCTAC) or 14, 15, 16 or 17 of the following continuous nucleotides.

[0039] This invention provides antisense oligonucleotides selected from the following:

[0040] CTTatGctttttatgGT (SEQ ID NO:6, Compound ID No 6_1),

[0041] CTTaTgctttttatgGT (SEQ ID NO:6, Compound ID No. 6_2),

[0042] CTtATgctttttatgGTT (SEQ ID NO:7, Compound ID No 7_1),

[0043] CTtAtgctttttatgGTT (SEQ ID NO:7, Compound ID No. 7_2),

[0044] CTtAtgctttttatGgTT (SEQ ID NO:7, Compound ID No. 7_3),

[0045] CTtAtgctttttatGGTT (SEQ ID NO:7, Compound ID No. 7_4),

[0046] GcttTttatggtTtCAC (SEQ ID NO:8, Compound ID No 8_1),

[0047] TATgcTttttatggtTTC (SEQ ID NO:9, Compound ID No. 9_1) and

[0048] AcCAAttttcatttCtAC(SEQ ID NO:18, Compound ID No 18_1)

[0049] The uppercase letters represent β-D-oxyLNA nucleosides, the lowercase letters represent DNA nucleosides, all LNA Cs are LNA 5-methylcytosine, and all inter-nucleoside bonds are thiophosphate nucleoside bonds.

[0050] The present invention also provides pharmaceutical salts of the antisense oligonucleotides of the present invention.

[0051] The present invention provides an antisense oligonucleotide selected from the groups listed in Table 1 or a pharmaceutical salt thereof.

[0052] Table 1. Compound List (Exemplary Antisense Oligonucleotides of the Invention) – HELM Annotation Format

[0053]

[0054]

[0055] Helm annotation index:

[0056] [LR](G) is a β-D-oxy-LNA guanine nucleoside.

[0057] [LR](T) is a β-D-oxy-LNA thymidine.

[0058] [LR](A) is a β-D-oxy-LNA adenine nucleoside.

[0059] [LR]([5meC] is β-D-oxy-LNA 5-methylcytosine nucleoside,

[0060] [dR](G) is a DNA guanine nucleoside.

[0061] [dR](T) is a DNA thymidine nucleoside.

[0062] [dR](A) is a DNA adenine nucleoside.

[0063] [dR]([C] is a DNA cytosine nucleoside,

[0064] [sP] represents the internucleotide bond between thiophosphate esters.

[0065] P represents the internucleotide bond between phosphodiester nucleotides.

[0066] Therefore, the present invention provides an antisense oligonucleotide selected from the group consisting of compound IDs #6_1, 6_2, 7_1, 7_2, 7_3, 7_4; 8_1 and 9_1.

[0067] The present invention further provides an antisense oligonucleotide having compound ID number 18_1.

[0068] In one embodiment, the antisense oligonucleotide is not the antisense oligonucleotide compound ID No 53_1 or 54_1 disclosed in WO 2019 / 193165 (see also Table 7 in the Examples section).

[0069] In one embodiment, the antisense oligonucleotide is not the antisense oligonucleotide compounds ID No 78_1 and 79_1 disclosed in WO 2019 / 193165 (see also Table 7 in the Examples section).

[0070] The present invention further provides a conjugate comprising the antisense oligonucleotide of the present invention and at least one conjugate portion covalently linked to the antisense oligonucleotide.

[0071] In some embodiments, the conjugation portion is capable of binding to desialyl glycoprotein receptors, such as human desialyl glycoprotein receptors. For example, the conjugation portion may include at least one desialyl glycoprotein receptor targeting portion selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-butyryl-galactosamine, and N-isobutyryl-galactosamine.

[0072] In some embodiments, the desialylate glycoprotein receptor targeting moiety is N-acetylgalactosamine (GalNAc). Therefore, the antisense oligonucleotides of the present invention can be conjugated to at least one conjugation moiety comprising at least one N-acetylgalactosamine (GalNAc) moiety, such as at least one conjugation moiety comprising at least one N-acetylgalactosamine (GalNAc) moiety, as described below. According to one aspect of the invention, the conjugation moiety is a GalNAc residue R as described below.

[0073] In some embodiments, the conjugate moiety is at least trivalent, such as a divalent, trivalent, or tetravalent GalNAc residue R. Preferably, the conjugate moiety is a trivalent GalNAc residue R.

[0074] As used herein, the term "trivalent GalNAc residue" refers to a residue containing three N-acetylgalactosamine moieties, preferably the three parts of the following formula.

[0075]

[0076] The conjugate moiety or GalNAc residue R can be linked to the antisense oligonucleotide via a linker L (such as a biolytic linker L). Therefore, the conjugate compound may contain a linker L located between the antisense oligonucleotide and the conjugate moiety or GalNAc residue R.

[0077] In some embodiments, the linker L comprises 1 to 10 linked nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides, such as 2 to 6 linked nucleosides, such as 2 to 5 linked nucleosides, such as 2 to 4 linked nucleosides. In some embodiments, the linker comprises two linked nucleotides. Thus, the nucleosides can be DNA nucleosides. Typically, nucleosides are linked via phosphodiester-nucleoside linkage. Furthermore, the linker L can be linked to an antisense compound via phosphodiester-nucleoside linkage.

[0078] Table 2 (HELM comment format) and Figures 1 to 8 , Figure 8_1 and Figure 10 Exemplary conjugates are provided in the document.

[0079] The present invention provides a conjugate selected from the group of conjugates listed in Table 2, or a pharmaceutical salt thereof.

[0080] Table 2. Compound Table (Exemplary Conjugates of the Invention) - HELM Annotation Format (For notes on HELM annotations, see the explanation in Table 1).

[0081]

[0082]

[0083] In the table above, [5gn2c6] is a GalNAc residue R with the following formula:

[0084]

[0085] It should be understood that R, as shown in the above figure and the table above, is... Figure 9 D1 and Figure 9 The mixture of the two stereoisomers shown in D2.

[0086] According to another aspect of the invention, R as shown in the above figure and used in the above table is as follows: Figure 9 The stereoisomer shown in D1.

[0087] According to another aspect of the invention, R as shown in the above figure and used in the above table is as follows: Figure 9 The stereoisomers shown in D1. The structures of the conjugates provided in Table 2 are as follows: Figures 1 to 8 and Figure 8_1 As shown.

[0088] This invention provides Figure 1 Conjugates or their medicinal salts.

[0089] This invention provides an antisense oligonucleotide of compound ID 6_1 or a pharmaceutical salt thereof.

[0090] This invention provides Figure 2 Conjugates or their medicinal salts.

[0091] This invention provides an antisense oligonucleotide of compound ID 6_2 or a pharmaceutical salt thereof.

[0092] This invention provides Figure 3 Conjugates or their medicinal salts.

[0093] This invention provides an antisense oligonucleotide of compound ID 7_1 or a pharmaceutical salt thereof.

[0094] This invention provides Figure 4 Conjugates or their medicinal salts.

[0095] This invention provides an antisense oligonucleotide of compound ID 7_2 or a pharmaceutical salt thereof.

[0096] This invention provides Figure 5 Conjugates or their medicinal salts.

[0097] This invention provides an antisense oligonucleotide of compound ID 7_3 or a pharmaceutical salt thereof.

[0098] This invention provides Figure 6 Conjugates or their medicinal salts.

[0099] This invention provides an antisense oligonucleotide of compound ID 7_4 or a pharmaceutical salt thereof.

[0100] This invention provides Figure 7 Conjugates or their medicinal salts.

[0101] This invention provides an antisense oligonucleotide of compound ID 8_1 or a pharmaceutical salt thereof.

[0102] This invention provides Figure 8 Conjugates or their medicinal salts.

[0103] This invention provides an antisense oligonucleotide of compound ID 9_1 or a pharmaceutical salt thereof.

[0104] This invention provides an antisense oligonucleotide of compound ID 18_1 or a pharmaceutical salt thereof.

[0105] This invention provides Figure 8_1 Conjugates or their medicinal salts.

[0106] Compound of formula (I)

[0107] The present invention also provides compounds of formula (I).

[0108]

[0109] in

[0110] n is 0 or 1

[0111] p is 0 or 1

[0112] The condition is that when n is 1, p is preferably 1.

[0113] And the condition is that when n is 0 and p is 0, R is preferably H.

[0114] L is a linker, preferably L is a linker containing 2 to 10 nucleosides or composed of them, such as 2 to 5 nucleosides.

[0115] R stands for a GalNAc residue, preferably a trivalent GalNAc residue.

[0116] A is an antisense oligonucleotide residue according to the present invention.

[0117] The term "antisense oligonucleotide residue" refers to the antisense oligonucleotide according to the present invention, which is via -(L) n -(OP(=O)(-OH)-) p - Linked to residue R via its 5' end, such as the antisense oligonucleotides shown in Table 6. Preferred antisense oligonucleotide residues are located at... Figure 1A , Figure 2A , Figure 3A , Figure 4A , Figure 5A , Figure 6A , Figure 7A and Figure 8A Described in the text. Figure 8_1A Further preferred antisense oligonucleotide residues are described in the text.

[0118] GalNAc residues R

[0119] R stands for GalNAc residue, preferably a trivalent GalNAc residue. As used herein, the term "GalNAc residue" refers to a residue containing at least one N-acetylgalactosamine (GalNAc) moiety, i.e., at least one part of the following formula.

[0120]

[0121] As used herein, the term "trivalent GalNAc residue" refers to a residue containing three N-acetylgalactosamine (GalNAc) moieties, preferably the three moieties of the following formula.

[0122]

[0123] Preferably, the GalNAc residues contain at least one (preferably three) GalNAc structural units having the following structure (L a ),

[0124]

[0125] Among them, the connector a The group is selected from alkyl groups, alkyl-oxy-alkyl groups, alkyl groups containing at least one phosphate diester bond, alkyl groups containing at least one amide bond, alkyl-oxy-alkyl groups containing at least one phosphate diester bond, and alkyl-oxy-alkyl groups containing at least one amide bond.

[0126] The term "alkyl" refers to a substituted or unsubstituted straight-chain or branched alkyl group, such as C1 to C20 alkyl groups, preferably C2 to C8, such as C2, C3, C4, C5, C6, C7, or C8 alkyl groups. Preferably, the alkyl group is unsubstituted, more preferably a straight-chain and unsubstituted alkyl group.

[0127] The term "alkyl-oxy-alkyl" refers to at least two alkyl groups linked by oxygen, preferably linked to an ethyl-oxy-ethyl group, such as -(CH2-O). x - A group having an integer x, preferably in the range of 2 to 20, more preferably in the range of 2 to 6, such as 2, 3, 4, 5 or 6, more preferably x is 3 or 5.

[0128] According to one aspect of the invention, the GalNAc structural unit (L a ) is selected from the following structure (L a ) group.

[0129] If there is more than one residue (L) a If it exists in GalNAc residues, such as the three residues in trivalent GalNAc residues, then all residues are preferably identical.

[0130] Most preferably, L a Having structure

[0131]

[0132] In this case, the conjugate portion R comprises multiple parts, such as preferably three GalNAc portions, R in addition to the GalNAc structural unit (L a In addition to ), it also includes multivalent, preferably tetravalent structural units (L). b ), structural unit (L a Preferably via -(L) n -(OP(=O)(-OH)-)p- p -Linked to antisense oligonucleotide residue A.

[0133] L b Preferably, it is selected from one of the following structures:

[0134]

[0135] X is O or S, and Z is O or NH, wherein n is 1 to 4, preferably 2 or 3, and more preferably 2.

[0136] More preferably, L b It has the following structure

[0137]

[0138] It should be understood that L b Having structure L b *or structure L b **or a mixture thereof. According to a preferred aspect, L b For L b * and L b **Mixture:

[0139]

[0140] Therefore, the conjugate portion R preferably contains the structure (L) a )3-L b - More preferably, R includes one of the following structures

[0141]

[0142] More preferably, structure

[0143]

[0144] Where L b Preferably L b * and L b ** mixture,

[0145] And where X is O or S, and Z is O or NH, and where n is 1 to 3, preferably 2, and L a As described above, L is preferred. a Choose from the following groups

[0146] and mixtures thereof, wherein preferably all residues within the GalNAc residues (L a )same.

[0147] If (L) a )3-L b for

[0148]

[0149] L a Better choose the group composed of the following items

[0150]

[0151] If (L) a )3-L b for

[0152] Preferably,

[0153]

[0154] L a Preferably

[0155]

[0156] Optionally, the conjugate portion R may additionally include a connector L. c Therefore, R preferably has a structure (L) a )3-L b -(L c ) c - An integer c that is either 1 or 0.

[0157] Such connector compounds are known to those skilled in the art and are appropriately selected to connect (L) a )3-L b Connected to the remainder of the compound, i.e. via -(L) n -(OP(=O)(-OH)-) p - Linked to antisense oligonucleotide residues.

[0158] According to L b The structure of L c Selected from the group consisting of: alkyl, alkyl-oxy-alkyl, amino-alkyl (-NH-alkyl-), amino-alkyl-oxy-alkyl, non-natural amino acid residues, and natural amino acid residues. According to one aspect of the invention, L c The lysine group may be substituted or unsubstituted.

[0159] According to one aspect of the invention, R is (L a )3-L b -(L c ) c , where c = 1 and (L a )3-L b for

[0160]

[0161] L c Preferably, it is an amino-alkyl or amino acid group, such as a substituted or unsubstituted lysine group, especially L... C For example, select a group consisting of the following items.

[0162]

[0163] amino and L b The carbonyl group is linked to form an amide bond. According to this aspect, the preferred residue R is... Figure 9 A1、 Figure 9 A2, Figure 9 C1, Figure 9 C2, Figure 9D1、 Figure 9 Depicted in D2. Therefore, according to one aspect of the invention, R is selected from... Figure 9 A1、 Figure 9 A2, Figure 9 C1, Figure 9 C2, Figure 9 D1 and Figure 9 The residues depicted in D2.

[0164] According to another aspect of the invention, R has a structure (L) a )3-L b -(L c ) c , where c is 0, and where (L a )3-L b for

[0165]

[0166] According to this aspect of the invention, the preferred residue R is in Figure 9 B1 and Figure 9 Described in B2.

[0167] According to another aspect of the invention, R has a structure (L) a )3-L b -(L c ) c With (L) a )3-L b

[0168]

[0169] And Z is 0. In this case, c is preferably 1, and L... c Preferably, it is an alkyl group, more preferably a C3-C6 alkyl group, even more preferably propyl, and most preferably a n-propyl group. According to this aspect, the preferred residue R is... Figure 9 E1、 Figure 9 F1 Figure 9 G1 and Figure 9 Depicted in H1. Therefore, according to one aspect of the invention, R is selected from... Figure 9 E1、 Figure 9 F1 Figure 9 G1 and Figure 9 The residues described in H1.

[0170] According to another aspect of the invention, R has a structure (L) a )3-L b -(L c ) c With (L) a )3-L b

[0171]

[0172] And Z is NH. In this case, c is preferably 1 and L c Preferably, it is an alkyl group, a group containing an amino acid, or a group having the following structure:

[0173]

[0174] In particular, in this case L c for

[0175]

[0176] According to this aspect of the invention, the preferred residue R is in Figure 9 Described in J1.

[0177] According to another aspect of the invention, R has a structure (L) a )3-L b -(L c ) c With (L) a )3-L b

[0178]

[0179] Furthermore, Z is NH, and c is 0. According to this aspect of the invention, the preferred residue R is... Figure 9 Described in I1.

[0180] According to one aspect of the invention, R is (L a )3-L b -(L c ) c , where c = 0 and where (L a )3-L b for

[0181]

[0182] According to this aspect of the invention, the preferred residue R is in Figure 9 L1 and Figure 9 Depicted in L2.

[0183] Therefore, R is preferably selected from... Figure 9 A1、 Figure 9 A2, Figure 9 C1, Figure 9 C2, Figure 9 D1、 Figure 9 D2、 Figure 9 E1、 Figure 9 F1 Figure 9 G1 Figure 9H1, Figure 9 I1、 Figure 9 J1, Figure 9 L1, Figure 9 The residues and mixtures described in L2, such as Figure 9 A1 and Figure 9 A2, Figure 9 C1 and Figure 9 C2 or Figure 9 D1 and Figure 9 A stereoisomer of D2; more preferably, R is selected from... Figure 9 D1、 Figure 9 The residues described in D2, more preferably R is Figure 9 D1 and Figure 9 A mixture of residues described in D2, such as Figure 9 D1 and Figure 9 A mixture of D2 with a molar ratio in the range of 10:90 to 90:10, such as in the range of 30:70 to 70:30, such as in the range of 45:55 to 55:45.

[0184] Therefore, compound (I) is preferably selected from... Figure 10 A1、 Figure 10 A2, Figure 10 C1, Figure 10 C2, Figure 10 D1、 Figure 10 D2、 Figure 10 E1、 Figure 10 F1 Figure 10 G1 Figure 10 H1, Figure 10 I1、 Figure 10 J1, Figure 10 L1, Figure 10 The compounds and mixtures thereof described in L2, such as those in Figure 10 D1、 Figure 10 Depicted in D2 Figure 10 A1 and Figure 10 A2, Figure 10 C1 and Figure 10 C2 or Figure 10 D1 and Figure 10 Stereoisomer mixtures of D2 and mixtures thereof, more preferably compound (I) is in Figure 10 D1 and Figure 10 The mixture of compounds depicted in D2, such as Figure 10 D1 and Figure 10 A mixture of D2 with a molar ratio in the range of 10:90 to 90:10, such as in the range of 30:70 to 70:30, such as in the range of 45:55 to 55:45.

[0185] Connector L

[0186] In the above formula, L is a connector as defined herein, preferably L is a connector comprising 2 to 10 nucleosides or composed thereof, such as 2 to 5 nucleosides, such as 2 nucleosides, wherein optionally, the nucleosides are phosphodiester-linked nucleosides.

[0187] The linker L may contain 1 to 10 linked nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides, such as 2 to 6 linked nucleosides, such as 2 to 5 linked nucleosides, such as 2 to 4 linked nucleosides. In some embodiments, the linker contains two linked nucleotides. Thus, the nucleosides may be DNA nucleosides. Typically, nucleosides are linked via phosphodiester-nucleoside bonding. Furthermore, the linker L may be linked to an antisense compound via phosphodiester-nucleoside bonding. Additionally, the linker L is linked to the conjugate moiety R via a suitable functional group, such as via an amide, amine, ether, ester, phosphodiester (-OP(=O)(-OH)-O-), or phosphothiodiester (-OP(=S)(-OH)-O-). It should be understood that L may optionally additionally contain an alkyl or alkyl-oxy-alkyl group between the nucleoside and the functional group linking L to R. In this case, the nucleoside is preferably linked to an alkyl group or an alkyl-oxy-alkyl group via a phosphodiester bond and then to R via a suitable functional group, such as an amide, amine, ether, ester, phosphodiester (-OP(=O)(-OH)-O-) or thiophosphate diester (-OP(=S)(-OH)-O-) bond.

[0188] According to a preferred embodiment, L is...

[0189]

[0190] antisense (A) oligonucleotide residues

[0191] A is an antisense oligonucleotide residue according to the present invention, such as the antisense oligonucleotide shown in Table 6, which is linked to R via its 5' primer end via -(L). n -(OP(=O)(-OH)-) p Preferably, A is selected from... Figure 1A , Figure 2A , Figure 3A , Figure 4A , Figure 5A , Figure 6A , Figure 7A and Figure 8A The residues depicted in or Figure 1A , Figure 2A , Figure 3A , Figure 4A , Figure 5A , Figure 6A , Figure 7A, Figure 8A and Figure 8_1A The antisense oligonucleotide residues described in the text.

[0192] According to another aspect of the invention, A is in Figure 8_1A The antisense oligonucleotide residues described in the text.

[0193] Therefore, compound (I) is preferably selected from... Figure 10 A1、 Figure 10 A2, Figure 10 C1, Figure 10 C2, Figure 10 D1、 Figure 10 D2、 Figure 10 E1、 Figure 10 F1 Figure 10 G1 Figure 10 H1, Figure 10 I1、 Figure 10 J1, Figure 10 L1, Figure 10 The compounds and mixtures thereof described in L2, such as Figure 10 A1 and Figure 10 A2, Figure 10 C1 and Figure 10 C2 or Figure 10 D1 and Figure 10 A stereoisomer mixture of D2; more preferably, compound (I) is selected from... Figure 10 D1 and Figure 10 The compounds and mixtures thereof depicted in D2, more preferably compound (I) is a mixture of compound 10D1 and compound 10D2, preferably A is selected from the antisense oligonucleotides shown in Table 6, and more preferably A is selected from Figure 1A , Figure 2A , Figure 3A , Figure 4A , Figure 5A , Figure 6A , Figure 7A and Figure 8A The residues depicted are antisense oligonucleotide residues. And L is a linker comprising 2 to 10 nucleosides or composed of them, such as 2 to 5 nucleosides, such as 2 nucleosides, wherein optionally the nucleosides are phosphodiester-linked nucleosides.

[0194] More preferably, where L is

[0195]

[0196] On the other hand, R is a residue with structure (I).

[0197]

[0198] L is a linker as defined herein, preferably L is a linker comprising 2 to 10 nucleosides or composed thereof, such as 2 to 5 nucleosides, such as 2 nucleosides, wherein optionally, the nucleosides are phosphodiester-linked nucleosides, more preferably L is

[0199]

[0200] and

[0201] A is an antisense oligonucleotide according to the present invention, such as the antisense oligonucleotide shown in Table 6.

[0202] According to one aspect of the invention, A is selected from... Figure 1A , Figure 2A , Figure 3A , Figure 4A , Figure 5A , Figure 6A , Figure 7A and Figure 8A The residues depicted in or Figure 1A , Figure 2A , Figure 3A , Figure 4A , Figure 5A , Figure 6A , Figure 7A , Figure 8A and Figure 8_1A The antisense oligonucleotide residues described in the text.

[0203] According to another aspect of the invention, A is in Figure 8_1A The antisense oligonucleotide residues described in the text.

[0204] In another aspect, the present invention provides pharmaceutical compositions comprising the antisense oligonucleotide of the present invention or the conjugate of the present invention and pharmaceutical diluents, carriers, salts and / or adjuvants.

[0205] This invention provides pharmaceutical salts of the antisense oligonucleotides of the present invention, or conjugates thereof. In some embodiments, the pharmaceutical salt is selected from the group consisting of sodium salts, potassium salts, and ammonium salts.

[0206] This invention provides a pharmaceutical solution of the antisense oligonucleotide or its conjugate of the invention, wherein the pharmaceutical solution comprises the antisense oligonucleotide or its conjugate of the invention and a pharmaceutical solvent, such as phosphate-buffered saline. Alternatively, the solvent may be water or a sodium chloride solution.

[0207] The present invention provides antisense oligonucleotides or conjugates thereof in the form of solid powders, such as lyophilized powders.

[0208] This invention provides pharmaceutical salts of the antisense oligonucleotides of the present invention, or conjugates thereof.

[0209] This invention provides pharmaceutical salts of antisense oligonucleotides or conjugates according to the invention, wherein the pharmaceutical salt is a sodium salt. Alternatively, the salt is a potassium salt.

[0210] The present invention provides pharmaceutical compositions comprising the antisense oligonucleotides or conjugates of the present invention, or salts of the present invention, as well as pharmaceutical diluents, solvents, carriers, salts and / or adjuvants.

[0211] This invention provides a method for inhibiting FUBP1 expression in target cells expressing FUBP1, the method comprising administering an effective amount of the antisense oligonucleotide of this invention, or the conjugate of this invention, or the salt of this invention, or the composition of this invention to the cells. This method may be an in vivo method or an in vitro method.

[0212] The present invention provides a method for treating and / or preventing HBV infection in a subject (such as a human), comprising administering a therapeutic or preventative amount of the antisense oligonucleotide of the present invention, or the conjugate of the present invention, or the salt of the present invention, or the composition of the present invention, such as for treating and / or preventing diseases selected from the group consisting of HBV infection, such as chronic HBV infection, and proliferative diseases such as cancer, particularly hepatocellular carcinoma.

[0213] In some embodiments, the antisense oligonucleotides of the present invention, or the conjugates of the present invention, or the salts of the present invention, or the pharmaceutical compositions of the present invention are used in the treatment and / or prevention of HBV infection, such as chronic HBV infection.

[0214] This invention provides the use of the antisense oligonucleotide of the invention, or the conjugate of the invention, or the pharmaceutical composition of the invention, or the salt of the invention in medicine. In another aspect, this invention provides a method for inhibiting the expression of FUBP1 in target cells expressing FUBP1 by administering an effective amount of the antisense oligonucleotide of the invention, or the conjugate of the invention, to said cells. In yet another aspect, this invention provides an in vivo or in vitro method for inhibiting the expression of FUBP1 in target cells by administering an effective amount of the antisense oligonucleotide of the invention, or the conjugate of the invention, to target cells expressing FUBP1. For example, the cells may be human cells, such as liver cells, such as hepatocytes. In one embodiment, the cells are hepatocellular carcinoma cells.

[0215] In another aspect, the present invention provides a method for reducing cccDNA in HBV-infected cells by administering an effective amount of the antisense oligonucleotide of the present invention, or a conjugate of the present invention, to the cells.

[0216] In another aspect, the present invention provides an in vivo or in vitro method for reducing cccDNA in HBV-infected cells by administering an effective amount of the antisense oligonucleotide of the present invention, or a conjugate of the present invention, to the cells.

[0217] On the other hand, the present invention provides methods for treating and / or preventing diseases selected from the group consisting of HBV infection, such as chronic HBV infection, and proliferative diseases such as cancer, particularly hepatocellular carcinoma.

[0218] In another aspect, the present invention provides antisense oligonucleotides for preparing medicaments, or conjugates of the present invention, or pharmaceutical compositions of the present invention, for treating and / or preventing diseases selected from the group consisting of: HBV infection, such as chronic HBV infection, and proliferative diseases such as cancer, particularly hepatocellular carcinoma.

[0219] In another aspect, the present invention provides antisense oligonucleotides for preparing antiviral drugs, or conjugates of the present invention, or pharmaceutical compositions of the present invention.

[0220] In another aspect, the present invention provides antisense oligonucleotides for preparing antitumor drugs, or conjugates of the present invention, or pharmaceutical compositions of the present invention.

[0221] The present invention provides antisense oligonucleotides of the present invention, or conjugates of the present invention, or pharmaceutical compositions of the present invention for the treatment and / or prevention of diseases selected from the group consisting of HBV infection, such as chronic HBV infection, and proliferative diseases such as cancer, particularly hepatocellular carcinoma.

[0222] sequence list

[0223] The sequence listings submitted with this application are incorporated herein by reference. If the sequence listings are inconsistent with this specification or the accompanying drawings, the information disclosed in this specification (including the drawings) shall be deemed correct.

[0224] Brief description of the attached figures

[0225] Figure 1 Compound 6_1 (SEQ ID NO:6) is conjugated to the GalNAc moiety via a phosphodiester-linked DNA dinucleotide.

[0226] Figure 1A Residue A of compound 6_1 (SEQ ID NO:6)

[0227] Figure 2 Compound 6_2 (SEQ ID NO:6) is conjugated to the GalNAc moiety via a phosphodiester-linked DNA dinucleotide.

[0228] Figure 2AResidue A of compound 6_2 (SEQ ID NO:6)

[0229] Figure 3 Compound 7_1 (SEQ ID NO:7) is conjugated to the GalNAc moiety via a phosphodiester-linked DNA dinucleotide.

[0230] Figure 3A Residue A of compound 7_1 (SEQ ID NO:7)

[0231] Figure 4 Compound 7_2 (SEQ ID NO:7) is conjugated to the GalNAc moiety via a phosphodiester-linked DNA dinucleotide.

[0232] Figure 4A Residue A of compound 7_2 (SEQ ID NO:7)

[0233] Figure 5 Compound 7_3 (SEQ ID NO:7) is conjugated to the GalNAc moiety via a phosphodiester-linked DNA dinucleotide.

[0234] Figure 5A Residues of compound 7_3 (SEQ ID NO:7)

[0235] Figure 6 Compound 7_4 (SEQ ID NO:7) is conjugated to the GalNAc moiety via a phosphodiester-linked DNA dinucleotide.

[0236] Figure 6A Residue A of compound 7_4 (SEQ ID NO:7)

[0237] Figure 7 Compound 8_1 (SEQ ID NO:8) is conjugated to the GalNAc moiety via a phosphodiester-linked DNA dinucleotide.

[0238] Figure 7A Residue A of compound 8_1 (SEQ ID NO:8)

[0239] Figure 8 Compound 9_1 (SEQ ID NO:9) is conjugated to the GalNAc moiety via a phosphodiester-linked DNA dinucleotide.

[0240] Figure 8A Residue A of compound 9_1 (SEQ ID NO:9)

[0241] Figure 8_1 Compound 18_1 (SEQ ID NO:18) is conjugated to the GalNAc moiety via a phosphodiester-linked DNA dinucleotide.

[0242] Figure 8_1A Residue A of compound 18_1 (SEQ ID NO:18)

[0243] Figure 9 An exemplary GalNAc section is shown. Figure 9 L1 and Figure 9 The compounds in L2 consist of the monomer GalNAc phosphoramide, which is added to the oligonucleotide as part of the synthesis while still on a solid support, X being S or O, Y being S or O, and n = 1 to 3 (see WO 2017 / 178656). Figure 9 B1, Figure 9 B2, Figure 9 D1 and Figure 9 D2 is also referred to in this document as GalNAc2 or GN2, which are respectively without and with a C6 connector.

[0244] Figure 10 An exemplary antisense oligonucleotide conjugate is shown. Figure 10 A1 and Figure 10 A2- Figure 10 D1 and Figure 10 The compound in D2 contains a dilysine branched molecule, a PEG3 spacer, and three terminal GalNAc carbohydrate moieties. Figure 10 A1、 Figure 10 A2, Figure 10 B1 and Figure 10 In compounds B2, the oligonucleotides are preferably directly linked to the desialylate glycoprotein receptor targeting the conjugate moiety, without the need for a linker. Figure 10 C1, Figure 10 C2, Figure 10 D1 and Figure 10 In the compound D2, the oligonucleotide is linked via a C6 linker to the desialyl glycoprotein receptor targeting the conjugate moiety. Figure 10 E1- Figure 10 The compound in J1 contains commercially available triploid branched molecules and spacers of varying lengths and structures, as well as three terminal GalNAc carbohydrate moieties. Figure 10 L1 and Figure 10 The compounds in L2 consist of the monomer GalNAc phosphoramide, which is added to the oligonucleotide as part of the synthesis while still on a solid support, X = S or O, Y is S or O and n = 1 to 3 (see WO2017 / 178656).

[0245] Figure 11Results of in vitro efficacy analysis of the anti-FUBP1 compound in HeLa cells are shown. FUBP1 mRNA levels were normalized and shown as a percentage relative to the control.

[0246] Figure 12 Target binding: As described in Example 3, four antisense oligonucleotide compounds were tested for FUBP1 mRNA in HBV-infected PHH cells. Each compound was delivered to cells weekly at a concentration of 10 μM for three weeks. The FUBP1 mRNA targeting KD was evaluated one week after the last treatment. Total RNA was extracted from cells using the MagNA Purerobot and MagNA Pure96 Cellular RNA Large Volume Kit, according to the manufacturer's protocol, and FUBP1 mRNA was quantified by TaqManqPCR. The figure shows the residual expression of the target mRNA compared to the negative control (NDC=1), with oligonucleotides tested at 10 μM. Data were normalized to the human GUSB reference gene, and the mean +SD of two biological replicates for each tested oligonucleotide is reported. 50% and 20% FC are highlighted in the figure. CMP ID NO:7_3 shows the optimal FUBP1 mRNA KD, with an 80% reduction in mRNA expression at 10 μM. Compared to existing oligomers (CMP ID NO: 35_1 and 50_1), CMP ID NO: 18_1 showed the strongest effect in reducing FUBP1 mRNA, similar to the oligonucleotide with CMP ID NO: 7_3. Both reduced target mRNA expression by approximately 80% at 10 μM compared to NDC (see Example 3 for more details).

[0247] Figure 13 In a single-dose mouse study, the in vivo liver PK / PD association of oligonucleotides with CMP ID Nos: 7_3 and 18_1 via phosphodiester-linked DNA dinucleotide conjugates to the GalNAc moiety was evaluated (Conj. = conjugate, see Example 4 for more details).

[0248] definition

[0249] HBV infection

[0250] The term "hepatitis B virus infection" or "HBV infection" is well-known in the field and refers to an infectious disease caused by the hepatitis B virus (HBV) that affects the liver. HBV infection can be acute or chronic. Chronic hepatitis B virus (CHB) infection is a global health problem affecting 248 million people worldwide. Approximately 686,000 deaths annually are attributed to HBV-related advanced liver disease and hepatocellular carcinoma (HCC) (GBD 2013; Schweitzer et al., 2015). The WHO predicts that without increased intervention, the number of people infected with CHB will remain at its current high level for the next 40 to 50 years, with a cumulative death toll reaching 20 million between 2015 and 2030 (WHO 2016). CHB infection is not a homogeneous disease with a single clinical presentation. Infected individuals experience several stages of CHB-related liver disease throughout their lives, and these stages form the basis for standard of care (SOC) treatment. Current guidelines recommend treatment only for selected CHB-infected individuals based on three criteria (serum ALT level, HBV DNA level, and liver disease severity) (EASL, 2017). This recommendation stems from the fact that SOCs (i.e., nucleoside (nucleotide) analogs (NAs) and pegylated interferon-α (PEG-IFN)) are not curative and require long-term administration, thus increasing their safety risks. NAs can effectively suppress HBV DNA replication; however, their effect on other viral markers is very limited / nonexistent. Two markers of HBV infection, namely hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA), are the primary targets of new drugs aimed at curing HBV. In the plasma of individuals with chronic hepatitis B (CHB), the number of HBsAg subviral (empty) particles exceeds that of HBV viral particles by 10³ to 10⁵ times (Ganem & Prince, 2014); this excess is believed to contribute to the immunopathogenesis of the disease, including the individual's inability to produce neutralizing anti-HBs antibodies, a serological marker observed after the resolution of acute HBV infection.

[0251] In some embodiments, the term "HBV infection" refers to "chronic HBV infection".

[0252] Furthermore, the term covers any HBV genotype infection.

[0253] In some embodiments, the patient to be treated is infected with HBV genotype A.

[0254] In some embodiments, the patient to be treated is infected with HBV genotype B.

[0255] In some embodiments, the patient to be treated is infected with HBV genotype C (tested in Example 3 of the Examples section).

[0256] In some embodiments, the patient to be treated is infected with HBV genotype D.

[0257] In some embodiments, the patient to be treated is infected with HBV genotype E.

[0258] In some embodiments, the patient to be treated is infected with HBV genotype F.

[0259] In some embodiments, the patient to be treated is infected with HBV genotype G.

[0260] In some embodiments, the patient to be treated is infected with HBV genotype H.

[0261] In some embodiments, the patient to be treated is infected with HBV genotype I.

[0262] In some embodiments, the patient to be treated is infected with HBV genotype J.

[0263] cccDNA (covalently closed circular DNA)

[0264] cccDNA is the viral genetic template located in the nucleus of infected hepatocytes, where it produces all the HBV RNA transcripts required for proliferative infection and plays a major role in viral persistence during the natural course of chronic HBV infection (Locarnini & Zoulim, 2010 Antivir Ther. 15 Suppl 3:3-14. doi:10.3851 / IMP1619). cccDNA serves as a viral reservoir and is the source of viral rebound after treatment cessation, thus requiring long-term (usually lifelong) therapy. Due to various side effects, PEG-IFN can only be administered to a small percentage of chronic hepatitis B (CHB).

[0265] Therefore, most CHB patients urgently need new treatments that can achieve a complete cure by degrading or eliminating HBV cccDNA.

[0266] compound

[0267] In this document, the term "compound" refers to any molecule capable of inhibiting FUBP1 expression or activity. Particular compounds of this invention are nucleic acid molecules, such as antisense oligonucleotides according to the invention or any conjugates comprising such nucleic acid molecules. For example, compounds herein can be nucleic acid molecules targeting FUBP1, particularly antisense oligonucleotides.

[0268] Oligonucleotides

[0269] As used herein, the term "oligonucleotide" is defined as a molecule comprising two or more covalently linked nucleosides, as commonly understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are typically produced in the laboratory, first through solid-phase chemical synthesis followed by purification and isolation. When referring to the sequence of an oligonucleotide, the sequence or order of the nucleobase portion of the covalently linked nucleotide or nucleoside or its modifications thereof is referred to. The oligonucleotides of the present invention are artificial and chemically synthesized, and are typically purified or isolated. The oligonucleotides of the present invention may contain one or more modified nucleosides, such as 2′ sugar-modified nucleosides. The oligonucleotides of the present invention may contain one or more modified internucleotide bonds, such as one or more phosphate thioester internucleotide bonds.

[0270] antisense oligonucleotides

[0271] As used herein, the term "antisense oligonucleotide" or "ASO" is defined as an oligonucleotide capable of regulating the expression of a target gene by hybridization with a target nucleic acid, particularly with a sequence of sequences on the target nucleic acid. Antisense oligonucleotides are not substantially double-stranded and therefore are not siRNA or shRNA. Preferably, the antisense oligonucleotides of the present invention are single-stranded. It should be understood that the single-stranded oligonucleotides of the present invention can form hairpin or intermolecular double-stranded structures (double strands between two molecules of the same oligonucleotide) as long as the degree of intra- or inter-sequence self-complementarity is less than 50% across the full length of the oligonucleotide.

[0272] In some embodiments, the single-stranded antisense oligonucleotides of the present invention may be free of RNA nucleosides.

[0273] Advantageously, the antisense oligonucleotides of the present invention comprise one or more modified nucleosides or nucleotides, such as 2′ sugar-modified nucleosides. Furthermore, it is preferred that the unmodified nucleoside is a DNA nucleoside.

[0274] Continuous nucleotide sequence

[0275] The term "continuous nucleotide sequence" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. This term is used interchangeably herein with the terms "continuous nucleobase sequence" and "oligonucleotide motif sequence." In some embodiments, all the nucleosides of the oligonucleotide constitute a continuous nucleotide sequence. In some embodiments, the oligonucleotide comprises a continuous nucleotide sequence, such as an FG-F'gapmer region, and may optionally comprise other nucleotides, such as nucleotide linker regions that can be used to attach functional groups (e.g., conjugate groups) to the continuous nucleotide sequence. Nucleotide linker regions may or may not be complementary to the target nucleic acid. In some embodiments, the nucleobase sequence of an antisense oligonucleotide is a continuous nucleotide sequence.

[0276] Nucleotides and nucleosides

[0277] Nucleotides and nucleosides are components of oligonucleotides and polynucleotides, and for the purposes of this invention, include both naturally occurring and non-naturally occurring nucleotides and nucleosides. In practice, nucleotides such as DNA and RNA nucleotides comprise a ribose moiety, a nucleobase moiety, and one or more phosphate ester groups (which are absent in nucleosides). Nucleosides and nucleotides may also be interchangeably referred to as “units” or “monomers.”

[0278] Modified nucleosides

[0279] As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside modified by introducing one or more modifications of a sugar moiety or (nucleo)base moiety compared to an equivalent DNA or RNA nucleoside. Advantageously, the one or more modified nucleosides of the antisense oligonucleotides of the present invention comprise modified sugar moieties. The term "modified nucleoside" may also be used interchangeably herein with the terms "nucleoside analogue" or modified "unit" or modified "monomer". Nucleosides having unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides modified in the base regions of DNA or RNA nucleosides are generally still referred to as DNA or RNA if Watson Crick base pairing is permitted.

[0280] Modified nucleoside interbonds

[0281] As commonly understood by those skilled in the art, the term "modified internucleotide bond" is defined as a bond other than a phosphodiester (PO) bond that covalently couples two nucleosides together. Therefore, the oligonucleotides of the present invention may contain one or more modified internucleotide bonds, such as one or more thiophosphate internucleotide bonds, or one or more dithiophosphate internucleotide bonds.

[0282] In some embodiments, at least 50% of the internucleotide bonds in the oligonucleotide or its continuous nucleotide sequence are phosphate thioesters, such as at least 60%, at least 70%, at least 75%, at least 80%, or at least 90% of the internucleotide bonds in the oligonucleotide or its continuous nucleotide sequence are phosphate thioesters. In some embodiments, all internucleotide bonds in the oligonucleotide or its continuous nucleotide sequence are phosphate thioesters.

[0283] In some advantageous embodiments, all internucleotide bonds in the continuous nucleotide sequence of the oligonucleotide are phosphate thioester bonds, or all internucleotide bonds in the oligonucleotide are phosphate thioester bonds.

[0284] Phosphophosphate bonds can exist in different tautomeric forms, as shown below:

[0285]

[0286] It should be recognized that, as disclosed in EP 2 742 135, antisense oligonucleotides may contain other nucleoside internucleotides (other than phosphodiester, thiophosphate and dithiophosphate), such as alkylphosphonate / methylphosphonate internucleotides, which, according to EP 2 742 135, may be tolerated, for example, in other ways, by the spacer region of DNA thiophosphate.

[0287] nucleobases

[0288] The term "nucleobase" includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization. In the context of this invention, the term "nucleobase" also encompasses modified nucleobases that may differ from naturally occurring nucleobases but function during nucleic acid hybridization. In this context, "nucleobase" refers to naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-natural variants. Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

[0289] In some embodiments, the nucleobase portion is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazo-uracil, 2-thiouracil, 2'-thio-thymidine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0290] The nucleobase moiety can be represented by a letter code for each corresponding nucleobase, such as A, T, G, C, or U, where each letter may optionally include a modified nucleobase with equivalent function. For example, in an exemplary oligonucleotide, the nucleobase moiety is selected from A, T, G, C, and 5-methylcytosine. Optionally, for an LNA gapmer, a 5-methylcytosine LNA nucleoside can be used.

[0291] Modified oligonucleotides

[0292] The term "modified oligonucleotide" describes an oligonucleotide comprising one or more sugar-modified nucleosides and / or modified internucleotide bonds. The term "chimeric oligonucleotide" is a term already used in the literature to describe oligonucleotides comprising sugar-modified nucleosides and DNA nucleosides. The antisense oligonucleotide of the present invention is preferably a chimeric oligonucleotide.

[0293] Complementarity

[0294] The term “complementarity” describes the ability of a nucleoside / nucleotide to pair with Watson-Crick bases. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). It should be understood that oligonucleotides may contain nucleosides with modified nucleosides, for example, 5-methylcytosine is often used instead of cytosine; therefore, the term complementarity covers Watson-Crick base pairing between unmodified and modified nucleosides (see, for example, Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).

[0295] As used herein, the term "complementarity percentage" refers (expressed as a percentage) the proportion of nucleotides in a continuous nucleotide sequence of a nucleic acid molecule (e.g., an oligonucleotide) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) spanning that continuous nucleotide sequence. Therefore, the percentage of complementarity is calculated by counting the number of aligned nucleosides that are complementary (forming Watson Crick base pairs) between two sequences (when compared to oligonucleotide sequences of the target sequence at 5'-3' and 3'-5'), dividing that number by the total number of nucleotides in the oligonucleotide, and then multiplying by 100. In this comparison, misaligned nucleosides / nucleotides (forming base pairs) are referred to as mismatches. Insertions and deletions are not permitted when calculating the percentage of complementarity for a continuous nucleotide sequence. It should be understood that chemical modifications of nucleosides (e.g., 5′-methylcytosine is considered identical to cytosine when calculating the percentage of complementarity) are disregarded as long as the functional ability of the nucleosides forming Watson Crick base pairs is preserved.

[0296] The term "perfect complementarity" refers to 100% complementarity.

[0297] identity

[0298] As used herein, the term "identity" refers (expressed as a percentage) the proportion of nucleotides in a nucleic acid molecule (e.g., an oligonucleotide) that are identical to a reference sequence (e.g., a sequence motif) across the continuous nucleotide sequence. Therefore, the identity percentage is calculated by counting the number of identical (matching) aligned nucleobases in the two sequences (in the continuous nucleotide sequence of the compound of the present invention and in the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide, and then multiplying by 100. Thus, the identity percentage = (number of matches × 100) / length of the aligned region (e.g., the continuous nucleotide sequence). Insertions and deletions are not permitted when calculating the identity percentage of a continuous nucleotide sequence. It should be understood that, in determining identity, chemical modifications of the nucleobases (e.g., 5-methylcytosine is considered identical to cytosine in calculating the identity percentage) are not considered, as long as the functional ability of the nucleobases forming Watson Crick base pairs is preserved.

[0299] Hybridization

[0300] As used herein, the term "hybridization" (or "hybridizing") should be understood as the formation of hydrogen bonds between base pairs on opposite strands of two nucleic acid chains (e.g., oligonucleotides and target nucleic acids), resulting in a double helix. The affinity between the two nucleic acid chains is the strength of the hybridization. It is typically expressed as the melting temperature (T0). m The temperature at which half of the oligonucleotides form a double helix with the target nucleic acid is described as T. Under physiological conditions, T... m It is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515–537). The standard-state Gibbs free energy ΔG° is a more accurate expression of binding affinity and is related to the dissociation constant of the reaction (K). d ) through ΔG°=-RTln(K dThe energy is related to the reaction between the oligonucleotide and the target nucleic acid, where R is the gas constant and T is the absolute temperature. Therefore, the very low ΔG° of the reaction between the oligonucleotide and the target nucleic acid reflects the strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with the reaction in which the water concentration is 1M, the pH is 7, and the temperature is 37°C. The hybridization of the oligonucleotide and the target nucleic acid is a spontaneous reaction, and for spontaneous reactions, ΔG° is less than zero. ΔG° can be measured experimentally, for example, using isothermal titration calorimetry (ITC) as described by Hansen et al., 1965, Chem. Comm. 36–38, and Holdgate et al., 2005, in Drug Discov Today. Those skilled in the art will know that commercial equipment is available for measuring ΔG°. ΔG° can also be numerically estimated using the nearest neighbor model as described in SantaLucia, 1998, Proc Natl Acad Sci USA. 95:1460–1465, with appropriate use of the derived thermodynamic parameters described in Sugimoto et al., 1995, Biochemistry 34:11211–11216 and McTigue et al., 2004, Biochemistry 43:5388–5405. To allow for the possibility of modulating its intended nucleic acid target through hybridization, for oligonucleotides of 10–30 nucleotides in length, the oligonucleotides of the present invention hybridize with the target nucleic acid at a ΔG° estimate below -10 kcal. In some embodiments, the extent or intensity of hybridization is measured according to the standard-state Gibbs free energy ΔG°. For oligonucleotides of 8–30 nucleotides in length, the oligonucleotides may hybridize with the target nucleic acid at a ΔG° estimate below -10 kcal, such as below -15 kcal, such as below -20 kcal, and such as below -25 kcal. In some embodiments, the oligonucleotide hybridizes with the target nucleic acid at a ΔG° estimate of -10 kcal to -60 kcal, such as -12 kcal to -40 kcal, such as -15 kcal to -30 kcal, or -16 kcal to -27 kcal, such as -18 kcal to -25 kcal.

[0301] target

[0302] As used herein, the term "target" refers to the mammalian protein "distal upstream element binding protein 1," alternatively referred to as "FUBP1," "FBP," or "hDH V." The Homo sapiens FUBP1 gene is located on chromosome 1, 77944055..77979435, complement (NC_000001.11, gene ID 1462). The FUBP1 gene encodes an ssDNA binding protein that activates the distal upstream element of c-myc and stimulates c-myc expression in undifferentiated cells. FUBP regulation of FUSE occurs through the binding of FUBP to a single strand of the non-coding strand. The FUBP1 protein possesses ATP-dependent DNA helicase activity. The amino acid sequence of human FUBP1 is known in the art and can be evaluated using UniProt; see, for example, UniProt entry Q96AE4 for human FUBP1, which is incorporated herein by reference.

[0303] target nucleic acid

[0304] According to the present invention, the target nucleic acid is a nucleic acid encoding mammalian FUBP1, and can be, for example, a gene, RNA, mRNA and precursor mRNA, mature mRNA, or cDNA sequence. This target can therefore be referred to as the FUBP1 target nucleic acid.

[0305] Suitable, the target nucleic acid encodes the FUBP1 protein, particularly mammalian FUBP1, such as the precursor mRNA encoded by the human FUBP1 gene or the mRNA sequences of SEQ ID NO:1, 2 and / or 3 provided herein. SEQ ID NO:1 is the sequence of human FUBP1 precursor mRNA. SEQ ID NO:2 and 3 are the sequences of human FUBP1 mRNA.

[0306] Table 3 lists the predicted exon and intron regions for SEQ ID NO:1.

[0307] Table 3. Exon and intron regions in human FUBP1 precursor mRNA.

[0308]

[0309] In some embodiments, the target nucleic acid may be cynomolgus monkey FUBP1 nucleic acid, such as mRNA or precursor mRNA.

[0310] In some embodiments, the target nucleic acid may be mouse FUBP1 nucleic acid, such as mRNA or precursor mRNA.

[0311] Table 4 provides an overview of the genomic sequences of FUBP1 in humans, cynomolgus monkeys, and mice. Table 5 provides an overview of the precursor mRNA sequences of FUBP1 in humans, monkeys, and mice, as well as the mature mRNA of human FUBP1.

[0312] In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NO:1, 2, 3, 4 and / or 5 or naturally occurring variants thereof (e.g., sequences encoding mammalian FUBP1).

[0313] In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NO:1, 2 and / or 3 or naturally occurring variants thereof (e.g., sequences encoding mammalian FUBP1).

[0314] In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NO:1 and 4 and 5 or naturally occurring variants thereof (e.g., sequences encoding mammalian FUBP1).

[0315] Table 4. Genome and assembly information of FUBP1 in multiple species.

[0316]

[0317] Fwd = forward strand. Rv = reverse strand. Genomic coordinates provide the precursor mRNA sequence (genomic sequence).

[0318] If the antisense oligonucleotides of the present invention are used in research or diagnosis, the target nucleic acid may be cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0319] For in vivo or in vitro applications, the therapeutic antisense oligonucleotides of the present invention are generally able to inhibit the expression of FUBP1 target nucleic acid in cells expressing FUBP1 target nucleic acid. The continuous nucleotide sequence of the antisense oligonucleotides of the present invention is generally complementary to conserved regions of the FUBP1 target nucleic acid, as measured over the entire length of the antisense oligonucleotide, optionally excluding one or two mismatches, and optionally excluding nucleotide-based linker regions that can connect the antisense oligonucleotide to optional functional groups (such as conjugates) or other non-complementary terminal nucleotides.

[0320] The target nucleic acid can be messenger RNA, such as precursor mRNA encoding mammalian FUBP1 protein, such as human FUBP1, such as human FUBP1 precursor mRNA sequences, such as the sequence disclosed as SEQ ID NO:1, cynomolgus monkey FUBP1 precursor mRNA sequence, such as the sequence disclosed as SEQ ID NO:4 or mouse FUBP1 precursor mRNA sequence, such as the sequence disclosed as SEQ ID NO:5 or mature FUBP1 mRNA, such as human mature mRNA disclosed as SEQ ID NO:2 or SEQ ID NO:3. SEQ ID NO:1-5, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:15 and SEQ ID NO:19 are DNA sequences, and it should be understood that the target RNA sequences have uracil (U) bases in place of thymidine (T) bases.

[0321] Table 5 provides additional information about exemplary target nucleic acids.

[0322] Table 5. Sequence details of FUBP1 across species.

[0323] Species RNA type Length (nt) SEQ ID NO Humans Precursor mRNA 35056 1 Humans Mature mRNA, variant 1 1968 2 Humans Mature mRNA, variant t 1935 3 Crab-eating macaques Precursor mRNA 39750 4 mice Precursor mRNA 26405 5

[0324] Note: SEQ ID NO:4 contains multiple NNNN regions, where sequencing cannot accurately refine the sequence, and therefore contains degenerate sequences. To avoid ambiguity, the compounds of this invention are complementary to the actual target sequences and are therefore not degenerate compounds.

[0325] In some embodiments, the target nucleic acid is SEQ ID NO:1.

[0326] In some embodiments, the target nucleic acid is SEQ ID NO:2.

[0327] In some embodiments, the target nucleic acid is SEQ ID NO:3.

[0328] In some embodiments, the target nucleic acid is SEQ ID NO:4.

[0329] In some embodiments, the target nucleic acid is SEQ ID NO:5.

[0330] In some embodiments, the target nucleic acid is SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0331] In some embodiments, the target nucleic acids are SEQ ID NO:1 and SEQ ID NO:4. Therefore, antisense oligonucleotides can target FUBP1 in humans and cynomolgus monkeys.

[0332] In some embodiments, the target nucleic acids are SEQ ID NO:1 and SEQ ID NO:5. Therefore, the antisense oligonucleotide targets human and mouse FUBP1.

[0333] In some embodiments, the target nucleic acids are SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:5. Therefore, the antisense oligonucleotides can target FUBP1 in humans, cynomolgus monkeys, and mice.

[0334] target sequence

[0335] As used herein, the term "target sequence" refers to a sequence of nucleotides present in a target nucleic acid that contains a nucleobase sequence complementary to the oligonucleotide or nucleic acid molecule of the present invention. In some embodiments, the target sequence comprises a region (i.e., a subsequence) on the target nucleic acid having a nucleobase sequence complementary to a continuous nucleotide sequence of an antisense oligonucleotide of the present invention. This region of the target nucleic acid may be interchangeably referred to as the target nucleotide sequence, the target sequence, or the target region. In some embodiments, the target sequence is longer than the complementary sequence of a single oligonucleotide and may, for example, represent a preferred region of the target nucleic acid that can be targeted by several antisense oligonucleotides of the present invention.

[0336] In one embodiment, the target sequence is a region within exon 14 of human FUBP1 mRNA (see Table 3 above).

[0337] In another embodiment, the target sequence is a region within exon 20 of human FUBP1 mRNA (see Table 3 above).

[0338] The antisense oligonucleotides of the present invention comprise a continuous nucleotide sequence that is complementary to or hybridizes to a region on a target nucleic acid (such as the target sequence described herein).

[0339] This document provides numerous target sequence regions, such as those defined by the regions of human FUBP1 precursor mRNA that can be targeted by the oligonucleotides of this invention (using SEQ ID NO 1 as a reference).

[0340] The oligonucleotides of the present invention comprise a continuous nucleotide sequence that is complementary to or hybridizes with a target nucleic acid (such as a subsequence of the target nucleic acid, such as the target sequence described herein).

[0341] Oligonucleotides comprise a continuous nucleotide sequence complementary to a target sequence present in a target nucleic acid molecule. The continuous nucleotide sequence (and therefore the target sequence) comprises at least 12 consecutive nucleotides, such as 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 nucleotides, such as 14 to 20 consecutive nucleotides, such as 14 to 18 consecutive nucleotides.

[0342] target sequence region

[0343] The inventors have identified a particularly effective sequence of the FUBP1 target nucleic acid, which can be targeted by the oligonucleotides of this invention.

[0344] In some embodiments, the target sequence is SEQ ID NO:10.

[0345] In some embodiments, the target sequence is SEQ ID NO:11.

[0346] In some embodiments, the target sequence is SEQ ID NO:15.

[0347] In some embodiments, the target sequence is SEQ ID NO:19.

[0348] SEQ ID NO 10:GTGAAACCATAAAAAGCATAAG

[0349] SEQ ID NO 11:AACCATAAAAAGCATAAG

[0350] SEQ ID NO 15:GTGAAACCATAAAAAGCATA

[0351] SEQ ID NO 19:GTAGAAATGAAAATTGGT

[0352] SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:15 and SEQ ID NO:19 are DNA sequences, and it should be understood that the target RNA sequence has uracil (U) bases that replace the thymidine base (T).

[0353] In some embodiments, the target sequence is the region of nucleotides 16184 to 16200 of SEQ ID NO:1.

[0354] In some embodiments, the target sequence is the region of nucleotides 16186 to 16203 of SEQ ID NO:1.

[0355] In some embodiments, the target sequence is the region of nucleotides 16188 to 16205 of SEQ ID NO:1.

[0356] In some embodiments, the target sequence is the region of nucleotides 16189 to 16205 of SEQ ID NO:1.

[0357] In some embodiments, the target sequence is the region of nucleotides 30536 to 30553 of SEQ ID NO:1.

[0358] target cells

[0359] As used herein, the term "target cell" refers to a cell that expresses a target nucleic acid. In some embodiments, the target cell may be in vivo or in vitro. In some embodiments, the target cell is a mammalian cell, such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell, such as a monkey cell or a human cell.

[0360] Typically, target cells express FUBP1 mRNA, such as FUBP1 precursor mRNA or mature FUBP1 mRNA. For example, target cells express human FUBP1 precursor mRNA, such as SEQ ID NO 1, or mature human FUBP1 mRNA containing exon 14 (or exon 20, such as SEQ ID NO: 2 or 3). For experimental evaluation, target cells expressing nucleic acids containing the target sequence can be used. For antisense oligonucleotide targeting, the polyadenosine (poly A) tail of FUBP1 mRNA is generally not considered.

[0361] The antisense oligonucleotides of the present invention are generally able to inhibit the expression of FUBP1 target nucleic acid in target cells expressing FUBP1 target nucleic acid, for example, in vivo or in vitro.

[0362] In addition, the target cells can be hepatocytes. In one embodiment, the target cells are HBV-infected primary human hepatocytes derived from HBV-infected individuals or HBV-infected mice (PhoenixBio, PXB-mouse) with humanized livers.

[0363] In one embodiment, the target cells may be infected with HBV. Furthermore, the target cells may contain HBV cccDNA. Therefore, the target cells preferably contain FUBP1 mRNA, such as FUBP1 precursor mRNA or mature FUBP1 mRNA, and HBV cccDNA.

[0364] In addition, target cells can be cancer cells, such as hepatocellular carcinoma cells.

[0365] Naturally generated variants

[0366] The term "naturally occurring variant" refers to a variant of the FUBP1 gene or transcript that originates from the same genetic locus as the target nucleic acid, but may differ, for example, due to the diversity of codons encoding the same amino acid caused by the degeneracy of the genetic code, or due to alternative splicing of the precursor mRNA, or the presence of polymorphisms such as single nucleotide polymorphisms (SNPs) and allelic variants. Based on the presence of sufficiently complementary sequences to the oligonucleotides, the oligonucleotides of the present invention can therefore target the target nucleic acid and its naturally occurring variants.

[0367] In some embodiments, the naturally occurring variant has at least 95%, such as at least 98% or at least 99%, homology with the mammalian FUBP1 target nucleic acid, such as a target nucleic acid selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, or 5. In some embodiments, the naturally occurring variant has at least 99% homology with the human FUBP1 target nucleic acid of SEQ ID NO: 1.

[0368] Suppression of expression

[0369] As used herein, the term "inhibition of expression" should be understood as the collective ability of oligonucleotides to inhibit the quantity or activity of FUBP1 in target cells. Inhibition of activity can be determined by measuring the levels of FUBP1 precursor mRNA or FUBP1 mRNA, or by measuring the levels of FUBP1 or FUBP1 activity in cells. Therefore, inhibition of expression can be determined in vitro or in vivo.

[0370] Typically, inhibition of expression is determined by comparing the inhibition of activity resulting from the administration of an effective amount of antisense oligonucleotide to target cells and comparing that level with a reference level obtained from target cells that have never been administered antisense oligonucleotide (control experiment) or a known reference level (e.g., the level of expression before the administration of an effective amount of antisense oligonucleotide, or a predetermined or otherwise known level of expression).

[0371] For example, control experiments can be conducted on animals or humans, or on target cells treated with a saline composition or a reference oligonucleotide (usually a randomized control).

[0372] The term inhibition (noun) or suppression (verb) can also be referred to as downregulation, reduction, inhibition, decrease, or reduction of FUBP1 expression.

[0373] Repression of expression can occur, for example, by degrading precursor mRNA or by recruiting oligonucleotides, such as gapmers, using RNase H.

[0374] High affinity modified nucleosides

[0375] A high-affinity modified nucleoside is a modified nucleoside that, when incorporated into an oligonucleotide, enhances the affinity of the oligonucleotide for its complementary target, as determined, for example, by melting temperature (Tm). The high-affinity modified nucleosides of the present invention preferably increase the melting temperature of each modified nucleoside by between +0.5°C and +12°C, more preferably between +1.5°C and +10°C, and most preferably between +3°C and +8°C. Many high-affinity modified nucleosides are known in the art and include, for example, many 2'-substituted nucleosides and locked nucleic acids (LNAs) (see, for example, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

[0376] Sugar modification

[0377] The oligomers of the present invention may contain one or more nucleosides having modified sugar moieties, wherein the modified sugar moieties are modifications of the sugar moieties compared to those found in DNA and RNA.

[0378] Many modified nucleosides with ribose moieties have been prepared, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0379] These modifications include those that modify the ribocyclic structure, for example, by replacing it with a hexose ring (HNA) or a bicyclic ring, typically a bicyclic ring with a biradicle bridge between the C2 and C4 carbon atoms on the ribocyclic ring (LNA), or an unconnected ribocyclic ring (e.g., UNA) typically lacking a bond between the C2 and C3 carbon atoms. Other sugar-modified nucleosides include, for example, bicyclic hexosinic nucleic acids (WO2011 / 017521) or tricyclic nucleic acids (WO2013 / 154798). Modified nucleosides also include those in which the sugar moiety is replaced with a non-sugar moiety, such as in the case of peptide nucleic acids (PNA) or morpholino nucleic acids.

[0380] Sugar modification also includes modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group that is naturally present in DNA and RNA nucleosides. For example, substituents can be introduced at the 2', 3', 4', or 5' positions.

[0381] 2′ sugar modified nucleosides

[0382] A 2' sugar-modified nucleoside is a nucleoside that has a substituent other than H or -OH at the 2' position (2' substituted nucleosides) or contains a 2' linking dimer that can form a bridge between the 2' carbon and the second carbon atom in the ribose ring, such as LNA (2'-4' bimer bridged) nucleoside.

[0383] In fact, considerable effort has been devoted to developing 2'-sugar-substituted nucleosides, and many 2'-substituted nucleosides have been found to possess beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can provide enhanced binding affinity and / or increased nuclease resistance to oligonucleotides. Examples of 2'-substituted modified nucleosides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-F-ANA nucleosides. For further examples, see, for instance, Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213 and Deleavey and Damha, Chemistry and Biology 2012, 19, 937. Below are schematic diagrams of some 2′-substituted nucleosides.

[0384]

[0385] In this invention, 2′-substituted sugar-modified nucleosides do not include 2′-bridged nucleosides like LNA.

[0386] Locked nucleoside (LNA nucleoside)

[0387] "LNA nucleosides" are 2′-modified nucleosides containing a C2′ and C4′ bimodal group (also known as a "2′-4′ bridge") that links the ribose ring of the nucleoside, restricting or locking the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridging nucleic acids or bicyclic nucleic acids (BNAs). When LNAs are incorporated into oligonucleotides of complementary RNA or DNA molecules, the locking of the ribose conformation is associated with enhanced hybridization affinity (double-strand stabilization). This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary double strand.

[0388] Non-limiting exemplary LNA nucleosides are disclosed in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO2008 / 150729, Morita et al. (Bioorganic & Med. Chem. Lett. 12, 73-76, Seth Other non-limiting exemplary LNA nucleosides are disclosed in Scheme 1 in et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81 and Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238 and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0389] Option 1:

[0390]

[0391] Specific LNA nucleosides are β-D-oxy-LNA, 6′-methyl-β-D-oxy-LNA such as (S)-6′-methyl-β-D-oxy-LNA (ScET) and ENA. A particularly advantageous LNA is β-D-oxy-LNA.

[0392] nuclease-mediated degradation

[0393] Nuclease-mediated degradation means that an oligonucleotide can centrally influence the degradation of a sequence when it forms a double strand with a complementary nucleotide sequence.

[0394] In some embodiments, the oligonucleotides may function via nuclease-mediated degradation of the target nucleic acid, wherein the oligonucleotides of the present invention are capable of recruiting nucleases, particularly endonucleases, preferably RNases (RNases), such as RNase H. An example of an oligonucleotide design that operates via a nuclease-mediated mechanism is an oligonucleotide that typically comprises a region of at least five or six consecutive DNA nucleosides, and has affinity-enhancing nucleosides, such as gapmers, attached to one or both sides of this region.

[0395] RNase H activity and recruitment

[0396] The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when forming a double strand with a complementary RNA molecule. WO01 / 23613 provides an in vitro method for determining RNase H activity, which can be used to determine the ability to recruit RNase H. An oligonucleotide is generally considered capable of recruiting RNase H if it has an initial rate (in pmol / L / min) when a complementary target nucleic acid sequence is provided to it, which is at least 5%, such as at least 10% or more than 20%, of the initial rate determined using the methodology provided in Examples 91 to 95 of WO01 / 23613 (incorporated herein by reference) with an oligonucleotide having the same base sequence as the modified oligonucleotide being tested but containing only DNA monomers with phosphate thioester bonds among all monomers in the oligonucleotide. For the purpose of determining RNase H activity, information can be found from Creative... Recombinant human RNase H1 was obtained by fusing a recombinant human RNase H1 with a His tag expressed in Escherichia coli.

[0397] Gapmer

[0398] The antisense oligonucleotide or its continuous nucleotide sequence of the present invention can be a gapmer, also known as a gapmer oligonucleotide or gapmer design. Antisense gapmers are generally used to inhibit target nucleic acids through RNase H-mediated degradation. A gapmer oligonucleotide contains at least three distinct structural regions: a 5' flanking region, a gap, and a 3' flanking region FG-F' in a "5->3" orientation. The "gap" region (G) contains a continuous DNA nucleotide that enables the oligonucleotide to recruit RNase H. The flanking region of the gap is a 5' flanking region (F) containing one or more sugar-modified nucleosides (preferably high-affinity sugar-modified nucleosides), and a 3' flanking region (F') containing one or more sugar-modified nucleosides (preferably high-affinity sugar-modified nucleosides). One or more sugar-modified nucleosides in regions F and F' enhance the affinity of the oligonucleotide for the target nucleic acid (i.e., affinity-enhancing sugar-modified nucleosides). In some embodiments, one or more sugar-modified nucleosides in regions F and F' are 2' sugar-modified nucleosides, such as high-affinity 2' sugar modifications, such as those independently selected from LNA and 2'-MOE.

[0399] In gapmer design, the 5' and 3' terminal nucleotides of the gap region are DNA nucleotides, located near the sugar-modified nucleotides in the 5'(F) or 3'(F') regions, respectively. Flanking regions can be further defined as those ends furthest from the gap region, i.e., the 5' end of the 5' flanking region and the 3' end of the 3' flanking region, containing at least one sugar-modified nucleotide.

[0400] Regions FG-F' form a continuous nucleotide sequence. The antisense oligonucleotides of the present invention, or their continuous nucleotide sequences, may include the gapmer region of formula FG-F'. In some embodiments, all internucleotide bonds between the nucleosides in the gapmer region of formula FG-F' are phosphate thioester nucleoside bonds.

[0401] The total length of the Gapmer design FG-F' can be, for example, 12 to 32 nucleotides, such as 13 to 24 nucleotides, such as 14 to 22 nucleotides, such as 15 to 20 nucleotides, such as 16 to 18 nucleotides. In some embodiments, the total length is 17 nucleotides.

[0402] For example, the gapmer oligonucleotide of the present invention can be represented by the following formula:

[0403] F 1-8 -G 5-16 -F′ 1-8 Such as

[0404] F 1-8 -G 7-16 -F' 2-8 or

[0405] F 4-8 -G 7-12 -F' 2-8 or

[0406] F 4-6 -G 7-11 -F' 2-6

[0407] The prerequisite is that the total length of the gapmer region FGF′ is at least 12, such as at least 14 nucleotides.

[0408] In one embodiment, the gapmer oligonucleotide of the present invention can be represented by the following formula:

[0409] F 4-6 -G 7-11 -F' 2-6

[0410] Preferably, the total length of the gapmer region FG-F' is at least 16 nucleotides, such as at least 17 or 18 nucleotides.

[0411] In one aspect of the invention, the antisense oligonucleotide or its sequential nucleotide sequence comprises or includes a gapmer of formula 5'-FG-F'-3', wherein regions F and F' independently comprise or consist of 1-8 nucleotides, wherein 1-4 nucleotides are modified with 2' sugars and define the 5' and 3' ends of regions F and F', and G is a region of 6 to 16 nucleotides, such as 7 to 12 nucleotides, capable of recruiting RNase H.

[0412] In some embodiments, all modified nucleosides in regions F and F′ are β-D-oxyLNA nucleosides. Furthermore, regions F or F′, or F and F', may optionally contain DNA nucleosides. Optionally, flanking regions F or F', or flanking regions F and F', may contain one or more DNA nucleosides (alternating flanking, see the definition of alternating flanking for more details).

[0413] The regions F, G, and F' are further defined below and can be incorporated into the FG-F' formula.

[0414] Gapmer - Region G

[0415] The gapmer region G (gap region) is a nucleoside region that allows the oligonucleotide to recruit RNase H, such as human RNase H1, typically a DNA nucleoside. RNase H is a cellular enzyme that recognizes the double strand between DNA and RNA and enzymatically cleaves the RNA molecule. A suitable gapmer may have a gap region (G) of at least 5 or 6 adjacent DNA nucleosides, such as 5-16 adjacent DNA nucleosides, such as 6-15 adjacent DNA nucleosides, such as 7-14 adjacent DNA nucleosides, such as 8-12 adjacent DNA nucleotides, such as a gap region (G) of 8-12 adjacent DNA nucleotides in length. In some embodiments, the gap region G may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 adjacent DNA nucleosides.

[0416] In some embodiments, the gap region G may consist of 12 or fewer (such as 7) consecutive DNA nucleotides. 8. 9, 10, or 11 consecutive DNA nucleotides, such as 9, 10, or 11 consecutive DNA nucleotides.

[0417] In some cases, one or more cytosine (C) residues in the gap region may be methylated (e.g., when DNA c is followed by DNA g). These residues are also labeled as 5-methyl-cytosine (C). me C). In some embodiments, the gap region G may consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 adjacent phosphate-thiolated DNA nucleosides.

[0418] In some embodiments, all nucleoside internucleotide bonds in the nick are thiophosphate bonds.

[0419] Gapmer - Flanking area, F and F'

[0420] The 5' DNA nucleoside in region F is immediately adjacent to that in region G. The 3' terminal nucleoside in region F is a sugar-modified nucleoside, such as a high-affinity sugar-modified nucleoside, or a 2'-substituted nucleoside, such as MOE nucleoside or LNA nucleoside.

[0421] The 3' DNA nucleoside of region F' is immediately adjacent to region G. The 5' terminal nucleoside of region F' is a sugar-modified nucleoside, such as a high-affinity sugar-modified nucleoside, or a 2'-substituted nucleoside, such as MOE nucleoside or LNA nucleoside.

[0422] Region F has a length of 1-8 consecutive nucleotides, such as 2-6, such as 4-6 consecutive nucleotides. In some embodiments, region F has a length of 4 consecutive nucleotides. In some embodiments, region F has a length of 5 consecutive nucleotides. In some embodiments, region F has a length of 6 consecutive nucleotides.

[0423] Preferably, the 5' terminal nucleoside of region F is a sugar-modified nucleoside. In some embodiments, both 5' terminal nucleosides of region F are sugar-modified nucleosides. In some embodiments, the 5' terminal nucleoside of region F is an LNA nucleoside. In some embodiments, both 5' terminal nucleosides of region F are LNA nucleosides.

[0424] Region F' has a length of 1–8 consecutive nucleotides, such as 2–6, or 2–5 consecutive nucleotides. In some embodiments, region F' has a length of 2 consecutive nucleotides. In some embodiments, region F' has a length of 3 consecutive nucleotides. In some embodiments, region F' has a length of 4 consecutive nucleotides. In some embodiments, region F' has a length of 5 consecutive nucleotides.

[0425] Preferably, the 3' terminal nucleoside of region F is a sugar-modified nucleoside. In some embodiments, both 3' terminal nucleosides of region F are sugar-modified nucleosides. In some embodiments, both 3' terminal nucleosides of region F are LNA nucleosides. In some embodiments, the 3' terminal nucleoside of region F' is an LNA nucleoside.

[0426] It should be noted that when the length of region F is one, it is preferably an LNA nucleoside. Furthermore, it should be noted that when the lengths of regions F and / or F' are two, the two nucleosides of regions F and / or F' are advantageously LNA nucleosides.

[0427] In some embodiments, the sugar-modified nucleosides in regions F and F' consist of only one type of sugar-modified nucleoside, such as only MOE, only β-D-oxyLNA, or only ScET. Such a design is also known as a uniform flanking or uniform gapmer design.

[0428] In some embodiments, all nucleosides in regions F or F′, or F and F', are LNA nucleosides, such as β-D-oxyLNA nucleosides. In alternative embodiments, all sugar-modified nucleosides in regions F and F' are LNA nucleosides, such as β-D-oxyLNA nucleosides, wherein regions F or F', or both F and F', may optionally contain DNA nucleosides (alternating flanking; see the definitions of these for more details).

[0429] In some embodiments, the 5' and 3' terminal nucleotides of regions F and F' are LNA nucleotides, such as β-D-oxyLNA nucleotides.

[0430] In some embodiments, the internucleotide bonds between regions F and G and / or between regions F' and G are phosphate thioester internucleotide bonds. In some embodiments, the internucleotide bonds between the nucleosides of regions F or F', F and F' are phosphate thioester internucleotide bonds.

[0431] LNA gapmer

[0432] An LNA gapmer is a gapmer in which one or both of regions F and F' contain or are composed of LNA nucleosides. A β-D-oxygenated gapmer is a gapmer in which one or both of regions F and F' contain or are composed of β-D-oxygenated LNA nucleosides.

[0433] In some embodiments, the LNA gapmer has the following formula: [LNA] 1-5 -[Region G]-[LNA] 1-5 , wherein region G is or contains a continuous DNA nucleoside region capable of recruiting RNase H.

[0434] MOE gapmer

[0435] A MOE gapmer is a gapmer in which regions F and F′ are composed of MOE nucleotides. In some embodiments, the MOE gapmer is designed as [MOE]. 1-8 -[Region G] 5-16 -[MOE] 1-8 Such as [MOE] 2-7 -[Region G] 6-14 -[MOE] 2-7 Such as [MOE] 3-6-[Region G] 8-12 -[MOE] 3-6 The region G has the definition as defined in the gapmer definition. MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) are widely used in this field.

[0436] Hybrid wing gapmer

[0437] The hybrid wing gapmer is an LNA gapmer in which one or both of regions F and F' contain a 2′-substituted nucleoside, such as a 2′-substituted nucleoside independently selected from the group consisting of: 2′-O-alkyl-RNA unit, 2′-O-methyl-RNA, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-alkoxy-RNA, MOE unit, arabinonucleotide (ANA) unit, and 2′-fluoro-ANA unit such as MOE nucleoside. In some embodiments in which at least one of regions F and F' or both regions F and F' contain at least one LNA nucleoside, the remaining nucleosides of regions F and F' are independently selected from the group consisting of MOE and LNA. In some embodiments in which at least one of regions F and F' or both regions F and F' contain at least two LNA nucleosides, the remaining nucleosides of regions F and F' are independently selected from the group consisting of MOE and LNA. In some embodiments of the hybrid wing, one or both of regions F and F' may further contain one or more DNA nucleosides.

[0438] Alternating flank gapmer

[0439] Flanking regions may contain both LNA and DNA nucleosides, and are referred to as “alternating flanking” because they contain alternating motifs of LNA-DNA-LNA nucleosides. A gapmer containing at least one alternating flanking is called an “alternating flanking gapmer.” Thus, an “alternating flanking gapmer” is an LNA gapmer oligonucleotide in which at least one flanking region (F or F') contains one or more DNA nucleosides in addition to an LNA nucleoside. In some embodiments, at least one of regions F or F', or both regions F and F', contains both LNA and DNA nucleosides. In such embodiments, flanking regions F or F', or both F and F', contain at least three nucleosides, wherein the 5' and 3' terminal nucleosides of regions F and / or F' are LNA nucleosides. Alternating flanking LNA gapmers are disclosed in WO2016 / 127002.

[0440] Alternating flanking regions may contain up to three consecutive DNA nucleotides, such as one to two, one, two, or three consecutive DNA nucleotides.

[0441] Alternating flanking regions can be annotated as a series of integers representing multiple LNA nucleotides (L) followed by multiple DNA nucleotides (D), for example, [L]. 1-3 -[D] 1-3 -[L] 1-3 Or [L] 1-2 -[D]1 -2 -[L] 1-2 -[D] 1-2 -[L] 1-2 In oligonucleotide design, these will often be represented as numbers, such that 2-2-1 represents 5'[L]2-[D]2-[L]3', and 1-1-1-1-1 represents 5'[L]-[D]-[L]-[D]-[L]3'. The lengths of the flanks (regions F and F') in oligonucleotides with alternating flanks are as described above for this region, such as 4 to 8, 5 to 6 nucleotides, or 4, 5, 6, or 7 modified nucleotides. It may be advantageous to have at least two LNA nucleotides at the 3' end of the 3' flank (F') to confer additional exonuclease resistance.

[0442] In one embodiment, the gapmer oligonucleotide of the present invention can be represented by the following formula:

[0443] F 4-6 -G 7-11 -F' 2-6 ,

[0444] Where F has [L] 1-3 -[D] 1-3 -[L] 1-3 The design, and F′ has [L] 1-2 -[D] 1-2 -[L] 2-4 Or [L] 2-6 Design

[0445] The prerequisite is that the total length of the gapmer region FGF′ is at least 16 nucleotides, such as 17 or 18 nucleotides.

[0446] Therefore, the gapmer oligonucleotide of the present invention may contain at least one alternating flanking wing. Typically, at least region F is an alternating flanking wing. In some embodiments, regions F and F' are alternating flanking wing. In some embodiments, region F is an alternating flanking wing and region F' is a uniform flanking wing (i.e., F' consists of only one type of sugar-modified nucleoside, such as only β-D-oxyLNA).

[0447] In some embodiments, the design of region F is selected from the designs of 3-2-1 (i.e., LLLDDL), 3-1-1 (i.e., LLLDL), 2-1-2 (LLDLL), 2-1-1 (LLDL), and 1-3-1 (i.e., LDDDL).

[0448] In some embodiments, region F' is designed as 1-1-3 (i.e., LDLLL) or 1-1-2 (i.e., LDLL). In some embodiments, region F is designed as LL, LLL, or LLLL.

[0449] Region D′ or D″ in oligonucleotides

[0450] In some embodiments, the oligonucleotides of the present invention may comprise or consist of the following: a continuous nucleotide sequence of an oligonucleotide complementary to a target nucleic acid, such as gapmer regions FG-F', and additional 5' and / or 3' nucleotides. The additional 5' and / or 3' nucleotides may be fully complementary to the target nucleic acid or may not be fully complementary. Such additional 5' and / or 3' nucleotides may be referred to herein as regions D' and D'.

[0451] For the purpose of conjugating a continuous nucleotide sequence (such as a gapmer) to a conjugate moiety or another functional group, the addition region D' or D'' can be used. When used to conjugate a continuous nucleotide sequence to a conjugate moiety, it can serve as a biolytic linker. Alternatively, it can be used to provide exonuclease protection or to facilitate synthesis or manufacturing.

[0452] Regions D' and D” can be attached to the 5' end of region F or the 3' end of region F', respectively, to generate the following formulas: D′-FG-F', FG-F'-D”, or D'-FG-F'-D”. In this case, FG-F' is the gapmer part of the oligonucleotide, while region D' or D” constitutes the individual part of the oligonucleotide.

[0453] Regions D′ or D” may independently contain or consist of one, two, three, four, or five additional nucleotides, which may or may not be complementary to the target nucleic acid. The nucleotides adjacent to the F or F′ regions are not sugar-modified nucleotides, such as DNA or RNA, or base-modified forms of these. Regions D′ or D” can be used as nuclease-sensitive biolyzable linkers (see definition of linker). In some embodiments, additional 5' and / or 3' nucleotides are linked by phosphodiester bonds and are DNA or RNA. WO2014 / 076195 discloses nucleotide-based biolyzable linkers suitable for use as regions D′ or D”, comprising, for example, phosphodiester-linked DNA dinucleotides. WO2015 / 113922 discloses the use of biolyzable linkers in polyoligonucleotide constructs, wherein they are used to link multiple antisense constructs (e.g., gapmer regions) within a single oligonucleotide.

[0454] In one embodiment, the oligonucleotide of the present invention, in addition to the continuous nucleotide sequence constituting the gapmer, also includes regions D' and / or D'.

[0455] In some embodiments, the oligonucleotide of the present invention may be represented by the following formula:

[0456] FG-F'; especially F 1-8 -G 5-16 -F' 2-8 Such as F 4-6 -G 7-11 -F' 2-6

[0457] D'-FG-F'-D", especially D' 1-3 -F 1-8 -G 5-16 -F' 2-8 , such as D' 1-3 -F 4-6 -G 7-11 -F' 2-6

[0458] FG-F'-D", especially F 1-8 -G 5-16 -F' 2-8 -D” 1-3

[0459] D'-FG-F'-D", especially D' 1-3 -F 1-8 -G 5-16 -F' 2-8 -D” 1-3

[0460] In some embodiments, the internucleotide bond between region D' and region F is a phosphodiester bond. In some embodiments, the internucleotide bond between region F' and region D' is a phosphodiester bond.

[0461] Conjugate

[0462] As used herein, the term "conjugate" refers to an oligonucleotide covalently linked to a nonnucleotide moiety (conjugate moiety or region C or third region). The conjugate moiety may be covalently linked to an antisense oligonucleotide, optionally via a linker group, such as region D' or D'.

[0463] Oligonucleotide conjugates and their synthesis are also discussed in Manoharan's *Antisense Drug Technology, Principles, Strategies, and Applications*, SCrooke, ed., Ch. 16, Marcel Dekker, Inc., 2001, and Manoharan, *Antisense and Nucleic Acid Drug Development*, 2002, 12, 103.

[0464] In some embodiments, the non-nucleotide portion (conjugated portion) is selected from the group consisting of carbohydrates (e.g., GalNAc), cell surface receptor ligands, drugs, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0465] Exemplary conjugate moieties include those capable of binding to the asialic acid glycoprotein receptor (ASGPR). In particular, trivalent N-acetylgalactosamine conjugate moieties are suitable for binding to ASGPR, see, for example, WO 2014 / 076196, WO 2014 / 207232 and WO 2014 / 179620. Such conjugates are used to enhance hepatic uptake of oligonucleotides.

[0466] In some embodiments, the conjugate is an antibody or antibody fragment having a specific affinity for the transferrin receptor, such as that disclosed in WO 2012 / 143379, which is incorporated herein by reference. In some embodiments, the nonnucleotide portion is an antibody or antibody fragment, such as an antibody or antibody fragment that facilitates the delivery of medicament across the brain-vascular barrier, particularly an antibody or antibody fragment targeting the transferrin receptor.

[0467] connector

[0468] A bond or linker is a connection between two atoms that links one target chemical group or segment to another via one or more covalent bonds. The conjugate portion can be directly or via a linker portion (e.g., a linker or tie). A linker can covalently link a third region, such as the conjugate portion (region C), to a first region, such as an oligonucleotide or continuous nucleotide sequence (region A) complementary to the target nucleic acid.

[0469] In some embodiments of the present invention, the conjugates or oligonucleotide conjugates of the present invention may optionally include a linker region (second region or region B and / or region Y) located between an oligonucleotide or continuous nucleotide sequence complementary to the target nucleic acid (region A or first region) and a conjugate portion (region C or third region).

[0470] A biolytic linker (region B) comprising physiologically unstable bonds or composed of such bonds is cleavable under conditions commonly encountered in mammals or similar conditions. Conditions under which a physiologically unstable linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidative or reducing conditions or reagents, and salt concentrations encountered in mammalian cells or similar salt concentrations. Intracellular mammalian conditions also include enzymatic activities commonly present in mammalian cells, such as enzymatic activities from proteolytic enzymes or hydrolases or nucleases. In one embodiment, the biolytic linker is sensitive to S1 nuclease cleavage. In some embodiments, the physiologically unstable linker (biolytic) comprises 1 to 10 linked nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 linked nucleosides, such as 2 to 6 linked nucleosides, such as 2 to 5 linked nucleosides, such as 2 to 4 linked nucleosides, wherein at least two consecutive bonds are biolytic, such as at least 3, 4, or 5 consecutive phosphodiester bonds. Preferably, the nucleoside is DNA or RNA.

[0471] In one embodiment, the linker between the oligonucleotide and the conjugate moiety is a physiologically unstable linker consisting of 2 to 5 consecutive phosphodiester-linked nucleosides, with at least two consecutive phosphodiester bonds at the 5' or 3' end of the consecutive nucleotide sequence of the antisense oligonucleotide.

[0472] In some embodiments, the physiologically unstable linker comprises or is composed of a DNA dinucleotide having a sequence selected from the group consisting of AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, wherein a phosphodiester bond exists between the two DNA dinucleotides, and at least one phosphodiester at the 5' or 3' end of the dinucleotide links an oligonucleotide to the dinucleotide or a conjugate portion to the dinucleotide of the nucleic acid molecule. For example, the linker may be a CA dinucleotide. In some embodiments, the physiologically unstable linker comprises or is composed of DNA trinucleotides of the sequences AAA, AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG, GGA, GGT, GGC, or GGG, wherein there is a phosphodiester bond between the DNA nucleosides and there may be another phosphodiester at the 5' or 3' end of the trinucleotide. Biolytic linkers containing phosphodiester are described in more detail in WO 2014 / 076195 (incorporated herein by reference). When compared with standards, in conjugate compounds with biolytic linkers, at least about 50% of the conjugate moiety is cleaved from the oligonucleotide, such as at least about 60%, such as at least about 70%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%.

[0473] Region Y refers to a linker that is not necessarily biodegradable but is primarily used to covalently link the conjugate portion (Region C or Third Region) to an oligonucleotide (Region A or First Region). Region Y linkers may contain repeating units such as ethylene glycol, amino acid units, or aminoalkyl chain structures or oligomers.

[0474] The oligonucleotide conjugates of the present invention may be composed of the following regioforms AC, ABC, ABYC, AYBC, or AYC. In some embodiments, the linker (region Y) is an aminoalkyl group (such as a C2-C36 aminoalkyl group), including, for example, C6 to C12 aminoalkyl groups. In some embodiments, the linker (region Y) is a C6 aminoalkyl group.

[0475] medicinal salt

[0476] The term "medicinal salt" refers to salts that retain the biological efficacy and properties of a free base or acid, which is not biologically or otherwise undesirable. These salts are formed using inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid (especially hydrochloric acid), and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and N-acetylcysteine. Furthermore, these salts can be prepared by adding inorganic or organic bases to a free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts formed with the following organic bases: primary amines, secondary amines, and tertiary amines. Substituted amines include naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyamine resins. The compounds of the present invention may also exist in zwitterionic form. Particularly preferred pharmaceutical salts of the compounds of formula (I) are salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and methanesulfonic acid.

[0477] treat

[0478] As used herein, the terms “treatment,” “treating,” “treats,” etc., generally refer to achieving a desired pharmacological and / or physiological effect. This effect is therapeutic in relation to the partial or complete cure of the disease and / or side effects attributable to the disease. As used herein, the term “treatment” covers any treatment of a subject’s disease, including: (a) suppressing the disease; or (b) improving (i.e., alleviating) the disease, i.e., leading to disease remission. Thus, compounds that improve and / or suppress HBV infection are compounds that treat HBV. Preferably, as used herein, the term “treatment” refers to a medical intervention for a pre-existing condition, such as HBV infection or cancer that has been defined and manifested.

[0479] prevention

[0480] The terms “prevention,” “protection,” or “avoidance” used herein refer to preventative treatment, i.e., measures or procedures whose purpose is to prevent rather than cure a disease. Prevention refers to achieving a desired pharmacological and / or physiological effect that has a preventative effect in completely or partially preventing the disease or its symptoms. Therefore, “prevention of HBV infection” as used herein includes preventing the occurrence of HBV infection in a subject, as well as preventing the onset of HBV infection symptoms. In this invention, the prevention of HBV infection from an HBV-infected mother to her child is particularly contemplated. The prevention of the transition from acute HBV infection to chronic HBV infection is also contemplated.

[0481] patient

[0482] For the purposes of this invention, a "subject" or "patient" can be a vertebrate. In the context of this invention, the term "subject" includes humans and other animals, particularly mammals and other organisms. Therefore, the means and methods provided herein are suitable for human treatment and veterinary applications. Thus, the subject herein can be an animal, such as a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bovine, horse, camel, or primate. Preferably, the subject is a mammal. More preferably, the subject is a human. In some embodiments, the patient suffers from a disease as referred to herein, such as HBV infection or cancer. In some embodiments, the patient is susceptible to said disease. Detailed Implementation

[0483] One aspect of the present invention is an enhanced antisense oligonucleotide or conjugate thereof targeting FUBP1 for the treatment and / or prevention of diseases selected from the group consisting of HBV infection, such as chronic HBV infection and proliferative diseases such as cancer, particularly hepatocellular carcinoma.

[0484] One embodiment of the present invention is the antisense oligonucleotide or conjugate thereof, which is capable of reducing HBV DNA, such as cccDNA, and HBV RNA transcripts, such as pgRNA, in infected cells (such as HBV-infected cells).

[0485] In another embodiment, the antisense oligonucleotide or its conjugate of the present invention can reduce HBsAg and / or HBeAg in individuals infected with HBV.

[0486] Another aspect of the invention is the use of the antisense oligonucleotides or conjugates thereof in the treatment and / or prevention of hepatitis B virus (HBV) infection, particularly chronic HBV infection, or in the treatment of cancer in which FUBP1 is overexpressed.

[0487] The antisense oligonucleotides of the present invention

[0488] The enhanced antisense oligonucleotides or their conjugates of the present invention may be excellent FUBP1 inhibitors because they can target FUBP1 transcripts and promote their degradation by RNase H cleavage.

[0489] One aspect of the present invention is an enhanced antisense oligonucleotide or a conjugate thereof for the treatment and / or prevention of HBV infection or for the treatment of cancer.

[0490] This section describes enhanced antisense oligonucleotides or their conjugates suitable for the treatment and / or prevention of HBV infection or for the treatment of cancer.

[0491] The antisense oligonucleotides or conjugates thereof of the present invention can inhibit the expression of FUBP1 in vitro and in vivo. Inhibition is achieved by hybridizing the antisense oligonucleotide with a target nucleic acid encoding FUBP1 or involved in the regulation of FUBP1. The target nucleic acid can be a mammalian FUBP1 sequence, such as a sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4 and / or 5.

[0492] Therefore, the oligonucleotide of the present invention is an antisense oligonucleotide that targets FUBP1.

[0493] In some embodiments, the antisense oligonucleotides or conjugates thereof of the present invention can regulate the expression of a target by inhibiting or downregulating its expression. Preferably, such regulation produces at least 20% inhibition of expression compared to the normal expression level of the target, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition compared to the normal expression level of the target. In some embodiments, using 25 μM of the antisense oligonucleotides or conjugates thereof of the present invention in PXB-PHH cells may be able to inhibit the expression level of FUBP1 mRNA by at least 50% or 60% in vitro. In some embodiments, using 25 μM of the antisense oligonucleotides or conjugates thereof of the present invention in PXB-PHH cells may be able to inhibit the expression level of FUBP1 protein by at least 50% in vitro, and this range of target reduction is preferred in terms of selecting antisense oligonucleotides that are well correlated with cccDNA reduction. Suitablely, assays for measuring FUBP1 RNA inhibition are provided in the examples (e.g., Examples 1 and 2). Target inhibition is triggered by hybridization between the continuous nucleotide sequence of the antisense oligonucleotide and the target nucleic acid. In some embodiments, the antisense oligonucleotide of the present invention comprises a mismatch between the antisense oligonucleotide and the target nucleic acid. Despite the mismatch, hybridization with the target nucleic acid may still be sufficient to exhibit the desired FUBP1 expression inhibition. The reduced binding affinity caused by the mismatch can preferably be compensated by an increase in the number of nucleotides in the oligonucleotide and / or an increase in the number of modified nucleosides capable of increasing binding affinity to the target, such as 2′ sugar-modified nucleosides present in the antisense oligonucleotide sequence, including LNA.

[0494] One aspect of the invention relates to enhanced antisense oligonucleotides of 12 to 30 nucleotides (e.g., 12 to 22, 16 to 20) nucleotides in length, comprising a continuous nucleotide sequence of at least 12 nucleotides in length, such as 14, 15, 16, or 17 nucleotides in length, having at least 90% complementarity, such as 100% complementarity, and a target sequence derived from nucleotides 16184-16205, such as target sequences selected from SEQ ID NO:1 16184-16200, 16186-16203, 16188-16205, and 16189-16205. In particular, antisense oligonucleotides capable of inhibiting FUBP1 expression, i.e., capable of reducing FUBP1 nucleic acid such as FUBP1 mRNA, are considered part of the invention.

[0495] In some embodiments, the antisense oligonucleotide of the present invention comprises a continuous nucleotide sequence of 12 to 22 nucleotides, such as 15 to 20 nucleotides, which is at least 90% complementary to the target nucleic acid of SEQ ID NO:10, such as being completely complementary.

[0496] In some embodiments, the antisense oligonucleotide comprises a continuous nucleotide sequence of 15 to 18 nucleotides in length, such as 17 or 18 nucleotides, which is at least 90% complementary to the target nucleic acid of SEQ ID NO:11, such as being completely complementary.

[0497] In some embodiments, the antisense oligonucleotide comprises a continuous nucleotide sequence of 15 to 18 nucleotides in length, such as 17 or 18 nucleotides, which is at least 90% complementary to the target nucleic acid of SEQ ID NO:18, such as being completely complementary.

[0498] In some embodiments, the antisense oligonucleotide comprises a continuous nucleotide sequence of 15 to 22 nucleotides, such as 15 to 18 nucleotides, such as 17 or 18 nucleotides, having at least 90% complementarity with the target nucleic acid, such as complete complementarity, wherein the target nucleic acid is selected from the following regions of SEQ ID NO:1: 16184-16205, 16184-16200, 16186-16203, 16188-16205, and 16189-16205. Alternatively, it may comprise a continuous nucleotide sequence of 15 to 22 nucleotides, such as 15 to 18 nucleotides, such as 17 or 18 nucleotides, having at least 90% complementarity with the target nucleic acid, such as complete complementarity, wherein the target nucleic acid is selected from the following regions of SEQ ID NO:1: 30536-30553.

[0499] In some embodiments, the antisense oligonucleotide comprises a continuous sequence of 12 to 30 nucleotides in length that is at least 90% complementary to a region or target sequence of a target nucleic acid, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% complementary.

[0500] It is preferred if the antisense oligonucleotide of the present invention or its continuous nucleotide sequence is completely complementary (100% complementary) to the region of the target nucleic acid, or in some embodiments may contain one or two mismatches between the oligonucleotide and the target nucleic acid.

[0501] In some embodiments, the antisense oligonucleotide sequence is 100% complementary to the corresponding target nucleic acid of SEQ ID NO:1.

[0502] In some embodiments, the antisense oligonucleotide or continuous nucleotide sequence of the present invention is at least 95% complementary to the target nucleic acids of SEQ ID NO:1 and SEQ ID NO:4, for example, completely (or 100%) complementary.

[0503] In some embodiments, the antisense oligonucleotide comprises a continuous nucleotide sequence of 15 to 22 nucleotides in length that is at least 90%, such as 100%, complementary to the corresponding target sequence present in SEQ ID NO:1, wherein the target sequence is selected from nucleotides 16184-16205, 16184-16200, 16186-16203, 16188-16205, 16189-16205, and 30536-30553 of SEQ ID NO:1.

[0504] In some embodiments, the sequential nucleotide sequence of the antisense oligonucleotide is at least 90% complementary to the target site sequence of SEQ ID NO:10, preferably 100% complementary.

[0505] In some embodiments, the sequential nucleotide sequence of the antisense oligonucleotide is at least 90% complementary to the target site sequence of SEQ ID NO:11, preferably 100% complementary.

[0506] In some embodiments, the sequential nucleotide sequence of the antisense oligonucleotide is at least 90% complementary to the target site sequence of SEQ ID NO:15, preferably 100% complementary.

[0507] In some embodiments, the sequential nucleotide sequence of the antisense oligonucleotide is at least 90% complementary to the target site sequence of SEQ ID NO:19, preferably 100% complementary.

[0508] In some embodiments, the continuous nucleotide sequence comprises a sequence of nucleobases selected from the group consisting of SEQ ID NO: 6, 7, 8, 9 and 18, or at least 14 continuous nucleotides thereof, such as 17 or 18 continuous nucleotides thereof.

[0509] In some embodiments, the antisense oligonucleotide or its continuous nucleotide sequence of the present invention comprises or consists of 10 to 30 nucleotides in length, such as comprising or consisting of 12 to 25, such as 11 to 22, such as 12 to 20, such as 14 to 18 or 16 to 18 continuous nucleotides in length.

[0510] In some embodiments, the antisense oligonucleotide or its sequential nucleotide sequence comprises, or is composed of, 22 or fewer nucleotides, such as 20 or fewer nucleotides, such as 18 or fewer nucleotides. For example, the antisense oligonucleotide or its sequential nucleotide sequence may comprise 14, 15, 16, or 17 nucleotides. It should be understood that any range given herein includes the endpoints of the range. Accordingly, if an oligonucleotide is described herein as comprising from 10 to 30 nucleotides, then both 10 and 30 nucleotides are included.

[0511] The present invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides of 12 to 24 nucleotides in length, such as antisense oligonucleotides of 12 to 18 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide sequence comprising at least 12, such as at least 13, such as at least 14, such as at least 15 or at least 16 continuous nucleotides present in SEQ ID NO:6.

[0512] The present invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides of 12 to 24 nucleotides in length, such as antisense oligonucleotides of 12 to 18 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide sequence comprising at least 12, such as at least 13, such as at least 14, such as at least 15 or at least 16 continuous nucleotides present in SEQ ID NO:7.

[0513] The present invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides of 12 to 24 nucleotides in length, such as antisense oligonucleotides of 12 to 18 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide sequence comprising at least 12, such as at least 13, such as at least 14, such as at least 15 or at least 16 continuous nucleotides present in SEQ ID NO:8.

[0514] The present invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides of 12 to 24 nucleotides in length, such as antisense oligonucleotides of 12 to 18 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide sequence comprising at least 12, such as at least 13, such as at least 14, such as at least 15 or at least 16 continuous nucleotides present in SEQ ID NO:9.

[0515] The present invention provides antisense oligonucleotides according to the invention, such as antisense oligonucleotides of 12 to 24 nucleotides in length, such as antisense oligonucleotides of 12 to 18 nucleotides in length, wherein the antisense oligonucleotide comprises a continuous nucleotide sequence comprising at least 12, such as at least 13, such as at least 14, such as at least 15, at least 16, at least 17 or 18 continuous nucleotides selected from SEQ ID NO 18.

[0516] In some embodiments, the continuous nucleotide sequence comprises or consists of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 consecutive nucleotides in length, such as 16, 17 or 18 consecutive nucleotides.

[0517] In some embodiments, the antisense oligonucleotide or its sequential nucleotide sequence comprises or consists of sequences selected from or composed of SEQ ID NO:6, 7, 8, 9 and 18.

[0518] In a preferred embodiment, the antisense oligonucleotide of the present invention comprises one or more sugar-modified nucleosides, such as one or more 2' sugar-modified nucleosides, which are independently selected from 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabinonucleotide (ANA), 2′-fluoro-ANA, and LNA nucleosides. It is preferred if one or more modified nucleosides are locked nucleic acids (LNAs).

[0519] In some embodiments, the continuous nucleotide sequence comprises LNA nucleoside.

[0520] In some embodiments, the continuous nucleotide sequence comprises LNA nucleoside and DNA nucleoside.

[0521] In some embodiments, the continuous nucleotide sequence comprises 2′-O-methoxyethyl (2′MOE) nucleotide.

[0522] In some embodiments, the continuous nucleotide sequence comprises 2'-O-methoxyethyl (2'MOE) nucleoside and DNA nucleoside.

[0523] Advantageously, the 3' terminal nucleotide of the antisense oligonucleotide or its continuous nucleotide sequence is a 2' sugar-modified nucleotide.

[0524] Preferably, the antisense oligonucleotide contains at least one modified nucleoside internucleotide bond, such as a thiophosphate or a dithiophosphate.

[0525] In some embodiments, at least one nucleoside-to-nucleotide bond in a continuous nucleotide sequence is a phosphate thioside-to-nucleotide bond.

[0526] In some embodiments, at least one internucleotide bond in the continuous nucleotide sequence is a dithiophosphate internucleotide bond.

[0527] In some embodiments, at least one internucleotide bond in the continuous nucleotide sequence is a phosphodiester internucleotide bond.

[0528] In some embodiments, all internucleotide bonds in a continuous nucleotide sequence are phosphate thioester internucleotide bonds.

[0529] In some embodiments, at least 75% of the internucleotide bonds in the antisense oligonucleotide or its continuous nucleotide sequence are phosphate thioester internucleotide bonds.

[0530] In some embodiments, all internucleotide bonds in an antisense oligonucleotide or its continuous nucleotide sequence are phosphate thioester internucleotide bonds.

[0531] In an advantageous embodiment of the invention, the antisense oligonucleotide of the invention is capable of recruiting RNase H, such as RNase H1. In some embodiments, the antisense oligonucleotide of the invention or its sequential nucleotide sequence is a gapmer.

[0532] In some embodiments, the antisense oligonucleotide or its sequential nucleotide sequence comprises or is composed of a gapmer of formula 5'-FG-F'-3'.

[0533] In some embodiments, region G consists of 6 to 16 DNA nucleotides, such as 7 to 12 DNA nucleotides. In some embodiments, region F contains 4 to 6 nucleotides and / or region F′ contains 2 to 6 nucleotides.

[0534] In some embodiments, regions F and F′ each contain at least one LNA nucleoside.

[0535] In some embodiments of the oligonucleotides of the present invention, all LNA nucleosides are β-D-oxyLNA nucleosides.

[0536] In some embodiments, the oligonucleotide of the present invention is an LNA gapmer with uniform flanks.

[0537] In some embodiments of the invention, the LNA gapmer is an alternating wing LNA gapmer. In some embodiments, the alternating wing LNA gapmer includes at least one alternating wing (such as wing F). In some embodiments, the alternating wing LNA gapmer includes one alternating wing (such as wing F) and one uniform wing (such as wing F′). In some embodiments, the alternating wing LNA gapmer includes two alternating winges. For example, the LNA gapmer may have a design selected from the following: 3-2-1-9-2, 3-1-1-10-2, 2-1-2-10-3, 2-1-1-11-3, 2-1-1-10-1-1-2, 2-1-1-10-4, 1-3-1-7-1-1-3, and 3-2-1-9-3. Alternatively, the LNA gapmer may have the following design: 1-1-3-9-1-1-2.

[0538] Table 6 lists the preferred designs for each motif sequence.

[0539] The present invention provides the following oligonucleotide compounds (Table 6):

[0540] Table 6: A list of oligonucleotide motif sequences (represented by SEQ ID NO) of the present invention, their design, and specific oligonucleotide compounds of the present invention (represented by CMP ID NO) designed based on the motif sequences.

[0541]

[0542] The heading "Oligonucleotide Compounds" in the table represents a specific design of the motif sequence. Uppercase letters represent β-D-oxyLNA nucleosides, lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, and all internucleotide bonds are phosphate thioester internucleotide bonds. The heading "Design" refers to the gapmer design, FG-F'. In gapmers with alternating flanking designs, the flanking annotations of oligonucleotides are a series of integers representing many β-D-oxyLNA nucleosides (L), followed by many DNA nucleosides (D). For example, a flanking design with a 2-2-1 motif represents LLDDL. Both flankings have β-D-oxyLNA nucleosides at both the 5' and 3' ends. A gap region (G) consisting of many DNA nucleosides lies between the flankings.

[0543] For some embodiments of the present invention, the oligonucleotide is selected from the group consisting of oligonucleotide compounds having CMP ID NO: 6_1, 6_2, 7_1, 7_2, 7_3, 7_4; 8_1 and 9_1 (see Table 6). For example, the compound may be a compound having CMP ID NO: 7_3.

[0544] In an alternative embodiment, the oligonucleotide is an oligonucleotide of a compound having CMP ID NO: 18_1 (see Table 6).

[0545] In all cases, the FG-F' design may further include regions D' and / or D'", as described under "Regions D' or D' in oligonucleotides" in the "Definitions" section. In some embodiments, the oligonucleotides of the present invention have, for example, one, two, or three phosphodiester-linked nucleoside units, such as DNA units, at the 5' end of the gapmer region. In some embodiments, the oligonucleotides of the present invention consist of two 5' phosphodiester-linked DNA nucleosides and the subsequent FG-F' gapmer region as defined above. Oligonucleotides containing phosphodiester-linked DNA units at the 5' or 3' end are suitable for conjugation and may further include the conjugation portion described herein. For delivery to the liver, the ASGPR targeting portion as the conjugation portion is particularly preferred; see the conjugation portion for more details.

[0546] Conjugate

[0547] Because HBV infection primarily affects hepatocytes in the liver, it is advantageous to conjugate the enhanced antisense oligonucleotide of the present invention to the conjugation portion, which will increase the delivery of the antisense oligonucleotide to the liver compared to the unconjugated antisense oligonucleotide. In one embodiment, the liver-targeting portion is selected from cholesterol or other lipids or conjugation portions capable of binding to the desialyl glycoprotein receptor (ASGPR).

[0548] In some embodiments, the present invention provides a conjugate comprising an antisense oligonucleotide of the present invention covalently linked to the conjugate portion.

[0549] The desialyl glycoprotein receptor (ASGPR) conjugate moiety comprises one or more carbohydrate moieties capable of binding to the desialyl glycoprotein receptor (ASGPR targeting moiety) with an affinity equal to or greater than that of galactose. The affinity of many galactose derivatives for the desialyl glycoprotein receptor has been studied (e.g., Jobst, ST and Drickamer, K. JB. C. 1996, 271, 6686) or readily determined using methods typical of the art.

[0550] In one embodiment, the conjugate portion comprises at least one desialylated glycoprotein receptor-targeting moiety selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-butyryl-galactosamine, and N-isobutyryl-galactosamine. Advantageously, the desialylated glycoprotein receptor-targeting moiety is N-acetylgalactosamine (GalNAc).

[0551] To generate the ASGPR conjugate moiety, an ASPGR targeting moiety (preferably GalNAc) can be attached to the conjugate scaffold. Typically, the ASGPR targeting moiety can be located at the same end of the scaffold. In one embodiment, the conjugate moiety consists of two to four terminal GalNAc moieties attached to spacers that link each GalNAc moiety to a branched molecule that can conjugate with an antisense oligonucleotide.

[0552] In another embodiment, the conjugated portion is monovalent, divalent, trivalent, or tetravalent relative to the desialylate glycoprotein receptor-targeting portion. Advantageously, the desialylate glycoprotein receptor-targeting portion comprises an N-acetylgalactosamine (GalNAc) portion.

[0553] GalNAc conjugate moieties may include, for example, those described in WO 2014 / 179620 and WO 2016 / 055601 and PCT / EP2017 / 059080 (incorporated herein by reference), as well as small peptides attached to the GalNAc moieties, such as Tyr-Glu-Glu-(aminohexylGalNAc)3 (YEE(ahGalNAc)3; a glycotripeptide that binds to the desialyl glycoprotein receptor on hepatocytes, see, for example, Duff et al., Methods Enzymol, 2000, 313, 297); lysine-based galactose clusters (e.g., L3G4; Biessen et al., Cardovasc. Med., 1999, 214); and cholane-based galactose clusters (e.g., carbohydrate recognition motifs of the desialyl glycoprotein receptor).

[0554] The ASGPR conjugate moiety, particularly the trivalent GalNAc conjugate moiety, can be attached to the 3′ or 5′ end of the oligonucleotide using methods known in the art. In one embodiment, the ASGPR conjugate moiety is attached to the 5′ end of the oligonucleotide.

[0555] In one embodiment, the conjugated moiety is trivalent N-acetylgalactosamine (GalNAc), such as Figure 9 A1、 Figure 9 A2, Figure 9 C1, Figure 9 C2, Figure 9 D1、 Figure 9 D2、 Figure 9 E1、 Figure 9 F1 Figure 9 G1 Figure 9 H1, Figure 9 I1、 Figure 9 J1, Figure 9 L1 and Figure 9 Those shown in L2; or the conjugate portion is Figure 9 A1 and Figure 9 A2 mixture; Figure 9 C1 and Figure 9 C2 mixtures or Figure 9 D1 and Figure 9 A mixture of D2. Especially trivalent N-acetylgalactosamine (GalNAc), such as... Figure 9 D1 or Figure 9 D2 or a mixture thereof.

[0556] In some embodiments, the conjugate is selected from the group consisting of:

[0557] 5'-GN2-C6 o c o a om C s T s T s a s t s G s c s t s t s t s t s t s a s t s g s G s T、

[0558] 5'-GN2-C6 o c o a o m C s T s T s a s T s g s c s t s t s t s t s t s a s t s g s G s T、

[0559] 5'-GN2-C6 o c o a o m C s T s t s A s T s g s c s t s t s t s t s t s a s t s g s G s T s T、

[0560] 5'-GN2-C6 o c o a o m C s T s ts A s t s g s c s t s t s t s t s t s a s t s g s G s T s T、

[0561] 5'-GN2-C6 o c o a o m C s T s t s A s t s g s c s t s t s t s t s t s a s t s G s g s T s T、

[0562] 5'-GN2-C6 o c o a o m C s T s t s A s t s g s c s t s t s t s t s t s a s t s G s G s T s T、

[0563] 5'-GN2-C6 o c o a o G s c s t s t s Ts t s t s a s t s g s g s t s T s t s m C s A s m C and

[0564] 5'-GN2-C6 o c o a o T s A s T s g s c s T s t s t s t s t s a s t s g s g s t s T s T s m C

[0565] 5'-GN2-C6 o c o a o A S c S m C S A S A S t S t S t S t S c S a S t S t S t S m C S tA S m C

[0566] In this context, uppercase letters represent β-D-oxyLNA nucleosides, lowercase letters represent DNA nucleosides, each LNA cytosine is 5-methylcytosine, the subscript 's' represents the bond between thiophosphate nucleosides, the subscript 'o' represents the bond between phosphodiester nucleosides, and GN2-C6 is... Figure 9 D1 and Figure 9 D2 shows trivalent N-acetylgalactosamine (GalNAc), such as... Figure 9 D1 or Figure 9 The trivalent N-acetylgalactosamine (GalNAc) shown in D2, or a mixture of both, is preferably linked via a phosphodiester bond at the 5' end of an oligonucleotide. Chemical diagrams representing certain molecules are shown below. Figures 1 to 8 and Figure 8_1 As shown.

[0567] In some implementations, the conjugate is as follows Figure 1 The conjugate shown.

[0568] In some implementations, the conjugate is as follows Figure 2 The conjugate shown.

[0569] In some implementations, the conjugate is as follows Figure 3 The conjugate shown.

[0570] In some implementations, the conjugate is as follows Figure 4 The conjugate shown.

[0571] In some implementations, the conjugate is as follows Figure 5 The conjugate shown.

[0572] In some implementations, the conjugate is as follows Figure 6 The conjugate shown.

[0573] In some implementations, the conjugate is as follows Figure 7 The conjugate shown.

[0574] In some implementations, the conjugate is as follows Figure 8 The conjugate shown.

[0575] In some implementations, the conjugate is as follows Figure 8_1 The conjugate shown.

[0576] Figures 1 to 8 and Figure 8_1 The compounds shown are presented in protonated form, meaning the S atom on the thiophosphate bond is protonated; it should be understood that the presence of protons will depend on the acidity of the molecule's environment and the presence of alternative cations (e.g., when the oligonucleotide is in salt form). Protonated thiophosphates exist in tautomeric form.

[0577] medicinal salt

[0578] The compounds according to the invention can exist in the form of their pharmaceutical salts. The term "pharmaceutical salt" refers to a conventional acid addition salt or base addition salt formed from a suitable non-toxic organic or inorganic acid or organic or inorganic base, while retaining the bioavailability and properties of the compounds of the invention. Acid addition salts include, for example, salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, aminosulfonic acid, phosphoric acid, and nitric acid, and salts derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, etc. Base addition salts include salts derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides such as tetramethylammonium hydroxide. The chemical modification of pharmaceutical compounds into salts to obtain improved physical and chemical stability, hygroscopicity, flowability, and solubility is a well-known technique among medicinal chemists. For example, Bastin describes this in *Organic Process Research & Development*, 2000, Vol. 4, pp. 427-435, or Ansel describes it in *Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed. (1995)*, pp. 196 and 1456-1457. For example, the pharmaceutical salt of the compounds provided herein may be a sodium salt.

[0579] In another aspect, the present invention provides pharmaceutical salts of antisense oligonucleotides or their conjugates, such as pharmaceutical sodium salts, ammonium salts or potassium salts.

[0580] Manufacturing method

[0581] In another aspect, the present invention provides a method for manufacturing the oligonucleotide of the present invention, the method comprising reacting nucleotide units to form covalently linked sequential nucleotide units contained in the oligonucleotide. Preferably, the method uses a phosphoramidite chemistry method (see, for example, Caruthers et al., 1987, Methods in Enzymology vol. 154, pages 287-313). In another embodiment, the method further comprises reacting the sequential nucleotide sequence with a conjugate moiety (ligand) to covalently link the conjugate moiety to the oligonucleotide. In another aspect, a method for preparing the compositions of the present invention is provided, the method comprising mixing the oligonucleotide or conjugated oligonucleotide of the present invention with a pharmaceutical diluent, solvent, carrier, salt and / or adjuvant.

[0582] Pharmaceutical Composition

[0583] In another aspect, the present invention provides pharmaceutical compositions comprising any of the aforementioned oligonucleotides and / or oligonucleotide conjugates or salts thereof, as well as pharmaceutical diluents, carriers, salts, and / or adjuvants. Pharmaceutical diluents include phosphate-buffered saline (PBS), and pharmaceutical salts include, but are not limited to, sodium, ammonium, and potassium salts. In some embodiments, the pharmaceutical diluent is sterile phosphate-buffered saline. Alternatively, the diluent may be water or a sodium chloride solution. In some embodiments, the oligonucleotide is used in the pharmaceutical diluent at a concentration of 50 μM to 300 μM solution.

[0584] Suitable formulations for use in this invention can be found in Remington's Pharmaceutical Sciences (17th ed., Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985). For a brief overview of drug delivery methods, see, for example, Langer (Science 249:1527-1533, 1990). WO 2007 / 031091 (incorporated herein by reference) provides other suitable and preferred examples of pharmaceutical diluents, carriers, and adjuvants. Suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations are also provided in WO 2007 / 031091.

[0585] In some embodiments, the antisense oligonucleotides or their conjugates or their pharmaceutical salts of the present invention are in solid form, such as powders or lyophilized powders.

[0586] In some embodiments, the antisense oligonucleotides or conjugates thereof of the present invention may be mixed with pharmaceutically active or inert substances to prepare pharmaceutical compositions or formulations. The composition and formulation of the pharmaceutical composition depend on many criteria, including but not limited to route of administration, disease severity, or dosage.

[0587] These compositions can be sterilized using conventional sterilization techniques or by aseptic filtration. The resulting aqueous solution can be used directly after packaging or lyophilized, with the lyophilized formulation mixed with a sterile aqueous carrier prior to application. The pH of the formulation is typically between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as 7 to 7.5. The resulting compositions in solid form can be packaged in multiple single-dose units, each unit containing a fixed amount of one or more of the aforementioned reagents, such as in sealed packages of tablets or capsules. The compositions in solid form can also be flexibly quantified in containers, such as in squeeze tubes designed for topical application of creams or ointments.

[0588] In some embodiments, the antisense oligonucleotide or its conjugate of the present invention is a prodrug. In particular, for antisense oligonucleotide conjugates, once the prodrug is delivered to the site of action, such as a target cell, the conjugate portion is cleaved from the oligonucleotide.

[0589] application

[0590] The enhanced antisense oligonucleotides of the present invention can be used as research reagents, for example, for diagnosis, treatment and prevention.

[0591] In research, these antisense oligonucleotides can be used to specifically regulate the synthesis of the FUBP1 protein in cells (e.g., in vitro cell cultures) and laboratory animals, thereby aiding in the functional analysis of the target or the assessment of its availability as a therapeutic intervention target. Typically, target regulation is achieved by preventing protein formation through degradation or inhibition of the mRNA that produces the protein, or by degradation or inhibition of the gene or mRNA that produces the protein.

[0592] If the antisense oligonucleotides of the present invention are used in research or diagnosis, the target nucleic acid may be cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0593] The present invention also includes a method for regulating FUBP1 expression in target cells expressing FUBP1 in vivo or in vitro, the method comprising administering an effective amount of the antisense oligonucleotide, conjugate thereof, or pharmaceutical composition of the present invention to the cells.

[0594] In some embodiments, the target cells are mammalian cells, particularly human cells. Target cells can be in vitro cell cultures or in vivo cells that form part of mammalian tissue. In a preferred embodiment, the target cells are located in the liver. Target cells can be hepatocytes.

[0595] One aspect of the present invention relates to the antisense oligonucleotides of the present invention, their conjugates, or pharmaceutical compositions used as pharmaceuticals.

[0596] In one aspect of the invention, the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the invention are capable of reducing cccDNA levels in infected cells and thus inhibiting HBV infection. Specifically, the antisense oligonucleotides or their conjugates are capable of affecting one or more of the following parameters: i) reducing cccDNA and / or ii) reducing pgRNA and / or iii) reducing HBV DNA and / or iv) reducing HBV viral antigens in infected cells.

[0597] For example, an antisense oligonucleotide or its conjugate that inhibits HBV infection can i) reduce the level of cccDNA in infected cells by at least 40%, such as 50%, 60%, 70%, 80%, or 90%, compared to a control; or ii) reduce the level of pgRNA by at least 40%, such as 50%, 60%, 70%, 80%, or 90%, compared to a control. The control can be untreated cells or animals, or cells or animals treated with an appropriate control.

[0598] Inhibition of HBV infection can be measured in vitro using HBV-infected primary human hepatocytes or in vivo using a humanized hepatocyte PXB mouse model (available from Phoenix Bio, see also Kakuni et al., 2014 Int. J. Mol. Sci. 15: 58-74). Inhibition of HBsAg and / or HBeAg secretion can be measured by ELISA, for example using the CLIA ELISA kit (Autobio Diagnostic) according to the manufacturer's instructions. Reduction of intracellular cccDNA or HBV mRNA and pgRNA can be measured by qPCR, for example, as described in the "Materials and Methods" section. Other methods to assess whether the test compound inhibits HBV infection include measuring HBV DNA secretion by qPCR, for example, as described in WO 2015 / 173208, or by Northern blotting, in situ hybridization, or immunofluorescence.

[0599] Due to the reduction in FUBP1 levels, the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention can be used to inhibit the development of HBV infection or treat HBV infection. In particular, compared with compounds that only reduce HBsAg secretion, the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention more effectively inhibit the development of chronic HBV infection or treat chronic HBV infection by destabilizing and reducing cccDNA.

[0600] Therefore, one aspect of the present invention relates to the use of the antisense oligonucleotides, conjugates thereof, or pharmaceutical compositions of the present invention for reducing cccDNA and / or pgRNA in HBV-infected individuals.

[0601] Another aspect of the invention relates to the use of the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the invention in inhibiting the development of chronic HBV infection or in treating chronic HBV infection.

[0602] Another aspect of the invention relates to the use of the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the invention in reducing the infectivity of HBV-infected individuals. In a particular aspect of the invention, the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the invention inhibit the development of chronic HBV infection.

[0603] Subjects treated (or preventively receiving) the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention are preferably human, more preferably HBsAg-positive and / or HBeAg-positive human patients, and even more preferably HBsAg-positive and HBeAg-positive human patients.

[0604] Therefore, the present invention relates to a method for treating HBV infection, wherein the method comprises administering an effective amount of the antisense oligonucleotide, its conjugate, or pharmaceutical composition of the present invention. The present invention further relates to a method for preventing cirrhosis and hepatocellular carcinoma caused by chronic HBV infection.

[0605] The present invention also provides the use of the antisense oligonucleotides, conjugates thereof, or pharmaceutical compositions of the present invention in the preparation of medicaments, particularly medicaments for treating HBV infection or chronic HBV infection or reducing the infectivity of HBV-infected individuals. In a preferred embodiment, the medicament is prepared in a dosage form for subcutaneous administration.

[0606] The present invention also provides the use of the antisense oligonucleotides, conjugates thereof, and pharmaceutical compositions of the present invention in the preparation of pharmaceuticals, wherein the pharmaceuticals are dosage forms for intravenous administration.

[0607] combination therapy

[0608] In some embodiments, the enhanced antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention are intended for use in combination with another therapeutic agent. The therapeutic agent may, for example, be a standard of care for the aforementioned disease or condition.

[0609] For example, the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention can be used in combination with other active substances, such as oligonucleotide-based antiviral agents (e.g., sequence-specific oligonucleotide-based antiviral agents), which act via antisense (including other LNA oligomers), siRNA (such as ARC520), aptamers, morpholino derivatives, or any other antiviral toxins, in a nucleotide sequence-dependent mode of action.

[0610] As a further example, the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention can be used in combination with other active substances, such as immunostimulatory antiviral compounds, such as interferons (e.g., pegylated interferon α), TLR7 agonists (e.g., GS-9620), or therapeutic vaccines.

[0611] As a further example, the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention can be used in combination with other active substances, such as small molecules, that have antiviral activity. These other active substances may be, for example, nucleoside / nucleotide inhibitors (e.g., entecavir or tenofovir disoproxil fumarate), capsidation inhibitors, or entry inhibitors (e.g., Myrcludex B).

[0612] In some embodiments, additional therapeutic agents may be HBV agents, hepatitis C virus (HCV) agents, chemotherapeutic agents, antibiotics, analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), antifungal agents, antiparasitic agents, antiemetics, antidiarrheal agents, or immunosuppressants.

[0613] Specifically, in relevant embodiments, additional HBV agents may be interferon α-2b, interferon α-2a, and interferon αcon-1 (pegylated and non-pegylated), ribavirin; HBV RNA replication inhibitors; second antisense oligomers; HBV therapeutic vaccines; HBV prophylactic vaccines; lamivudine (3TC); entecavir (ETV); tenofovir disoproxil fumarate (TDF); telbivudine (LdT); adefovir; or HBV antibody therapy (monoclonal or polyclonal).

[0614] In other specific related embodiments, additional HCV agents may be interferon α-2b, interferon α-2a, and interferon αcon-1 (pegylated and non-pegylated); ribavirin; Pegasys; HCV RNA replication inhibitors (e.g., ViroPharma's VP50406 series); HCV antisense agents; HCV therapeutic vaccines; HCV protease inhibitors; HCV helicase inhibitors; or HCV monoclonal or polyclonal antibody therapies.

[0615] application

[0616] The enhanced antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention are formulated, measured, and administered in accordance with good medical practice. Factors to be considered in this context include the specific mammal being treated, the individual patient's clinical condition, the site of delivery of the agent, the method of administration, the timing of administration, the patient's age and sex, and other factors known to a practicing physician. In this document, an "effective amount" (also referred to as a "therapeutic effective dose") means the amount of compound that will elicit a biological or medical response in a subject being sought by a physician or other clinician. The "effective amount" of the oligonucleotides, conjugate compounds, or pharmaceutical compositions of the present invention will be determined by such considerations and is the minimum amount required to inhibit HBsAg and / or HBeAg. For example, this amount may be below an amount toxic to the receptor's cells or the mammal as a whole.

[0617] In some embodiments, the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention are administered at doses ranging from 0.1 mg / kg to 15 mg / kg, such as 0.2 mg / kg to 10 mg / kg, or such as 0.25 mg / kg to 5 mg / kg. Administration may be once weekly, once every two weeks, once every three weeks, or even once monthly.

[0618] The antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention can be applied topically (e.g., to the skin, inhaled, to the eyes, or to the ears), or enterically (e.g., orally or via the gastrointestinal tract), or parenterically (e.g., intravenously, subcutaneously, or intramuscularly).

[0619] In a preferred embodiment, the antisense oligonucleotides, their conjugates, or pharmaceutical compositions of the present invention are administered via a parenteral route, including intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion. In one embodiment, the active oligonucleotide or oligonucleotide conjugate is administered intravenously. For GalNAc-conjugated compounds, subcutaneous administration may be advantageous to delay ASGP receptor saturation.

[0620] Embodiments of the present invention

[0621] The following embodiments of the present invention may be used in conjunction with any other embodiments described herein. The definitions and explanations provided above (particularly in the sections “Summary of the Invention,” “Definitions,” and “Detailed Description”) are applicable mutatis mutandis to the following.

[0622] 1. An antisense oligonucleotide comprising a continuous nucleotide sequence that is at least 90% complementary to FUBP1 nucleic acid, such as being fully complementary, wherein the antisense oligonucleotide is capable of inhibiting the expression of FUBP1, such as human FUBP1, in cells.

[0623] 2. The antisense oligonucleotide of Example 1, wherein

[0624] a) The continuous nucleotide sequence is at least 90% complementary to the region within exon 14 of human FUBP1, such as complete complementarity (see Table 3), or

[0625] b) The continuous nucleotide sequence is at least 90% complementary to the region within exon 20 of human FUBP1, such as complete complementarity (see Table 3).

[0626] 3. The antisense oligonucleotides of Examples 1 and 2, wherein

[0627] a) A continuous nucleotide sequence that is completely complementary to the region of nucleotides 16184 to 16205 of the human FUBP1 precursor mRNA as shown in SEQ ID NO:1, such as the region selected from nucleotides 16184 to 16200, nucleotides 16186 to 16203, nucleotides 16188 to 16205, and nucleotides 16189 to 16205 of SEQ ID NO:1, or

[0628] b) The continuous nucleotide sequence is completely complementary to the region of nucleotides 30536 to 30553 of the human FUBP1 precursor mRNA as shown in SEQ ID NO:1.

[0629] 4. An antisense oligonucleotide according to any one of Examples 1 to 3, wherein a) the continuous nucleotide sequence is completely complementary to SEQ ID NO:10 and / or SEQ ID NO:11 or b) the continuous nucleotide sequence is completely complementary to SEQ ID NO:19.

[0630] 5. The antisense oligonucleotide according to any one of Examples 1 to 4, wherein the antisense oligonucleotide is 12 to 30 nucleotides in length, such as 12 to 22 nucleotides in length, such as 16 to 20 nucleotides in length.

[0631] 6. The antisense oligonucleotide according to any one of Examples 1 to 4, wherein the continuous nucleotide sequence is a continuous sequence of at least 12 nucleotides, such as 14, 15, 16, 17 or 18 nucleotides.

[0632] 7. The antisense oligonucleotide according to Example 6, wherein the continuous nucleotide sequence is a continuous sequence of 17 or 18 nucleotides.

[0633] 8. An antisense oligonucleotide according to any one of Examples 1 to 7, wherein the sequential nucleotide sequence is 100% identical to the sequence selected from the group consisting of SEQ ID NO: 6, 7, 8, 9 and 18; or at least 15 sequential nucleotides thereof.

[0634] 9. An antisense oligonucleotide according to any one of Examples 1 to 8, wherein one or more modified nucleosides are contained in a continuous nucleotide sequence.

[0635] 10. The antisense oligonucleotide according to Example 9, wherein one or more modified nucleosides in the continuous nucleotide sequence are 2' sugar-modified nucleosides.

[0636] 11. The antisense oligonucleotide according to Example 10, wherein one or more 2' sugar-modified nucleosides are independently selected from the group consisting of: 2′-O-alkyl-RNA, 2′-O-methyl-RNA, 2′-alkoxy-RNA, 2′-O-methoxyethyl-RNA, 2′-amino-DNA, 2′-fluoro-DNA, arabinonucleotide (ANA), 2′-fluoro-ANA and LNA nucleosides.

[0637] 12. The antisense oligonucleotide according to any one of Examples 9 to 11, wherein one or more modified nucleosides are LNA nucleosides, such as oxy-LNA having the following 2'-4' bridge -O-CH2-.

[0638] 13. The antisense oligonucleotide according to Example 12, wherein one or more modified nucleosides are β-D-oxy-LNA.

[0639] 14. An antisense oligonucleotide according to any one of Examples 1 to 13, wherein at least one nucleoside bond in the continuous nucleotide sequence is a phosphate thioside bond.

[0640] 15. The antisense oligonucleotide according to any one of Examples 1 to 14, wherein at least one nucleoside bond in the continuous nucleotide sequence is a phosphate thioside bond.

[0641] 16. The antisense oligonucleotide according to any one of Examples 1 to 15, wherein at least one nucleoside bond in the continuous nucleotide sequence is a phosphodiester nucleoside bond.

[0642] 17. The antisense oligonucleotide according to Example 16, wherein all nucleoside-to-nucleotide bonds in the continuous nucleotide sequence are phosphate-thioside-to-nucleoside bonds.

[0643] 18. The antisense oligonucleotide according to any one of Examples 1 to 17, wherein the antisense oligonucleotide is an antisense oligonucleotide capable of recruiting RNase H, such as RNase H1.

[0644] 19. The antisense oligonucleotide according to Example 18, wherein the antisense oligonucleotide or its continuous nucleotide sequence comprises or includes a gapmer of formula 5'-FG-F'-3'.

[0645] 20. The antisense oligonucleotide according to Example 19, wherein the length of region G is 6 to 16 DNA nucleosides, such as 7 to 12 DNA nucleosides, such as 7 to 11 DNA nucleosides.

[0646] 21. An antisense oligonucleotide according to any one of Examples 18 to 20, wherein regions F and F′ each contain at least one LNA nucleoside, for example, wherein regions F and F′ each contain at least one LNA nucleoside.

[0647] 22. An antisense oligonucleotide according to any one of Examples 18 to 21, wherein the length of region F is 1 to 8 DNA nucleosides, such as 4 to 6 DNA nucleosides.

[0648] 23. An antisense oligonucleotide according to any one of Examples 18 to 22, wherein the length of region F is 1 to 8 DNA nucleosides, such as 2 to 6 DNA nucleosides.

[0649] 24. An antisense oligonucleotide according to any one of Examples 18 to 23, wherein the antisense oligonucleotide or its continuous nucleotide sequence is derived from Formula F 4-6 -G 7-11 -F′ 2-6 It constitutes or includes it, and preferably, the gapmer includes at least one alternating flank.

[0650] 25. The antisense oligonucleotide according to any one of Examples 1 to 24, wherein the antisense oligonucleotide is selected from the group consisting of antisense oligonucleotides comprising:

[0651] CTTatGctttttatgGT(SEQ ID NO:6),

[0652] CTTaTgctttttatgGT (SEQ ID NO: 6),

[0653] CTtATgctttttatgGTT (SEQ ID NO:7),

[0654] CTtAtgctttttatgGTT (SEQ ID NO:7),

[0655] CTtAtgctttttatGgTT(SEQ ID NO:7),

[0656] CTtAtgctttttatGGTT(SEQ ID NO:7),

[0657] GcttTttatggtTtCAC(SEQ ID NO:8),

[0658] TATgcTttttatggtTTC(SEQ ID NO:9) and

[0659] AcCAAttttcatttCtAC(SEQ ID NO:18)

[0660] The uppercase letters represent β-D-oxyLNA nucleosides, the lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, and all internucleotide bonds are thiophosphate internucleotide bonds.

[0661] 26. A conjugate comprising: an antisense oligonucleotide according to any one of Examples 1 to 25, and at least one conjugate moiety covalently linked to the antisense oligonucleotide.

[0662] 27. The conjugate according to Example 27, wherein the conjugate portion comprises at least one desialylate glycoprotein receptor targeting moiety selected from the group consisting of: galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-butyryl-galactosamine, and N-isobutyryl-galactosamine.

[0663] 28. The conjugated compound according to Example 27, wherein the desialyl glycoprotein receptor targeting moiety is N-acetylgalactosamine (GalNAc).

[0664] 29. The conjugate compound according to Example 27 or 28, wherein the conjugate portion is monovalent, divalent, trivalent, or tetravalent relative to the desialyl glycoprotein receptor targeting portion.

[0665] 30. The conjugate compound according to Example 29, wherein the conjugate portion consists of two to four terminal GalNAc portions and spacers connecting each GalNAc portion to a branched molecule that can be conjugated with an antisense compound.

[0666] 31. The conjugate compound according to Example 30, wherein the spacer is a PEG spacer.

[0667] 32. The conjugate compound according to any one of Examples 26 to 31, wherein the conjugate moiety is a trivalent N-acetylgalactosamine (GalNAc) moiety.

[0668] 33. The conjugate compound according to any one of Examples 26 to 32, wherein the conjugate portion is selected from... Figure 9 A1、 Figure 9 A2, Figure 9 C1, Figure 9 C2, Figure 9 D1、 Figure 9 D2、 Figure 9 E1、 Figure 9 F1 Figure 9 G1 Figure 9 H1, Figure 9 I1、 Figure 9 J1, Figure 9 L1 and Figure 9One of the trivalent GalNAc components in L2.

[0669] 34. The conjugate compound according to Example 33, wherein the conjugate portion is Figure 9 D1 or Figure 9 The trivalent GalNAc moiety or mixture thereof in D2.

[0670] 35. The conjugate compound according to any one of Examples 26 to 34, wherein the conjugate compound comprises a linker located between the antisense oligonucleotide and the conjugate moiety.

[0671] 36. The conjugate compound according to Example 35, wherein the linker comprises or consists of two to five consecutive phosphodiester-linked nucleosides, such as two consecutive phosphodiester-linked nucleosides, such as phosphodiester-linked nucleosides ca.

[0672] 37. The conjugate according to any one of Examples 26 to 36, wherein the conjugate is selected from the group consisting of:

[0673] 5'-GN2-C6 o c o a o m C s T s T s a s t s G s c s t s t s t s t s t s a s t s g s G s T,

[0674] 5'-GN2-C6 o c o a o m C s T s T s a s T s g s c s t s t s t s t s t s a s t s g s Gs T、

[0675] 5'-GN2-C6 o c o a o m C s T s t s A s T s g s c s t s t s t s t s t s a s t s g s G s T s T、

[0676] 5'-GN2-C6 o c o a o m C s T s t s A s t s g s c s t s t s t s t s t s a s t s g s G s T s T、

[0677] 5'-GN2-C6 o c o a o m C s T s t s A s t s g s c s t s t s t s t s t s a s t s G s g s T s T、

[0678] 5'-GN2-C6 o c o a o m C s T s t s A s t s g s c s t s t s t s t s t s a s t s G s G s T s T,

[0679] 5'-GN2-C6 o c o a o G s c s t s t s T s t s t s a s t s g s g s t s T s t s m C s A s m C,

[0680] 5'-GN2-C6 o c o a o T s A s T s g s c s T s t s t s t s t s a s t s g s g s t s T s T s m C and

[0681] 5'-GN2-C6 o c o a o A S c S m C S A S A S t S t S t S t S c S a S t S t S t S m C S tA S m C

[0682] Preferably, the uppercase letters represent β-D-oxyLNA nucleosides, the lowercase letters represent DNA nucleosides, and each LNA cytosine is 5-methylcytosine. m c represents 5-methylcytosine DNA, where the subscript 's' represents phosphate thioester nucleoside bonding, the subscript 'o' represents phosphodiester nucleoside bonding, and GN2-C6 represents... Figure 9 The trivalent N-acetylgalactosamine (GalNAc) shown, such as Figure 9 D1 or Figure 9 The trivalent N-acetylgalactosamine (GalNAc) shown in D2, or a mixture thereof, is preferably bound via a phosphodiester bond at the 5' end of the oligonucleotide.

[0683] 38. Conjugate shown in Figure 1 middle.

[0684] 39. Conjugates are shown in Figure 2 middle.

[0685] 40. Conjugate shown Figure 3 middle.

[0686] 41. Conjugate shown Figure 4 middle.

[0687] 42. Conjugates are shown in Figure 5 middle.

[0688] 43. Conjugates are shown in Figure 6 middle.

[0689] 44. Conjugates are shown in Figure 7 middle.

[0690] 45. Conjugates are shown in Figure 8 middle.

[0691] 46. ​​Conjugates are shown in Figure 8_1 middle.

[0692] 47. A pharmaceutical salt of an oligonucleotide of any one of Examples 1 to 25, or a conjugate according to any one of Examples 26 to 46.

[0693] 48. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of Examples 1 to 25, a conjugate according to any one of Examples 26 to 46, or a pharmaceutical salt according to Example 48, and a pharmaceutical diluent, solvent, carrier, salt, and / or adjuvant.

[0694] 49. An in vivo or in vitro method for regulating FUBP1 expression in target cells expressing FUBP1, the method comprising administering to the cells an effective amount of an antisense oligonucleotide according to any one of Examples 1 to 25, a conjugate according to any one of Examples 26 to 46, a pharmaceutical salt according to Example 48, or a pharmaceutical composition according to Example 48.

[0695] 50. A method of treating or preventing a disease, comprising administering to a subject suffering from or susceptible to said disease a therapeutically or preventively effective amount of an antisense oligonucleotide according to any one of Examples 1 to 25, a conjugate according to any one of Examples 26 to 46, a pharmaceutical salt according to Example 47, or a pharmaceutical composition according to Example 48, wherein the disease is hepatitis B virus (HBV) infection and / or cancer.

[0696] 51. The antisense oligonucleotide according to any one of Examples 1 to 25, the conjugate according to any one of Examples 26 to 46, the pharmaceutical salt according to Example 47, or the pharmaceutical composition according to Example 48, for use in medicine.

[0697] 52. The antisense oligonucleotide according to any one of Examples 1 to 25, the conjugate according to any one of Examples 26 to 46, the pharmaceutical salt according to Example 47, or the pharmaceutical composition according to Example 48, for the treatment or prevention of hepatitis B virus (HBV) infection and / or cancer.

[0698] 53. Use of the antisense oligonucleotide according to any one of Examples 1 to 25, the conjugate according to any one of Examples 26 to 46, the pharmaceutical salt according to Example 47, or the pharmaceutical composition according to Example 48 for the preparation of a medicament for the treatment or prevention of hepatitis B virus (HBV) infection and / or cancer.

[0699] 54. The method according to Example 50, the antisense oligonucleotide, conjugate, pharmaceutical composition or pharmaceutical salt used according to Example 52, or the use according to Example 53, wherein the disease is hepatitis B virus (HBV) infection, such as chronic HBV infection.

[0700] 55. The method according to Example 50, the antisense oligonucleotide, conjugate, pharmaceutical composition or pharmaceutical salt used according to Example 52, or the use according to Example 53, wherein the disease is cancer, such as hepatocellular carcinoma.

[0701] 56. The antisense oligonucleotide according to any one of Examples 1 to 25, the conjugate according to any one of Examples 26 to 33 and 45, the pharmaceutical salt according to Example 47 or the pharmaceutical composition according to Example 48, the use according to claim 53, or the method according to Examples 54 and 54, wherein the antisense oligonucleotide is AcCAAttttcatttCtAC (SEQ ID NO:18).

[0702] 57. The antisense oligonucleotide according to any one of Examples 1 to 25, the conjugate according to any one of Examples 26 to 33 and 42, the pharmaceutical salt according to Example 47 or the pharmaceutical composition according to Example 48, the use according to claim 53, or the method according to Examples 54 and 54, wherein the antisense oligonucleotide is CTtAtgctttttatGgTT (SEQ ID NO:7).

[0703] Example

[0704] Foreword

[0705] Overexpression and mutation of FUBP1 have been known to be associated with cancer for many years. In particular, significant overexpression of FUBP1 in human hepatocellular carcinoma (HCC) supports tumor growth and is associated with poor patient prognosis.

[0706] HBV cccDNA in infected hepatocytes is responsible for persistent chronic infection and reactivation. It serves as a template for all viral subgenomic transcripts and pregenomic RNA (pgRNA) to ensure that newly synthesized viral progeny and the cccDNA pool are replenished through nuclear capsid recycling.

[0707] In WO 2019 / 193165, FUBP1 was shown to be associated with cccDNA stability. This understanding provides an opportunity for the destabilization of cccDNA in HBV-infected subjects, which in turn creates an opportunity for the complete cure of patients with chronic HBV infection.

[0708] In this study, over 2000 antisense oligonucleotides targeting human FUBP1 were screened. During this screening, compounds particularly effective at targeting human FUBP1 were identified. Specifically, nine alternating flanking gapmer LNA oligonucleotides were identified that target a region within exon 14 of human FUBP1 and conferred significant downregulation of human FUBP1 in vitro. Additionally, one alternating flanking gapmer LNA oligonucleotide was identified that targets a region within exon 20 of human FUBP1 and also conferred significant downregulation of human FUBP1. An overview of the nine identified compounds is provided in Table 6 above.

[0709] The target sequences of the identified compounds overlapped with those of CMP ID NO 53_1 and 54_1 disclosed in WO 2019 / 193165. These two compounds inhibited FUBP1 in HeLa cells by approximately 70% at 5 μM. However, the nine identified compounds were clearly more effective, as they inhibited FUBP1 in HeLa cells by approximately 25% to 35% at 3.3 μM, or by approximately 27% at 5 μM (CMP ID NO: 18_1). Furthermore, they were more effective than CMP ID NO 50_1 in targeting FUBP1 in HeLa cells, which was the best compound in WO 2019 / 193165 (see Example 1).

[0710] Table 7 below provides an overview of prior art compounds 35_1, 50_1, 53_1, 54_1, 78_1, and 79_1 under WO 2019 / 193165. These compounds are gapmers with uniform flanking. CMP ID NO: 50_1 is the best compound in PHH cells, and CMP ID NO: 35_1 is the best compound in HeLa cells. CMP ID NOs 53_1 and 54_1 are the closest compounds to CMP ID NOs: 6_1, 6_2, 7_1, 7_2, 7_3, 7_4; 8_1, and 9_1. CMP ID NOs 78_1 and 79_1 are the closest compounds to CMP ID NO: 18_1.

[0711] Table 7: List of control oligonucleotide compounds (as disclosed in WO 2019 / 193165)

[0712]

[0713] For compounds: uppercase letters represent LNA nucleosides (β-D-oxy-LNA nucleosides were used), all LNA cytosines are 5-methylcytosine, and lowercase letters represent DNA nucleosides. All inter-nucleoside bonds are phosphate thioside bonds.

[0714] Example 1: Testing the in vitro efficacy of antisense oligonucleotides targeting human FUBP1 mRNA in HeLa cells.

[0715] The ability of antisense oligonucleotides targeting FUBP1 to reduce FUBP1 mRNA expression in human HeLa cells obtained from ECACC (catalog number 93021013) was tested.

[0716] HeLa cells were grown in EMEM (Sigma, catalog number M2279) supplemented with 10% fetal bovine serum (Sigma, catalog number F7524), 2 mM glutamine (Sigma, catalog number G7513), 0.1 mM NEAA (Sigma, catalog number M7145), and 0.025 mg / ml gentamicin (Sigma, catalog number G1397). Cells were digested with trypsin every 5 days, washed with phosphate-buffered saline (PBS) (Sigma catalog number 14190-094), and then incubated with 0.25% trypsin-EDTA solution (Sigma, catalog number T3924) at 37°C for 2–3 minutes. Cells were homogenized before seeding.

[0717] For experimental use, 2500 cells per well were seeded into 96-well plates (Nunc catalog number 167008) with 190 μL of growth medium. Approximately 24 hours after seeding to the final customized concentration, ASO dissolved in PBS was added. The cells were incubated for 3 days without any changes to the culture medium.

[0718] After incubation, cells were harvested by removing the culture medium and then adding 125 μL of RLT lysis buffer (Qiagen 79216) and 125 μL of 70% ethanol. RNA was purified according to the manufacturer's instructions (Qiagen RNeasy 96 kit) and eluted in a final volume of 200 μL of DNase-free / RNase-free water (Gibco).

[0719] RNA was heat-shocked at 90°C for 40 seconds to unwind the RNA:LNA double strand, transferred directly to ice, and centrifuged before use. For a one-step qPCR reaction, the qPCR-mixture (from QauntaBio's qScript) was... TM XLE 1-step Low ROX (catalog number 95134-500) was mixed with two IDT probes (final concentration 1X) to generate the master mixture. Taqman probes were purchased from IDT:FUBP1:Hs.PT.58.26883775 (primer-probe ratio 2, FAM) or ThermoFisher Scientific:GUSB:4326320E. The master mixture (6 μL) and RNA (4 μL, 1 ng / μL to 2 ng / μL) were then plated in a qPCR plate. Mix in optical 384 wells (4309849). After sealing, rapidly rotate the plate at 1000g for 1 minute at room temperature, then transfer to a Viia™ 7 system (Applied Biosystems, Thermo) and use the following PCR conditions: 50°C for 15 minutes; 95°C for 3 minutes; 40 cycles: 95°C for 5 seconds, then decrease the temperature by 1.6°C / sec, followed by 60°C for 45 seconds. QuantStudio was used. TM Data analysis using Real-time PCR software.

[0720] qPCR data were captured and raw data quality control was performed in Quantstudio7 software.

[0721] The data is then imported into an E-Workbook, where a BioBook template is used to capture and analyze the data. Analyze the data using the following steps:

[0722] 1. Calculate the quantity using the △△Ct method (quantity = 2^(-Ct) * 1000000000)

[0723] 2. Normalize the number to the calculated number of housekeeping gene assays run in the same well. Relative target number = Number - Target / Number - Housekeeping Genes

[0724] 3. RNA knockout in each well was calculated by dividing by the average of all PBS-treated wells on the same plate. Normalized target count = (Relative target count / [Average target count]_pbs_wells) * 100

[0725] 4. The final data is displayed as the percentage of untreated (PBS) wells.

[0726] 5. For the concentration-response experiment, a curve was fitted based on the RNA knockout value of each compound (steps 3-4) (8 or 10 concentrations, depending on the dilution model). The curves were fitted using a 4-parameter sigmoid dose-response model from Biobook.

[0727] Table 8 shows the relative FUBP1 mRNA expression levels, expressed as a percentage relative to the control; that is, the lower the value, the greater the inhibitory effect. Furthermore, the results... Figure 11 As shown in the image.

[0728] Table 8: In vitro efficacy of anti-FUBP1 compounds in HeLa cells. FUBP1 mRNA levels were normalized to GUSB and are shown as a percentage relative to the control.

[0729]

[0730] *Control compound, nd: not detected.

[0731] **CMP ID NO:17_1 is as follows: ATgctTtttatggtttCA (SEQ ID NO:17), and CMP ID NO:16_1 is as follows: TTAtgctttttatggTTT (SEQ ID NO:16), where uppercase letters represent β-D-oxyLNA nucleosides, lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, and all inter-nucleoside bonds are phosphate thioester nucleoside bonds. CMP ID NO:17 targets nt 16185 to 16202 of SEQ ID NO:1. CMP ID NO:16 targets nt 16187 to 16204 of SEQ ID NO:1.

[0732] ***Data from WO 2019 / 193165

[0733] The experiments with the control compound were conducted separately.

[0734] Example 2: Testing the in vitro efficacy of antisense oligonucleotides targeting human FUBP1 mRNA in primary human hepatocytes (PXB-PHH).

[0735] Fresh primary human hepatocytes (PXB-PHH) harvested from humanized mice (uPA / SCID mice) (referred to as PHH in this paper) were obtained from Phoenix Bio Co., Ltd. (Japan) in 96-well format and cultured in modified hepatocyte clonal growth medium (dHCGM). dHCGM is a DMEM medium containing 100 U / ml penicillin, 100 μg / ml streptomycin, 20 mM Hpeps, 44 mM NaHCO3, 15 μg / ml L-proline, 0.25 μg / ml insulin, 50 nM dexamethasone, 5 ng / ml EGF, 0.1 mM Asc-2P, 2% DMSO, and 10% FBS (Ishida et al., 2015).

[0736] The cells were cultured at 37°C in a humid atmosphere with 5% CO2. The culture medium was changed twice a week until harvest.

[0737] Uninfected cells were treated once at 5 μM and harvested after 7 days. In all treatments, oligonucleotide compounds were administered to cells in dHCGM medium at a final volume of 120 μl / well. Experiments for RNA measurement were performed in a biologically replicate manner.

[0738] FUBP1 RNA was then subjected to real-time PCR. Total mRNA was extracted from cells using the MagNA Pure robot and MagNA Pure 96Cellular RNA Large Capacity Kit (Roche, #05467535001) according to the manufacturer's protocol. mRNA expression levels were quantified by qPCR using the QuantStudio12K Flex (Applied Biosystems), TaqMan RNA-to-CT1-Step Kit (Applied Biosystems, #4392938), and a human GusB endogenous control (Applied Biosystems, #Hs00939627_m1) in two technical replicates. mRNA expression was analyzed using the comparative cycle threshold 2-ΔΔCt method, which was normalized for the reference gene GusB and untreated cells. The TaqMan primers used for GusB RNA and FUBP1 RNA quantification are shown in the table below.

[0739] Table 9: Primers for GusB RNA and FUBP1 RNA quantification

[0740] parameter source FUBP1 ThermoFisher – Analytics ID: Hs00900762_m1 GusB <![CDATA[ThermoFisher – Analytical ID: Hs00939627_m1 >

[0741] Table 10 shows the relative FUBP1 mRNA expression levels of eight compounds (CMP ID Nos: 6_1, 6_2, 7_1, 7_2, 7_3, 7_4; 8_1 and 9_1; CMP ID Nos: 78_1 and 79_1) in PXB-PHH cells, expressed as a percentage relative to the control; lower values ​​indicate greater inhibitory effect. The FUBP1 mRNA expression level of CMP ID No: 18_1 in PXB-PHH cells was analyzed in Example 3.

[0742] Table 10: In vitro efficacy of anti-FUBP1 compounds in PXB-PHH cells. FUBP1 mRNA levels were normalized to GUSB and are shown as a percentage relative to the control.

[0743]

[0744] Conclusions drawn from Examples 1 and 2

[0745] The data in Examples 1 and 2 show that, as shown in Table 6, targeting FUBP1 with LNA ASO results in an effective reduction of FUBP1.

[0746] Example 3: Further analysis of CMP IDs NO:7_3 and 18_1

[0747] The following describes additional experiments conducted using two of the nine identified compounds: CMP IDsNO: 7_3 and 18_1. In these experiments, these two compounds were compared with two existing compounds that yielded the best results in WO 2019 / 193165.

[0748] Materials and methods

[0749] Primary human hepatocytes (PXB-PHH)

[0750] According to Example 2, fresh primary human hepatocytes (PXB-PHH) were cultured, except that a 24-well format was used.

[0751] ASO sequences and compounds

[0752] Table 11 provides an overview of the compounds tested in Example 3:

[0753] Table 11: Human FUBP1 sequences targeted by ASO

[0754]

[0755] *See Table 6 for compounds according to the present invention.

[0756] **See Table 7: Comparative Compounds Disclosed in WO 2019 / 193165**

[0757] HBV infection and oligonucleotide treatment

[0758] Upon arrival, PHH cells were infected with purified inoculum (genotype C) derived from chronic patients at MOI 110 by incubating PHH medium with HBV in 4% (v / v) PEG for 16 hours. Cells were then washed three times with PBS and cultured in fresh PHH medium under a humid atmosphere containing 5% CO2. Four days post-infection, cells were treated with FUBP1 LNA at a final concentration of 10 μM (see Table 11), repeated twice, or with PBS as a drug-free control (NDC). On the day of treatment, the old medium was removed from the cells and replaced with 400 μL / well of fresh PHH medium. For each well, 100 μL of 50 μM of each FUBP1 LNA or PBS as an NDC was added to 400 μL of PHH medium. The same treatment was repeated three times on days 4, 11, and 18 post-infection. The original cell medium was replaced every three days with fresh cell culture medium on days 7, 14, and 21 post-infection.

[0759] Real-time PCR of intracellular HBV pgRNA and FUBP1 mRNA

[0760] After cell viability was determined, cells were washed once with PBS. Total RNA was extracted from cells using the MagNA Purerobot and MagNA Pure 96Cellular RNA Large Capacity Kit (Roche, #05467535001) according to the manufacturer's protocol. FUBP1 mRNA and viral pgRNA expression levels were quantified by qPCR using two technical replicates with the QuantStudio 12KFlex (Applied Biosystems), the TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems, #4392938), and a human GusB endogenous control (Applied Biosystems, #Hs00939627_m1). The relative expression of FUBP1 mRNA and viral pgRNA was analyzed using the comparative cycle threshold 2-ΔΔCt method, normalized for the reference gene GusB and untransfected cells. Table 12 lists the TaqMan primers used for quantification of GusB RNA, FUBP1 RNA, and HBV pgRNA.

[0761] Table 12: TaqMan primers for quantification of GusB gene, FUBP1 RNA, and HBV pgRNA

[0762] parameter source FUBP1 ThermoFisher – Analytics ID: Hs00900762_m1 HBV pgRNA Customized: AILJKX5 GusB ThermoFisher – Analytics ID: Hs00939627_m1

[0763] result

[0764] The relative FUBP1 mRNA expression levels of the tested compounds are shown in Table 13 and Figure 12 As shown in the table and Figure 13 It can be concluded that, compared with NDC, the two compounds of the present invention (CMP ID NO: 7_3 and 18_1) reduced the expression of target mRNA by approximately 80%. Their effect on FUBP1 mRNA levels is much stronger than that of prior art compounds (CMP ID NO: 50_1 and 35_1).

[0765] Table 13: In vitro efficacy of anti-FUBP1 compounds in PXB-PHH cells. FUBP1 mRNA levels were normalized to GUSB and are shown as a percentage relative to the control.

[0766]

[0767] Table 14 shows the pgRNA levels in HBV-infected PHH cells treated with different concentrations of antisense compounds. The table shows that downregulation is related to the concentration of the antisense compound. At a concentration of 10 μM, the lowest pgRNA level was observed for CMP ID NO: 7_3. Furthermore, the highest pgRNA level was observed for the prior art compound with CMP ID NO: 35_1. CMP ID NO: 18_1 downregulated HBV pgRNA in a similar manner to the prior art compound CMP ID NO: 50_1.

[0768] Table 14: In vitro efficacy of anti-FUBP1 compounds in HBV-infected PXB-PHH cells: pgRNA. pgRNA levels were normalized to untreated cells (NDCs) and are shown as a percentage relative to the control.

[0769]

[0770] Cells were also tested weekly at a concentration of 2 μM for three weeks. At 2 μM, CMP ID NO:7_3 showed the optimal FUBP1 mRNA KD, with a 50% reduction in mRNA expression. Therefore, the effect is concentration-dependent (as an 80% reduction was observed at 2 μM). Furthermore, CMP ID NO:18_1 showed a similar effect on target mRNA expression levels compared to prior art oligonucleotides (2 μM).

[0771] Example 4: FUBP1 ASO in vivo PK / PD

[0772] In a single-dose mouse study, C57BL / 6 mice were used to evaluate the in vivo liver PK / PD association of oligonucleotides with CMP ID Nos: 7_3 and 18_1 conjugated to the GalNAc moiety via phosphodiester linkage (for the structure of the conjugate, see, for example...). Figure 5 and Figure 8_1 Mice were administered 3 mg / kg subcutaneously, with administration terminated at different time points. Fubp1 mRNA knockout, compound exposure, and PKPD were measured as described below.

[0773] Materials and methods

[0774] Tissue sample processing

[0775] Material supplier Catalog Number Eppendorf 2ml tube Eppendorf 0030 123.344 5mm tungsten carbide beads Qiagen 69989 MagNaPure LC RNA Isolation Tissue Buffer Roche Applied Science 03604721001

[0776] Liver samples were frozen in 2 ml round-bottom Eppendorf tubes and homogenized 2 x 1.5 min in MagNa pure buffer (Roche) on a TissueLyser II (Qiagen) after adding 5 mm homogenization beads. Once fully homogenized, the homogenate was incubated at room temperature (RT) for 30 min to complete tissue lysis. Due to the buffer thiocyanate and mercaptoethanol content, all steps of the homogenization process were performed in a flow chamber. After lysis, the homogenate was centrifuged at 17,000 g for 3 min.

[0777] Dilute the homogenate to approximately 20 mg tissue per 400 μL to avoid overloading the MagNA pure instrument. Use 350 μL of homogenate for RNA extraction on the MagNA pure 96 instrument (for subsequent qPCR analysis). Use the remaining aliquots of the homogenate for hELISA analysis.

[0778] Hybrid ELISA

[0779] The following oligonucleotides and (full LNA phosphodiester) ELISA probes were used for hELISA analysis. All design, synthesis and identification were performed at Roche Innovation Center Copenhagen A / S.

[0780]

[0781]

[0782] Before hELISA analysis, homogenize at room temperature and vortex before use. Dilute samples at least 10-fold in 5×SSCT buffer.

[0783] Run appropriate standards matched to the sample matrix and dilution factor on each plate, and prepare them in parallel with the samples using relevant oligomers (from formulations that have undergone quality and property checks). Incorporate the standard for each compound into the sample cell from the unloaded sample. Ensure the incorporation concentration is within approximately 10 times the oligonucleotide content of the sample.

[0784] Add the sample and standard to the desired dilution plate setup and perform a dilution series. Add 300 μL of sample / standard with capture detection solution to the first well and 150 μL of capture detection solution to the remaining wells.

[0785] Perform a two-fold dilution series of standards and samples by sequentially transferring 150 μL of liquid. Reserve 2–4 wells as blanks (for capturing the detection solution only). For best results, a two-fold sample dilution series of at least 6 wells is recommended.

[0786] Incubate the sample in the dilution plate at room temperature for 30 minutes. Transfer 100 μL of liquid from the dilution plate to the streptavidin plate. Incubate the plate at room temperature for 1 hour while gently stirring (plate shaker). Aspirate and wash the wells three times with 300 μL of 2x SSCT buffer.

[0787] Add 100 μL of anti-DIG-AP diluted 1:4000 in PBST (prepared on the same day) to each well and incubate with gentle stirring at room temperature for 1 hour. Aspirate and wash the wells three times with 300 μL of 2x SSCT buffer.

[0788] Add 100 μL of substrate (AP) solution (freshly prepared) to each well. After incubating with gentle stirring for 30 minutes, measure the intensity of the color development at 615 nm using a spectrophotometer.

[0789] The raw data was exported from the reader (Gen5 2.0 software) to Excel format and further analyzed in Excel. Standard curves were generated using GraphPad Prism 8 software and a logistic 4PL regression model.

[0790] The data point report is the average of technical repetitions.

[0791] RNA purification

[0792] All samples were purified using MagNA Pure 96 Instrument (Roche) according to the manufacturer's protocol.

[0793]

[0794] 350 μL of tissue homogenate was transferred to a MagNaPure 96 processing kit. Remaining lysates were stored for subsequent oligonucleotide exposure analysis. RNA was purified using the MagNa Pure 96 with the Cellular RNA Large Volume Kit, following protocol “RNA Tissue FF Standard LV 3.1”. RNA was eluted in 50 μL of elution buffer (from the kit, 05467535001).

[0795] RNA concentrations and A260 / 280 ratios of approximately 2.0 were determined for all samples using an Eon microplate spectrophotometer (BioTek Instruments). Based on these concentrations, samples were normalized to 25 ng / μL by dilution in DNase- and RNase-free water and further diluted to a working concentration of 2.5 ng / μL.

[0796] The samples were then used as input for a one-step qPCR analysis. Measurement details are shown below.

[0797] qPCR analysis

[0798] Use the following materials to run qPCR in one-step qPCR format:

[0799]

[0800] RNA preparation for qPCR analysis

[0801] Throughout all steps of this protocol, the reaction was kept cooled to avoid unwanted RT enzyme activity. The diluted RNA was then heat-shocked at 90°C for 40 seconds to unwind the RNA:ASO double strand and placed on ice. Prior to analysis, the RNA sample was centrifuged to the bottom of the well.

[0802] A standard curve was run on each plate and used for quantification and amplification efficiency measurements. 4 μL of a 10 ng / μL PBS sample was used as input to a 10 μL reaction. A 2-fold dilution series was prepared in RNase-free water to form a 7-point standard curve.

[0803] Two separate mouse Fubp1 assays and four control assays were performed in a double reaction, with two technical replicates per animal.

[0804] For qPCR, follow these steps:

[0805] For each qPCR well, a stock master mixture was prepared, comprising 5 μL of XLT one-step master mixture, 0.5 μL of probe mixture 1 (20x), and 0.5 μL of probe mixture 2 (20x). 6 μL of the stock master mixture was added to each well of a 384-well plate (MicroAmp Optical 384-well plate - Applied Biosystems 4309849).

[0806] From the RNA dilution plate, add 4 μL of diluted RNA (2.5 ng / μL) to each well of the master mixture. Seal the plate and vortex. Centrifuge the plate at high speed for 3 minutes. Keep the qPCR reaction cooled until transferred to a qPCR instrument (LifeTechnology Via7; software: QuantStudio v.1.3) and set to run the following program: incubate at 50 °C for 15 minutes, then at 95 °C for 3 minutes, with a temperature change rate of 1.9 °C / s. This was followed by 40 cycles of 95 °C for 5 seconds and 60 °C for 45 seconds, with a temperature change rate of 1.6 °C / s.

[0807] All samples were analyzed in the same run, thus minimizing technical variability.

[0808] qPCR data processing

[0809] qPCR data were reviewed in Quantstudio software (Applied Biosystems). Possible abnormal wells were identified and removed based on irregularities in the amplification curves. After reviewing each plate, an export file was generated containing the number of samples calculated from the ct values ​​based on the standard curve of each qPCR assay, and analyzed using Excel.

[0810] Overall, the standard curves were of high quality, with efficiency between the recommended 95% and 105%, indicating high analytical performance.

[0811] Four distinct HK genes (Gusb, Rplp0, Rps29, and Tbp) were examined, and their geometric mean was used for normalization. HK gene stability was assessed prior to inclusion using the method published by Vandesompele et al. (Vandesompele et al., 2002). With the four HK genes, paired HK gene variants were below the recommended threshold of 0.15 for all tissues.

[0812] The percentage of remaining Fubp1 is calculated as follows: the number from each Fubp1 qPCR assay is normalized to the geometric mean of the HK assays, and then further divided by the mean of the untreated group to give the percentage of remaining mRNA. The average of the two percentage results of remaining Fubp1 mRNA is used as the final reading.

[0813] PKPD plotting and calculation

[0814] Liver tissue exposure values ​​were calculated as nmol of compound per gram of tissue (nmol / g). These were further transformed using a log10 conversion, and plotted against the percentage of remaining Fubp1 mRNA. Figure 13 GraphPad Prism 8 was used to fit the nonlinear regression curve (4PL regression model, constraint at top = 100). The best-fit estimated PKPD IC50 was calculated by the software (regression IC50: conjugate of CMP ID 18_1: 0.092 nmol / g; conjugate of CMP ID 7_3: 0.068 nmol / g).

[0815] Results: Both conjugates tested exhibited good PK properties. The conjugate of CMP ID 7_3 was slightly superior to the conjugate of CMP ID 18_1 in terms of early action on the target KD. sequence list <110> Hofmeister Roche Ltd. <120> Enhanced oligonucleotides for regulating FUBP1 expression <130> P36078 <150> EP 20182437.2 <151> 2020-06-26 <160> twenty two <170> PatentIn version 3.5 <210> 1 <211> 35056 <212> DNA <213> Homo sapiens <400> 1 gcgcaagaat gtaatagagc ttcgacggcc gccattttct ttctttctta gctgttagct 60 gagaggaagt ctctgaacag gcggcagcgg ctctttatagt gcaaccatgg cagactattc 120 aacgtgcct cccccctctt ctggctcagc tggtggcggt ggtggcggcg gtggtggtgg 180 aggagttaac gacgctttca aagatgcact gcagagagcc cggcaggtaa gtgtggaccg 240 cgcggcggaa tcccgaaagc tcacggtaat tggccgctga ctgagtaggc cgctaccctt 300 aagcgcatga ggaagaggaa agaggtgttc ttccgggctg aaatgtgaag agacacgttt 360 ccccatgttg gtaataacga ttagagacca gaacccagtt ttgtgttctt ggtgcctaat 420 ccacttagaa ccccgacgcg tgctacgcaa agaaggcctg aagtctttct cccgcttctg 480 cggcactcgc gtgtctccag tgagctagtt tagataaaga tcctcttcca ggggataaag 540 cgcagttagt ttcacacaat ttaatggaag gttctggtaa tgagtttggg aaagaactag 600 ggtctgtcct ggagccatag caagggaagg gatttatcat taaagtagcc tttacagctc 660 atttccgtc tctctcgcaa ttaaaaccgc tttcagtacc attcaccgtc acacctctac 720 aaggaaggga cttgaaagca gccttttct gggcgggatt tacgtgtcag tctgttccac 780 cagtccgccc cccttatttc tcaaaatggc ctcaggccca ttataccaga ggtttcaatt 840 tgaatctgcc tctcagttca gagtcgtaaa ctgaccagac ctctttgtat tacgtagtgc 900 gtgcatttgc cctgaaggca ccacttccc agacgaaagc tgttaaaata gtgcgtgtat 960 tccaggaaaaaaaagatac cttaatttga actttacatt cttagatagt cccctatatat 1020 attack tcaaatgta tggtgttggt atagatttgc atgtaagcaa aagaatccta 1080 ttctctgtga cacatgcat attgtactag gtgctgggca tttttacta gtttaagct 1140 aatgataatt agaaaccagt gttgtgctgt gtttcgttg cattaggagt tcacttagtt 1200 aactttttac cgggacagtt gaaggaacat tgagtcaaa ttagaattca taaaatccgt 1260 tgtaacacat ctaatgtgaa cgcattataa acatgtacct gtacttttt ataccagaa 1320 attackaggag tagtcaacaa aaggtcatca ttatattag ttctgggtt ttttccacgt 1380 aatttagaa attctgaca tgtttagca caagcatata actatgacaa acacctcttag 1440 cgtgttttat tagatt tgtaaaactt agggaacta ttttac tggaaccaac 1500 tattttatt taccagacc agcacattgg atttattggt atcgcacact gtaggtagat 1560 actggagttt tgttttgttt tgtttgagac ggagtctccc tctgtcgccc agactggagt 1620 gcagtggtgc gatctcgtcc cactgcaacc tctgcctccc ggcttcaagc aattctcctg 1680 cctcagcctc ccgagtagct gggattacag gcgcccgcca ccatgcccgg ctaatttttt 1740 gtgtttttt tgtttgtttg tttgttttca gtagagacag ggtttcacca tgctggccag 1800 gctggtctcg aactcctgac ctcgtgatcc gcccgtcttg gcctcccaaa gctgggatta 1860 caggcgtgag ccgctgcgcc cggccttgtt ttttttcgtt tgtttgtttt aatgcatgaa 1920 ttgtttccta ctaagaagct atgatatagt tccttgacca aatgcagatg aacaggatta 1980 tctgattaat actttaacga gagcagacaa aatatggata tttaattcat ccacttgctt 2040 tataagtgtt tatagagttt gttaggaggg gatgcccaat ttcctaagta aaggtgatat 2100 atgagcaaga cattataaaa tgaaagagag tttggccagt gaagaaaga agcagcatgc 2160 gcagaggtgt ggggacttga gggaatggca aggacccaga agtgaagtcg ctaagaatg 2220 ggggtgggag gacgtgaaac gaggcaagga aaggtacaag cggatccaga tactggaaga 2280 ccttgtgttg caatgcttta attaaaaatt ggatttcagg ccgggtacgg tggctcacgc 2340 ctgtagtccc agcactttgg gaggctgagg caggcagatc acttgaagtc ggagtttaag 2400 accagcctgg ccaacatggt gaaaccccat cttacaaaa aatacaaaaa gtagccgggc 2460 ttggtggcct gtgcctgtag tcctagctac ttgggaggct gaggcacaag aaccgcttca 2520 acctgggaag tggaggttgc agtgagtgga gatcacgctg ctgcactcca gcctggtcat 2580 agagcgagac cctgtctcaa aaaaaaaaaa attggatttt attattaggc taagccataa 2640 tttctttata cttcttaaaa tatagaaatt gttggcaaat tgtgaaattt attgttgtat 2700 aatgctaata gatcagttgt cctgatgtct ttgctgtaat gatttcttta taaaaatgat 2760 cttaaatctg agctccctaa ctttagtttt tgcctggaat tacccattac atttgatgat 2820 atctctaaat gtcagttgta gctgttactc tgatgatata agtggaatat acagaagcgt 2880 acttagacaa aagttaggtt aatatctgaa ctacttcctc cttgtgtatt taagagaata 2940 ttgacttaag tttctagaat cctcaactaa tcctaagttt atttctttg tctagaatac 3000 tatgctgttt ttgtttttgg aaggaagaga tataggcata gtttcctgct ctcaaggagc 3060 ttcaaaggct gtaccagtgg ggatgccatt ggtattttta gctggatagt tgttattcag 3120 aaaagcagga taattaatta tgattcctgg tccgtacctg gtaatgccag taatgttaac 3180 tctagctggt tgttgacatc tggtcattta gttgccaatc ttcttttttt ttttctggct 3240 tttatgtgaa atttttagat ttttataata tcctgagcta aattcaacac agggacacca 3300 gattgctgct ttagttcagg gtttccagcc tgtgcactta agaaatttat ttttgtgtat 3360 atcaagctgt aactccagag attgggattg tttgattggg tctttagcag tggtactaat 3420 agcaacttct gtctctagaa cattggaaaa ttaaaatgtg tttatctacc gtttttttcc 3480 tcgaggttat atgaaggtag aaatgaatca gactagatga ttagctaagc gagactatta 3540 accctcatcc cttcccctct agaacactat gaaattagtc attatgtatt cgatccttct 3600 tgcagtctct tctctgacag ttataaaagt gatttaggct gcataatgtt gtttgaatga 3660 aatgaaaata tagactagag ctgttttttt ttttatttcc atcagtctct tcagtgaaaa 3720 ctaacatttg agcatgattc tttttttaaa tcattttgtg acagtttagc aaggcttgtg 3780 ataagcaagt tatggtatgg taatatttct agtgtccacg tttcttcaca tgtctggtgt 3840 atgggaacta ctaactccat caggaccttg cctatagtag gtactcaaca tttactgaat 3900 taaatcaata aacattttta atgaattaca gtacaagtca gacctctgta tctgtgggct 3960 ctgcatctgc aaattcagcc aaccatggat cagaaatatt agaaaaatgg aagaacagtc 4020 cagcaataca agtaatatga ataaaaacaa tacaacaact atgtacattg tatcaggtat 4080 tataagtaat ttagagatgc tttaagtata ctgaaggatt tgcgtaggtt atatgcagat 4140 actgtaccat tttatataag gaacttgagc atctgtggat tttggtattt gcatggttcc 4200 tggaaccaat cccccaggga tactgaggga ctatagttga tcataccacc tgattttaga 4260 gattttctga gtctcagaag ttaattaagt aaactacaat agtctgttct taacctcgga 4320 ggatacattc caagaacctc agtgaatatc tgaaaccaca gatagtattg aatccaatat 4380 atacacggta atattttttc ctatacatat gtatctataa agtttaaatt ctaaatcaga 4440 cacagtatta acgataataa taaattagtg acagactggg catagtggtt cacacctata 4500 atctttacac tttaactag acgttgtctt tgaaaagaaa tcagctagcc aaggtggctc 4560 atggctgtaa tcctagtgtt ttgagaagct aagtcaggaa gattgcttga gcccaggagt 4620 ctgagaccac cctaggcaac atggtgaaac cctgtttcta taaaaaatac CAaaaatag 4680 gctgggcgcg gtggctcacg cctgtaatct cagcacttg ggaggctgag gcgggtggat 4740 cacgaggtca ggagatgag accgtcctgg ctaacacggt gaaacccgt cttactaaa 4800 atacaaaaa attagccgggg agtagtgggc gcctgtagtc ccagctactc aggagactga 4860 ggcaggagaa tggcgtgaac ccgggaggtg gagcttgcag tgaccgaga tcgcgccact 4920 gcatgccagc ctgggcaaca gagcgagact ccatctcaaa aaaaaaaaa aaaaaaccaa 4980 aaaattagcc agacgtggtg gtgcttgcct gtagtaccag ctatccagaa ggctgaggtg 5040 ggaggattgc ttgaacttgg gaggtcaagt ctagaatgtt gatagttg ggtcctttat 5100 gtagttgcat aagtgagcca tgatcgtgcc actccactac atccttgggc aacagcctga 5160 ccctgtctca aaattttaat ttaattaaaa aaataaaata gaacaattac aacaatacac 5220 tgtattactg gacaagaagg gcaaatttaa aaaaaattaa accaatatgc tataataagt 5280 tatatgaatg aggggaccct ccctacccc agaatatctg attgtactat atcataggta 5340 actgaaaccg tgaagagcaa aaactgaaga taaagagact actgtgtctt ttaagttttct 5400 tttcaactcc caaattcttg gatttctcac ctcttggctt cctcaataga ggtgagaaat 5460 gttaaagtag tgaaaacagg aaaaataact tactcattca agaagtagat aatggtccag 5520 atggaaagct tgaattattt ttgtaaaact aaaattaaat aaagtagcca ggcatggtgg 5580 cttacgcctg taatcccagc actttgggag gctgaggcgg gtggatcact tgcggtcagg 5640 agttcaagac cagcctggcc aacaaggtga aaccctgtct atactaaaaa tacaaaaatt 5700 agctgagcat ggtggcgggc gcctgtaatc ccagctactc gagaggctga ggcaggagaa 5760 tcgcttgaac ttggggggcg gacattgcag tgagcccaga tcacgccact gcactctagt 5820 ctgggtaaca tcttgagact ccatctcaaa tataataat aattaaataa agtaaaaagt 5880 ttcccacacc tcataaatgt ctaataaaaa ttgaatatgt tgagttcaag tactctgaaa 5940 aaggattga atatagttgg aggttggttt ttaggaatta ctattttct taaattaact 6000 atccttgtag tcacctagga attgtgtatt ttctagat cttagaaaat tatcaaatct 6060 acagttcatt ttgttttttc agtttttttt ttttttttaa gagatggagt cttgctgtat 6120 tagcgttgaa ctcctggcct cagccagttc tcccatctca gcttctgaag tagctggggc 6180 tgcaggtgcc actgagcctg gcttctttat tggtattttt attaaacact tttctctaat 6240 gtctttgtaa cagttctcag tttttgaaat gctgttactg tttctttagt gtgaactgtc 6300 aactttcatt ttttcttttc ttttcttttc ttttcttttt ttcttttttg agacagagtc 6360 tcgctctgtc acccagactg gagtgcagtg gtgcgatctt ggctcactgc gacctctgcc 6420 tcccgggttc aagtgattct cctgcctgag cctcccgagt agctgggaatt acaggtgcgc 6480 accactgtgc ctggctaatt tttttttttt ttttttgtat ttttagtaga ggtggtgttt 6540 caccatgtca gtcaggctgg tcttgaactc ctgaactcat gatcccccccc gccctgcctc 6600 ggcctcccaa agtgctggga ttacaggcat gagcaccac gcctggcctc agctttcatt 6660 ttcatttggt tagtttttga actattcagt gggtaaagtt gtataaataa gtgtcttttc 6720 tctgtataga agtgtcttgg agttcaagga gtgctgcttt gcaaactcat agagtattta 6780 taaaagctaa ctgcagaagg tattcatagg ctaaaccgtt tcctattctt ggtagcacca 6840 ttttctctgg cctgaaatac tttccttcta ctattagtgc ctgtcgatac ccagcagtgt 6900 atttactttc ctgaggaaca attcaaatgc taagtgcttt aagacctaag ggtggaaaag 6960 cagtgttttc aggcattatt aggaaaataa gatttaaatt agacacccag aaacaaaaac 7020 aggtttgtaa ttggtaaagt gaaagatggt taaagaaggt tagattgacc aaagcgagaa 7080 tttacctttt tttttttttt ttttttgaga cagagtctca tgccgttgcc caggctggag 7140 tgcagtggcg tgatcttggc tcaccgtaac ctccacttcc tgggttcaag cagttctccc 7200 acctgagcct cccgagtagc tgggtgacat gcgccaccac gctcagctaa tttctttgta 7260 tttttagtag agactgggtt tctccatgtt ggtcaggctg gtctcgaact cctgacctca 7320 gtgatctgcc cgccttggcc tcccaaaatg ctgggattac aggcatgagc cactgtgtcc 7380 ggctgagagt gtaccttttttttttttt caagcaatct agtacttgat cctaataatc 7440 tttgtggtag gtgtttgcat ttttagatga ggaaaaggga aatctatgag tcctaggaaa 7500 tacagttggt atatgggaac tgatatgtaa ttagacttaa gtgatccatg ttgaatttat 7560 gacttaagca cttaactata atcttaacct ctccagttgt ctgatgaagt tagtatatgg 7620 gaactgatac atagacttaa gtgatccatg ttgaatttat gacttcagca cttaactata 7680 attttaccct ctccagttgt ctgatgataa taaaaacttg aagcagttat ccatattgggg 7740 atctctttgg ggaatcccag tcaccaaaag ttaggttttc tttaatattt tttcatggaa 7800 gatttcaaat atactcaaaa ttgaaagaat tatataataa attctcatga gcccatcaca 7860 catcaataat gaatgtacag cattgcagtg tggagcttgg cctattgctg accactcagc 7920 aatgtggcag aaccactcca tgattcccca tggaaatggg aactacttcg gttgtccttt 7980 tagaaaaa ttcagtaagt atctgctgat tgtgccctac ttgtgacttg aagccaggtt 8040 tttttttttg ttttttattt tttttgtttt gttttgttttg taacagtctt gctctgtcat 8100 ccaagagggg catgatattg gtgcactgca acctccacct cctgggttca agtgattctc 8160 gtgcctcagc ctcccgagta gctgggacta tgggcgtgca ccaccacacc tggctaattt 8220 ttgtatttag tagagatgga gtttcatcat gttgcccagg ctgctctcga actcctgagc 8280 tcaagcaatc tacccacctc cacctcccaa agtgctaaga ttacaggcat gagccaccat 8340 gacagcaaag ctgggtattt cttaaattgg ttcagtcagg tgcaataat tatttgccct 8400 actctaaaaat ttaaaaatct tctaagaatt atggttttgc agctgaggtt tttttaagac 8460 tcgagctccc tggactccta catatatcct tagaacaaca ttgtccaata gaagtacaat 8520 gtgagccaca tgttttgttt aacccagcat atccaaaata ttaccccctt tgcatgtgct 8580 taatataaaa atttaagatg atttatattc caggttttca atattcagtg agtaatttta 8640 cacttagagc aagtatcatt tcagactagt cacattttga gtactcaata accacatatg 8700 gctagtggct accttactag atagcatagc ctttgagtcc cacaaagtgt tcacattcta 8760 tgtgtttaca cacatctttt gaagtgtcat gacagagcca gataggatcc agattttctt 8820 taaatctggt ctctcttccg gattcctagt tgattacttc tttgttgctt cttataggga 8880 tgtatcagaa gtttaataat cttatgatta ttatgttaac tgcctgaagt attaatggta 8940 9000 tttcacatat acatgtatca ggaagtaat gggggtaatt tagtaatgaa tagtataataa 9060 tagtttatga tggtattttt ctttttttt tttagattgc agcaaaaatt ggaggtgatg 9120 cagggacatc actgaattca aatgactatg gttatgggg acaaaaaaga cctttagaag 9180 atggaggtaa gttatactct aagtatttta aattgttttt cagagtgttt agttgaagtg 9240 attctcggta tttttctgtt attttattga gatattaact tttattaataa ggttgttaaa 9300 attgtaagct gtatattggc ctaaaagggg ggaaagaaaa ctagacaagg tagtaaaat 9360 ttgaaaaa ttttttaaaa aattttttaa aaaaggaagt tttgtcttaa tagaaaaaa 9420 tttattttcc ctcttttagg attgtgccag attgaaagtt tgcacagacg tcttgttaat 9480 aatattaaaa aacatataaa ttgcttagaa gacacttcac tggttttact catacgtgaa 9540 tggattttaa tatgctgtat tttcgtcatt tttctatttc caattgcacc ttaaaggttg 9600 aaattcctat agtttgctac tctagtgtgt tgcaggttat accatttttt ttttaatgtt 9660 ctttactttc agtacttttg tgtttcacgt ttagctttaa acctgtggat taaaacagtg 9720 gatttacagt gctatgtatt tttaaaaatc gtaatcgttg aagcttctga acttagaagt 9780 ctgcatgtat tttttgtttt aggtttgata tatgagtttt gatacatttt ctttttaccc 9840 ttttttttaa agggaggatt ctcactgagg ctatagaatg tatttgtagc ttttgaccag 9900 gagaacttgg tttcctttta tttaagtgtc tttcatattt atagtgaggt ttttaatgta 9960 gaaaaaaaat gagctaatga tgcttcagat gttgtatgta atgtattctt tttattttat 10020 gtgtagatgg ctcttggaca agtccgagca gtacaacaca ctgggaggga atgccctctc 10080 cttttaaagg caggaatttt tatttattac ctgtgttcag tatgtaaacg tgaaataaac 10140 cagtggattc ttaaatggac acaaatattt cttggattat gtgtctgcgc atattttatt 10200 tttgctgcac aacattctga tgtttaatca tttaagtttg aaggggggag gagaatgtag 10260 tactttgagc tataggttgt ctgttccaag gtatgcattg tattcatctg tgtaatggat 10320 ttaggtgaag gtagtcatgt agttgctttg agattttatt ttttgctaa agttttatgc 10380 agtgaaatgt ttgtttataa ataatagaac agtttaggtt gagattgcct tgtaatgttg 10440 tggggggtt ttttgttttt ttttttttgg gcatagcttt gtggtcactg tcagatacac 10500 tttaatatgt cagatttttg tagtttgata ggctttctc cccccagtct tcagttcctg 10560 aggtggaagc atcattagcc tttagcatgt gatattttgc tagtaatgga cctaaagtac 10620 gttgtctttgt gtcattctaa tgtgcttaac atacattaag gtcagtgatt tcttaagaat 10680 cagataacta ttttaatgtc tgtgcatctt ttgaacgtga agagaatgaa gtatcgtttc 10740 ttttttagat tactgagttg gggttgaatt ttggcagttt tggttcaaat gataaaccat 10800 accttcagat atttcaataa atgtttatat tgttatttat tcttgtttgg gaggggagaa 10860 gctttgtact taattggcaa aaaattaaaa gacacttaat tttgcagatc aaccagatgc 10920 taagaaagtt gctcctcaaa atgactgtaa gtattccttt taaactgggt caaaagctaa 10980 agtaacaatt tcaatgttaa gattttgttc atattattgg gtatctttga agttagttgg 11040 tttatgagta ttttggatca gctagcctga atttctttgt aaatatatac ctttttctcc 11100 tattttacaa tctgtcccat taattgtggc cgggtatatg taaagattgg ttgctgaatt 11160 attttacata tttaacaact ccaattcttg atctatactt gtacaacttg aaaaaaggaaa 11220 ttttttgtt ctgtgccatt gctaaataa tgtcttccct ttcattggct ctcttgcccc 11280 tgtcaatgcc aataaata ttgtgtaaaa aatttgactc tcttcaaggt gttgtctgtg 11340 cattagggag agatttttga atgtttgatg tgattgtgtt gttaaatata tagtaaatt 11400 taaattttga attttgttt tattttttt taattttttg atagcttttg gaacacagtt 11460 accaccgatg catcagcagc aaaggtatag tcacaagatt ttcaaaaagt actctgcaag 11520 ttttggttga gctgtatgta aaaacacaac cacattggtg tatattgaat atgtgtctgt 11580 gtattttttg gtgtacctag ttcatatcac tccccttggg aaggtaccat aaagtgatga 11640 tttttctttt gagtgagaaa aatttgtgat ttggagagat aggtggaatt aaccacattt 11700 tagaagaaca ggggtgaatt agagtaactg ttaagatgac attctctaaaa ctccacttca 11760 acttctttac agttaatgcc ttcagactgt tccattcatc atcccttctt cacttgatgt 11820 gtcatcttaa atttcttaat ttaactactc aagtaataag atcatatttt ttgacatgag 11880 tctgagccta gaaccttagt ttaagccatt gggagacatt agacttccat ttttattaat 11940 agattatctt ttattgttaa acaaagtatc tttcattgaa ggaaaatggt gctttctgtt 12000 atttcttagc agatctgtaa tgacagaaga atacaaagtt ccagatggaa tggttggatt 12060 ftaagtaa cttgattttt aaagttttga aaacatgatc aaaacatact ttagaatctt 12120 tcaaccaaaa aaaaaatttt tttttttcta actagtaatt ggcagaggag gtgaacagat 12180 ctcacgcata caacaggaat ctggatgcaa aatacagata gctcctggta atgttacatt 12240 ctcatggtat tttcagtgtg actagaaaac tagctttttt ttttttttaa gccttttagt 12300 aacaataatg ctacttttaa tcttttgacc tgaagttatc tgtttgtttt aaattgtaga 12360 cagtggtggc cttccagaaa ggtcctgtat gttaactgga acacctgaat ctgtccagta 12420 agtttgaaaa atcttaaaaa tctacttaag taacaacagc agaactcttt gaattttgtc 12480 tcttctcttt gttactgctt tattttacac tgtggtttcg ctgccacctt ccctcaaagt 12540 cctccaactc ctttgaagtt tatgcctcat gcctttctca ggtggggttc atcatctgaa 12600 tcattaaaca cagaaaatgg taaaacaac tccatatcta ctccagtctc tacttgtaaa 12660 12720 acattaagct caagtggact gttgtgttgc ctgcatttcc ctagttccat tcacttttcc 12780 actcctctcc caggctcttt aatactgtat ttcccccacct ccaaatcttc agcatctaac 12840 cccacgctct cccacttaag cttatttact gagaaaatgg aagcaaatga taagaagctt 12900 ttctttttcc ctaccactaa acctaccagc cttcatttt cctctgttca catagtactc 12960 aggtaattgc tttccttttg tgttcgagtg cctaaagcca gccctttctt cctactgaag 13020 gttcagcttg cagttgtact ttcttctgca ttgttagttc tccctcatta ctaggttttt 13080 tcttttcttt tttttgagac gaagtctcgc tctgttgcca ggctggagtg caggggcacg 13140 atctcggctc actgcaacct ccgcctcccg agtagctggg actaccggtg catgccacca 13200 cacccagcta atttttgtat ttttttagta gagacagggt ttcaccatgt tggccgggat 13260 ggtctcaatc tcttgacctc gtgatccacc cacctcggcc tcccaaagtc ctgggattac 13320 aggcgtgagc cacttccccc agcctgattt tctttatggc actttccaaa taatgtgtta 13380 ttcatttgac atgttatttt tacttattta atgaaatgaa gccactctag caggaaccct 13440 gtttctttga gtgctattac cccattacct agaatagcac ctgcacatag ttgatattta 13500 aatatttgtt gaatgaataa ttgtagcata tgagtaagca aaatggtagt ttaaaaatgt 13560 aaataaatca tttagttctt ggaagaatca gtttaattct gagataactt tagcattaga 13620 gttctttctt ggaaattttg gactattctt aaaaataaaa attgtatatc tagaaaattt 13680 ttttgcataa tctctcaatc tttgaccctt gatggcattt tctttcagtt aaaagtaaaa 13740 gcattgttaa agttagcatc aaggcaccta atcctgaact gggataggag gagtacttgg 13800 ttatattgtt ttatatttct ctatttgaat aagcttgggt atgctacagc ttactattta 13860 aatattaatt tgttaacagg tcagcaaaac ggttactgga ccagattgtt gaaaaaggaa 13920 gaccagctcc tggcttccat catggcgatg gaccgggaaa tgcagttcaa gaaatcatga 13980 ttccagctag caaggcagga ttagtcattg gaaaaggggg agaaactatt aaacagcttc 14040 aggtattgtt atttttgtga aatggctact tttgatctgt tttgatgccc atttttgtcc 14100 acttcctttt gttaatatat attatttcta tgattgtaac aggaacgggc tggagttaaa 14160 atggttatga ttcaagacgg gccgcagaac actggtgctg acaaacctct taggattaca 14220 ggagacccat ataaagttca agtaaactta actttatact ttataaagaa agagtgggtt 14280 gaatggggtt gggcaaaata tgcatgaata attaaaatgt tttgagacat gctttctaaa 14340 ttagctaact tttctgctt tagcaagcca aggaaatggt gttagagtta attcgtgatc 14400 aaggcggttt cagagaagtt cggaatgagt atgggtcaag aataggagga aatgaaggga 14460 tagatgtaag taaaaatacc cattcagaaa tggttgtatg ctaattcata aatataatag 14520 tgttttctgt tttgtgttaa gtagctctaa cattgttatc cttttatttc acctttatac 14580 tttagaatac agaattctat atatcttgtt accctattta ctataaatat agaattatat 14640 gtacttttat gatttgaggc agattttcag gaaatggcgc ttttttaaaa tacttttttt 14700 tactttaaac cctgagaagc tagctttctt aatacttagt cttttttaca taaggtcccc 14760 attccaagat ttgctgttgg cattgtaata ggaagaaatg gagagatgat caaaaaaaata 14820 caaaatgatg ctggtgttcg cattcagttt aagccaggtg agtacatata ataatcttgt 14880 aagtgttggc agcagtgagt tttgacatac atttattgtt taattaattt tgtttctttg 14940 ttttgaagat gatgggacaa cacccgaaag gatagcacaa ataacaggac ctccagaccg 15000 atgtcaacat gctgcagaaa ttattacaga ccttcttcga agtgttcagg tttgatagaa 15060 agttaacatt ttcattttt gtttttatgg aaaagtattt tccttcatga aatctgaagt 15120 tacccttata tcagagtctg cttgatgatg ctttattaat ggagaaaagtt taaattgctt 15180 taaggtaaag atcttggagc aggagaact actaccttaa gtgctacctt atttactctt 15240 gtattttaaa aaatgttat acttattagg gccagttcat ctctacattt ctgattcagg 15300 tatatgagag ctgggaaaaa taaacttaat aattttatca tgaaacaaaa gtatttctgt 15360 gctgactctt cgttcttgtc tttccctctc tataggctgg taatcctggt ggacctggac 15420 ctggtggtcg aggaagaggt agaggtcaag gcaactggaa catgggacca cctggtggac 15480 tacaggaatt taattttatt gtgccaactg ggaaaaactgg attaataata ggaaaaggca 15540 atgtatttta aactcttaat gttttaacac attattcatt tttctggaca ctttctgttg 15600 ctgtcgtaaa caagtggcaa tgcttttct ctgaccgtat tttagtagaa aagaattctt 15660 atgttaatat gtaacaagta aaacataaat gagggatctc atgtatattt agaagaaagag 15720 caggatttta atcttactag cttctagaga aagcgaacta agagataatt attagcataa 15780 gaaatgtctt ttgacccaaa aagtggtttg agtgtttttg tttgtgcatt ttggtttttc 15840 ccgactcata ttttaaaaat ttgaatgttt ataagtgtat tagtttatat ttacactgct 15900 tttaaaagca gttaattcaa atattttatt ataatcacat taagtttatg tttaaacata 15960 ctaagtaat gtaaatgtat tttaagagaa gcatgaaatg cttcctaaaa tttgattttc 16020 agtgtagaat attaaatgaa aaatcttaat acaatattgt caattaggat actgaccaaa 16080 ccatattttt aatggcccat ttaattgtga ccattttctt ctaaatagct cctagtacac 16140 ccttgaaacc tttagagaaa ttactgtctt ttgatttag gaggtgaaac cataaaaaagc 16200 ataagccagc agtctggtgc aagaatagaa cttcagagaa atcctccacc aaatgcagat 16260 cctaatatga agttatttac aattcgtggc actccacaac agatagacta tgctcggcaa 16320 ctcatagaag aaaagattgg tgtgagtata ctttaaactt ttaattttta gtgtagaccc 16380 ttagactgta gttaaattaa gacgtttatt caaatacatc aaaggaaaat gtatcattac 16440 tagtcagcat ttatagattt catgatatgt ataatagata caacgtgaag attttccagc 16500 aatgaaaata acctaattaa atgtgtagtt acaggttttg agaacaacct tacatttggg 16560 tgtggctaga taagaggagg gtagtgttac ctgtaggcat gatattagtg ttggtgtagg 16620 attgtggaac atacttgaag gacatagtta acggggaattc attatttatt aagattttac 16680 tctactgaac cccagcgagg caaacaagat aaatcagata catctgccac tctacagtag 16740 aaattcataa atcctaggtt ttggactggc tcacagatca tctgggggta ttaaaatgta 16800 gactatttgg gtgcttccca ctcctacaca tatatacag agacagacct gactcaaaat 16860 gtctgggata gggcccagca tctaatttta catagatgtt tgggtgattc tggtgcacag 16920 acaaatttgg aaatttttcc tccaggaaag ttttctgtta gaacaaaaaa gtatgaaacg 16980 ctttgactgc ttttttgtaa gtgaggcaga cagtgtctta ctggagtttt taacacaaag 17040 tgtgcagggg gcatcttaaa ttattagaat tgcctaggaa aaattatttt tggtgtttgc 17100 catgctgtga atggggtgcg tgcaaaacaa ggttgacact gtttctgtca acttgggaag 17160 acaaaaatag atgtacaaaa gacgcttaag gaacgtctta caaaatgtac acaaacattg 17220 tgagttctgt tgtatggata gtttaatggt tcaaaaaatg aagaggatgt ttgtagagta 17280 atgatagagg ttgatgccat ggcaaaaaaa tgaatagcac tcatcttggt tttttttt 17340 acagtattag cctaacatgc atgcgtcagg atttctgttg gattcatgga aaaacaagat 17400 agattgtttt ggagaagcaa tttggtgtgg tgattaagag catggactct gtagtcgggt 17460 tgcctcagtt cagaattctg tgaatcattt actggttctg tgaccttgga gaagttactc 17520 aggcttttct gtgccttggt ttcctcctgt aaaat your taatggtatc tacttcatag 17580 agttgtaggg attaatgaa tgttcatgtg tgtgtcactt aaaagaatgc ctgccacata 17640 accctaaaaa atgttgctac tttttcagta ttatttttac tacttggaaa gaataggtca 17700 tggatagtaa gtgagagatg acagcaattg gagatttaaa gagacaagtt aataaaaaga 17760 actttaaagg tcatgaagtt tagtttccct attttgcagt gagaaattta caacaaaatg 17820 tagtgttagc attttgtcca acatgtcgcc tggttgtgtt aactactcag aaggagcatt 17880 ttaggacagt taagtgtaat gtctttgttg gcttaaccaa aagaaatca gttaagcgtt 17940 attaatgatg tggcatgcat gcatgataag gataaaat atcctgattt attgaaggaa 18000 ttaaaaaggg aatttttgtg ctattaaaca tcatgataca tgaaaggcca aaaaggatat 18060 aaattattga tctgaatggg attttagtga cagaaatagg ttgtgaggtg gattttagtt 18120 tcatagtgaa acaactagct attaacgtta tcagtgaaat gttcagaaga cgatgataca 18180 ggacccaagc ggtttgggaa tatattgtca agacggttgt ctcatgttag gcaagtagat 18240 18300 aaaagtaaga atgataaaag gaagggaac tactgctgtc cgcaaacaag ctaaaggaa 18360 tttaattgct aatttaatt taatttaatt taaattgcat ttaattgcta ctgctattga 18420 ttttagtgaa ttttacatgt ctcattatta tggcagatga aatagtttt gcaaaatgaa 18480 tgaggaaagg aaggaaaacc taaaatttgt tatttgtgac tataagaggg tagaaatgga 18540 tgattattg gccgtagagt tgtttaacca ttactgtgtt tttctgattt ttctatgtca 18600 tcctttttt tgtagggccc agtaaatcct ttagggccac ctgtacccca tgggccccat 18660 ggtgtcccag gcccccatgg acctcctggg cctccagggc ctggaactcc aatgggacca 18720 tacaaccctg caccttataa tcctggacca ccaggcccgg ctcctcagta agtattgggt 18780 ttagttctgg gctttcccca aagattctag ttttgggact gttttttatg ctgatttttc 18840 ttttcagtgg tcctccagcc ccatatgctc cccagggatg gggaaatgca tatccacact 18900 ggcagcagca ggctcctcct gatccaggta gaagatgctt attatttgtg tgttatctgt 18960 attattttcc actcctgtta cattattaaa ttttctagtg ttgattctac atttgtatgc 19020 atcaccttca ctcactttac tctttcaaca gtgttaggca ctgcctctac cccagtgtat 19080 aggactgaca tgaatatgag ctctgctttt atggaatttc tttctacttg cctttggctt 19140 atgagttgat acagtagaat gataaaagct aaaagctgca ggaaagagca cagtgtcata 19200 ggttttggat accagtgctg tcaaatgtgt agtatgttca ttgtgacatt atctgtggaa 19260 aatagtttt tacttattta gaaaagtatg tgataggggc tgggcactga ggctcacgtc 19320 tgtaatccca cctattcaga agtctgaggc tgaggcttga gaccagcctg ggcaatatgg 19380 caagacccat ctctaaaaaa atttgtttta aattagccgg gcagggtggt atgtgcctgt 19440 acttgaggct gaggtgggag gattgcttga gcctagaagt tctagattgt attgagctgt 19500 gggcacacca ttgcactccc ctgggcaaca gaatgggatc ccatctctta aaaaattatg 19560 tatatatgta acagtctata taaatatata tatataacag tccaacagag tgttaagtat 19620 tggggcatta aacttccaaa ttgtcaaata agatatctgt tctagtactc ctcatatgac 19680 aacttcatgt gagtaaaaat caggcctgta tttaataact gcatgctaaa gcccaaatac 19740 gttaattat tttctatatt cagatttatt tttatcctat tatattctgg catctttcca 19800 taccctgtag tttgctttcc atcttggtca aagagtcatt cttgaaacc aattaacatt 19860 tatttatgat ccttttttct cacctgctac ctacctttta ggcctctttc actcgtgtag 19920 tttcaaggaa aatatactca attatggaat actttctaca cataatacat ttatcccaaa 19980 aaaacttgaa gtaatttatg taaatgaaat tgcttgatta acttcatagg aagtgtgctg 20040 tttggaatat gatgacacag catgacagtt accaagcatg acttgagttg tgctaggata 20100 taggtgtgca aagttggggt tgtgttctat agcaaaagaa tgcactccca gcgtaagcaa 20160 cactgatgga aggggctcac agggtacagg atataatact cttaacaact aatttttgga 20220 gaatgaaagg gcttttcttt ccctcttgtt ggctgattgg gatggtataa ttaatgggat 20280 tgaagagttt gagtaggtta aaaggcagat tcatattggg taacttggat ctgccaggat 20340 tgtatttttg agcactactg ggtggttagc atgattgagg aaaaatgatg ggaataagaa 20400 gtggaagtgg tctttgtatc acaagttgaa tttctcactt ggagtagtag tgaagtcact 20460 actgtaagag ctggtcagtg aatgtggttt cagcatggcc tttgggcaag aagtaaccca 20520 tttaactaaa accagctggt tggccccact cagatttatc aaagggttac tgggtccctg 20580 ggggtggata ttgcttatat tagacttaga atagcatact gttttaatat tatatgaact 20640 aaaatgtttc tttaaaaaaa gagtggtctg ttaatggatt tatgtagtgg tcaagaattt 20700 agacttcaga gtcaaataaa cctatatcag tcctagtcct acagtttact aattgtgaga 20760 tgtcaagcaa gtttttgaac tcctctaagc ctctgttttc ttatctataa attaataaat 20820 gaatgaatcg ggttgagtga atatttagta aattcttagt acatactagt tatttgtaac 20880 tgtgagactg gttttttggt atggttttca catttgggag tagaaatacc acttcctaaa 20940 gtctgtttta tctcaaattc tctatccagg catagtgtaa agtgaaatac ctagatttct 21000 tgattaatat acagataatg gccagacgcc atggctaaaa cctgtgacgc tagcacttcg 21060 gaaggctgag gcgggcggat cacttgaggt caggagttgg agaccagcct ggcaaacatg 21120 gcgaaaccct gtctctacta aaaatacaaa aattagctgg atgtggtggc aggtgtctgt 21180 aatcccagct acttaggagg ctgagacagg agaactcctt gagaattgct ccactgccct 21240 ccagcctggg caacagagtg agacactca tctcaaaaa aaaaaaaaaa tacagataat 21300 gawactattg agatgtaa acatccaca taaaaaagca gtacattgggg cattgagaa 21360 aagtttggca gtgtcctaa taacacctta caattatatt accttgcaat tttcttttc 21420 taggaatttct tgctcatttt tctcaactt gactaactt tattaagcag ctgagaattg 21480 ctactgttca gatgaaggt atatagaaa atttaaaagt tttattgta tggtattcct 21540 agtataaaag acatcaagt tttctggt ttcagaga tgaaggtc tcaactgtt 21600 cgcactcag ttgatgtggg agatgagtga gggtcagtca agtgtagagg agacaacata 21660 gctaaaagcc gagacacgggg catagtgat tctgaaagt acagcactg tgttgtggct 21720 ggagcttggg tgttgagag atacaacca agggagagat gaggctgaaa tggaaggat 21780 aagccagggg cttcaggctg ttaagatgt tgaatgag gctgtactta agctttgaac 21840 atcctgaga tgcagagaa tgagaattt caagtaggat gatcagt ttatacatt 21900 caatgtgaaa taggatgaaa tgtggctaat cttttttaag attttat tttctcttca 21960 ttgaaaataa ggacaaagtt cattgttcta aaatatttt ctttcttata caggttagtc 22020 cctagaaatg tttcttta gtcatcttct agatagagct gtttgtgctt gaggcgaaac 22080 caatttagaa aaaaaacagg gcacaggatg gtttgagaca gagcattgga tttggagcca 22140 aaagaccttt attcatatcc cagttatgcc actcagtagt ttttaacta ggaaagtcac 22200 ttagtctctg agacttggtt ttctttaata aaagctgatg gtgacacaa aaatataga 22260 aaatttaagt ttcatatac tttttat gttctagtta attggggg ctgtatattt 22320 gccagagagc tgggcggggg gtgtgtgt gtgtgtgtga gagagaatta cagacttcac 22380 atgcaaacct tgaactttca tttatttag ctaggcagg aacggatcca attcagcag 22440 cttgggctgc ttattacgct cactattc aacagcaagc acagccacca ccagcagcccc 22500 ctgcaggtgc accactaca actcaaacta atgacaagg taactacaga acttattgta 22560 tgtgaaagcc aaagttgtg cttggaatta tatatgaagt acatcactgt ataataccta 22620 aaattctga cattatttaa ttataattta agcagacttt tcctttttta aattgttact 22680 ttgaaatagt ttcaaacttt cataaaagtt gtaagaagaa tacccggagc acccagatag 22740 cccagctgtt aatgttttat tcccttgttt actctgttac catctatatc atacatgtgt 22800 atctgtattt atttaaata tccattataa ttagttctga aggaagctaa ttatatccag 22860 cataattagt tctgaaagaa atcactgaag actaaactgt agacatgatg tcctgttggg 22920 tacttcttca aaaatacata accatcatac agccctccaa atcagtaagt caacattgat 22980 atattaagtt cttatatca ttatggattt ctgatttttg cagtaggtta tatttttaaa 23040 gtattttat aattttgata atcagattat cctggatttg gccagtcagg gagtatattc 23100 agggtggtgc ctatatcctt ttgaaatacc tgtcattctt ttaagcactt catactgtc 23160 tggcacagta agattgttat tttgtgcttt ctgggctcca gccctcgagt cagctatttc 23220 ttcaaggagc tcttattcct tttagtagag tatggtagtt agaaacgaga cttgagtatg 23280 cttgttgcta ctgaggtgta attgcttcta gcttctttca acaggcagaa ctaggaaata 23340 tatttacata catgcatacc tacatacaca caccaaaaaa cacataaaca tcgatatatg 23400 tatatttt taaaaactat gttcatatca ccacatccgt ttcagcattt tggggtttc 23460 aagccttttc ctttttgta cttgtttaca aacgtgagaa acctggtgtc ctcagtgtat 23520 ttccttattt gatagcatt tacttatatg ttcacataa ccagtcttca aacaggttgg 23580 ttttctttc tgtccaccac ctctgtaccc ccagtacctt ctatctttgg cactgttagg 23640 gatgccacca ccacatagta cttccctcct accccactg tcacattgca ggcccctgcc 23700 agctcctgca cccaaggaaa cggccaaat tgccttttaa aactttaat tctgtttttt 23760 gttttgtttt gcttgaaca ggagatcagc agaatccagc cccagctgga caggttgatt 23820 ataccaaggc ttgggaag tactacaaga aaatggtat gttttaca tttcttgaaa 23880 atacatactt aattaattg aaaaaatta ttctctcag gagagaata attgataaa 23940 atcactggac ttgtaaacat atcacag ttgtaaatta tagtttta atttgtggtt 24000 atatggcaag gaaattttttt tttctaattg catttgtcaa ccagttatta attgaaacta 24120. 24120. 24120. 24120. 24120. 24120. 24120. 24120 fathers cagtctaaat gacataaggt taattttaa tgtgtcaggc gaaaattgtg tgtgatacca ttatttttgc tgcaagata gcaggtaga agtaatctgc agtgacgga agtaaccaag tgatggaacc agaatctggc ttccaagagg gtctgagtcc caagcttgtc tcccaaattt gtctctttag gaccatttg gaacctgata ctatacttct ggacaaatca ctatattca gctgcttttg ctcttagtca tttaaaatta ttacatacca cagctagatg tcacaaatga aagctaaatt ggtaagcttg gttatccttc actagcagaa aaagaaccta tagtggtag agttttgtca tagcg gtctacttgg gattttcaat gttttctttg 24540 ccaagaatg tttctcattc tccacagaaa gaaaatttc cagaaggtg atgattttaa 24660. tcttctagat gtaaaattac atatacctga tgataaagtt gttttgcaca actggtttct ttttaaaga aaattgtttt tcctcttaga atggcttcct aggagagtca tgttccgtct ctttctgagg ctttaacaga ttatgtttt gtgacctagc ttaggcagat ctacagtagc 24780 tacaattcgg caaaaagaaa cttttaactt aaaaacagca tactctgatt aaggttggtt 24840 acataattta ttttctgaac tgggatcctt tttagaatga atagggatgc tattaataat 24900 catgccctga cagcattgtg gtcaggacct gtaatcaact taactttaat taaatagcat 24960 caccatttta aaagacttga gcatgagcca cgtgcggtgg cacacacttg tagtcccagc 25020 tgcttgggag gctaaggtgg aggattcct tgaacacagg agattgaggc cagccttggc 25080 atcatagtga gacctggtca cgtgtttggg ttaaaaaatca ctaacttcaa cttcttttt 25140 ctcaatgggt aatgtcccct agatagggtc cctagttat attaagtagg taaaaataaa 25200 ggcttgttaa tggattttagg taattatgga ggaatgagtt tggcttctgt gctttttt 25260 accatattga ttattgttaa tatggccatt aatacattta ctgtttagtc ttttttgttt 25320 tactttttat gttttactca aaatgagtgg gtgggtggaa ttctaatttt tattgttaag 25380 ggaagacatt ttaccttgtc tttaattttt tatttttttt taccatttcc ctgccagtta 25440 gagatactat actatactgt cttgaatcct ctgtaggaaa acatggcata gaaataatta 25500 aataataatt agatgttaaa tataatgct gtatgactaa agaacctcct taccccacct 25560 tttctgttgt tgttctgtag taccacaata atcacttgtt aattttattt atttatttat 25620 ttgagaggga gtctcactct cgcccaggct ggagtgcggt ggcgcaatct cagctcactg 25680 caacctccgc ctcccaggtt caagcgattc tccggcctca tactcccaag tagctgggat 25740 tacaggcgcc tgctaccaca tccggctgat ttttgtata tttagtagat acgggctttc 25800 atcatgttgg ccaagctggt ctcaaactcc tgacctcagg tgatccacct gcctcagcct 25860 tccaaagtgt tgggattatg ggtgtgagcc actgcaccca gcctcacctg ttaattttat 25920 gagcaaaaca gattagttgg gcaagtcctt cacatgcata tctcgttgtt gttgtttttt 25980 ttaagacgga gtcttggtct tactccccaa gcaatggtgc gatttcgtct cagtgcaacc 26040 tctgcttcct gggttcaagc tattctcctg cctcaacttc ccaagtagct gggattacag 26100 gcgcccaacc acacccagct aatttttgta tttttagtag agacagagtt tcaccatgtt 26160 ggccaggctg gtctcgaact cctgacctca ggtgatccac ccgcctcagc ctcgcaaagt 26220 gttgggatta caggtgtgag ccactgcgcc tggccacatg tgtatatctt aaaggaaata 26280 atgctaggta atttagtcag gtgcttgatg agcatttgtc atcatcatac ggagtcaatt 26340 tgtctttttc tataaaagtt ctttttgtaa atgattagtt gcttaactgc tttaatttct 26400 tctaggtacc ttacctgtca tcaggatctt tttaccacaa ataagaaacc ttaaagcata 26460 aaacttggtt cagtcttcac atttcttaaa tggaaggaga aagggaataa tagggagata 26520 tctttatctc aaagccaact gttgttgact tttccagtgg caggggtatg atgctaggac 26580 ttcagatttc ctgattcccc atcctagtgc cccttttgcc aaactaggca ggctttcaca 26640 gcttttggag cctaatttaa gtttttcttg ttaagaacag aaaactccat tcatagattt 26700 taatttgtca ttatttgctt atttactgaa aaaaaaaaaa aactattgaa aacaggtgtg 26760 ggttaagttt cccaaaatta ggtttatatt tcaaacaat tctaagttcc caggataac 26820 accaaactaa gaggataaat ttttatttt tttttttta ttttatttt ttgagatgga 26880 gcctttccct gtcgcccagg ctgaagtgca gtggcatgat ctcggctcac tgcagcatc 26940 cacctcccag gttgaagcaa ttctctgcc tcagcctccc aagtagagaa atcttgata 27000 agatttcaga gggcttataag gaacagcagt aagatgggt gttttaag ctagaataca 27060 gggattttt taagaccct aagagaacaaaaggtttc aaatagaaa ggagtaattc 27120 ttgtttggag aagtgaagac atttgggactt gaggaaggcc attatgataa 27180 ggactgatgt ggtagactct gaaccctgaa gatctgta ccttaagcct aaggaggggt 27240 agagtgagga cttgtgggaa aggtacagtt atatgggag acactgagt gtgaaaatct 27300 attcatgcaa ggctggctct taggctctaa tagaatcttg caagctact ctagagcaag 27360 tcttttatct ttgggatgaa agaatttaa ggctaaactt tatacattat aattataaa 27420 ctataataa tattagataa tgttctactg aagtataaac tcaactatct ggcatcaac 27480 agtaactaag ccatgatcac accactgcca ctgtgttctg gcctaggtga cagagcaaga 27540 ccctgtctca aaacaaaatc cagtaactaa agagaggaat aaggggagcg caaggtaagg 27600 cagtacatgt ctgaaggggc agggaaagag ttctttctct acttccaact gggcaaaata 27660 aatcacattt gccctttaga ttggaagagt gaaataagct tttcttgaac atcttttaag 27720 attggagtgt caaatattac caacttattt accaagaatt tggtttttct taaagtctaa 27780 agtgtttact attagctttc cacagggata taggagtttg cagaggttgt agttttttaa 27840 gggaacatca atgaatttc ttgatgcata tgcctgtttc taccatttta cattgttaat 27900 ttgcctctaa aatgagtaac tcttacaatg gggtttaaaa cctgaagact attgattgct 27960 accttgatca ggtttggttt caagtgtgca ccctgtaaga gacaatgtgt ggttttattg 28020 ctattgtcac accagttttt tagatattga aacctgtttc agatttgctt gaattgtgtc 28080 ttgtaagagg aaaatgttaa acttacttcc tctttgagaa cagttattta tagaagacag 28140 gaaaatatga gaatttttaa tagtatatga gagttctctg ttacccaaga aaagagggtt 28200 tttttcaggc atttttaaag aatcataaat cttaaattct ttcactcagt tgctttgagt 28260 ctgtgacctg ttttacaatg gtgatagact gctttctgaa actatgaaat tggtcttgtt 28320 ggcagcatcc tacaaacata aaaagagctt cctgtgtgtc catgctccag tatttttgtc 28380 tatggtagtt atttcacaaa gccaagcgag ttacaaacga aataaaatag tgcttaagta 28440 aagaaactga ataggagaac atatactctc tcttctcaat tttttattta gaaaattata 28500 taccttcaga aattggggaa ataaggcatc agacactcag cttcatcatt ttttaaaagt 28560 ttgctgtatg cgctttatat gtatgttttt tgtgtatgaa ccatttcaaa gtaagttgca 28620 gacaagacag tttgctctaa ataaactcaa tgtctgggca cagtggttga cgcctgtaat 28680 cccaggactt tgggaggcca aggcaggtgg atcacttgag tccaggagtt caggaccagc 28740 ctggacaaca tggcaaaacc ccatctctac aaaaaaagag aaaaatgtca gctaggcatg 28800 gtgggcctgt agtcccagct actctagagg ctgaggtggg aggatcacct gagccctggg 28860 aggttgaggc tgcagtgagc catgattgca ccgctgcact ccagcctggg tggcagagtg 28920 agaccctgtc tgaaaaaaa taccctcaat attcacaa acatacagac tatattaaa 28980 tttccatagt tgtcaggaat atgtcttttg tcgtctgtgt gttttgttta attcagagta 29040 cattcagat tcatgcgttg cattcattg ctatatttct ttaatctct aattcagcaa 29100 agtcacctta tttggaaaaa gacctgattg tgaagctga gtttagtg tttcattga 29160 tcttgttctt tattccttgt caggttactt ttaatacat tgtgttgtaa ttggaataaa 29220 ttattaac ttcacatc ttattatgtg tgtgtgtaca tacacttat aggttactaa 29280 tcaggaaaaa gtcttggcta ggtcttaata cagtctttaa atcattgcct ttaagtgggc 29340 ttaagtttt tcaaaatgt tctttttgta attctggaat cgaattaaga ttatgcctaa 29400 atctttacct tccttagcta aagcagtgtg gatttggggt tgatctgtt tttttactaa 29460 taatgacgct ctagaacta aagttaacga tattattaa ggcaaaaga aagagcgtt 29520 cttttttttt ttctaattgc agagtagttt cctgacacta ctaaatgaat attttaaata aaacaggagt aattctgacc ctctgtgctt ttgtcttata acctgtactt acagtggatg 29640 tttttata ttaagtttt ggggtttttt tttttggtca acaagggcaa acacaagatt attcaaaggc ttgggagaaa attcaaaggc agcaaggtat tgtttttatt agcaatgaga tgttgggctt atattgtgg ttacagcaac aaagttcttt tttttcaaag gtcaggcagt tcctgctccg actggggctc ctccaggtgg tcagccagat tatagtgcag cctgggctga gtattataga caacaagcag cctattatgc ccagacaagt ccccaggga tgccacagca tcctccagca cctcaggtat aatgtaattg ctaatttgtt gattctact ccagtctgtt ttctgcatgt ttactgtttg tctgtttggg agtgtttgcc ttttaaattt ttatctggca 30060 aagtataata actatttaaa tgaagtacta cggtgtattg tttgggtttt tttgtttttt attgcttt ccagcatctg agtggtgaat atttctgcaa tgcctttgat tttaaaaata aattttcttc ccccagggat ttgcaaatca tgcaagaagc caccaccatt tatattaacc actttcttt tctttaagga ttcactcctg aattagctcc atttcagga ttttctttaa 30300 ctttttgtgt atttcttatg tatctctct gcacaggggcc aataaga agtggacaat 30360 acagtatttg cttcattgtg tgggggaaa aaacctttgt taatatatg gatgcagacg 30420 acttgatgaa gatctttatt ttgttttgg tttaaatag tgttttcctttt tttttttt 30480 ttttgaaaa tgtacaaaat atctatcact actgatagga ggttaatatt tctgtgtaga 30540 aatgaaaattt gtttgtttt tagtatttag tgtagatgta cacattccag caatgtatt 30600 tgcaattg tggttgatgc ttgtgatat aaatgtactt ttcaatgta tactttcact 30660 tttaaaatgc ctgttttgtg ctttacaata aatgatatga aacctcctgt gtcggtaagt 30720 tggatatgtg ggtatttaaa ggattcataa tttcttagca atgataaatt agatacata 30780 tacacaaata tataagcttt cccatgaaa tattgagtttt ttaacactg gcatgttttt 30840 ccccccttgc agtatagtgg tagattggag gatctttcc attattgta tggctcttt 30900 cagcacaagt aatcctgata tcttcatttt ttttccttct gtttgattaa aaactgcatg 30960 tgtgtacaat gatcttttgg catacttcca ttgcattaac agtgaaattt ccttttatac 31020 atgaccactg tttcagacct gtactgctgc tataacagtt aacctttctg ttcttaattt 31080 gataatactt gatttccaag actgtttcgg cataactaat tttaaacagt tttcagatag 31140 tgaatatgag tagtctaata agaacagttt ttttccatgt gaagcaactc tttcaatgta 31200 tataatgtta gtgtgtttct ttctaaattt aggatagaaa agtgaatagt gtgcaaaaag 31260 tatagctaca ttgcatctgc cattgaaaca taaatggggt atggaaacgt tcaagctttt 31320 tttttttctt tatgcagtat agataagctt tgttttgtaa atgcacaagt ccaatcattg 31380 aatcaactta atttttttat gtacttgaag tcattttatt actctttaac actcatgctg 31440 aagttctgat attttgttga aatccattgt tttactcttt gcatatttgt tggctctttg 31500 catattaata tattagacta catgcaaata cagtctgtct tgccattgtc tgttgaagtg 31560 caggtttgat ccagccagta tagaactagc tctgtagggg tgaggaggac tgtgctgtgt 31620 atcatccttg attgtgttcc ttcaaggagc attgcactgt aagtacatca gaatgacaaa 31680 ttgatgaact gcaacagtat cttttgtca atgttccaca taatgcaaat gccatacgtt 31740 gtgtgaatat tatgttggaa tacagtgctg atacttgga aaaccataac tgcctcttaa 31800 tttaacatag aataatacat agttctgtat ttttttaaa gtgagcttaa tgggtaagta 31860 ttttttatat gctttagcta tagctaaaga aaactgatac ttaacaaagt tgaatagtat 31920 tattcactgg tgctcctaaa atattgtttt tcagtgtaaa atatgcatat cttctatatt 31980 tatatgaaa gtcttgaaat gtatcagaca gaaggggatt tcagtttgca aataatgagc 32040 aatgtagcaa ttttaacaca tttcataaat atatattttg tcattggtgg agagcaccat 32100 ttgttgtttt gaatacttt taaaggaaga ggtacaagga cataaatgtt gagattacct 32160 acaggatgga aatagcagta cagttcattg tagatatttt gaaatgtttt tgattgtttt 32220 atataaccta gagtgacttc ccttaccctt atttagatct gcatatatag ttctagtatg 32280 aagtttaata gttaaggagt tagctatttg ttatctttaa gagtagggta ttgacgtgaa 32340 caattgcagt atttgcatg atactgtttt atagatgacc ttttaggaaa gtggtgcatt 32400 tattaattga actgaagaag tagttcagtt gaattcagta tcataattca caaattggag 32460 gctgttgatt ttgattcatt taaggtttaa aatctttatt aattgcaaac agtgcaatta 32520 tttatacttc acagtgcctt cccagacctt ccaccttagg ttctgctgca aaaagcacca 32580 ggtaagcaca acctaaggac atatataaat aaatatttca atacattaat gttgtccctg 32640 tgaggtttt gtggttgtgt attcaaaggc aatctgctac tgcttcccca aaatgtattt 32700 tgttatttta tgctaccatc ttagtggaaa gtctgtaagt tgttaaagca actgtttaca 32760 tttctgggta atgtttttta ttttacttttt tttttttat taagacaaga aaatgatgag 32820 tagattgctg cagtaattga actacatcca aatctttttg tattttttcc ccaaatatag 32880 aagtgttaat attaagaaag gacaattaca cagttttcaa gatttaggaa atcacttgtt 32940 tagaaacttc aacagccttc acaatctgtt ttatatgatg gacagaaaat ttctttgccc 33000 tccaaaatta taatttcttt atttttttct tattctttaa ctataataat tcagtagga 33060 tattatggtt tagatttta ttatgattt ttcttagac aaagttata tgctgaagaa 33120 ggaaaaagtt aaggcagt atgttttgat aaaaggcatg tgcatcagtg aaatgttaac 33180 tgtatagcaa ataacctttc ataatctgta gcaatcagta ttttctgat ttaatatat 33240 tttaatact gacgctgcat ttaattttt tgccagttta aaatgttttgt gtgtttttat 33300 agatgatttt aactggtaca tattttgagt taagttgaat gtatgaagc agcatcttat 33360 cagttttgtt tattcgattt ctaaaatgtg ctgatccttt taaaactcct gcttatctct 33420 gcaacaaaga aaatattca aaatactgc cttcattttc accacagtg ctgaagagatgc 33480 tgcaagcacc aaatcatagc tcataaaatc aggtcctgag atagttaccc AAagagga 33540 atcctttgag tgtatgccat tggtgagccg atgagcatgg accatagaag ggctcaatgt 33600 agaaggtaa attggcaat caataattgag aaattgaaa tgtattccca tacatatat 33660 ggtatagggt gtaatgtacc tgcttttgat cacttttcat tttaaagtgc tattcacttg 33720 atcttaaatg ttccatgaac tgttaaattt cttaagttac atagttacta caccacattt 33780 atgtgtatgt tatgttttaa tagtcaatga taggtatgta caattgataa tataaagggg 33840 ctcattgaaa cttgagagcc tgttgagttt tggttagttg tagattgcat ttttataaaa 33900 aaaaatacag atagattgat gataatagat attggggcat tgtttctgtc tcatgagaat 33960 tcttttattc attaccataa gccttcactg atactataag cattatttta aatgacgctg 34020 atcttaagtc tgaaataaat ggaaagcaga aaaggtgagc cagttgattt gaatgcattg 34080 gatattagtg ttagaaacaa tgtatagttt agattgaaac tgaactgact tatttagcac 34140 ttaaacaaaa atttgacaat gtttttagtt ttttttaaga cagcttagtg tggtgatact 34200 tagaattcta tggtttgatg tttcttttag aaatgagaag tagattttta ttttttaata 34260 taaaaaatgg ttttaatact aaaactagta atttgatact agttgtttat aaacattgta 34320 aaatatatct tttaaacaaa ttatcttggt agttaattca taagggtggg tttgggtagg 34380 atagcagag tactttcaga gggaaagggg agtcattcag aagtgatagc attttatttg 34440 tttgaatact ctgccagtaa aatcagctgt acttagaaag ttatctgttg tgtagaataa 34500 tgatgtagag tttactaatc agtgaggatg tcttgttttt atttctgca aactctgcct 34560 cactttaaaa tgcattataa caatacctaa ttaaagataa ttttggctct gaaagttacc 34620 ttattttttg ttgagttagt gacttcattt ttcttgccac aatatagct tttgagggat 34680 ttttttaaat tggtgcttt aataagcaaa taaatcccag ggttttatt tcttcagtga 34740 taccctata gaaactctta aatgtatttg cgcatatata tatatatatt ttcttatgca 34800 tgctcgatgc atttcgtcc tgagaaaaat gttctctaca gaaactaccc gtgtgtaaaa 34860 agaagattgg cttaaaatgg ctactgtgat gggaacagtg tcttagggag atgcagcttg 34920 gacttgaggt aaattgaata ctttacaact gtggtttaga gtttgcttta atgacattgt 34980 atgtaaaagg tcacatgatt gctgtaattt tgtattcatt atggtttcct caataaatgt 35040 buy ctatta 35056 <210> 2 <211> 1968 <212> DNA <213> Homo sapiens <400> 2 atggcagact attcaacagt gcctcccccc tcttctggct cagctggtgg cggtggtggc 60 ggcggtggtg gtggaggagt taacgacgct ttcaaagatg cactgcagag agcccggcag 120 attgcagcaa aaattggagg tgatgcaggg acatcactga attcaaatga ctatggttat 180 gggggacaaa aaagaccttt agaagatgga gatcaaccag atgctaagaa agttgctcct 240 caaaatgact cttttggaac acagttacca ccgatgcatc agcagcaaag cagatctgta 300 atgacagaag aatacaaagt tccagatgga atggttggat tcataattgg cagaggaggt 360 gaacagatct cacgcataca acaggaatct ggatgcaaaa tacagatagc tcctgacagt 420 ggtggccttc cagaaaggtc ctgtatgtta actggaacac ctgaatctgt ccagtcagca 480 aaacggttac tggaccagat tgttgaaaaa ggaagaccag ctcctggctt ccatcatggc 540 gatggaccgg gaaatgcagt tcaagaaatc atgattccag ctagcaaggc aggattagtc 600 attggaaaag ggggagaaac tattaaacag cttcaggaac gggctggagt taaaatggtt 660 atgattcaag acggggccgca gaacactggt gctgacaac ctcttaggat tacaggagac 720 ccatataaag ttcacaagc caggaatg gtgttagagt taattcgtga tcaggcggt 780 ttcagagaag ttcggaatga gtatgggtca agaataggag gaatgaagg gatagatgtc 840 cccattccaa gatttgctgt tggcattgta ataggaaa atggagagat gatcaaaaaa 900 atacaaaatg atgctggtgt tcgcattcag tttaagccag atgatgggac aacacccgaa 960 aggatagcac aaatacagg acctccagac cgatgtcac atgctgcaga attackattaca 1020 gaccttctc gaagtgttca ggctggtaat cctggtggac ctggacctgg tggtcgagga 1080 1140 tttattgtgc siactgggaa aactggatta atataggaaaggaggtga aaccataaaa 1200 agcataagcc agcagtctgg tgcaagaata gaacttcaga gaatcctcc accaatgca 1260 gatcctaata tgaagttatt tacaatcgt ggcactccac aacagataga ctatgctcgg 1320 caaccatag aagaaaagat tggtggccca gtaaatcctt tagggccacc tgtacccat 1380 gggccccatg gtgtcccagg cccccatgga cctcctgggc ctccagggcc tggaactcca 1440 atgggaccat acaaccctgc accttataat cctggaccac caggcccggc tcctcatggt 1500 cctccagccc catatgctcc ccagggatgg ggaaatgcat atccacactg gcagcagcag 1560 gctcctcctg atccagctaa ggcaggaacg gatccaaatt cagcagcttg ggctgcttat 1620 tacgctcact attatcaaca gcaagcacag ccaccaccag cagcccctgc aggtgcacca 1680 actacaactc aaactaatgg acaaggagat cagcagaatc cagccccagc tggacaggtt 1740 gattatacca aggcttggga agagtactac aagaaaatgg gtcaggcagt tcctgctccg 1800 actggggctc ctccaggtgg tcagccagat tatagtgcag cctgggctga gtattataga 1860 caacaagcag cctattatgc ccagacaagt ccccagggaa tgccacagca tcctccagca 1920 cctcagggat ttgcaaatca tgcaagaagc caccaccatt tatattaa 1968 <210> 3 <211> 1935 <212> DNA <213> Homo sapiens <400> 3 atggcagact attcaacagt gcctcccccc tcttctggct cagctggtgg cggtggtggc 60 ggcggtggtg gtggaggagt taacgacgct ttcaagatg cactgcag agcccggcag 120 attgcagcaa aaattggagg tgatgcaggg acatcactga attcaatga ctatggttat 180 gggggacaaa aaagaccttt agaagatgga gatcaccag atgctaagaa agttgctccct 240 caaaatgact cttttggaac acagttacca ccgatgcatc agcagcaag cagatctgta 300 atgacagaag atacaagt tccagatgga atggttggat tcatattgg cagaggaggt 360 gaacagatct cacgcataca acaggaatct ggatgcaaaa tacagatagc tcctgacagt 420 ggtggccttc cagaaggtc ctgtatgtta acggacac ctgaatctgt ccagtcagca 480 aaacggttac tggaccagat tgttgaaaaa ggaagaccag ctcctggctt ccatcatggc 540 gatggaccgg gaatgcagt tcaagaatc atgattccag ctagcaggc aggattc 600 attggaaag ggggagaac tattaacag cttcaggaac gggctggagt taaaatggtt 660 atgattcaag acggggccgca gaacactggt gctgacaac ctcttaggat tacaggagac 720 ccatataaag ttcacaagc caggaatg gtgttagagt taattcgtga tcaggcggt 780 ttcagagaag ttcggaatga gtatgggtca agaataggag gaatgaagg gatagatgtc 840 cccattccaa gatttgctgt tggcattgta ataggaaa atggagagat gatcaaaaaa 900 atacaaaatg atgctggtgt tcgcattcag tttaagccag atgatgggac aacacccgaa 960 aggatagcac aaatacagg acctccagac cgatgtcac atgctgcaga attackattaca 1020 gaccttctc gaagtgttca ggctggtaat cctggtggac ctggacctgg tggtcgagga 1080 1140 tttattgtgc siactgggaa aactggatta atataggaaaggaggtga aaccataaaa 1200 agcataagcc agcagtctgg tgcaagaata gaacttcaga gaatcctcc accaatgca 1260 gatcctaata tgaagttatt tacaatcgt ggcactccac aacagataga ctatgctcgg 1320 caaccatag aagaaaagat tggtggccca gtaaatcctt tagggccacc tgtacccat 1380 gggccccatg gtgtcccagg ccccatgga cctcctgggc ctccaggggcc tggaactcca 1440 atgggaccat acaccctgc accttataat cctggaccac caggcccggc tcctcatggt 1500 cctccagccc catatgctcc ccagggatgg ggaaatgcat atccacactg gcagcagcag 1560 gctcctcctg atccagctaa ggcaggaacg gatccaaatt cagcagcttg ggctgcttat 1620 tacgctcact attatcaaca gcaagcacag ccaccaccag cagcccctgc aggtgcacca 1680 actacaactc aaactaatgg acaaggagat cagcagaatc cagccccagc tggacaggtt 1740 gattatacca aggcttggga agagtactac aagaaaatgg gtcaggcagt tcctgctccg 1800 actggggctc ctccaggtgg tcagccagat tatagtgcag cctgggctga gtattataga 1860 caacaagcag cctattatgc ccagacaagt ccccagggaa tgccacagca tcctccagca 1920 cctcagggcc aataa 1935 <210> 4 <211> 39750 <212> DNA <213> Cynomolgus monkey <220> <221> misc_feature <222> (1246)..(1266) <223> n is a, c, g or t <220> <221> misc_feature <222> (2077)..(2131) <223> n is a, c, g or t <220> <221> misc_feature <222> (2666)..(2690) <223> n is a, c, g, or t <220> <221> misc_feature <222> (3304) (3345) <223> n is a, c, g, or t <220> <221> misc_feature <222> (3869)...(3870) <223> n is a, c, g, or t <220> <221> misc_feature <222> (6076)..(6076) <223> n is a, c, g, or t <220> <221> misc_feature <222> (6091)...(6093) <223> n is a, c, g, or t <220> <221> misc_feature <222> (6159)...(6183) <223> n is a, c, g, or t <220> <221> misc_feature <222> (6259)..(6277) <223> n is a, c, g, or t <220> <221> misc_feature <222> (6869)...(6906) <223> n is a, c, g, or t <220> <221> misc_feature <222> (7072)..(7139) <223> n is a, c, g, or t <220> <221> misc_feature <222> (7714)..(7714) <223> n is a, c, g, or t <220> <221> misc_feature <222> (11374)...(11394) <223> n is a, c, g, or t <220> <221> misc_feature <222> (11454)...(11491) <223> n is a, c, g, or t <220> <221> misc_feature <222> (12562)..(12562) <223> n is a, c, g, or t <220> <221> misc_feature <222> (12699)...(12742) <223> n is a, c, g, or t <220> <221> misc_feature <222> (12793)...(12794) <223> n is a, c, g, or t <220> <221> misc_feature <222> (13648)...(13656) <223> n is a, c, g, or t <220> <221> misc_feature <222> (14247)...(14261) <223> n is a, c, g, or t <220> <221> misc_feature <222> (14474) (20234) <223> n is a, c, g, or t <220> <221> misc_feature <222> (21758)..(21789) <223> n is a, c, g, or t <220> <221> misc_feature <222> (23223)..(23225) <223> n is a, c, g, or t <220> <221> misc_feature <222> (24454)..(24512) <223> n is a, c, g, or t <220> <221> misc_feature <222> (24563)..(24589) <223> n is a, c, g, or t <220> <221> misc_feature <222> (25270)...(25338) <223> n is a, c, g, or t <220> <221> misc_feature <222> (25723)..(25734) <223> n is a, c, g, or t <220> <221> misc_feature <222> (25857)..(25857) <223> n is a, c, g, or t <220> <221> misc_feature <222> (26517)...(26523) <223> n is a, c, g, or t <220> <221> misc_feature <222> (27589)..(27590) <223> n is a, c, g, or t <220> <221> misc_feature <222> (27660)..(27688) <223> n is a, c, g, or t <220> <221> misc_feature <222> (28733)..(28733) <223> n is a, c, g, or t <220> <221> misc_feature <222> (30266)..(30266) <223> n is a, c, g, or t <220> <221> misc_feature <222> (30317)...(30371) <223> n is a, c, g, or t <220> <221> misc_feature <222> (30453)...(30768) <223> n is a, c, g, or t <220> <221> misc_feature <222> (30833)...(30837) <223> n is a, c, g, or t <220> <221> misc_feature <222> (30906)...(30912) <223> n is a, c, g, or t <220> <221> misc_feature <222> (30997)..(30998) <223> n is a, c, g, or t <220> <221> misc_feature <222> (31271)...(31562) <223> n is a, c, g, or t <220> <221> misc_feature <222> (31697)..(31697) <223> n is a, c, g, or t <220> <221> misc_feature <222> (31707)...(31782) <223> n is a, c, g, or t <220> <221> misc_feature <222> (32257)..(32257) <223> n is a, c, g, or t <220> <221> misc_feature <222> (33477)..(33478) <223> n is a, c, g, or t <220> <221> misc_feature <222> (37353)..(37427) <223> n is a, c, g, or t <400> 4 gagatttcgt tccttcagtc tccacccctt tacggcacga tggtcgcgca agaatgtgat 60 acagcttcga cggccgccat tttctttctt tcttagctgt tagctgagag gaagtctctg 120 aacgggcggc agcggctagc gtagtgtaac catggcagac tattcaacag tgcctccacc 180 gtcttctggc tcagctggtg gcggcggtgg cggcggtggt ggtggaggag ttaacgacgc 240 tttcaaagat gcactacaga gagcccggca ggtaagtgtg gaccgcgcgg cggaatcccg 300 aaagctcacg gtaattggcc gctgactgag taggccgcta ccctttagcg catgaggaag 360 aggaaagagg tgtccttccg ggctgaaatg tgaggagaca cgtttcccct tgttggtaat 420 aaagattaga gaccagaact cagttttgtg ttcttggtgt gtaatccact tagaaccccg 480 acgcgtgcta cgcaaaggcc tgaagtcttt ctcccgcttc tgcggcactc gtgtgtcgcc 540 agcgagctag cttagcctcc ccttttcctc gagatgaaga tcctcttcca ggggataaag 600 cgcaggtagt ttcacacaat ttaatggaag gttctggtaa tcagtttggg aaagaactag 660 ggtcggtctc ctggagccat agcaagggaa gggatttgtc gttaaagtag cctttacagc 720 tcattccgt tccctcgc attaaaacc gctttccgta cctttcacct tctcacctct 780 acaaggaagg gacttgaaag ccgtcttttt ctgggcggga tttacgcgtc agtctgttct 840 aacgtcagt cccccttatt tctcaaaatg gcctcaggcc cattatacca gaggtttcaa 900 tttgaatctg cctctctgtt aagagtcgta aactgaccag acctctttgt attacgtagt 960 gcgtacattt gccctgaaga caccactttc ccagacgaaa gctgttaaaa tagtgcgagt 1020 attccaggaa atagagat ttcttaattt gaactttaca ttttagata gtcccctaat 1080 atatttaaaa ttcaaatg tattggtgttg gtaggattt gcatgtaagc aaagaattc 1140 tattctctat gandacatgc atgttgtact aggtgctgga catttttact agtgttaagc 1200 taatggtagg tagaaccag tgttgtgctg tgttcatt gcattnnnnnnnnnnnn 1260 nnnnnncggt acagttaaag gaacattgag tcaaatcag aattcataa atccgttgta 1320 acatacctaa tgtgaacaca ttatcacat gtacctgtac ttgtttataa ccagagatat 1380 tagtagt caaaaagg tattcattaa tatttctt gcggttttt cacataattt 1440 aagaaattct gaacatgttt agcagcaagc atttgtataa ctatgacaaa cactgttagc 1500 gcgttttatt agatgattt gtaaaactta actattgtat ttattggaac caactatttt 1560 attactag aaccagcaca ttggattact tagtatcaa cactgtaggt agatactgga 1620 gttttgtttt gttttgtttg agacggagtc tccctctgtc gcccaggctg gattggagta 1680 gcgtggtgcg atctcgtccc actgcaacct ccgcctccgg cttcaagcaa ttctcctgcc 1740 tcagcctccc gagtggctgg aattacaggc gcccgccacc gtgcccggct aattttttgt 1800 tttgtttttg ttttttaagt agagacaggg tttcaccatg ctggccaggc tggtctcgaa 1860 ctcctgacct cgtgatccgc ccgccttgac ctcccaaagt gctgcgatta cagcagtgag 1920 cccggccttt tttgttgttg ttgtttaaat gcatgaattg tttcctacta agaagctatg 1980 atatagttcc ttgaccaaat gcagatgaac aggattatct gataaattga ttaacgagag 2040 cagacaaaat acgggtattt aattcatctg ctcattnnnn nnnnnnnnnn nnnnnnnnnn 2100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngttgggagg ggatacccag cttcctaagt 2160 aaaggagtga tatttgagca aggcattata gaataaatga gagtttggcc agtagggaaa 2220 agaagcagca tgcggagagg tatgggacgt gggggaatga caagaaccca gaattgaagt 2280 cactagaaga tggtggtggg aggacgtgaa acaaggcaag gaaaggtaca agcggatcca 2340 gatactggaa gactttgtgt tgcaatgctt taatgaaaaa ttggattcg ggccgggttc 2400 ggtggctcat gcctgtaatc ccagcacttt gggaggctga ggcagacaga tcacttgaag 2460 tcggggttta agaccagcct ggccaacatg gtgaaacccc atctctacaa aaaatacaaa 2520 aggtagccgg gcttggtggc ctgtgcctgt agttccagct acttgggagg ctgaggcaca 2580 agaattgcct gaacctggaa agtggaggta gcagtgagtg gagatcatgc tgctgcactc 2640 cagcctgggc atagagcgag accctnnnnn nnnnnnnnn nnnnnnnnn ggttttatta 2700 ttaggctaag ccataatttc tttctacttt ttaaaataca gaaattgcca tcaaactgaa 2760 atttattgtc ttatatgcta ataggtcagt tgttctgtct ttactgtaat gatttcttta 2820 tgaaaatgat cttaaatctg agattcctaa ctttggcttt taactagaat tacccattac 2880 atttgatgat gtctttaaat gtcagttgta gctattagtc tgaataatg tgtggaatat 2940 acagaagggt atttagacag aagttaggtt aacatctgaa ctacttcctc cttgcgtatt 3000 taagagaata ttgagttagg tttctagaat cctcaactaa ctctaagttt atttctttg 3060 tctagaatac tatgctgttt ttgtttttgg aaggaagaga tataagaaca gtttgctgct 3120 ctcaaggagc ttcaaaggct gtaccagtgg ggatgccatt ggtatattta gctggatagt 3180 tgttattcag aaaagcagga caagtaattt tgattcctgg tccgtacttg gtaatgtcag 3240 taatgttaac tctagctggt tgttgacatc tggtcattta gttgccaatt cctttttttt 3300 tttnnnnnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn nnnntattc tgagctcaat 3360 tcaacacagg gacaccaggt tgctgctttt gttcatggtt tccagcctgt gcacttaaga 3420 aatttatttt tatgtatttc aagctgtaac tccagagatt gggattgttt gattgcgtcc 3480 ttagcagtga tactaatagc aacttctgtc tctagaacat tggaaaatta aaatgtattt 3540 atctaccgtt ttttcccttg aggttatatg aaggtagaaa tgaatcagac tagatgatta 3600 gctaagcaaa actgttaacc ctcatccctt ccccttaga caactatgaa attagtcagt 3660 atgtattcga tccttcttgc aatctcttct ctgacaatta taaaagtgat tcaggctgca 3720 taatgttgtt tgaatgaaat gaaaatacag actagagctg tttttggtttt tgtttttttt 3780 tttatttacc atcagtctgt tcagtgaaaa ctaacattta agcatgattc ttttaaaaa 3840 tcatttgtg acagtttagc agggcttgnn tgataagcaa actatggtat ggtaatatttt 3900 ctagtgtgca cgtttcttca catgtctggt atatgggaac tctaactcca tcaggacttt 3960 gcctatagta ggtactcagc atttactgaa ttaaatcaat aaacattttt gatgaattaa 4020 agtacaagtc agacctctgt gtctgtgggc tctgcatctg caaattcagc caactgtgga 4080 tcaaaaatat tagaaaaatg gaatgacggt caacaatac aagtaatacg aatgaaaaca 4140 atacaactat gtacattgta tcaggtatta tagtaattt agagatgctt tagtatacc 4200 aaaggatttg cataggttgt atgcagatac tgtaccattt tgtgtaagga acttgagcat 4260 ctgtggattt tggtatttgc atggttcctg gaaccaatcc ctcagggaca ctgagggact 4320 atagttggtc ataccacctg atttagaga ttttctgatc ctcagaagtt aattaagtaa 4380 actacagtag tctgttctta acctcggagg atacattcca agaacctcag tgaatatctg 4440 aaaccacata tagtattgaa tccgatatat acacggtaat atttttcct atacatgtgt 4500 atctataaag tttaaattct aaattagaca tagtattaac aataataata aattagaaaa 4560 agactgggca tagtggttca cgcctataat cttaacactt taacttttaa ctatgacgtt 4620 gtcttgaaaa agaaatcagc cagccaaggt ggctcatgtc tgtaatccta gtgttttgag 4680 aagctcagtc aggaagtttg tttgaaccca ggaatccgag accaccctag gcaacatggt 4740 gaaaccttgt ttctataaaa aaaaaccaaa aaatgcca gacctggtgg tgcatgcctg 4800 tagtaccagc tacccaggag gctgaggtgg gaggagtgct tgaacttggg aggtcaagtt 4860 tacaatgttg acaatgttgg gtcctttacg tagttgtgta agtgagccat gatcatgcca 4920 ctgcactaca gccttgggca acagcctgac cctgtctcaa aattttaatt taattttaaa 4980 aatgaatag aacaattaca acaatacgct gtattactga caagaagggc aaatttttaa 5040 aaaaccaata tgctgtaata agttatatga ataaggggat cctcccctac cccagaatat 5100 ctgattgtac tataccgtag gtaactgcaa ccgtggagag caaaaactga agatactgtg 5160 tcttaagttt ctttttcaac tcccaaattc ttggatttct cacgtcttgg cttcctcagt 5220 agaggtgaga aatgctaaaa cagtgaaaac aggaaaaata acttactcat tcaagaagtc 5280 gattatggtc cagatggaaa atttgaatta tttttgtaaa actaaactaa agtagccagg 5340 caccgtggct cacgcctgta atctcagcac tttgggaggc tgaggcgggt ggatcacttg 5400 aggtcaggaa ttcaagacca gcctggccaa caaggtgaaa ccctgtctct actaaaaata 5460 caaaaaattag tcaggcatgg tggcgggcac ctgtagtccc ggctacttgg gaggctgagg 5520 tgggagaatc gcttgaacct ggggtgtgga cattgcagtg agcccagatc acgccactgc 5580 actccagcct gggcaacatt gtgagactac aaaaaatata atagtaagta aagtaaaaag 5640 tttcccatac ttgataaatg tctaataaaa attgaatatg ttctaggact ctgaaaaagg 5700 agttgaatat agttggaggt tggtttttag gaattatttt tcttaaatta attatccttg 5760 tagtcaccta ggaattgtat attttctgtt gatcttagaa aattgatcaa atctatagtt 5820 cattttgttt tttcaatttt tttttaaaga gatggggtct tactgtatta atgttgaact 5880 cctggcctca accaggtctc ccacctcagc ttctgaagta actgggattg caggtgccac 5940 tgagccaggc tcctttattg gtatttttat taaaagcttt tctctaatgt ctttgtaaca 6000 gttctcaatt tttgaaatgg tgttactcat tctttagagt aaactgtcaa ctttcatttt 6060 cttttttttt tttttntttt ttctttcttt nnnttgagac ggaatctcgc tctgtcaccc 6120 aggctggagt gtagtggtgg gatctcggct cactgtgann nnnnnnnnnn nnnnnnnnnn 6180 nnntctcctg cctcagcctc cagagtagct ggaattaacg ggcacgtgac accgtgccca 6240 gctacttttt ttttttttnn nnnnnnnnnn nnnnnnngat gggggtttca ccatgttggt 6300 caggctggtc tcgaactcca gacgtcaggt gatccaccca cctcagcctc ccaaagtgct 6360 ggtattacag gcatgagcaa cagcgcccag cctcaacttt cattttcatt tggttagttt 6420 ttgaactatt cagtgggtaa ggttgtataa atgtcttttc tctgtataga agtttcttgg 6480 agttcaagga gtgctacttt gcaaactcat agagtattta taaaagctaa ctgcagaagg 6540 tgttcatagg ctaaaccgtt tcctattctt ggtagcacca ttttctctgg cctgaaatac 6600 tttccctcta ctattagtgt ctgtcagtgc ccagcagtgt atttactttc ctgaggaaca 6660 attcaaatgc taagtgcttt aagacctaag ggtggaaaag cagtgttttc aggcattatt 6720 aggaaaataa gattttaatt agacacccag aaacaaaaac aggtttgtaa ttggtaaagt 6780 gaaagatggt taaagaaggt tagattgacc aaagcttag ttttcttttt tttcttttttt 6840 tttttttttt ttttgagac aggtctcnn nnnnnnnnn nnnnnnnnn nnnnnnnnn 6900 nnnnnnggct agctgcaacc tccacttcct gggttcaaac agttctccca cctgagcctc 6960 ctgagtagct gcgaggcatg tgtcaccatg ctcagccatt ttttgtgtgt ttttagtaga 7020 gactgagttt ctccatgttg gtcaggctgg tctcgaactc ttgagctcag gnnnnnnnnn 7080 nnnnnnnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn 7140 gtacctttt ttaatcgagc aatctagttc ttgatcctaa tagtctttgt ggtgggtgtt 7200 tgcattttta gatgaggaaa agggaaatct aggagtccta ggaaatacag ttggtatatg 7260 ggaactgata cgtaattaga cttaagcaat ccatgttgaa tttgtgactt aagcacttaa 7320 ctataaattt accctcca gttgtctgat gataatcaaa acttgaagca gttatccata 7380 ttgggatctc tttggggaat cccagtcacc aaaagttagg ttttctttaa tattttttca 7440 tggaagtttt caaatatact caaaattgaa agaattatgt aataaattct catgagccca 7500 tcacacatct tcaataatga atgtacagca ttgcagtgtg gagcttggcc tattgctgac 7560 cactcatcaa tgtggcagaa ccactccatg gttccccatg gaaatgggag ctacttcagt 7620 tctcttttta cagaaaaatt caataaatat ctactgattg tgccctactt gtgacttgaa 7680 gccaggtttg tttgttgttg tttttctttt tttntttttt tttttttttt actagtcttg 7740 ctctgtcacc cgagctacag tgcaatggca tgatatcagt gcactgcaac ctccagctcc 7800 tgggttcaag tgattctctt gcctcagcct cccgagtagc tgggactatg ggcgtgcacc 7860 acaataccta gctaattttt gtatatagta gagatagagc ttcatcatgt tgcccaggct 7920 gctctcgaac tcctgagctc aagcagtcta cccacctcca cctcccaaag tactaagatt 7980 acaggcatga gccaccatgc cagcaaagct gggtatttct taaatttgtt cagtcaggtg 8040 caagaaatta tttgccctac tctgaaagtt aaaaatattc taagagaatt atggtttcgc 8100 agctggggtt ttttaagact tgagctcctg gggctcctgc atatatcctt agaacaacat 8160 tgtccagtag aagtacaatg taagccatgt tttgtttaac ccagcatatc caaaatatta 8220 ccccttttgc atgtgcttaa tataaaaaaat taagatgatt tctattccag gttttcaata 8280 ttcagtgagt aattttacac ttagagcaag tgtcatttca gactagtcac attttgagta 8340 ctcagtaacc acatatgggc tagtgctacc ttgctatagata gcatagcctt tgggtcccac 8400 aaagtgttca cattctctgt ttacacacat cttttgaagt gtcatgagag agagccagat 8460 aggatccaga tttctttat atctgtttgg tctgtcttac ggattcctag ttgattattt 8520 ctttgttgct tcttataggg atgtatcaga agcttaataa actcatgatt attatgttaa 8580 ctgcctgaag tattaatttt agcaacaaat tgaactataa ttttaatttt tcaagtaaat 8640 tttcttatgt gatactaagt atttcatata tacatgtatt agaagtaat gggagtttag 8700 taatgaatag tatatatagtag tttgtgatta tatttttctg tttttttttt ttagattgca 8760 gcaaaaattg gaggtgatgc tgggacatca ctgaattcaa atgactatgg ttacggggga 8820 caaaaaagac ctttagaaga tggaggtaag ttatactcat acgtatttta aattgttttt 8880 cagagtgttt agttgaagtg gttctcagta ttttttgtta ttctattgag acattacttt 8940 tattataaga ttgttaaaac tgtaaggtgt atattggcct aaaggggggg aagagaccta 9000 gagaaggtaa gtaaaatttc agtcaaccag aaagttttgt cttactagaa aacagtttat 9060 tttccctctt ttaggattgt gccagattga aagtttgcac agacgtcttg ttaataatat 9120 taaaaaaaca aaaatataaa ttgcttagaa gacacttcac tggttttact catacatgaa 9180 tggattttaa tatgctgtgt tttcgtcatt tttctatttg caattacact ttaaaggtcg 9240 aaattcgtat tgtttgctac tctagtgtgt tgcaggttat accgtttttt ttaatgttct 9300 ttgcttttag tacttttgtg tttcacgttt agctgtaaac ctatagatta aaacagtggg 9360 ttcacagtgc tttgtagttt taaaaatcat aatcattgaa gcttctgaac ttagaagtct 9420 gcatgtattt tttgttttag gtttgatgta tgagttttga tacattttct ttttaccctt 9480 ttttttaaag gaaggattct cactgaggct atagaatgta tttgtagctt ttgaccagga 9540 gaactttgtt tcctttattt aagtgtcttt catattata gtgaggtttt taatataaaa 9600 aaaaaaaatg agctaatgat gcttcagatg ttgtagtaa tgtattcttt ttattttag 9660 tgtagatggc tcttggacaa gtccgagcag tacacac tgggagga tgccctctcc 9720 ttttaaaggc aggaattttt attattacc tgtgttcagt atgtaaacgt gaaataaacc 9780 agtggattct taaatggaca caatattc ttggattg tgtctgaatt ttgctgcac 9840 aacattctga tggttaatca tttaagttg aagggggg gagaatgtag tactttgagc 9900 tataggttgt ctgttccaag gtatgcattg tattcatctg tgtaatggat ttagatgaag 9960 gtagtcatgt agttgccttg agttttttg ttttttgt ttttttgct aaagttttat 10020 acagtgaaat gtttgtttat aaatataga acagtttaag ttgagattgc cttgtaatgt 10080 tgtggggttt gtttttttt ttaattttt ggggcatagc ttgtggtca ctgtccgata 10140 tactcaactt ttaatatgtc agatttttgt agtttgatc gctttttcc ccccagtctt 10200 cagttcctga ggtggaagca tcattagcct ttagcatgtg atattttgct agtaatggac 10260 ctaaagtact ttgtcttgta tcattctaat gtgcataaca tacattaagg tcagtgattt 10320 gttaagaatc agataacat tttaatgtct gtgcatcttt tgaatgtgaa gagaatgaag 10380 tatagttct tttttagat actgagtttg tgttgaattt tggcagtttt ggttcaatg 10440 aaaccgta ccttcagata ttcataaa tgtttatatt tattctgtt tgggagggga 10500 gaagctttgt acttaatttgg taaaaaatta aagacactt aatttgcag atcaccaga 10560 tgctaagaaa gttgctccctc aaaatgactg taagtattcc ttttaactg gtcaaagc 10620 tatattaga ttttgttcat attattgggt atctttgaag ttagttggtt tatgagtatt 10680 ttggatcagc tagcctgaac ttctttgta atatgtacct ttttctccta ttttacaatc 10740 tgtcccatta attgtggcca ggtatatgta aggattggtt gccgaattat tttacatatt 10800 taacaactcc aattcttgat ctacgcttgt acaacttga aaaggaaattt atttgttct 10860 gtgccattgc tatataatg tcttccttt cattggctct cttaccctg tcagtgccaa 10920 tataaatatt ctgtataaaa tttgactc ttcaggtgt tgtctgtgca ttaggaaag 10980 gtttttgaat gtttgatgtg attttgttgt taaatacata agtaacttta aattttgaat 11040 tttcgtttta tttttattta attttttgat agcttttgga acacagttac caccgatgca 11100 tcagcagcaa aggtatagtc acaaactttt caaaaagtac tctgcaagtt ttggttgagc 11160 tgcatgtaaa aacacaacca cattggtata tattgaatat gtgtctgtat tttttggtgt 11220 acctagttca tatcactccc cttgggaagg taccataaag tgatgatttt tcttttgagt 11280 gagaaaaatt tgtgatttgg agagagagag ggaattagcc acagtttaga agaacagggg 11340 tgaattagag taactgttaa gatgacattc tctnnnnnnn nnnnnnnnnn nnnnacagtt 11400 catgccttca gactgttcca ttgatcatcc cttcttcact tgatgtatca tctnnnnnnn 11460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nattttttga catgagtctg agcatagaac 11520 cttagtttaa gccattggga gacattagac ttccattttt attaatagat tatcttttat 11580 ttgtaaacaa agtatctttc actgaaggaa aaatggtact ttctgttact ttttagcaga 11640 tctgtaatga cagagaata caagttcca catggaatgg ttggatttag tagtactt 11700 gatttttaaa gttttgaaa catgatcaa acatacttta gatctttca accaaaaaa 11760 ctttttttt ctaactagta attggcag gaggtgaca gatctcacgc atacacagg 11820 aatctggatg caaatacag atagctcctg gtaatgttac attctctgg tattttcaga 11880 gtgactagaa aagtagcttt ttttttct tttaggtttc tagtactaat aatgctgcct 11940 ttaatctttt gacctgaagt tctatttg ttttaattg tagacagtgg tggccttcca 12000 gaaaggtcct gtagttaac tggaacct gatctgtcc agtaagtttg aaatcttta 12060 aaatggactt aaagtaacaa cgggagaact ctttgaattt tctctctgct ctttgttact 12120 gctttattt acactactct tcgttgcct ccttccctcc caagtccctct gactccttg 12180 aagtttatgc ctcatgcctt tctcattag ggtttcat taaacacaga aaatggttaa 12240 aacaacttca tatctactcc agtctctact tacaaagcga agtgtagcct ggaggagaat 12300 gcgcagtaat gttgactggt vakacctaa actcagacat taagctcaag tggactgttg 12360 tgttgtctgc atttccctag ttccattcac ttttccattc ctctccaagg ctctttaata 12420 ctatatttcc ccatctccaa atcttcagca tctaacccca cctctctcct tcttaagctt 12480 atttacggag aaaatggaaa cgaatgataa gaagcttttc ttttccccta ccgctaaacc 12540 taccagcctt catttttttt cnctgttcac atagtactca ggtaattgct ttcctttgtg 12600 ttcatgtgcc taaagccagc cctttcttcc tattgaaggt tcagcctgca gttgtacttt 12660 cttctgcgtt attagttccc cccctttact agatttttnn nnnnnnnnnn nnnnnnnnnn 12720 nnnnnnnnnn nnnnnnnnnn nntgcaggtg catgccacca cacccagcta atttttgtat 12780 ttttttagta ganncagggt ttcaccatgt tggccaggat ggtgtcaatg tcttgacctc 12840 gttatccacc cgcctcggcc tccccaagtc ctgggattat aggcgtgagc cactgccccc 12900 agcctgattt tctttgtagc actttccaaa taatacgtta ttcatttgac atgttatttt 12960 tacttatttt aatgaaacga agccactcca gcaggaaccc tgtctctttg agtgttacca 13020 ccccattacc taaaatggca cttgcacatg gttgatattt aagtatttgt tgaatgaata 13080 attgtagcat atgagtaagt aaaatggtag tttaaaaatg taaataaata aatcttttag 13140 ttcttggaag aatcagttta attctgagat aactttagca ttagagttct tggaaattgt 13200 ggactattct taaaaataaa aattgtatat ctagaaaatt tattgcctaa tctctcaatc 13260 tttgaccctt gatggcattt tctttcagtt aaaagtaaaa acattgttaa agttagcatt 13320 aaggcaccta atcctgaatt ggggtaggag gagtacttgg ttacattgtt ttgtatttct 13380 ctatttgaat aaacttgggt atgctgcaac ttactattta aatattaatt tgttaacagg 13440 tcagcaaaac ggttactgga ccagattgtt gaaaaaggaa gaccagctcc tggcttccat 13500 catggcgatg gaccgggaaa tgcagttcaa gaaatcatga ttcctgctag caaggcagga 13560 ttagtcattg gaaagggggg agaaactatt aaacagcttc aggtattgtt atgtttgtga 13620 aatggctact tttggtctgt tttgatgnnn nnnnnngtct gctcccttt gttaatatgt 13680 attatttct atgattataa caggaacggg cgggagttaa aatggttatg attcaagacg 13740 ggccgcaaaa cactggtgct gacaaacctc ttaggattac aggagaccca tataaagttc 13800 aagtaaactt aactttatac tttataaaga aagagtttgg gttgaatggg gttgggcaaa 13860 atatgcatga ataattaaaa tgttttgaga cgtgctttct aaattagcta actttttcta 13920 ctttagcaag ccaaggaaat ggtgttagag ttaattcgtg atcaaggtgg tttcagagaa 13980 gttcggaatg agtatgggtc aagaatagga ggaaatgaag ggatagatgt aagtaaaat 14040 acccattcag aaatggttgt atgctaattc gtaaatatag tagtgttttc tgttttgtgt 14100 taaatagctc taacattgtt atcctttat ttcaccttta tactttagaa tacagaatta 14160 agtattttat atcttgtcac cctatttgct ataaataataa aattatatgt actattatga 14220 tttgaggcag attttcagga aatggtnnnn nnnnnnnnnn ncttttttt actttaaacc 14280 ctgagaagct agttttctta atactcagtc ttttttacat aaggtcccca ttccaagatt 14340 tgctgttggc attgtaatag gaagaaacgg agagatgatc aaaaaaaatac aaaatgatgc 14400 tggtgttcga attcagttta agccaggtga gtgcatataa taatcttgta agtgttggca 14460 gcaatgagtt ttgnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 15960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17340 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17520 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17640 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17700 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 17940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19260 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19740 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 20220 nnnnnnnnnn nnnnacaccc atttactgtt taattaattt tgtttctttg ttttgaagat 20280 gatgggacaa cacctgagag gatagcacaa ataacaggac ctccagaccg atgtcaacat 20340 gctgcagaaa ttattacaga ccttcttcga agtgttcagg tttgatagaa agttaacatt 20400 ttcatttttt gtttttatgg aaaaatactt tccttcatga aatctgaagt ttcctctata 20460 tcagagtctg cttgatgatg ctttattaat ggagaaagtt taaattgttt taaggtaaag 20520 atcttggagc aggaagaact actaccttaa gtgctacctt atttactctt taatttaaaa 20580 aaatgttatt acttatgatt atttgggccg gttcatctct acatttatgg ttcaggtatt 20640 tgagagctgg gaaaaataga cataattaat tttacatga aaaaagtat ttctgtgctg 20700 atttttctt tcttgtctat tcctctat aggctgtaa tcctggtgga cctggacctg 20760 gtggtcgagg agaggtaga ggtcaggca actggaacat gggaccacct ggtggactac 20820 aggaatttaa ttttattgtg ccaactggga aactggatt ataatagga aaggcaatg 20880 tattttaaac tcttaatgtt taacacatta ttcattttc tggacactt tctgttgct 20940 gttgtaaaca agtggcaatg ctttttctct ggctgtgttt tagtagaaaa gcattcttat 21000 gttaatacgt aacaagtaa acataatga aggatctaat gtatattat aaaagagca 21060 ggattttaat cttactagct tcatagagaaa gtgaactaag atattatta gcatagaaa 21120 ggtcttttga cccaaaaagt tgtttgagtg ttttgttg tgcattttgg ttttcccga 21180 ctcatatttt aaaaatttga atgtttataa gtgtattagt tattattat ctgcttttaa 21240 aagcagtttta ttcaatatt ttcattaat cacattaagg ttagtttta acataccaag 21300 taaatgtaaa tgtattttaa gagaagcatg aaatgcttcc taaatttag atttaagtg 21360 tagatatta atgaaaat cttaataca tactgtcaag tagatactg actgaccaaa 21420 ccatgttttt aatggcctat ttaattgtga ccatttctt ctaaatagct tctagtatac 21480 ccttgaaacc tttagagaaa ttactgtctt ttattttagg aggtgaaacc aaaaagca 21540 taagccaaca gtctgtgca agaatagagc ttcagagaaa tcctccacca aatgcagatc 21600 ctaatatgaa gttattaca attcgtggca ctccacaca gatagatt gctcggcaac 21660 tcatagaaga aaagattggt gtgagtatac tttaaacttt taatttag tgtagaccct 21720 tagattgtag ttaaattaag acgtttattc gatacannn nnnnnnnn nnnnnnnnn 21780 nnnnnnnnt tatagatttc atgataccta taatagatac atgtgaaga ttttccagca 21840 atgaaaataa cctaattaaa tgtgcagtta caggtttga gaacacctt acgttttgagt 21900 gtggatagat aggaggtgc agcatcata ttagtgttat tgtaggattg tggaacatac 21960 ttgaaggaca cagttaatgg gattcatta tttattaaga ttttactata ctgaacccca 22020 gcaaggcaaa caagataaat cagatgcatc ttccgctctg cagtagaaat tcgtaaatcc 22080 tagctttg actggctcac aaatcatctg ggttttaaaa tgtagactat ttgggtgctt 22140 ccaactccta cacataatat acagagacag acctgactca aaatgtctgg gatagggcc 22200 agcatctgat tttacacaga tgtttgggtt ttttcttcc agaaaagtttt tctgttagaa 22260 caaaaagtat aaaaagcttt gactgcttt ttgtaaatga ggcagatggt gtcttactgg 22320 agtttttaac acaaagtgtg cagggagcat cttaaattat taatcagaat ttcctaggaa 22380 aaattagttt tggtgtttgc catgctgtga atggagtgca tgcaaaacaa ggttgacgct 22440 gttttctgtg tacttgggaa gacaaaaata gatgtactaa agatgcttaa ggaacatctt 22500 ataaaatgta cacaaacatt gtaagttctg ttgtatggat agtttaattg ttcaaaaaat 22560 gaagatgttt gtggagtaat gataggaggt tgatgccata gcaaaaaaaa atgaatagca 22620 catcttggtt tttgatttta cagtattagc cttacatgca tggatcagga tttctgttgg 22680 attcatggta aaacagata gattgtttta gagaagcaat ttggtgtggt gattaagagc 22740 acggactctt ttcttgaat catttactgg gtctgtgacc tggagagt tgctcaggct 22800 tttctgtgcc ttggttcct cctataaaat gaggataatt gtatctattt catagagttg 22860 taggatta atgaacgttc acctgtgtgt cacttaaag aatgcctggc acatagccct 22920 aaaaaatgtt gctacttttt cagtattatt tttacttt ggaaagaata gatatgt 22980 aaataagtga gagatgacag cattgga cttaagaga caagttaata aaatactt 23040 taaaggtcat gaagtttagt ttccttatt tgcagtgaga atttacaac aaaatagt 23100 gttaccattt tgtccagcat gtcgcctggt tgtgttaact accagaagg agcattttag 23160 gaggagttaag tgtaatgtct tgttggctt aaaaacag aaatcagtta agcattatta 23220 atnnngatgt ggcatgcatg catgatagg ataaaata tcctgattta ttgaggact 23280 taaaaaggga atgtttgtgc tgtttagaat tatgatacat gaaaggccaa aagatata 23340 aattattgat ctgaatgggg ttcagtgac agaataggt tgtgaggtgg agttttggtt 23400 aatagtgaac caactagctg ttaacattat caatgaagtg ttcagaagac aattgatgat 23460 ataggaccca agcagtttgg gaatatattg tcaagatggt tgtcttatgt taggcaagta 23520 gatgtagagagaagctga agataaagac ctgatttctg tgattaagat gaaaaaagat 23580 agtaaaagaa agaattataa aaggaaaggg atctactgct gtcgcagaca agctaaagaa 23640 aatttaattg ctaattttaa tttaatttaa attgcattta attgctaccg ctattgattt 23700 tagtaaattt cacatgtctc cttattgg cagataaaat agtttttgca aaatgaatga 23760 ggaaaggaag gaaaacctaa aatttgttgt ttgtgactat aagagggtag aaatgggaaga 23820 ttatttgtcc atagagttgg tttgctgatt tttctatgtc ttcctttttt tttttgtagg 23880 gcccagtaaa tcctttaggg ccacctgtac cccatgggcc ccatggcgtc ccaggccccc 23940 atggacctcc tgggcctcca gggcctggaa ctccaatggg accatataac cctgcacctt 24000 ataatcctgg accaccaggc ccagctcctc agtaagtatt gggtttagtt ctgggctttc 24060 ccccaaagat tctagtttt ggactacatt tttatactga atttcttct cagtggtcct 24120 ccagccccat acgctcccca gggatgggga aatgcatatc cacactggca gcagcaggct 24180 cctcctgatc caggtaaaaa gatgcttatc atttgtgtgt tagctgtatt gttttcact 24240 cgtgttacat tattaaattt tctagtgttg attctacatt tgtatgcctc accttcactc 24300 actctactct ttcaacagcg ttaggcactg cctctacccc agtgtatata gaactgacat 24360 gaatatgatc tctgctttta tgaaattcct ttcagcttgc atttggcttt cttatgagtc 24420 gatatagcag aatgataaaa actaaaagct gcannnnnnn nnnnnnnnn nnnnnnnnn 24480 nnnnnnnnnnn nnnnnnnnn nnnnnnnnn nnaatatgta gtgtgttaat tgtgacatta 24540 tctatgaaaa aatagttttt acnnnnnnnn nnnnnnnnn nnnnnnnng ggcactgagg 24600 ctcacgtctg taatcccacc tgttcagaag tcgaggctga ggcttgagac cagcctaggc 24660 aacatggcaa gacccatctc taaaaaaatt tgttttaaat tagccagcca gggtggtatg 24720 tgtctgtatt tctagctgct cagaaggctg gagggggggc ggggattgct tgaggctaga 24780 agttctagat tgtattgagc tgtgggcgca ccactgcact cccctgggca acagaatggg 24840 accccatctc ttaaaaaata tatatatatg tatatataaa acagtcttac agagtgttaa 24900 gtatttgggc attaaactcc caaattgtca aataagatac cggttctagt actcctcata 24960 atgacaactt catgtgagta aaaatcaggc ctgtatttaa taactgcatg ctaaaaccca 25020 aatacattta attattttct atattcagat ctgtattttc gatatgtatc tattatatcc 25080 tgtactattc tggcgtcttt cataccctg tagttgctt tccatcttgg tcaaagagtc 25140 attctttgaa accagttaac atttatttat gatcttttttt tttcacctgc tacctacctt 25200 ttaggcctct ttcaccagag tagtttcaag gaaatatac tcaattatgg attactttct 25260 acacataatn nnnnnnnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn nnnnnnnnn 25320 nnnnnnnnnnn nnnnnnnntg ctgtttggaa tatgatgaca cagcatgata gttaccaaat 25380 aatcacaact tccattaatt caaggaatga cttgagttgt gctaggatat aggtatgcaa 25440 agttggggtt gtattctata gcaaaagaat gcactcccag cataccaaca ctgatagaag 25500 ggctcacaag gtacagaata taatactgta aacacgaatt tttggagaat gaaagggctt 25560 tgcttttcct cttgttggct aattgggatg gtataattaa gtaatgagat tgaagagttt 25620 gagtaggtta agagacagat tcatactggg taacttggat ctgccaggat tgtatttttg 25680 agcactactg ggtggttagc atgattgaga aaaaatgatg ggnnnnnnnn nnnnaagtgg 25740 tctttgtatc acatgttgag tttcccactt ggagtagtag tgaagtcact actataaaag 25800 ctggtcagtg aatgtggttt cagcatggcc tttgggcaag aagtaaccca tttaaancca 25860 gctggttggc cccactcaga tttatcaaag ggttactggg tctctggggt ggatattgct 25920 tatattagac ttagaatgac gtaacgtttt aatgtatgaa ctaaaatatt tctttaaaaa 25980 aagagtggtc tgttacggat ttatgtagtg gtcaagaatt tagacttcag agtcaaatag 26040 acctagatca gtcctagtcc tacagtttac taatggtgag atgtcaggca agtttttgaa 26100 ctcctctaag cctgttttct tatctataaa ttgatcaatg aatgaatctt gggttgagtg 26160 aatatttagt aaattcttag tacatactag ttatttgtga gactggtact tcagtatggt ttacacgttt gggtgtagaa ataacacttc ctaaagtctg ttttatctca aattctctgt ccaggcatag tgtaaagtga fathercctaga tttcttgatt fathercaga fatherccgg gcgccatggc taacacctgt aatcctagta ctttggggaga ctgaggcggg cggttcactt gaggtcagga gttggagacc agcctagcca acatggcga aacctgtctc tactaaaaat acaaaaatta gctgggtgtg gtggcgggtg cctgtaatgc cagctacttg ggaggcnnnn 26520. 26580. nnnggagaat tgcttgagaa tcgctccact gcattccagc ctgggcaaca gagtgagaca cttcatctca aaaataata fatheraccg fathergacac tattgagata tgtaaacatc caggatacaa aagcagtaca ttgggcaatt gagaaagct tggagggtgt cctaaaaaca ccttacaatt atattacctt gtagttttct ttttctacaa attctcactc ctttttctca aacttgacta acctttgtta agcagctgag aattgctact gttcagaatg aaagcataat agaaattta aaagttttaa ttgtatgata ttcctagtat aaaggacaga atcaagtttt tttgtggttt ctagaagatt gagaggtctc aaactgtttg cacttcagtt gatgtggggag atgagtgagg gtcagtcaag tgtagagga acatagctaa aagctgagac atgggcatag tgatttctga aaagtacaag cactgtgttg tggctggagc ttgggtgtta aagagataca aatgaagga gaggtgaggc tgaatgga aggataagcc aggggattca gactgttaa gatgttgaat gataggctgt acttaagctt tgaccatcct gaagattcta gagaatgaag aatttcaagt aggatgacat caagtttata acattcaatg tgaaatagga tgaaatgtgg ctaatctttt ttttaagatt tttatatttt ctcttcattg aaaatagga caaagttcat tgttctaaaa tattgtttc tttcttatgc aggttagccc ctagaaatgt ttttcttaag tcatcttcca gatagagctg tttgtgcttg aggcgaacc aatttagaaa aaaacaaggg cacaggatgg tttgagacag agcattggat ttggagccaa aagaccttta ttcatatccc agttatgcaa ctcagtagtt tttaacctgg gaaagtcact tggtctctga gccttgtttt ctttaataaa agctgatggt aacaacaaaa ataatagaaa atataaaann tttcatgtac 27600 ttttttttaa tgttctagtt aattttggga gctgtatatt tgccagagag ctgggggggn 27660 nnnnnnnnn nnnnnnnnn nnnnnnnngt gagaattaca gacttcaaca tgcaaacctt 27720 gaactttcat ttattctagc taaggcagga acggatccaa attcagcagc ttgggctgct 27780 tattatgctc actattatca acagcaagca cagccaccac cagcagcccc tgcaggtgca 27840 ccaactacaa ctcaaactaa tggacaaggt aactaaagaa cttatatgtg aaagtcaaaa 27900 cttgtgcttg gaattata tgaagtacat cactgtataa tacctaaaat ttctaacatt 27960 attaattat aatttaagca gacttttcct ttttaaatt gttacctgga aatagtttca 28020 aactttcata aaagttataa gaataccagg agccacccat ataaccagct gttaatgttt 28080 tatcccttgt ttactctaat accgtctata tcatacaagt gtatgggtgt gtatttattt 28140 taaatatcca ttataattag ttctgaagga aactaattat atccagtata attagttctg 28200 28260. aaagaatca tttaagacta aactatagac acaatgtcct gttgggtact tcttcaaaaa father tcatacagcc ctccaatca gtaagtcaac attachment tagctcttt atatcattat ggatttctga tttttgcagt gggttatatt ttttgaatt ttttataatt 28440. ttgataatca gactatccta gattttggcca gtcagggagt atattcaggg tggtgcctat atccttttga aatatctgtc gttattttaa gcacttccat actgtctggc acagtgagat tgttattttg tgttttatgg gctccaaccc ttaagtcagc tatttcttca aggagctctg attcctttta gtagtagtatg gtagttagaa acgaggcttg actatgcttg ttgctactga ggtgtaattg cttctaactt ctttcagcag acagaactag gaaatatt tacatacatg catacctaca tacacatgcc aaaaaacaca taacattga tatgtgtata tantttttaa aactatgttc attack attcatttca gcattttggg gttttcaagc cttttccttt tctgtacttg tttacaaatg gtgagaaacc tggtatcctc agtgtatctc cttattttac atacatttac ttatatgttc to attack gtcttccaaa caggttggtt ttcttttctg tccaccacct ctgtaccccc agtaccttct atctttggca ccagtaggga tgccaccacc 28980 acatagtacc ttcctcctac ccccactgt cacattgcag gcccctgcca gctcctgcac 29040 ccaaggaaac agctaaatt gccttttaa aactttaatt ctgttttttct gttttgtttt 29100 gttttgtttt gcttgatcaa ggagatcagc agaatccagc cccagctgga caggttgatt 29160 ataccaaggc ttgggaag tactacaaga aaatggtat gttttaca tttcttgaaa 29220 atacatactt aattaattg aaacacaagg tattctctc agaagagaa tattgaata 29280 aaatcactgg actcgtaaac ataccagac agttgcaat tatagtttt aaatttgtgg 29340 ttatatagca aggaaatatt tttcttcta attgcattg tcaccagtt atttattgaa 29400 actagaaattt tcctcactgg cacatacagt atttacatta ctatacttttt gatagacaca 29460 gttatata ttagtctgag tgacatagg ttaatttta atgtgtcagg tgaaaatgga 29520 ttgtgtgtga taccattatt ttgctgcaa gataagcagg tagaagtaa tctgtagtga 29580 gggaaagtaa ctaagtgatg gaaccagaat ctggcttcca agagggtctg agtcccaagc 29640 ttgtctccca aatttgtctc tttagggacc atttggaacc tggtactata cttctggaca 29700 aatcactatg tttcaactgc ttttgctctt taaaaattat tacataccac agctagatgt 29760 cacaaacgaa aactaaattg gtaagcttgg ttatccttca ctagcagaaa aagaatctat 29820 aggtggtagt tttgtcataa gagatcggtc tacttgggat tctcagtgta agtttcaatg 29880 ttttctttgc caaagaatgt ttctcattct ccgcagaaag aaaaatttcc agaagggtga 29940 tgattttaat ctttaggtg taaaattata tatacttgat gataaagatg ttctgcacaa 30000 ctggtttctt tttaaagaaa gaaaaaattg tttttcctca tagagtggct tcctaggaga 30060 gtcatattcc atctatttct gagactataa cagattaata tatgtttttg tgacgtagct 30120 taggcagatc tacagtagct gaaattccgc aaaaagaaac ttttaactta aaaacagcat 30180 actctgatta aggttggtta cataatatat tttctgaaca gggatctttt ttagaatgaa 30240 tagggatgct attaatcatg tcctgncggg cgcggtggct cacgcctgta atcccagcac 30300 tttgggaggc cgaggcnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30360 nnnnnnnnnn ncgtctctac taaaaataca aaaaattaac cgggcgcgat ggcgggcgcc 30420 tgtagtccca gctactcagg agcctgaggc agnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnta aaaatcttca 30780 acttctattt tctcaatggg taatgttccc tagatatggt ccctagttat tannnnntta 30840 agtagataaa aataaaggct tgttaatgga tttagctaat tactgaggaa tgagtttgac 30900 ttctgnnnnn nnttttacct tattgattat ttgtaatatg gccgttaata catttactgt 30960 ttagtctttt atgttttact tttttatgtt ttactcnntg agtgggtggg ttgaattctg 31020 atttttattg ttaagggaag aaattctaat tttcattgtt aagggaagaa attgtcttta 31080 ttgtctttaa tttttttttt caccatttcc ctgccagtta gagatactgt gctatactgt 31140 cttaaatcct ctgtaggaaa acatggcata gaaataatta aataataata ctgcatgact 31200 aaagaacctc cttaccccac cttttctatc atttttctgt agtaccacaa taatcacctg 31260 ttaattttat nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31320 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 31560 nntcacctgt taattttatg agcaaaatag attagttgaa aacagatgta ggttaagttt 31620 tccaaaattg gctttatatt ttaaagacaa ttctaaattc cccaggataa caccaaacta 31680 agttatttt eat eat eat nnnnnnnnnnnnnnnn 31740 nnnnnnnnnnnnnnnnnnnnnnn nnggttcaag caatcttct 31800 gcctcagcct cccaagtaga gaaatcttg atgagatttc agagagttat aaggaatggc 31860 agcaatctaa agatggtat ttttaaagct agaatagaag gattttt aagagactct 31920 tagagaca agcaggtttt caagtagaa aggagtatt cttttttga gaagtgaga 31980 catttgagaa aagtttgact taggaggc ccttacata aggactg tagtagactc 32040 tgaaccctta agaatctggt accttaagcc taaaggagggg tagagtgaga cttgtaggaa 32100 aggtacaagt ataggaag acaacgagt gtgaaaatct attcaagcaa ggctgactct 32160 taggcttaa taaaatctta caggctact gtagagcaag tcttatct tttggatgaa 32220 agaatgtaa ggctaaactt tattagatta attatnaa ctataataa tattagataa 32280 tgttctactg aagtgtaac tcaactgtct tgaatcaac agtaactaag ccatgatcac 32340 accactacca ctgtgttcca gcctaggtga cagagcaaga ccctgtctca aaacaaaatc 32400 caataactaa agagaataat aaggggagca aggtaaggca gtacatgtct gaaagggcaa 32460 ggaaagagtt ctttaaagtc taaagtgttt actattagct ttccacaggg atataggagat 32520 ttgcagacgt tgtagtttt taagggaaca ttgatggaat tttcttgatg aatatgcctg 32580 tttctaccat cttacatcta aaatgagtaa ctctcacagt gggtttaaaa cctgaagaat 32640 attgattgct accttgatca ggtttggttt caagtgtgca gcttgtaaga gaaaacgtgt 32700 ggttttattg ctgttgttac acgagagtat ttggcagttt tttaaatatt gaaacctgtt 32760 tcagatttgc ttgaattgtg tcttgtaaga ggcaaatgtt cctgtttgat taacagttct 32820 ttatagaaga caggaaata tgagaatttt taatagtcta tgagagttct ctgttaccca 32880 agaaaagagg gtttgtttca tgcattttta aagaatcata aatcttaaat tctttcactc 32940 agttgctttg attctgtcac ctattttaca atggtgatgg acaggtttct gaaactagga 33000 aattggcctt gttggcaaca tcatacaaac ataaaaacag cttcctatac gtccatgctc 33060 cagtactttt gtctgtggta attatttcac aaagccaagt gagttacaa cgaataaaa 33120 tagtgcttaa gtaaagcaac tgaataggaa aacacatctc tcttctcaat ttttatta 33180 gaaaattata taccttcaga aattggggaa attaggtacc actcagctc attcatcatt 33240 tttttaaagt ttgctgtatg tgctttatat gtacgttttt ctgtatgaac catttcaatg 33300 taagttgcag acagacagt tggctctaaa gaactcaat gtctggacac agtgattcac 33360 gcctgtaatc ccaggactt tgggagacca aggcaggtgg atcactttag cccaggatt 33420 aaagaccagc ctggacaacg ctgcaaacc ccatctctgc aaaaaagag aaaaatnnca 33480 gctaggcatg gtgggcttgt agtcccagct acttagagg cagaagtggg aggatcatct 33540 gagccgtggg aggtcgaggc tgcagcgagc catgaatgca ccgctgcact ccagcctggg 33600 tggcatagtg agaccctgtc tgaaataaa taccttcaat agtatcacaa aaatacagac 33660 tatattaaa tttccatagt tgtcaggaat atgtctttcg tcgtctgtgt gtttgttaaa 33720 ttcagagtgc attcaaagat tcgtgcattg catttcattg ttatatttct ttagtctctt 33780 aattcagcaa agtcatctca tttggagaaa gacctgatcg tgaaagctga gttatgtgtt 33840 tcattgatct tgttctttat tccttgtcac gttactttta ataccattgt gttgtaattg 33900 gaataaatta ttgatacttt taacatctta ctactaatct ctgtgtgtgt gtacatgtac 33960 atgtacatac actttatagg ttactaatca ggaaaaagtc ttggctaggt cttaaaataa 34020 taaaatcttt aaatcattgc aattaagtgg gttttttgtt tttaattctg gaatccaatc 34080 aagattatgc ctaaatcttt accttcctta gctaaagcag tgtggatttg gggttgattt 34140 tgttttttta ctaataatga cgctctagaa ctaaacatta tgattaatta taaggcaaaa 34200 agaaaataaa gagcgttttt tttttatttt agagtagtttt cctgacacta ctaatgaata 34260 ttttaaataa aacaggagta attctgaccc tctgtgcttt tgtcttatga ttgtaattta 34320 atattaaatt tagggggtttt ttttaggtca acaagggcag acacaagatt attcaaaggc 34380 ttgggaggaa tattacaaga agcaaggtat tgtttttact ggaaatgagg tgttggactt 34440 ttaattgtgg ttacagcaac aaaattcttt tttttcaaag gtcaagcagt tcctgctccg 34500 actggggctc ctccaggtgg tcagccagat tatagtgcag cctgggctga gtattataga 34560 caacaagcag cctattatgc ccagacaagt ccccagggaa tgccacagca tcctccagca 34620 cctcaggtat aatgtaattg ctaatttgtt gatttctact ccagtctgtt ttctgcatgt 34680 ttactgtttg tctgtttggg agtgtttgcc ttttaaattt ttatctggca aagtataact 34740 tatttaaatg aagtactacg gtgtattgtt tgggtttttt tgtttgtttt ttataatgtt 34800 ttccggcatc tgagtgctga atatttctgc aatgcctttg attttaaaaa taaattttct 34860 tcccccaggg atttgcaaat catgcaagaa gctaccacca tttatattaa ccagtttttc 34920 tttcttaaag gattcactcc tgaattagct ccatttcaag gattttcttt aactttttgt 34980 gtatttctta tgtatctctt ctgcacaggg ccaataataa gaagtggaca atacagtatt 35040 tgcttcattg tgtgggggaa aaaaaccttt gttaaatata tggatgcaga cgacttgatg 35100 aagatcttaa ttttgttttt ggtttaaaat agtgtttttt tttgtttttt tttgtttttt 35160 ttttttgcaa atgtacaaaa tatctatcac tactgatagg aggttaatat ttctgtgtag 35220 aaatgaaaat tggtttgttt ttagtatta gtgtagatgt acacattcca gcaaatgtat 35280 ttgcaattat gtggttgatg ctttgtgata taaatgtact ttttcaatgt atactttcac 35340 ttttaaaatg cctgttttgt gctttacaat aaatgatatg aaacctcctg tgtcggtaag 35400 ttggatatgt gggtatttaa aggattcata atttcttagc aatgataaat taagatacat 35460 atacacaaat atataagctt tccccatgaa atattgagtt tttaaacact ggcatgtttt 35520 tccccccttg foottagtg gtagattgga ggatcttttc catttatgt atttggctct 35580 ttcagcacaa gtaatcctgg tatcttcatt tttttcctt tgtttgatta aaaactgcat 35640 gtgtgtacaa tgatcttctg gcatacttcc attgcattaa cagtgaaatt tcctttttat 35700 acatgaccac tgtttcagac ctgtactgct gctataacag ttaacctttc tgttcttaat 35760 ttgataatac ttgatttcca agactgtttc ggcatacta attttaaaca gttttcagat 35820 agtgaatatg agtagtctaa tagacagt ttttccat gtaaagcaac tctttcaatg 35880 tatataag tgtgtttctt tctaaattta ggatagaaaa gtgaatagtg tgcaaaagt 35940 atagctacat tgcatctgcc attgaaacat aaatggggta tggaacgtt caagcttttt 36000 tttttttttt aagcagtata gataagcttt gttttgtaaa tgcacaagtc caatcattga 36060 atcaacttaa ttttttag tacttgaagt cattttatta ctcatgctga 36120 agttctgata ttttgttgaa atccattgtt ttactcttg catatttgtt ggctctttgc 36180 atattatat attagactac atgcaatac agtctgtctt gccattgtct gttgaagtgc 36240 aggttgatc cagccagtat agaactagct ctgtaggggt gaggaggact gtgctgtgta 36300 tcatccttga tgtgttcct tcaggagca ttgcactgta agtacatcag atgacaaat 36360 tgatgaactg cacagtatc ttttgtcaa tgttccacat aatgcaaatg ccatactttg 36420 tgtgaatatt atgttggaat acagtgctga tatcttgga aaccatact gcttcttaat 36480 ttaacataga ataatacata gttctgtatt tttttaag tgagcttaat gggtaagtat 36540 tttttatag ctttagctat agctaagaa aactgatact taacaaagtt gatagtatt 36600 attcactggt gctcctgaaa tattgtttt cagtgtaaaa tatgcatata ttctatattt 36660 atatgaaag tcttgaatg tatcagaagg gatttcagtt tgcaataat gagcaatgta 36720 gcaattttaa cacattcat aaatatatat tttgtcattt gtggggagca ccatttgttg 36780 ttttgaatat actttaaagg agaggtaca aggacataa tgttgagatt acctacagga 36840 tggaaatagc agtacagttc attagata tttgaatg ttttgattg ttttatataa 36900 cctagagtga cttcccttac ccttatttag atctggatat atagttctag ttgaagttt 36960 atagttaag gagttagcta ttgttatct ttaagagtag ggtattgacg tgagcaattg 37020 cagtatttg catgatactg ttttagat gaccttttag gaagtggtg catttattaa 37080 ttgaactgaa gaagtagttc agttgaatttc agtatcataa ctcacaatt ggaggctgtt 37140 gattttgatt cattttaggt ttaaaatctt tattaattgc aacagtgca attattatta 37200 cttcacagtg ccttcccaga ccttccacct taggttctgc tgcaaaaagc accaggtaag 37260 cacaacctaa ggacatatat aaataatat ttcagtacat taatgttgtc cctgtgaggt ttttgtggtt gtgtattcaa aggcaatctg ctnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnncca tcttagtgga aagtctgtaa gttgttaaag caactgttta catttctggg taatgttttt atttacgttt ttttttttta tttaagacaa gaaaatgatg agtagattgc tgcagtaatt gatctacatc caaatctttt tgtatttttt ccccaaatat agagtgtta agtagttag agcccaatta cacagttttc aagatttagg aaatcacttg ttcagaaact tcaacagcct tcacaatctg 37740. ttttatatga tggacagaaa atttctttgc cctccaaaac tatattct ttattttttt cttaactat aataattcag tagtagatt atgggttaca attttatt gttttttctg agacaaaagt fathergctag aaaaggaaaa father gcagtatgtt ttgataaaag gcatgtgcat cagtgaaatg ttaactgtac aggaataac ctttcataat ctgtagcaat 37920 footttttt ctgatttaat attttaataa ctgacgctgc atttatataa tttttttgcc 37980 agtttaaaat gtttgtgtgt ttttatagat gattttaact ggtacatatt ttgagttaag 38040 atgaatgtat gaagcagca tcttaccagt tttgtttat caatttctaa aatgtgctga 38100 tccttttaaa actcctgctt atctctgcaa caaagaaaaa tattcaaaaa tactgccttc 38160 attttcacac acagtgctga agatgctca agcaccaaat catagctcaa taaaatcagg 38220 tcctgagata gttaccta aagaggaatc ctttgagtgt atgccattgg tgagccgatg 38280 agcatggacc atagaagggc tcaatgtaga aggtaaaatt ggcaaatcat aattgagaaa 38340 tatgaaatgt attcccatac samaatggt atagggtgta atgtacctgc ttttgatcac 38400 ttttcatttt aaagtgctat tcacttgatc ttaaatgttc catgaactgt taaatttctt 38460 aagttacata gttattacac cacatttatg tgtatgttat gttttaatag tcaatgatag 38520 gtatgtaaat ataaagggac tcattgaaac ttgagagcct gtcgagtttt ggttagttgt 38580 agattgcatt tttattaaaa aaatatagat agatgaatga taatagatat tggggcactg 38640 tttctgtctc atgagaattc ttttattcat taccataagc cttcactgat aatataagca 38700 ttatttaaa tgacgctggt cttaaatctg aaataaatgg aaagcagaaa aggtgagcca 38760 gttgatttga atgcattgga tattagtgtt agaaacaatg tagattttag attgaaattg 38820 aactgacttt attagcact taaacaaaa...

Claims

1. An antisense oligonucleotide selected from the group consisting of the following: CTtAtgctttttatGgTT, AcCAAttttcatttCtAC, CTTatGctttttatgGT, CTTaTgctttttatgGT, CTtATgctttttatgGTT, CTtAtgctttttatgGTT, CTtAtgctttttatGGTT, GcttTttatggtTtCAC, and TATgcTttttatggtTTC, The uppercase letters represent β-D-oxyLNA nucleosides, the lowercase letters represent DNA nucleosides, all LNA Cs are 5-methylcytosine, and all nucleoside bonds are thiophosphate nucleoside bonds.

2. The antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide is CTtAtgctttttatGgTT.

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

4. A conjugate comprising the antisense oligonucleotide according to claim 1, and at least one conjugate moiety covalently linked to said antisense oligonucleotide. The conjugate portion is or a mixture thereof, The conjugate contains a linker located between the antisense oligonucleotide and the conjugate moiety, and The linker consists of two to five consecutive phosphodiester-linked nucleosides.

5. The conjugate according to claim 4, wherein the conjugate is selected from:

6. A pharmaceutical salt of an antisense oligonucleotide according to any one of claims 1 to 3 or a conjugate according to any one of claims 4 to 5.

7. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1 to 3, a conjugate according to any one of claims 4 to 5, or a pharmaceutical salt according to claim 6, and a pharmaceutical diluent, solvent, salt, and / or adjuvant.

8. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1 to 3, a conjugate according to any one of claims 4 to 5, or a pharmaceutical salt according to claim 6, and a pharmaceutical diluent, a carrier, a salt, and / or an adjuvant.

9. Use of the antisense oligonucleotide according to any one of claims 1 to 3, the conjugate according to any one of claims 4 to 5, the pharmaceutical salt according to claim 6, or the pharmaceutical composition according to claim 7 or 8 in the preparation of a medicament for treating or preventing hepatitis B virus infection.

10. Use in the preparation of a medicament for treating or preventing an effective amount of the antisense oligonucleotide according to any one of claims 1 to 3, the conjugate according to any one of claims 4 to 5, the pharmaceutical salt according to claim 6, or the pharmaceutical composition according to claim 7 or 8, in a subject suffering from or susceptible to a disease, wherein the disease is hepatitis B virus infection.

11. Use of the antisense oligonucleotide according to any one of claims 1 to 3, the conjugate according to any one of claims 4 to 5, the pharmaceutical salt according to claim 6, or the pharmaceutical composition according to claim 7 for the preparation of a medicament for the treatment or prevention of hepatitis B virus infection.

12. The use according to any one of claims 9-11, wherein the hepatitis B virus infection is a chronic hepatitis B virus infection.

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