Use of COPS3 inhibitor for treating hepatitis b virus infection

Abstract

To provide a COPS3 inhibitor to be used in treating HBV infection particularly chronic HBV infection.SOLUTION: Provided is use of a COPS3 inhibitor for destabilizing a cccDNA (covalently closed circular DNA) such as an HBV cccDNA. Also provided is a nucleic acid molecule which is complementary with COPS3, and can reduce a COPS3 mRNA level. A pharmaceutical composition and use thereof in treating HBV infection are also included.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to COPS3 inhibitors for use in the treatment and / or prevention of hepatitis B virus (HBV) infection, particularly chronic HBV infection. The present invention particularly relates to the use of COPS3 inhibitors for destabilizing cccDNA such as HBV cccDNA. The present invention also relates to nucleic acid molecules that are complementary to COPS3 and capable of reducing the expression of COPS3, such as oligonucleotides containing siRNA, shRNA, and antisense oligonucleotides. The present invention also includes pharmaceutical compositions and their use in the treatment and / or prevention of HBV infection.

Background Art

[0002] Hepatitis B is an infectious disease caused by the hepatitis B virus (HBV), a small liver-tropic virus that replicates via reverse transcription. Chronic HBV infection is an important factor for severe liver diseases such as cirrhosis and hepatocellular carcinoma. Current treatments for chronic HBV infection are based on the administration of pegylated interferon type 1 or nucleos(t)ide analogs, such as lamivudine, adefovir, entecavir, tenofovir disoproxil, and tenofovir alafenamide, which target the viral polymerase, a multifunctional reverse transcriptase. The success of treatment is usually measured as the disappearance of hepatitis B surface antigen (HBsAg). However, since hepatitis B virus DNA remains in the body after infection, complete HBsAg clearance is rarely achieved. The persistence of HBV is mediated by the episomal form of the HBV genome, which is stably maintained in the nucleus. This episomal form is called "covalently closed circular DNA" (cccDNA). cccDNA serves as a template for all HBV transcripts, including the pregenomic RNA (pgRNA), which is a viral replication intermediate. The presence of several copies of cccDNA would be sufficient to reactivate end-stage HBV infection. Current treatments for HBV do not target cccDNA. However, the elimination of cccDNA would be necessary for the cure of chronic HBV infection (reviewed by Nassal, Gut. 2015 Dec;64(12):1972-84. doi:10.1136 / gutjnl-2015-309809).

[0003] COP9 (constitutive photomorphogenesis 9)The COP9 signalosome is a protein complex with isopeptidase activity. It catalyzes the hydrolysis of the NEDD8 protein from the cullin subunit of the Cullin-RING ubiquitin ligase and is responsible for the deacetylation of the Cullin-RING ubiquitin ligase. Furthermore, it can bind to the deacetylated cullin-RING complex, thereby keeping the complex in an inactive form. Therefore, the COP9 signalosome functions as an inactivator of the Cullin-RING ubiquitin ligase. In mammals, the signalosome is involved in various processes such as signal transduction, protein stability, protein phosphorylation, cell cycle regulation, and apoptosis. The COP signal transduction body is found in all eukaryotes. In humans, the COP9 signalosome contains eight subunits and has a size of approximately 350 kDa. All subunits seem to be essential for the complete function of the signalosome. (Lingaraju et al. (2014) Nature. 512(7513):161-5. doi:10.1038 / nature13566. PMID 25043011).

[0004] COPS3( COP COP9 signalosome subunit 3 )is the third subunit of the signalosome and maintains the integrity of the complex. It has been shown to bind to the striated muscle-specific β1D integrin tail, and its intracellular localization changes in differentiated skeletal muscle cells. Other names for COPS3 are JAB1-containing signalosome subunit 3, signalosome subunit 3, CSN3, and SGN3. Some various publications have described the downregulation of COPS3 in target cells using RNAi-based techniques, and some of these publications are cited below.

[0005] Some various publications have described the downregulation of COPS3 in target cells using RNAi-based techniques, and some of these publications are cited below.

[0006] Ba et al. described the use of shRNA for the down-regulation of COPS3 in C2C12 cells. Down-regulation of COPS3 was accompanied by destabilization of several COP9 subunits and increased nuclear NF-κB localization and decreased growth rate (Ba et al., BMC Pharmacol Toxicol. 2017 Jun 17;18(1):47. doi:10.1186 / s40360-017-0154-5).

[0007] Kim et al. examined the effect of siRNA-mediated knockdown of each subunit of the COP9 signalosome in oocytes. COPS3 knockdown resulted in a metaphase I arrest, disruption of maturation-promoting factor (MPF) activity, and reduced degradation of anaphase-promoting complex / cyclosome (APC / C) substrates (Kim et al., PLoS One. 2011;6(10):e25870. doi:10.1371 / journal.pone.0025870).

[0008] Yoneda-Kato et al. showed that myeloid leukemia factor 1 controls p53 by suppressing COP1 via the COP9 signalosome subunit 3. Specifically, reduction of COPS3 protein levels by siRNA suppressed MLF1-induced G1 arrest and impaired activation of p53 by genotoxic stress (Yoneda-Kato et al., The EMBO Journal (2005) 24, 1739-1749. doi:10.1038 / sj.emboj.7600656).

[0009] COPS3 further plays a role in cancer. For example, Pang et al. showed that knockdown of COPS3 by shRNA inhibits lung cancer tumor growth in nude mice. (Pang et al., J Cancer. 2017 Apr 9;8(7):1129-1136.doi:10.7150 / jca.16201). Similarly, Yan et al. demonstrated that siRNA-mediated silencing of the COPS 3 gene decreases the proliferation and migration of HOS cells and may be related to metastasis (Yan et al., Cancer Gene Therapy (2011) 18, 450-456). Yu et al. showed that knockdown of COPS3 expression using shRNA in hepatocellular carcinoma cell lines (SMMC-7721 and Hep3B) exhibits in vitro growth inhibition and in vivo tumor weight reduction in xenograft mice (Yu et al., Cancer Chemother Pharmacol (2012) 69:1173-1180, DOI 10.1007 / s00280-011-1810-x).

[0010] To the best knowledge of the inventors, COPS3 has not been identified as a cccDNA-dependent factor regarding the stability and maintenance of cccDNA, and no molecule that inhibits COPS3 has been suggested as a cccDNA destabilizing agent for the treatment of HBV infection. Objectives of the Invention

[0011] The present invention shows that there is a relationship between the inhibition of COPS3 (COP9 signalosome subunit 3 or constitutive photomorphogenesis 9 signalosome subunit 3) in HBV-infected cells and the reduction of cccDNA, and is related to the treatment of HBV-infected individuals. The objective of the present invention is to identify a COPS3 inhibitor that reduces cccDNA in HBV-infected cells. Such a COPS3 inhibitor can be used for the treatment of HBV infection.

[0012] The present invention further identifies novel nucleic acid molecules that can inhibit the expression of COPS3 in vitro and in vivo. SUMMARY OF THE INVENTION

[0013] The present invention relates to oligonucleotides targeting nucleic acids that can regulate the expression of COPS3 and treat or prevent diseases related to the function of COPS3.

[0014] Accordingly, in a first aspect, the present invention provides a COPS3 inhibitor for the treatment and / or prevention of hepatitis B virus (HBV) infection. In particular, a COPS3 inhibitor that can reduce HBV cccDNA and / or HBV pregenomic RNA (pgRNA) is useful. Such inhibitors are preferably nucleic acid molecules 12 to 60 nucleotides in length that can reduce COPS3 mRNA.

[0015] In a further aspect, the present invention relates to nucleic acid molecules 12 to 60 nucleotides in length, such as 12 to 30 nucleotides, comprising a continuous nucleotide sequence of at least 12 nucleotides, particularly 16 to 20 nucleotides, that is at least 90% complementary to mammalian COPS3, such as human COPS3, mouse COPS3, or cynomolgus monkey COPS3. Such nucleic acid molecules can inhibit the expression of COPS3 in cells that express COPS3. Inhibition of COPS3 allows for a decrease in the amount of cccDNA present in the cell. The nucleic acid molecule can be selected from single-stranded antisense oligonucleotides, double-stranded siRNA molecules, or shRNA nucleic acid molecules (particularly chemically generated shRNA molecules).

[0016] A further aspect of the present invention relates to single-stranded antisense oligonucleotides or siRNAs that inhibit the expression and / or activity of COPS3. In particular, modified antisense oligonucleotides or modified siRNAs comprising one or more 2'-sugar-modified nucleosides and one or more phosphorothioate linkages that reduce COPS3 mRNA are advantageous.

[0017] In a further aspect, the present invention provides a pharmaceutical composition comprising a COPS3 inhibitor of the present invention, such as an antisense oligonucleotide or siRNA of the present invention, and a pharma- ceutically acceptable excipient.

[0018] In a further aspect, the invention provides a method of modulating COPS3 expression in a target cell expressing COPS3 in vivo or in vitro by administering to said cell an effective amount of a COPS3 inhibitor of the invention, such as an antisense oligonucleotide or composition of the invention. In some embodiments, COPS3 expression is reduced in the target cell by at least 50%, or at least 60%, compared to levels without any treatment or treated with a control. In some embodiments, the target cell is infected with HBV and the cccDNA of HBV-infected cells is reduced by at least 50%, or at least 60%, in the HBV-infected target cell compared to levels without treatment or treated with a control. In some embodiments, the target cell is infected with HBV and the cccDNA of HBV-infected cells is reduced by at least 25%, such as at least 40%, in the HBV-infected target cell compared to levels without treatment or treated with a control. In some embodiments, the target cells are infected with HBV and pgRNA in HBV-infected cells is reduced by at least 50%, or at least 60%, or at least 70%, or at least 80% in HBV-infected target cells compared to levels without treatment or treated with a control.

[0019] In a further aspect, the present invention provides a method for treating or preventing a disease, disorder, or dysfunction associated with the in vivo activity of COPS3, comprising administering to a subject suffering from said disease, disorder, or dysfunction ... The method includes administering to a subject suffering from or susceptible to the disease a therapeutically or prophylactically effective amount of a COPS3 inhibitor of the invention, such as an antisense oligonucleotide or siRNA of the invention.

[0020] Further aspects of the invention are conjugates of the nucleic acid molecules of the invention and pharmaceutical compositions comprising the molecules of the invention, in particular conjugates that are liver targeted, such as GalNAc clusters.

Brief Description of the Drawings

[0021]

Figure 1A

Figure 1B

Figure 1C

Figure 1D

Figure 1E

Figure 1F

Figure 1G

Figure 1H

Figure 1I

Figure 1J

Figure 1K

Figure 1L

[0022] The two different diastereoisomers shown in each of Figures 1A - D are the result of the conjugation reaction. Thus, a pool of a particular antisense oligonucleotide conjugate can contain only one of the two different diastereoisomers, or a pool of a particular antisense oligonucleotide conjugate can contain a mixture of the two different diastereoisomers. **DETAILED DESCRIPTION OF THE INVENTION**

[0023] Definitions HBV infection The terms "hepatitis B virus infection" or "HBV infection" are generally known in the art and refer to an infectious disease caused by the hepatitis B virus (HBV) and affecting the liver. HBV infection can be either acute or chronic. Chronic hepatitis B virus (CHB) infection is a global disease burden affecting 248 million people worldwide. Approximately 686,000 deaths annually are attributable to HBV-related end-stage liver disease and hepatocellular carcinoma (HCC) (GBD 2013; Schweitzer et al., Lancet. 2015 Oct 17;386(10003):1546-55). The WHO has predicted that, without further intervention, the number of people infected with CHB will remain at current high levels over the next 40 to 50 years and that a cumulative 20 million people will die between 2015 and 2030 (WHO 2016). CHB infection is not a homogeneous disease presenting specific clinical symptoms. Infected individuals progress through several stages of CHB-related liver disease over their lifetime. These disease stages also form the basis for treatment by the standard of care (SOC). Current guidelines recommend treating only specific individuals infected with CHB based on three criteria: serum ALT level, HBV DNA level, and severity of liver disease (EASL, 2017). This recommendation is due to the fact that the SOC, namely nucleos(t)ide analogs (NA) and pegylated interferon-alpha (PEG-IFN), are not curative and must be administered for long periods, thereby increasing the safety risk. NA effectively suppresses HBV DNA replication. However, it has very limited / no effect on other viral markers. The hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA), two features of HBV infection, are the main targets of new drugs aimed at curing HBV. In the plasma of CHB patients, HBsAg subviral (empty) particles outnumber HBV virions by a factor of 103 to 105 (Ganem & Prince, N Engl J Med. 2004 Mar 11;350(11):1118-29).That excess is thought to contribute to the immunopathogenesis of the disease, including individuals who cannot express neutralizing anti-HBs antibodies, which are serological markers observed after the resolution of acute HBV infection.

[0024] In some embodiments, the term "HBV infection" refers to "chronic HBV infection".

[0025] Furthermore, this term encompasses infection by any HBV genotype.

[0026] In some embodiments, the patient to be treated is infected with HBV genotype A.

[0027] In some embodiments, the patient to be treated is infected with HBV genotype B.

[0028] In some embodiments, the patient to be treated is infected with HBV genotype C.

[0029] In some embodiments, the patient to be treated is infected with HBV genotype D.

[0030] In some embodiments, the patient to be treated is infected with HBV genotype E.

[0031] In some embodiments, the patient to be treated is infected with HBV genotype F.

[0032] In some embodiments, the patient to be treated is infected with HBV genotype G.

[0033] In some embodiments, the patient to be treated is infected with HBV genotype H.

[0034] In some embodiments, the patient to be treated is infected with HBV genotype I.

[0035] In some embodiments, the patient to be treated is infected with HBV genotype J.

[0036] cccDNA (covalently closed circular DNA) cccDNA is the genetic template of the HBV virus that exists in the nuclei of infected hepatocytes, generates all HBV RNA transcripts necessary for productive infection, and is involved in viral persistence during the natural course of chronic HBV infection (Locarnini & Zoulim, Antivir Ther. 2010;15 Suppl 3:3-14.doi:10.3851 / IMP1619). cccDNA acts as a viral reservoir and is the source of viral rebound after treatment discontinuation, requiring long-term and sometimes lifelong treatment. PEG-IFN can only be administered to a small subset of CHB due to its various side effects.

[0037] Therefore, there is a strong need for new therapeutic methods that can bring about a complete cure defined by the degradation or removal of HBV cccDNA to the majority of CHB patients.

[0038] Compound As used herein, the term "compound" means any molecule capable of inhibiting the expression or activity of COPS3. Specific compounds of the present invention are nucleic acid molecules such as RNAi molecules or antisense oligonucleotides according to the present invention, or any conjugate containing such nucleic acid molecules. For example, herein, the compound can be a nucleic acid molecule targeting COPS3, particularly an antisense oligonucleotide or siRNA.

[0039] Oligonucleotide As used herein, the term "oligonucleotide" is defined as generally understood by those skilled in the art as a molecule containing two or more covalently linked nucleosides. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers.

[0040] The oligonucleotides described in the specification and claims are generally therapeutic oligonucleotides less than 70 nucleotides in length. The oligonucleotide can be, or can include, a single-stranded antisense oligonucleotide, or can be other nucleic acid molecules such as, for example, CRISPR RNA, siRNA, shRNA, aptamer, or ribozyme. Therapeutic oligonucleotide molecules are typically made in the laboratory by solid-phase chemical synthesis followed by purification and isolation. However, shRNA is often delivered to cells using a lentiviral vector and then transcribed to produce single-stranded RNA, which will form an RNA stem-loop (hairpin) RNA structure that can interact with the RNA interference machinery (including the RNA-induced silencing complex: RISC). In one embodiment of the invention, the shRNA is a chemically generated shRNA molecule (independent of cell-based expression from a plasmid or virus). When referring to the sequence of an oligonucleotide, the sequence or order of the nucleobase portions of the covalently linked nucleotides or nucleosides, or modifications thereof, is referred to. Generally, the oligonucleotides of the present invention are artificial, chemically synthesized, and typically purified or isolated. However, in some embodiments, the oligonucleotides of the present invention are shRNAs transcribed from a vector upon entry into a target cell. The oligonucleotides of the present invention can include one or more modified nucleosides or nucleotides.

[0041] In some embodiments, the oligonucleotides of the present invention include, or consist of, a length of 10 to 70 nucleotides, such as 12 to 60, such as 13 to 50, such as 14 to 40, such as 15 to 30, such as 16 to 25, such as 16 to 22, such as 16 to 20 consecutive nucleotides. Thus, the oligonucleotides of the present invention can have a length of 12 to 25 nucleotides in some embodiments. Alternatively, the oligonucleotides of the present invention can have a length of 15 to 22 nucleotides in some embodiments.

[0042] In some embodiments, the oligonucleotide or its contiguous nucleotide sequence comprises or consists of 24 or fewer nucleotides, such as 22, such as 20 or fewer nucleotides, such as 18 or fewer nucleotides, such as 14, 15, 16, or 17 nucleotides. It should be understood that any range provided herein includes the endpoints of the range. Thus, when a nucleic acid molecule is described as comprising 12 to 25 nucleotides, both 12 nucleotides and 25 nucleotides are included.

[0043] In some embodiments, the contiguous nucleotide sequence comprises or consists of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotide lengths.

[0044] The oligonucleotide(s) is for regulating the expression of a target nucleic acid in a mammal. In some embodiments, nucleic acid molecules such as siRNA, shRNA, and antisense oligonucleotides are typically for inhibiting the expression of the target nucleic acid(s).

[0045] In one embodiment of the invention, the oligonucleotide is selected from RNAi agents such as siRNA or shRNA. In another embodiment, the oligonucleotide is a single-stranded antisense oligonucleotide such as a high-affinity modified antisense oligonucleotide that interacts with RNase H.

[0046] In some embodiments, the oligonucleotide of the invention may comprise one or more modified nucleosides or nucleotides such as, for example, 2'-sugar modified nucleosides.

[0047] In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages.

[0048] In some embodiments, the oligonucleotide can be conjugated to a non-nucleoside moiety (conjugate moiety).

[0049] A library of oligonucleotides should be understood as a collection of variant oligonucleotides. The purpose of a library of oligonucleotides can be various. In some embodiments, a library of oligonucleotides is composed of oligonucleotides having overlapping nucleic acid base sequences that target one or more mammalian COPS3 target nucleic acids for the purpose of identifying the most potent sequences within the library of oligonucleotides. In some embodiments, a library of oligonucleotides is a library of oligonucleotide design variants (daughter nucleic acid molecules) of a parent or ancestral oligonucleotide, and the oligonucleotide design variants retain the core nucleic acid base sequence of the parent nucleic acid molecule.

[0050] Antisense oligonucleotide As used herein, the term "antisense oligonucleotide", or "ASO", is defined as an oligonucleotide that can regulate the expression of a target gene by hybridizing to a target nucleic acid, particularly a contiguous sequence on the target nucleic acid. Antisense oligonucleotides are not basically double-stranded and thus are not siRNA or shRNA. Preferably, the antisense oligonucleotides of the present invention are single-stranded. The single-stranded oligonucleotides of the present invention are understood to be capable of forming hairpin or intermolecular duplex structures (duplexes between two molecules of the same oligonucleotide) as long as the degree of complementarity within or between themselves is less than 50% over the entire length of the oligonucleotide.

[0051] Advantageously, the single-stranded antisense oligonucleotides of the present invention do not contain RNA nucleosides to reduce nuclease resistance.

[0052] Advantageously, the oligonucleotides of the present invention contain one or more modified nucleosides or nucleotides, such as 2'-sugar modified nucleosides. Further, it is advantageous that the unmodified nucleosides are DNA nucleosides.

[0053] RNAi molecule As used herein, the term "RNA interference (RNAi) molecule" refers to a short double-stranded oligonucleotide containing RNA nucleosides that mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC), where the RNA-induced silencing complex interacts with the argonaute, a catalytic RISC component. RNAi molecules regulate, e.g., inhibit, the expression of a target nucleic acid in a cell, e.g., a cell in a subject such as a mammalian subject. RNAi molecules include single-stranded RNAi molecules (Lima at al 2012 Cell 150:883) and double-stranded siRNAs, as well as short hairpin RNAs (shRNAs). In some embodiments of the invention, the oligonucleotides of the invention, or contiguous nucleotide sequences thereof, are RNAi agents such as siRNAs.

[0054] siRNA The term "small interfering ribonucleic acid" or "siRNA" refers to small interfering ribonucleic acid RNAi molecules. This is a class of double-stranded RNA molecules, also known in the art as short interfering RNAs or silencing RNAs. siRNAs typically include a sense strand (also called the passenger strand) and an antisense strand (also called the guide strand), each strand being 17-30 nucleotides in length, typically 19-25 nucleosides in length, with the antisense strand being complementary to the target nucleic acid (suitably a mature mRNA sequence), e.g., at least 95% complementary, e.g., fully complementary, and the sense strand being complementary to the antisense strand, so that the sense and antisense strands form a duplex or duplex region. The siRNA strands can form blunt-ended duplexes or, preferably, the 3' ends of the sense and antisense strands may form 3' overhangs of, e.g., 1, 2, or 3 nucleosides, which is similar to the product generated by Dicer that forms the RISC substrate in vivo. Effective extended forms of Dicer substrates are described in U.S. Patent No. 8,349,809 and U.S. Patent No. 8,513,207, which are incorporated herein by reference. In some embodiments, both the sense and antisense strands have 2nt 3' overhangs. Thus Thus, the double-stranded region can be, for example, 17 to 25 nucleotides in length, for example, 21 to 23 nucleotides in length.

[0055] Once inside the cell, the antisense strand is incorporated into the RISC complex that mediates target degradation or target inhibition of the target nucleic acid. siRNA typically contains modified nucleosides in addition to RNA nucleosides. In one embodiment, the siRNA molecule may also be chemically modified using modified internucleotide linkages and 2'-sugar modified nucleosides, such as 2'-4'-bicyclic ribose modified nucleosides (LNA and cET or 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-substituted modifications such as 2'-fluoro-ANA). In particular, 2'-fluoro, 2'-O-methyl, or 2'-O-methoxyethyl can be incorporated into the siRNA.

[0056] In some embodiments, all of the nucleotides of the siRNA sense (passenger) strand can be modified with 2'-sugar modified nucleosides such as LNA (see, e.g., WO 2004 / 083430, WO 2007 / 085485). In some embodiments, the passenger strand of the siRNA can be discontinuous (see, e.g., WO 2007 / 107162). Incorporation of thermally labile nucleotides occurring in the seed region of the antisense strand of siRNA has been reported to be useful for reducing the off-target activity of siRNA (see, e.g., WO 2018 / 098328). Preferably, the siRNA contains a 5'-phosphate group or a 5'-phosphate mimic at the 5'-end of the antisense strand. In some embodiments, the 5'-end of the antisense strand is an RNA nucleoside.

[0057] In one embodiment, the siRNA molecule further comprises at least one phosphorothioate or methylphosphonate internucleoside linkage. The phosphorothioate or methylphosphonate internucleoside linkage can be at the 3' end of one or both strands (e.g., the antisense strand or the sense strand), or the phosphorothioate or methylphosphonate internucleoside linkage can be at the 5' end of one or both strands (e.g., the antisense strand or the sense strand), or the phosphorothioate or methylphosphonate internucleoside linkage can be at both the 5' and 3' ends of one or both strands (e.g., the antisense strand or the sense strand). In some embodiments, the remaining internucleoside linkages are phosphodiester linkages. In some embodiments, the siRNA molecule comprises one or more phosphorothioate internucleoside linkages. In the siRNA molecule, the phosphorothioate internucleoside linkage can reduce nuclease cleavage in the RICS, and thus it is advantageous that not all internucleoside linkages in the antisense strand are modified.

[0058] The siRNA molecule can further comprise a ligand. In some embodiments, the ligand binds to the 3' end of the sense strand.

[0059] For biodistribution, the siRNA can be conjugated to a targeting ligand and / or formulated into lipid nanoparticles.

[0060] Other aspects of the invention relate to pharmaceutical compositions comprising these dsRNAs, such as siRNA molecules suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering a dsRNA molecule, such as the siRNA of the invention, for the treatment of various disease states disclosed herein, for example.

[0061] shRNA The term "short hairpin RNA" or "shRNA" generally refers to a molecule having a nucleotide length between 40 and 70, such as a nucleotide length between 45 and 65, such as a nucleotide length between 50 and 60, which forms a stem-loop (hairpin) RNA structure, which is thought to interact with an endonuclease known as Dicer that processes dsRNA into short interfering RNAs of 19 - 23 base pairs having a characteristic 2-base 3' overhang, and is then incorporated into the RNA-induced silencing complex (RISC). When bound to an appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing. shRNA oligonucleotides may be chemically modified using modified internucleotide linkages and 2'-sugar modified nucleosides, such as 2'-4'-bicyclic ribose modified nucleosides (LNA and cET or 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-fluoro-ANA, including such 2'-substituted modifications).

[0062] In some embodiments, the shRNA molecule comprises one or more phosphorothioate internucleotide linkages. In RNAi molecules, phosphorothioate internucleotide linkages can reduce nuclease cleavage in the RICS, and thus it is advantageous that not all internucleotide linkages in the stem-loop of the shRNA molecule are modified. Phosphorothioate internucleotide linkages can be advantageously placed at the 3' and / or 5' ends of the stem-loop of the shRNA molecule, particularly in portions of the molecule that are not complementary to the target nucleic acid. However, the regions of the shRNA molecule that are complementary to the target nucleic acid can also be modified at the first 2 - 3 internucleotide linkages in the portions that are predicted to become the 3' and / or 5' ends after cleavage by Dicer.

[0063] Contiguous nucleotide sequence The term "continuous nucleotide sequence" refers to a region of a nucleic acid molecule that is complementary to a target nucleic acid. This term is used interchangeably herein with the terms "continuous nucleic acid base sequence" and "oligonucleotide motif sequence". In some embodiments, all of the nucleotides of an oligonucleotide constitute a continuous nucleotide sequence. In some embodiments, the continuous nucleotide sequence is included in the guide strand of an siRNA molecule. In some embodiments, the continuous nucleotide sequence is part of an shRNA molecule that is 100% complementary to the target nucleic acid. In some embodiments, an oligonucleotide includes a continuous nucleotide sequence, such as an F-G-F' gapmer region, and optionally further nucleotide(s), for example, a nucleotide linker region that can be used to attach a functional group (e.g., a conjugate group for targeting) to the continuous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. In some embodiments, the nucleic acid base sequence of an antisense oligonucleotide constitutes a continuous nucleotide sequence. In some embodiments, the continuous nucleotide sequence is 100% complementary to the target nucleic acid.

[0064] Nucleotides and Nucleosides Nucleotides and nucleosides are the building blocks of oligonucleotides and polynucleotides and, for the purposes of the present invention, include both naturally occurring nucleotides and nucleosides and non-naturally occurring nucleotides and nucleosides. By nature, nucleotides such as DNA nucleotides and RNA nucleotides include a ribose sugar moiety, a nucleic acid base moiety, and one or more phosphate groups (not present in nucleosides). Nucleosides and nucleotides can also be referred to interchangeably as "units" or "monomers".

[0065] Modified Nucleosides As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside modified by the introduction of one or more modifications to the sugar moiety or the (nucleic acid) base moiety, as compared to an equivalent DNA or RNA nucleoside. Advantageously, one or more of the modified nucleosides include a modified sugar moiety. The term modified nucleoside may also be used interchangeably with the terms "nucleoside analog", or modified "unit", or modified "monomer". Nucleosides having an unmodified DNA or RNA sugar moiety are referred to herein as DNA or RNA nucleosides. Nucleosides having a modification in the base region of a DNA or RNA nucleoside are still generally referred to as DNA or RNA if they are capable of Watson-Crick base pairing.

[0066] Modified internucleoside linkage The term "modified internucleoside linkage" is defined as is generally understood by those skilled in the art as a linkage other than a phosphodiester (PO) linkage that covalently joins two nucleosides together. Thus, the oligonucleotides of the invention can include one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages or one or more phosphorodithioate internucleoside linkages.

[0067] In the oligonucleotides of the invention, it is advantageous to use phosphorothioate internucleoside linkages.

[0068] Phosphorothioate internucleoside linkages are particularly useful due to nuclease resistance, beneficial pharmacokinetics, and ease of manufacture. In some embodiments, at least 50% of the internucleoside linkages of an oligonucleotide or a continuous nucleotide sequence thereof are phosphorothioates, and at least 60%, such as at least 70%, such as at least 75%, such as at least 80% or such as at least 90% of the internucleoside linkages of the oligonucleotide or its nucleotide sequence are phosphorothioates. In some embodiments, all of the internucleoside linkages of an oligonucleotide or a continuous nucleotide sequence thereof are phosphorothioates.

[0069] In some advantageous embodiments, all of the internucleoside linkages of a continuous nucleotide sequence of an oligonucleotide are phosphorothioates, or all of the internucleoside linkages of the oligonucleotide are phosphorothioate linkages.

[0070] As disclosed in European Patent No. 2 742 135, it is recognized that antisense oligonucleotides may contain other internucleoside linkages (other than phosphodiester and phosphorothioate), such as alkylphosphonate / methylphosphonate internucleoside linkages, which according to European Patent No. 2 742 135 may be resistant, for example, within a gap region of another DNA phosphorothioate.

[0071] Nucleobase 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 in nucleic acid hybridization. In the context of the present invention, the term "nucleobase" includes modified nucleobases that may differ from naturally occurring nucleobases but are functional in nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, and non-naturally occurring variants. Such variants are described, for example, in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.37 1.4.1. as described in

[0072] In some embodiments, the nucleobase moiety is modified by changing a purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or pyrimidine, such as isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2’-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0073] The nucleobase moiety can be denoted by the letter code of each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter can optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotide, the nucleobase moiety is selected from A, T, G, C, and 5-methylcytosine. Optionally, in an LNA gapmer, 5-methylcytosine LNA nucleosides can be used.

[0074] Modified oligonucleotide The term "modified oligonucleotide" describes an oligonucleotide that contains one or more sugar-modified nucleosides and / or modified internucleoside linkages. The term "chimeric" oligonucleotide is a term used in the literature to describe an oligonucleotide that contains modified nucleosides and DNA nucleosides. The antisense oligonucleotides of the present invention are preferably chimeric oligonucleotides.

[0075] Complementarity The term "complementarity" or "complementary" refers to the ability of nucleosides / nucleotides to form Watson-Crick base pairs. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). Oligonucleotides may contain nucleosides with modified nucleobases, for example 5-methylcytosine is often used in place of cytosine, and thus it will be understood that the term complementarity encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, for example, Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.37 1.4.1).

[0076] As used herein, the term “% complementary” refers to the percentage of nucleotides in a contiguous nucleotide sequence of a nucleic acid molecule (e.g., an oligonucleotide) that is complementary to a reference sequence (e.g., a target sequence or sequence motif) over a contiguous nucleotide sequence. Thus, the percentage of complementarity is calculated by counting the number of aligned nucleic acid bases that are complementary (from Watson-Crick base pairs) between two sequences (when the target sequence 5'-3' and the oligonucleotide sequence from 3'-5' are aligned), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleic acid bases / nucleotides that do not align (form base pairs) are referred to as mismatches. Insertions and deletions are not tolerated in the calculation of % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of nucleic acid bases are ignored so long as the functional ability of the nucleic acid base to form Watson-Crick base pairing is retained (e.g., 5'-methylcytosine is considered identical to cytosine for purposes of calculating % identity).

[0077] The term “fully complementary” refers to 100% complementarity.

[0078] Identity As used herein, the term "identity" refers to the percentage (expressed as a percentage) of nucleotides in a continuous nucleotide sequence within a nucleic acid molecule (e.g., an oligonucleotide) that is identical to a reference sequence (e.g., an array motif) across the continuous nucleotide sequence. Thus, the percentage of identity is calculated by counting the number of identical (matching) aligned nucleic acid bases between two sequences (in the continuous nucleotide sequence of a compound of the invention and the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, percent identity = (number of matches × 100) / length of the aligned region (e.g., continuous nucleotide sequence). Insertions and deletions are not allowed in the calculation of the percentage of identity of a continuous nucleotide sequence. It will be understood that in determining identity, chemical modifications of nucleic acid bases are ignored as long as the functional ability of the nucleic acid base to form Watson Crick base pairs is retained (e.g., 5-methylcytosine is considered identical to cytosine for the purposes of calculating % identity).

[0079] Hybridization As used herein, the terms "hybridize" or "hybridizing" are to be understood to mean that two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) form a duplex by forming hydrogen bonds between base pairs on opposing strands. The affinity of the bond between two nucleic acid strands is the strength of hybridization. This is often described by the melting temperature (T m ), which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. Under physiological conditions, T m is not strictly proportional to affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° more accurately represents the binding affinity and is given by ΔG° = -RTln(K d ) for the dissociation constant (K d) is associated, 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 a reaction having an aqueous concentration of 1 M, a pH of 7, and a temperature of 37 °C. The hybridization of the oligonucleotide to the target nucleic acid is a spontaneous reaction, and in the case of a spontaneous reaction, ΔG° is less than zero. ΔG° can be measured experimentally, for example, by isothermal titration calorimetry (ITC), as described in Hansen et al., 1965, Chem. Comm. 36 - 38 and Holdgate et al., 2005, Drug Discov Today. Those skilled in the art will know that commercially available devices are available for ΔG° measurement. ΔG° can also be numerically estimated by using the nearest neighbor model described by SantaLucia, 1998, Proc Natl Acad Sci USA. 95: 1460 - 1465. In order to have the potential to regulate its intended nucleic acid target by hybridization, the oligonucleotide of the present invention hybridizes to the target nucleic acid with an estimated ΔG° value of less than -10 kcal for oligonucleotides 10 - 30 nucleotides in length. In some embodiments, the degree or strength of hybridization is measured by the standard state Gibbs free energy ΔG°. The oligonucleotide can hybridize to the target nucleic acid with an approximate ΔG° value of less than -10 kcal, such as less than -15 kcal, such as less than -20 kcal, and such as less than -25 kcal for oligonucleotides 8 - 30 nucleotides in length. In some embodiments, the oligonucleotide is -10 to -60 kcal, such as -12 to -40, such as -15 to -30 kcal or -16 to -27 kcal, such as -18 to -25 k Hybridize to the target nucleic acid with an estimated ΔG° value in the range of cal.

[0080] Target nucleic acid According to the present invention, the target nucleic acid is a nucleic acid encoding mammalian COPS3, and can be, for example, a gene, RNA, mRNA, and pre-mRNA, mature mRNA or cDNA sequence. Therefore, the target can be referred to as a COPS3 target nucleic acid.

[0081] Suitably, the target nucleic acid encodes a COPS3 protein such as the human COPS3 gene encoding the pre-mRNA or mRNA sequence provided herein as SEQ ID NO: 1, 4, 5, 6, 7, 8 and / or 9, particularly encoding mammalian COPS3.

[0082] The therapeutic nucleic acid molecule of the present invention can target, for example, the exon region of mammalian COPS3 (particularly siRNA and shRNA, and antisense oligonucleotides), or can target, for example, any intron region of COPS3 pre-mRNA (particularly antisense oligonucleotides). The human COPS3 gene encodes 18 transcripts, 6 of which encode proteins and are therefore potential nucleic acid targets.

[0083] Table 1 lists the predicted exon and intron regions of SEQ ID NO: 1, that is, the human COPS3 pre-mRNA sequence.

Table 1

[0084] Suitably, the target nucleic acid encodes a COPS3 protein such as human COPS3 (see, for example, Tables 2 and 3) that provides an overview of the genomic sequences of human, cynomolgus monkey and mouse COPS3 (Table 2), the pre-mRNA sequences of human, monkey and mouse COPS3, and the mature mRNA of human COPS3 (Table 3), particularly encoding mammalian COPS3.

[0085] In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 6, 7, 8, and / or 9, or natural variants thereof (such as the sequence encoding mammalian COPS3). [Table 2]

[0086] When using the nucleic acid molecules of the present invention for research or diagnosis, the target nucleic acid can be cDNA, or a synthetic nucleic acid derived from DNA or RNA.

[0087] For in vivo or in vitro applications, the therapeutic nucleic acid molecules of the present invention can typically inhibit the expression of the COPS3 target nucleic acid in cells expressing the COPS3 target nucleic acid. In some embodiments, the cells contain HBV cccDNA. The continuous sequence of nucleobases of the nucleic acid molecules of the present invention is typically complementary to the conserved region of the COPS3 target nucleic acid, when measured over the length of the nucleic acid molecule, optionally excluding one or two mismatches, and optionally, a nucleotide-based linker region that can bind to any functional group such as conjugating an oligonucleotide, or other non-complementary terminal nucleotides. The target nucleic acid is a pre-mRNA encoding a mammalian COPS3 protein such as human COPS3, for example, a human COPS3 pre-mRNA sequence such as that disclosed as SEQ ID NO: 1, a monkey COPS3 pre-mRNA sequence such as that disclosed as SEQ ID NO: 2, or a mouse COPS3 pre-mRNA sequence such as that disclosed as the SEQ ID NO: 3 target nucleic acid, or a mature COPS3 mRNA such as human mature mRNA disclosed as SEQ ID NOs: 4, 5, 6, 7, 8, or 9. SEQ ID NOs: 1-9 are DNA sequences. It will be understood that the target RNA sequence has uracil (U) bases instead of thymidine (T) bases.

[0088] Further information regarding exemplary target nucleic acids is provided in Tables 2 and 3. [Table 3] Note Sequence number 2 includes regions of multiple NNNN where array determination cannot accurately purify the array and thus includes degenerate arrays. To avoid doubt, the compounds of the invention are complementary to the actual target sequence and are thus not degenerate compounds.

[0089] In some embodiments, the target nucleic acid is SEQ ID NO: 1.

[0090] In some embodiments, the target nucleic acid is SEQ ID NO: 2.

[0091] In some embodiments, the target nucleic acid is SEQ ID NO: 3.

[0092] In some embodiments, the target nucleic acid is SEQ ID NO: 4.

[0093] In some embodiments, the target nucleic acid is SEQ ID NO: 5.

[0094] In some embodiments, the target nucleic acid is SEQ ID NO: 6.

[0095] In some embodiments, the target nucleic acid is SEQ ID NO: 7.

[0096] In some embodiments, the target nucleic acid is SEQ ID NO: 8.

[0097] In some embodiments, the target nucleic acid is SEQ ID NO: 9.

[0098] In some embodiments, the target nucleic acid is SEQ ID NO: 1, 4, 5, 6, and / or 8.

[0099] In some embodiments, the target nucleic acid is SEQ ID NO: 1, 4, and / or 5.

[0100] Target sequence As used herein, the term "target sequence" refers to the sequence of nucleotides present in a target nucleic acid that includes a nucleic acid base sequence complementary to an oligonucleotide or nucleic acid molecule of the invention. In some embodiments, the target sequence consists of a region on a target nucleic acid having a nucleic acid base sequence complementary to a contiguous nucleotide sequence of an oligonucleotide of the 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 the nucleic acid molecule of the invention and may represent, for example, a preferred region of a target nucleic acid that can be targeted by some nucleic acid molecules of the invention.

[0101] In some embodiments, the target sequence is a sequence selected from the group consisting of human COPS3 mRNA exons, such as e1, e2, e3, e4, e5, e6, e7, e8, e9, e10, e11, and e12 (see, for example, Table 1 above).

[0102] Accordingly, the present invention provides an oligonucleotide comprising a contiguous sequence that is at least 90% complementary, such as completely complementary, to the exon region of SEQ ID NO: 1 selected from the group consisting of e1 - e12 (see Table 1).

[0103] In some embodiments, the target sequence is a sequence selected from the group consisting of human COPS3 mRNA introns, such as i1, i2, i3, i4, i5, i6, i7, i9, i10, and i11 (see, for example, Table 1 above).

[0104] Accordingly, the present invention provides an oligonucleotide comprising a contiguous sequence that is at least 90% complementary, such as completely complementary, to the intron region of SEQ ID NO: 1 selected from the group consisting of i1 - i11 (see Table 1).

[0105] In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 10, 11, 12, and 13. In some embodiments, the continuous nucleotide sequence referred to herein is at least 90% complementary, such as at least 95% complementary, to the target sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, and 13. In some embodiments, the continuous nucleotide sequence is completely complementary to the target sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, and 13.

[0106] The nucleic acid molecule of the present invention comprises 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.

[0107] The target nucleic acid sequence to which the therapeutic nucleic acid molecule is complementary or hybridizes generally comprises a stretch of at least 10 consecutive nucleic acid bases. The continuous nucleotide sequence is 12 to 70 nucleotides, such as 12 to 50, such as 13 to 30, such as 14 to 25, such as 15 to 20, such as 16 to 18 consecutive nucleotides.

[0108] In some embodiments, the nucleic acid molecule of the present invention targets the regions shown in Table 4 or 5.

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

[0109] In some embodiments, the target sequence is selected from the group consisting of target regions 1A to 1846A as shown in Table 4 above.

Table 5-1

Table 5-2

Table 5-3

[0110] In some embodiments, the target sequence is selected from the group consisting of target regions 1C to 178C as shown in Table 5 above.

[0111] Target cell As used herein, the term "target cell" refers to a cell that expresses a target nucleic acid. For the therapeutic uses of the present invention, it is advantageous if the target cell is infected with HBV. In some embodiments, the target cell can 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 woodchuck cell, or a primate cell, such as a monkey cell (e.g., cynomolgus monkey cell) or a human cell.

[0112] In a preferred embodiment, the target cell expresses COPS3 mRNA such as COPS3 pre-mRNA or COPS3 mature mRNA. The polyA tail of COPS3 mRNA is typically ignored in antisense oligonucleotide targeting.

[0113] Furthermore, the target cell may be a hepatocyte. In one embodiment, the target cell is a primary human hepatocyte infected with HBV, either derived from an HBV-infected individual or from a mouse with a humanized liver (PhoenixBio, PXB mouse) infected with HBV.

[0114] According to the present invention, the target cell may be infected with HBV. Furthermore, the target cell may contain HBV cccDNA. Thus, it is preferred that the target cell contains COPS3 mRNA such as COPS3 pre-mRNA or COPS3 mature mRNA and HBV cccDNA.

[0115] Naturally occurring variant The term "naturally occurring variant" refers to variants of the COPS3 gene or transcript that originate from the same locus as the target nucleic acid but may differ, for example, due to the degeneracy of the genetic code resulting in multiple codons encoding the same amino acid, or due to alternative splicing of the pre-mRNA or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs), and allelic variants. Based on the presence of a sufficient complementary sequence to the oligonucleotide, the oligonucleotide of the invention can thus target the target nucleic acid and its naturally occurring variants.

[0116] In some embodiments, a naturally occurring variant has at least 95%, such as at least 98% or at least 99% homology to a mammalian COPS3 target nucleic acid, such as the target nucleic acid of SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, a naturally occurring variant has at least 99% homology to the human COPS3 target nucleic acid of SEQ ID NO: 1. In some embodiments, a naturally occurring variant is a known polymorphism.

[0117] Inhibition of expression As used herein, the term "inhibition of expression" refers to inhibition of expression of COPS in a target cell. The expression or activity of a COPS3 (COP9 signalosome subunit 3) inhibitor should be understood as a general term of the ability of the COPS3 (COP9 signalosome subunit 3) inhibitor to inhibit, i.e. reduce, the amount or activity of COPS3. The inhibition of expression or activity can be determined by measuring the level of COPS3 pre-mRNA or COPS3 mRNA, or by measuring the level of COPS3 protein or activity in the cell. The inhibition of expression can be determined in vitro or in vivo. Advantageously, the inhibition is evaluated with respect to the amount of COPS3 before administration of the COPS3 inhibitor. Alternatively, the inhibition is determined by reference to a control. The control is generally understood to be an individual or target cell treated with a saline composition, or an individual or target cell treated with a non-targeting oligonucleotide (mock).

[0118] The term "inhibit" or "inhibiting" may also be referred to as down-regulating, reducing, suppressing, decreasing, lowering, or diminishing the expression or activity of COPS3.

[0119] Inhibition of COPS3 expression can occur, for example, by degradation of pre-mRNA or mRNA, using nucleic acid molecules that function via the RNase H mobilizing oligonucleotides such as gapmers, or via RNA interference pathways such as siRNA or shRNA. Alternatively, the inhibitors of the present invention can bind to the COPS3 polypeptide and inhibit the activity of COPS3 or prevent its binding to other molecules.

[0120] In some embodiments, inhibition of the expression of the COPS3 target nucleic acid or the activity of the COPS3 protein results in a decrease in the amount of HBV cccDNA in the target cells. Preferably, the amount of HBV cccDNA is decreased as compared to the control. In some embodiments, the decrease in the amount of HBV cccDNA is at least 20%, at least 30% as compared to the control. In some embodiments, the amount of cccDNA in HBV-infected cells is decreased by at least 50%, such as 60% as compared to the control.

[0121] In some embodiments, inhibition of the expression of the COPS3 target nucleic acid or the activity of the COPS3 protein results in a decrease in the amount of HBV pgRNA in the target cells. Preferably, the amount of HBV pgRNA is decreased as compared to the control. In some embodiments, the decrease in the amount of HBV pgRNA is at least 20%, at least 30% as compared to the control. In some embodiments, the amount of pgRNA in HBV-infected cells is decreased by at least 50%, such as 60% as compared to the control.

[0122] Sugar modification The oligonucleotides of the present invention can include one or more nucleosides having a modified sugar moiety, i.e., a modification of the sugar moiety as compared to the ribose sugar moiety found in DNA and RNA.

[0123] Numerous nucleosides having modifications of the ribose moiety have been prepared mainly for the purpose of improving certain properties of oligonucleotides such as affinity and / or nuclease resistance.

[0124] Such modifications include, for example, those in which the ribose ring structure is modified by replacing it with a hexose ring (HNA), or a bicyclic ring having a biradical bridge between the C2 and C4 carbons of a ribose ring (LNA), or typically a non-bridged ribose ring lacking a bond between the C2 and C3 carbons (e.g., UNA). Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acid (International Publication No. WO 2011 / 017521) or tricyclic nucleic acid (International Publication No. WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety is replaced by a non-sugar moiety, for example in the case of peptide nucleic acid (PNA) or morpholino nucleic acid.

[0125] Sugar modifications also include modifications made by changing a substituent on the ribose ring to a group other than hydrogen, or to the 2'-OH group that is naturally present in DNA and RNA nucleosides. The substituent can be introduced, for example, at the 2', 3', 4', or 5' position.

[0126] High-affinity modified nucleosides High-affinity modified nucleosides are modified nucleotides that, when incorporated into an oligonucleotide, increase the affinity of the oligonucleotide for its complementary target, as measured, for example, by the melting temperature (T m m). The high-affinity modified nucleosides of the present invention preferably have a range of +0.5 to +12 °C, more preferably +1.5 to +10 °C, and most preferably +3 to +8 oIt results in an increase in the melting temperature in the range of C. Numerous high-affinity modified nucleosides are known in the art, 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).

[0127] 2'-sugar modified nucleoside A 2'-sugar modified nucleoside is a nucleoside having a substituent other than H or -OH at the 2'-position (2'-substituted nucleoside), or a nucleoside containing a 2'-bonded biradical capable of forming a crosslink between the 2'-carbon and a second carbon on the ribose ring, such as an LNA (2'-4'-biradical crosslinked) nucleoside.

[0128] In fact, much attention has been focused on the development of 2'-sugar substituted nucleosides, and numerous 2'-substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides. For example, 2'-modified sugars can confer 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 example, 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. The following are examples of some 2'-substituted modified nucleosides. [Chemical formula]

[0129] Regarding the present invention, the 2'-substituted sugar-modified nucleosides do not include 2'-bridged nucleosides such as LNA.

[0130] Locked nucleic acid nucleoside (LNA nucleoside) An "LNA nucleoside" is a 2'-sugar-modified nucleoside that contains a biradical (also referred to as a "2'-4' bridge") that binds C2' and C4' of the ribose sugar ring of the nucleoside, which restricts or locks the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNA). The locking of the ribose conformation is associated with an improvement in hybridization affinity (duplex stabilization) when LNA is incorporated into oligonucleotides of complementary RNA or DNA molecules. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0131] Non-limiting and exemplary LNA nucleosides are described in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett. 12, 73-76, Seth et al. J. Org. Chem. 2010, Vol 75(5) pp. 1569-81, Mitsuoka et al., Nucleic Acids Research 2009, 37(4), 1225-1238, and Wan and Seth, J. Medical Chemistry 2016, 59, 9645-9667.

[0132] Specific examples of the LNA nucleosides of the present invention are shown in Scheme 1 (where B is as defined above). Scheme 1

Chemical formula

[0133] Specific LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxy LNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET) and ENA.

[0134] RNase H Activity and Recruitment The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when forming a duplex with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for determining RNase H activity that can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is tested in pmol / l / min when a complementary target nucleic acid is provided, having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers and having phosphorothioate linkages between all monomers of the oligonucleotide, and is considered to be able to recruit RNase H when it has at least 5%, for example at least 10% or more than 20% of the initial rate determined when using the methodology provided by Examples 91-95 of WO 01 / 23613 (incorporated herein by reference). For use in the determination of RHase H activity, recombinant human RNase H1 is available from Creative Biomart® (recombinant human RNase H1 fused with a His tag expressed in E. coli). For use in the determination of RHase H activity, recombinant human RNase H1 is available from Creative Biomart® (recombinant human RNase H1 fused with a His tag expressed in E. coli).

[0135] Gapmer The antisense oligonucleotide of the present invention or its continuous nucleotide sequence may be a gapmer, and may also be referred to as a gapmer oligonucleotide or a gapmer design. Antisense gapmers are typically used for the inhibition of target nucleic acids via RNase H-mediated degradation. A gapmer oligonucleotide comprises at least three distinguishable structural regions, a 5'-flank, a gap, and a 3'-flank, F-G-F', in a 5'->3' orientation. The "gap" region (G) comprises a continuous stretch of DNA nucleotides that enables the oligonucleotide to recruit RNase H. The gap region is flanked by a 5'-adjacent region (F) comprising one or more sugar-modified nucleosides, preferably high-affinity sugar-modified nucleosides, and a 3'-adjacent region (F') comprising one or more sugar-modified nucleosides, preferably high-affinity sugar-modified nucleosides. One or more of the sugar-modified nucleosides in regions F and F' improve the affinity of the oligonucleotide for the target nucleic acid (i.e., these are affinity-improving sugar-modified nucleosides). In some embodiments, one or more of the sugar-modified nucleosides in regions F and F' are 2'-sugar-modified nucleosides, such as, for example, selected independently from LNA and 2'-MOE, such as, for example, high-affinity 2'-sugar modifications.

[0136] In gapmer design, the most 5' and 3' nucleosides of the gap region are DNA nucleosides and are positioned adjacent to the sugar-modified nucleosides of the 5' (F) or 3' (F') regions, respectively. The flanks may further be defined by having at least one sugar-modified nucleoside at the end most distant from the gap region, i.e., the 5'-end of the 5'-flank and the 3'-end of the 3'-flank.

[0137] The region F-G-F' forms a continuous nucleotide sequence. The antisense oligonucleotide of the present invention, or its continuous nucleotide sequence, may comprise a gapmer region of the formula F-G-F'.

[0138] The total length of the gapmer design F-G-F' can be, for example, 12 to 32 nucleosides, such as 13 to 24, such as 14 to 22 nucleosides, such as 15 to 20, such as 16 to 18 nucleosides.

[0139] As an example, the gapmer oligonucleotide of the present invention can be represented by the following formula: F 1-8 -G 5-18 -F’ 1-8 , for example F 1-8 -G 7-18 -F’ 2-8 However, the total length of the gapmer region F-G-F' is conditioned to be at least 12, for example at least 14 nucleotides in length.

[0140] In one aspect of the present invention, the antisense oligonucleotide or its contiguous nucleotide sequence consists of or comprises a gapmer of the formula 5'-F-G-F'-3', wherein the regions F and F' independently contain or consist of 1 to 8 nucleosides, 1 to 4 of which are 2'-sugar modified, define the 5' and 3' termini of the F and F' regions, and G is a region of 6 to 18 nucleosides capable of recruiting RNase H. In some embodiments, the G region consists of DNA nucleosides.

[0141] In some embodiments, the regions F and F' independently consist of or comprise a contiguous sequence of sugar-modified nucleosides. In some embodiments, the sugar-modified nucleosides of the region F can be independently selected from 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, LNA units, arabinonucleic acid (ANA) units, and 2'-fluoro-ANA units.

[0142] In some embodiments, regions F and F’ independently contain both an LNA and a 2’-substituted sugar-modified nucleotide (mixed wing design). In some embodiments, the 2’-substituted sugar-modified nucleotide is independently selected from the group consisting of 2’-O-alkyl-RNA units, 2’-O-methyl-RNA units, 2’-amino-DNA units, 2’-fluoro-DNA units, 2’-alkoxy-RNA units, MOE units, arabinonucleic acid (ANA) units, and 2’-fluoro-ANA units.

[0143] In some embodiments, all modified nucleosides in regions F and F’ are LNA nucleosides independently selected from, for example, beta-D-oxy-LNA, ENA, or ScET nucleosides, and region F or F’, or F and F’ may optionally contain DNA nucleosides. In some embodiments, all modified nucleosides in regions F and F’ are beta-D-oxy-LNA nucleosides, and region F or F’, or F and F’ may optionally contain DNA nucleosides. In such embodiments, the flanking region F or F’, or both F and F’ contain at least three nucleosides, and the most 5’ and 3’ nucleosides of the F and / or F’ region are LNA nucleosides.

[0144] LNA gapmer An LNA gapmer is a gapmer that contains or consists of LNA nucleosides in one or both of regions F and F’. A beta-D-oxy gapmer is a gapmer that contains or consists of beta-D-oxy-LNA nucleosides in one or both of regions F and F’.

[0145] In some embodiments, the LNA gapmer has the formula: [LNA] 1-5 -[region G] 6-18 -[LNA] 1-5 where region G is as defined in the definition of gapmer region G.

[0146] MOE gapmer The MOE gapmer is a gapmer in which regions F and F’ consist of MOE nucleosides. In some embodiments, the MOE gapmer is designed [MOE] 1-8 -[Region G] 5-16 -[MOE] 1-8 , for example [MOE] 2-7 -[Region G] 6-14 -[MOE] 2-7 , for example [MOE] 3-6 -[Region G] 8-12 -[MOE] 3-6 , for example [MOE]5-[Region G] 10 -[MOE]5, and region G is as defined in the definition of the gapmer. The MOE gapmer having a 5-10-5 design (MOE-DNA-MOE) is widely used in the art.

[0147] Region D’ or D’’ in the oligonucleotide In some embodiments, the oligonucleotide of the present invention comprises, or consists of, a continuous nucleotide sequence of an oligonucleotide that is complementary to a target nucleic acid such as a gapmer region F-G-F’, and additional 5’ and / or 3’ nucleosides. The additional 5’ and / or 3’ nucleosides may or may not be completely complementary to the target nucleic acid. Such additional 5’ and / or 3’ nucleosides may be referred to herein as regions D’ and D’’.

[0148] The addition of region D’ or D’’ can be used for the purpose of conjugating a continuous nucleotide sequence, such as a gapmer, to a gate moiety or another functional group. When used for conjugation, the continuous nucleotide sequence having a conjugate moiety can serve as a biodegradable linker. Alternatively, it can be used to provide exonuclease protection or to facilitate synthesis or manufacture. Regions D’ and D’’ are each attached to the 5’ end of region F or the 3’ end of region F’ to form the following formulas D’-F-G-F’, F-G-F’-D’’ or

[0149] It is possible to generate a design of D’-F-G-F’-D’’. In this case, F-G-F’ is the gapmer portion of the oligonucleotide, and the regions D’ or D’’ constitute separate portions of the oligonucleotide.

[0150] The region D’ or D’’ independently contains or consists of 1, 2, 3, 4, or 5 additional nucleotides, which may or may not be complementary to the target nucleic acid. The nucleotides adjacent to the F or F’ region are not sugar-modified nucleotides such as DNA or RNA, nor are they base-modified versions thereof. The D’ or D’’ region can serve as a nuclease-sensitive biodegradable linker (see definition of linker). In some embodiments, the additional 5’ and / or 3’ terminal nucleotides are linked by phosphodiester bonds and are DNA or RNA. Nucleotide-based biodegradable linkers suitable for use as the region D’ or D’’ are disclosed in International Publication No. WO 2014 / 076195, which includes, by way of example, phosphodiester-linked DNA dinucleotides. The use of biodegradable linkers in polyoligonucleotide constructs is disclosed in International Publication No. WO 2015 / 113922, and they are used to link multiple antisense constructs (e.g., gapmer regions) within a single oligonucleotide.

[0151] In one embodiment, the oligonucleotide of the present invention includes the regions D’ and / or D’’ in addition to the continuous nucleotide sequence that constitutes the gapmer.

[0152] In some embodiments, the oligonucleotide of the present invention can be represented by the following formula: F-G-F’; particularly F 1-8 -G 5-18 -F’ 2-8 D’-F-G-F’, particularly, D’ 1-3 -F 1-8 -G 5-18 -F’ 2-8 F-G-F’-D’’, particularly, F1-8 -G 5-18 -F’ 2-8 -D’’ 1-3 D’-F-G-F’-D’’, particularly, D’ 1-3 -F 1-8 -G 5-18 -F’ 2-8 -D’’ 1-3 。

[0153] In some embodiments, the internucleoside linkage located between region D’ and region F is a phosphodiester linkage. In some embodiments, the internucleoside linkage located between region F’ and region D’’ is a phosphodiester linkage.

[0154] Conjugate As used herein, the term conjugate refers to an oligonucleotide covalently attached to a non-nucleotide moiety (conjugate moiety or region C or third region). The conjugate moiety may be covalently attached to the antisense oligonucleotide, optionally via a linker group such as region D’ or D’’.

[0155] For oligonucleotide conjugates and their synthesis, see Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., Ch. 16, M arcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, which are incorporated herein by reference in their entireties.

[0156] In some embodiments, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of a carbohydrate (e.g., galactose or N-acetylgalactosamine (GalNAc)), a cell surface receptor ligand, a prodrug, a hormone, a lipophilic substance, a polymer, a protein (e.g., an antibody), a peptide, a toxin (e.g., a bacterial toxin), a vitamin, a viral protein (e.g., a capsid), or a combination thereof.

[0157] Exemplary conjugate moieties are those that can bind to the asialoglycoprotein receptor (ASGPR). In particular, conjugate moieties of trivalent N-acetylgalactosamine are suitable for binding to ASGPR; see, for example, WO 2014 / 076196, WO 2014 / 207232, and WO 2014 / 179620, which are incorporated herein by reference. Such conjugates serve to enhance the uptake of oligonucleotides into the liver.

[0158] Linker A bond or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds. The conjugate moiety can be attached to the oligonucleotide directly or via a linking moiety (e.g., a linker or a tether). The linker serves to covalently attach a third region, e.g., a conjugate moiety (region C), to a first region, e.g., an oligonucleotide or a continuous nucleotide sequence (region A) complementary to the target nucleic acid.

[0159] In some embodiments of the invention, the conjugate or oligonucleotide conjugate of the invention can optionally include a linker region (second region or region B and / or region Y) located between an oligonucleotide or continuous nucleotide sequence (region A or first region) complementary to the target nucleic acid and a conjugate moiety (region C or third region).

[0160] Region B refers to a biodegradable linker that contains or consists of a physiologically labile bond that can be cleaved under conditions normally encountered or similar to those encountered in the mammalian body. Conditions under which a physiologically labile linker undergoes chemical conversion (e.g., cleavage) include pH, temperature, oxidation or reduction conditions, or chemical conditions such as agents, as well as salt concentrations similar to those found or encountered in mammalian cells. Intracellular conditions in mammalian cells also include the presence of enzyme activities normally present in mammalian cells, such as proteolytic enzymes, hydrolytic enzymes, or nucleases. In one embodiment, the biodegradable linker is susceptible to S1 nuclease cleavage. In a preferred embodiment, the nuclease-sensitive linker comprises 1 to 5 nucleosides, such as 1, 2, 3, 4, or 5 nucleosides, more preferably 2 to 4 nucleosides, and most preferably at least 2 consecutive phosphodiester bonds, such as at least 3 or 4 or 5 consecutive phosphodiester bonds, in 2 or 3 linked nucleosides. Preferably, the nucleoside is DNA or RNA. Biodegradable linkers containing phosphodiesters are described in more detail in International Publication No. WO 2014 / 076195, which is incorporated herein by reference.

[0161] Region Y refers to a linker that is not necessarily biodegradable but mainly serves to covalently attach the conjugate moiety (Region C or the third region) to the oligonucleotide (Region A or the first region). The Region Y linker can include a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. The oligonucleotide conjugate of the present invention can be constructed from the following local elements A-C, A-B-C, A-B-Y-C, A-Y-B-C, or A-Y-C. In some embodiments, the linker (Region Y) is an aminoalkyl group, such as a C2-C36 aminoalkyl group containing a C6-C12 aminoalkyl group. In some embodiments, the linker (Region Y) is a C6 aminoalkyl group. The oligonucleotide conjugate can be constructed from the following local elements A-C, A-B-C, A-B-Y-C, A-Y-B-C, or A-Y-C. In some embodiments, the linker (Region Y) is an aminoalkyl group, such as a C2-C36 aminoalkyl group containing a C6-C12 aminoalkyl group. In some embodiments, the linker (Region Y) is a C6 aminoalkyl group.

[0162] Treatment As used herein, the term "treatment" refers to both the treatment of an existing disease (e.g., a disease or disorder mentioned herein) or the prevention of a disease, i.e., prophylaxis. Thus, it will be appreciated that the treatments referred to herein may, in some embodiments, be prophylactic. Prevention can be understood as preventing the conversion of HBV infection to chronic HBV infection or preventing serious liver diseases such as cirrhosis and hepatocellular carcinoma caused by chronic HBV infection.

[0163] patient For the purposes of the present invention, a "subject" (or "patient") can be a vertebrate. In the context of the present invention, the term "subject" includes both humans and other animals, particularly mammals, and other organisms. Thus, the means and methods provided herein are applicable to both human therapy and veterinary use. Preferably, the subject is a mammal. More preferably, the subject is a human.

[0164] As described elsewhere herein, the patient to be treated may be suffering from HBV infection such as chronic HBV infection. In some embodiments, a patient suffering from HBV infection may be suffering from hepatocellular carcinoma (HCC). In some embodiments, a patient suffering from HBV infection does not have hepatocellular carcinoma.

[0165] Mode for Carrying Out the Invention HBV cccDNA in infected hepatocytes is involved in persistent chronic infection and reactivation and serves as the template for all viral subgenomic transcripts and pregenomic RNA (pgRNA), ensuring both newly synthesized viral progeny and cccDNA pool replenishment via intracellular nucleocapsid recycling. In the context of the present invention, it has been shown for the first time that COPS3 is associated with cccDNA stability. This knowledge creates an opportunity to destabilize cccDNA in HBV-infected subjects and opens the door to the complete cure of chronic HBV patients.

[0166] One aspect of the present invention is a COPS3 inhibitor for the treatment and / or prevention of hepatitis B virus (HBV) infection, particularly chronic HBV infection.

[0167] The COPS3 inhibitor can be, for example, a small molecule that specifically binds to the COPS3 protein, and the inhibitor prevents or reduces the binding of the COPS3 protein to cccDNA.

[0168] An embodiment of the present invention is a COPS3 inhibitor that can reduce cccDNA and / or pgRNA in infected cells such as HBV-infected cells.

[0169] In a further embodiment, the COPS3 inhibitor can reduce HBsAg and / or HBeAg in vivo in an HBV-infected individual.

[0170] COPS3 inhibitor for use in the treatment of HBV Without being bound by theory, COPS3 is involved in the stabilization of cccDNA in the cell nucleus through direct or indirect binding to cccDNA, and by preventing the binding / association of COPS3 with cccDNA, cccDNA is destabilized and more prone to degradation is considered. Thus, one embodiment of the present invention is a COPS3 inhibitor that interacts with the COPS3 protein and prevents or reduces its binding / association to cccDNA.

[0171] In some embodiments of the present invention, the inhibitor is an antibody, antibody fragment or small molecule compound. In some embodiments, the inhibitor can be an antibody, antibody fragment or small molecule that specifically binds to a COPS3 protein such as the COPS3 protein encoded by SEQ ID NO: 1, 4, 5, 6 or 7.

[0172] The nucleic acid molecule of the present invention Therapeutic nucleic acids are potentially excellent COPS3 inhibitors because they can target COPS3 transcripts and promote their degradation via either the RNA interference pathway or RNase H cleavage. Alternatively, oligonucleotides such as aptamers can also act as inhibitors of COPS3 protein interactions.

[0173] One aspect of the invention is a COPS3-targeted nucleic acid molecule for use in the treatment and / or prevention of hepatitis B virus (HBV) infection. Such nucleic acid molecules can be selected from the group consisting of single-stranded antisense oligonucleotides, siRNA molecules, and shRNA molecules.

[0174] This section describes novel nucleic acid molecules suitable for use in the treatment and / or prevention of hepatitis B virus (HBV) infection.

[0175] The nucleic acid molecules of the invention can inhibit the expression of COPS3 in vitro and in vivo. Inhibition is achieved by hybridizing an oligonucleotide to a target nucleic acid encoding COPS3 or involved in the regulation of COPS3. The target nucleic acid can be a mammalian COPS3 sequence. In some embodiments, the target nucleic acid can be a human COPS3 pre-mRNA sequence such as the sequence of SEQ ID NO: 1 or a human COPS3 mRNA sequence selected from SEQ ID NOs: 4-9. In some embodiments, the target nucleic acid can be a cynomolgus monkey COPS3 sequence such as the sequence of SEQ ID NO: 2.

[0176] In some embodiments, the nucleic acid molecules of the invention can regulate a target by inhibiting or downregulating its expression. Preferably, such regulation results in at least 20%, more preferably at least 30%, at least 40%, at least 50%, at least 60% inhibition compared to the normal expression level of the target. In some embodiments, the nucleic acid molecules of the invention can inhibit the expression level of COPS3 mRNA by at least 60% or 70% in vitro by transfecting 25 nM of the nucleic acid molecule into PXB-PHH cells, and this range of target reduction is advantageous in selecting nucleic acid molecules that have a good correlation with cccDNA reduction. Suitably, the examples provide assays that can be used to measure COPS3 RNA or protein inhibition (e.g., Example 1 and the "Materials and Methods" section). Target inhibition is caused by hybridization between the continuous nucleotide sequence of an oligonucleotide, such as the guide strand of an siRNA or the gapmer region of an antisense oligonucleotide, and the target nucleic acid. In some embodiments, the nucleic acid molecules of the invention contain mismatches between the oligonucleotide and the target nucleic acid. Despite the mismatches, hybridization to the target nucleic acid can still be sufficient to exhibit the desired inhibition of COPS3 expression. The decrease in binding affinity resulting from the mismatches can be advantageously compensated for by an increase in the number of nucleotides in the oligonucleotide complementary to the target nucleic acid and / or an increase in the number of modified nucleosides that can increase the binding affinity to the target, such as the number of 2'-sugar modified nucleosides containing LNA present within the oligonucleotide sequence.

[0177] One aspect of the invention relates to nucleic acid molecules 12 to 60 nucleotides in length, comprising a continuous nucleotide sequence at least 12 nucleotides in length, such as at least 12 to 30 nucleotides in length, that is at least 95% complementary, such as fully complementary, to a mammalian COPS3 target nucleic acid, particularly a human COPS3 nucleic acid. These nucleic acid molecules can inhibit the expression of COPS3.

[0178] One aspect of the present invention relates to a nucleic acid molecule having a length of 12 to 30 nucleotides, comprising a contiguous nucleotide sequence that is at least 90% complementary, such as completely complementary, to a mammalian COPS3 target sequence, for example, having a length of 12 to 30 nucleotides.

[0179] A further aspect of the present invention relates to a nucleic acid molecule according to the present invention comprising a contiguous nucleotide sequence having a length of 12 to 20 nucleotides that is at least 90% complementary, such as completely complementary, to the target nucleic acid of SEQ ID NO: 1.

[0180] In some embodiments, the nucleic acid molecule comprises a contiguous sequence having a length of 12 to 30 nucleotides, which is at least 90% complementary, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or 100% complementary to a region of the target nucleic acid or target sequence.

[0181] It is advantageous if the nucleic acid molecule or its contiguous nucleotide sequence is completely complementary (100% complementary) to the target nucleic acid, or in some embodiments, may contain one or two mismatches between the oligonucleotide and the target nucleic acid.

[0182] In some embodiments, the oligonucleotide sequence is 100% complementary to the target nucleic acid regions of SEQ ID NO: 1 and / or SEQ ID NOs: 4, 5, 6, 7, 8, and / or 9.

[0183] In some embodiments, the nucleic acid molecule or contiguous nucleotide sequence of the present invention is at least 90% or 95% complementary, such as completely (or 100%) complementary, to the target nucleic acids of SEQ ID NOs: 1 and 2.

[0184] In some embodiments, the oligonucleotide or continuous nucleotide sequence of the present invention is at least 90% or 95% complementary, for example, completely (or 100%) complementary, to the target nucleic acid of SEQ ID NO: 2 and SEQ ID NO: 4, 5, 6, 7, 8, or 9.

[0185] In some embodiments, the oligonucleotide or continuous nucleotide sequence of the present invention is at least 90% or 95% complementary, for example, completely (or 100%) complementary, to the target nucleic acids of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0186] In some embodiments, the continuous sequence of the nucleic acid molecule of the present invention is at least 90% complementary, for example, completely complementary, to the region of SEQ ID NO: 1 selected from the group consisting of target sequences 1A to 1846A shown in Table 4.

[0187] In some embodiments, the continuous sequence of the nucleic acid molecule of the present invention is at least 90% complementary, for example, completely complementary, to the region of SEQ ID NO: 1 selected from the group consisting of target sequences 1C to 203C shown in Table 5.

[0188] In some embodiments, the nucleic acid molecule of the present invention has a length of 12 to 60 nucleotides, for example, 1 3 to 50, for example, 14 to 35, for example, 15 to 30, for example, 16 to 22 continuous nucleotides in length, or consists of them. In a preferred embodiment, the nucleic acid molecule has a length of 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides in length or consists of it.

[0189] In some embodiments, the continuous nucleotide sequence of the nucleic acid molecule that is complementary to the target nucleic acid has a length of 12 to 30, for example, 13 to 25, for example, 15 to 23, for example, 16 to 22 continuous nucleotides in length, or consists of them.

[0190] In some embodiments, the oligonucleotide is selected from the group consisting of antisense oligonucleotides, siRNAs, and shRNAs.

[0191] In some embodiments, the continuous nucleotide sequence of the siRNA or shRNA that is complementary to the target nucleic acid comprises or consists of 18 to 28, such as 19 to 26, such as 20 to 24, such as 21 to 23 consecutive nucleotides in length.

[0192] In some embodiments, the continuous nucleotide sequence of the antisense oligonucleotide complementary to the target nucleic acid comprises or consists of 12 to 22, such as 14 to 20, such as 16 to 20, such as 15 to 18, such as 16 to 18, such as 16 to 17, 18, 19 or 20 consecutive nucleotides in length.

[0193] It is understood that the continuous oligonucleotide sequence (motif sequence) can be modified, for example, to increase nuclease resistance and / or binding affinity for the target nucleic acid.

[0194] The pattern in which modified nucleosides (such as high-affinity modified nucleosides) are incorporated into the oligonucleotide sequence is generally referred to as oligonucleotide design.

[0195] The nucleic acid molecules of the present invention can be designed using modified nucleosides and RNA nucleosides (especially siRNA and shRNA molecules) or DNA nucleosides (especially single-stranded antisense oligonucleotides). It is advantageous to use high-affinity modified nucleosides.

[0196] In an advantageous embodiment, the nucleic acid molecule or continuous nucleotide sequence comprises one or more sugar-modified nucleosides, such as 2'-sugar-modified nucleosides, such as 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-fluoro-ANA and LNA nucleosides, independently selected from the group consisting of one or more 2'-sugar-modified nucleosides. It is advantageous if one or more of the modified nucleosides (plural possible) are locked nucleic acids (LNA).

[0197] In some embodiments, the continuous nucleotide sequence comprises LNA nucleosides.

[0198] In some embodiments, the continuous nucleotide sequence comprises LNA nucleosides and DNA nucleosides.

[0199] In some embodiments, the continuous nucleotide sequence comprises 2'-O-methoxyethyl (2'MOE) nucleosides.

[0200] In some embodiments, the continuous nucleotide sequence comprises 2'-O-methoxyethyl (2'MOE) nucleosides and DNA nucleosides.

[0201] Advantageously, the most 3'-terminal nucleoside of the antisense oligonucleotide, or its continuous nucleotide sequence, is a 2'-sugar modified nucleoside.

[0202] In a further embodiment, the nucleic acid molecule comprises at least one modified internucleoside linkage. Suitable internucleoside modifications are described in the "Definition" section of "Modified internucleoside linkages".

[0203] Advantageously, the oligonucleotide comprises at least one modified internucleoside linkage such as phosphorothioate or phosphorodithioate.

[0204] In some embodiments, at least one internucleoside linkage in the continuous nucleotide sequence is a phosphodiester internucleoside linkage.

[0205] It is advantageous if at least 2 to 3 internucleoside linkages at the 5'-end or 3'-end of the oligonucleotide are phosphorothioate internucleoside linkages.

[0206] For a single-stranded antisense oligonucleotide, it is advantageous if at least 75%, for example all, of the internucleoside linkages within the continuous nucleotide sequence are phosphorothioate linkages. In some embodiments, all of the internucleoside linkages in the continuous sequence of the single-stranded antisense oligonucleotide are phosphorothioate linkages.

[0207] In advantageous embodiments of the invention, the antisense oligonucleotides of the invention can recruit RNase H, such as RNase H1. Advantageous structural designs are, for example, the gapmer designs described in the "Definitions" section of "Region D' or D'' in the oligonucleotide". In the present invention, it is advantageous if the antisense oligonucleotide of the present invention is a gapmer of the F-G-F' design.

[0208] In all cases, the F-G-F' design may further include regions D' and / or D'' as described in the "Definitions" section of "Region D' or D'' in the oligonucleotide".

[0209] The present invention provides an antisense oligonucleotide according to the present invention, such as antisense oligonucleotides 12 - 24, for example 12 - 18 nucleosides in length, comprising a continuous nucleotide sequence comprising at least 14, for example at least 15, for example 16, consecutive nucleotides present in SEQ ID NO: 23.

[0210] The present invention provides an antisense oligonucleotide according to the present invention, such as antisense oligonucleotides 12 - 24, for example 12 - 18 nucleosides in length, comprising a continuous nucleotide sequence comprising at least 14, for example at least 15, for example 16, consecutive nucleotides present in SEQ ID NO: 24.

[0211] The present invention provides an antisense oligonucleotide according to the present invention, for example, a nucleoside having a length of 12 to 24, for example 12 to 18, which comprises a continuous nucleotide sequence containing at least 14, for example at least 15, for example 16 continuous nucleotides present in SEQ ID NO: 25.

[0212] The present invention provides an LNA gapmer according to the present invention comprising or consisting of the continuous nucleotide sequence shown in SEQ ID NO: 23, 24 or 25. In some embodiments, the LNA gapmer is an LNA gapmer having CMP ID NO: 23_1, 24_1 or 25_1 in Table 7.

[0213] In a further aspect of the present invention, a nucleic acid molecule such as an antisense oligonucleotide, siRNA or shRNA of the present invention can be directly targeted to the liver by covalently binding it to a conjugate moiety capable of binding to the asialoglycoprotein receptor (ASGPr), for example a divalent or trivalent GalNAc cluster.

[0214] Conjugate Since HBV infection mainly affects hepatocytes in the liver, it is advantageous to bind a COPS3 inhibitor to the conjugate moiety to increase the delivery of the inhibitor to the liver as compared to the unbound inhibitor. In one embodiment, the liver targeting moiety is selected from a moiety containing cholesterol or other lipids, or a conjugate moiety capable of binding to the asialoglycoprotein receptor (ASGPR).

[0215] In some embodiments, the present invention provides a conjugate comprising a nucleic acid molecule of the present invention covalently bound to a conjugate moiety.

[0216] The asialoglycoprotein receptor (ASGPR) conjugate moiety comprises one or more carbohydrate moieties capable of binding to an asialoglycoprotein receptor (ASPGR targeting moiety) with an affinity equal to or greater than that of galactose. The affinities of a number of galactose derivatives for the asialoglycoprotein receptor have been studied (see, e.g., Jobst, S.T. and Drickamer, K. JBC 1996, 271, 6686), or can be readily determined using methods typical in the art.

[0217] In one embodiment, the conjugate moiety comprises at least one asialoglycoprotein receptor targeting moiety selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine, and N-isobutanoylgalactosamine. Advantageously, the asialoglycoprotein receptor targeting moiety is N-acetylgalactosamine (GalNAc).

[0218] To generate the ASGPR conjugate moiety, an ASPGR targeting moiety (preferably GalNAc) can be attached to a conjugate scaffold. Generally, the ASPGR targeting moiety can be at the same terminus of the scaffold. In one embodiment, the conjugate moiety consists of 2 to 4 terminal GalNAc moieties attached to a spacer that attaches each GalNAc moiety to a branching molecule that can be attached to an antisense oligonucleotide.

[0219] In a further embodiment, the conjugate moiety is monovalent, divalent, trivalent, or tetravalent with respect to the asialoglycoprotein receptor targeting moiety. Advantageously, the asialoglycoprotein receptor targeting moiety comprises an N-acetylgalactosamine (GalNAc) moiety.

[0220] Examples of the GalNAc conjugate moiety include those described in WO 2014 / 179620, WO 2016 / 055601, and PCT / EP2017 / 059080 (incorporated herein by reference), and small peptides to which a GalNAc moiety such as Tyr-Glu-Glu-(aminohexyl GalNAc)3 (YEE(ahGalNAc)3; a glycotripeptide that binds to the asialoglycoprotein receptor on hepatocytes, see, e.g., Duff, et al., Methods Enzymol, 2000, 313, 297) is attached; lysine-based galactose clusters (e.g., L3G4; Biessen, et al., Cardovasc. Med., 1999, 214); and collagen-based galactose clusters (e.g., carbohydrate recognition motifs for the asialoglycoprotein receptor).

[0221] 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.

[0222] In one embodiment, the conjugate moiety is trivalent N-acetylgalactosamine (GalNAc) such as that shown in Figure 1. In one embodiment, the conjugate moiety is the trivalent N-acetylgalactosamine (GalNAc) of Figure 1A-1 or Figure 1A-2, or a mixture of both. In one embodiment, the conjugate moiety is the trivalent N-acetylgalactosamine (GalNAc) of Figure 1B-1 or Figure 1B-2, or a mixture of both. In one embodiment, the conjugate moiety is the trivalent N-acetylgalactosamine (GalNAc) of Figure 1C-1 or Figure 1C-2, or a mixture of both. In one embodiment, the conjugate moiety is the trivalent N-acetylgalactosamine (GalNAc) of Figure 1D-1 or Figure 1D-2, or a mixture of both.

[0223] Manufacturing method In a further aspect, the present invention provides a method for producing an oligonucleotide of the present invention, which comprises reacting nucleotide units to thereby form covalently linked contiguous nucleotide units consisting of oligonucleotides. Preferably, this method uses phosphoramidite chemistry (see, for example, Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313). In a further embodiment, this method further comprises reacting a contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide. In a further aspect, a method for producing a composition of the present invention is provided, which comprises mixing an oligonucleotide or a conjugated oligonucleotide of the present invention with a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0224] Pharmaceutical salts The compounds according to the present invention may exist in the form of their pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" refers to conventional acid addition salts or base addition salts that retain the biological effectiveness and properties of the compounds of the present invention.

[0225] In a further aspect, the present invention provides pharmaceutically acceptable salts of nucleic acid molecules or conjugates thereof, such as pharmaceutically acceptable sodium salts, ammonium salts, or potassium salts.

[0226] Pharmaceutical composition In a further aspect, the present invention provides a pharmaceutical composition comprising any of the compounds of the present invention, particularly the aforementioned nucleic acid molecule and / or nucleic acid molecule conjugate or a salt thereof, and a pharmaceutically acceptable diluent, carrier, salt and / or adjuvant. Pharmaceutically acceptable diluents include phosphate buffered saline (PBS), and pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts. In some embodiments, the pharmaceutically acceptable diluent is sterile phosphate buffered saline. In some embodiments, the nucleic acid molecule is used in a pharmaceutically acceptable diluent at a concentration of a solution of 50 to 300 μM.

[0227] Formulations suitable for use in the present invention can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief overview of methods of drug delivery, see, for example, Langer (Science 249:1527-1533, 1990). WO 2007 / 031091 provides further suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (incorporated herein by reference). Appropriate dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, prodrug formulations are also provided in WO 2007 / 031091. For a brief overview of methods of drug delivery, see, for example, Langer (Science 249:1527-1533, 1990). WO 2007 / 031091 provides further suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (incorporated herein by reference). Appropriate dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, prodrug formulations are also provided in WO 2007 / 031091.

[0228] In some embodiments, the nucleic acid molecule or nucleic acid molecule conjugate of the present invention, or a pharmaceutically acceptable salt thereof, is in solid form, such as a powder, such as a lyophilized powder.

[0229] The compounds, nucleic acid molecules or nucleic acid molecule conjugates of the present invention can be mixed with pharmaceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the preparation of pharmaceutical compositions depend on many criteria including, but not limited to, the route of administration, the degree of the disease, or the dosage administered.

[0230] These compositions may be sterilized by conventional sterilization techniques or may be passed through a sterilizing filter. The resulting aqueous solution can be packaged for use as is or lyophilized, and the lyophilized preparation is combined with a sterile aqueous carrier prior to administration. The pH of the preparation will typically be from 3 to 11, more preferably from 5 to 9 or 6 to 8, most preferably from 7 to 8, for example from 7 to 7.5. The resulting solid form compositions can be packaged in multiple single-dose units, each containing a fixed amount of the above agent or group of agents, such as in a sealed package of tablets or capsules. The solid form compositions can also be packaged in flexible amount containers such as squeezable tubes designed for topically applicable creams or ointments.

[0231] In some embodiments, the nucleic acid molecule or nucleic acid molecule conjugate of the present invention is a prodrug. In particular, with respect to the nucleic acid molecule conjugate, the conjugate moiety is cleaved from the nucleic acid molecule when the prodrug is delivered to the site of action, e.g., the target cell.

[0232] Administration The compounds, nucleic acid molecules or nucleic acid molecule conjugates, or pharmaceutical compositions of the present invention can be administered locally (e.g., to the skin, by inhalation, to the eye, or to the ear, etc.), or enterally (orally or through the gastrointestinal tract), or parenterally (intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly, or intrathecally, etc.).

[0233] In preferred embodiments, the oligonucleotides or pharmaceutical compositions of the present invention are administered by a parenteral route including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion. In one embodiment, the active nucleic acid molecule or nucleic acid molecule conjugate is administered intravenously. In another embodiment, the active nucleic acid molecule or nucleic acid molecule conjugate is administered subcutaneously.

[0234] In some embodiments, the nucleic acid molecules, nucleic acid molecule conjugates or pharmaceutical compositions of the present invention are administered at a dose of 0.1 to 15 mg / kg, such as 0.2 to 10 mg / kg, such as 0.25 to 5 mg / kg. Administration can be once a week, once every two weeks, once every three weeks, or even once a month.

[0235] The present invention also provides the use of a COPS3 inhibitor, such as the nucleic acid molecule or nucleic acid molecule conjugate of the present invention, in the manufacture of a medicament in a dosage form for subcutaneous administration.

[0236] Combination therapy In some embodiments, the inhibitor of the present invention, such as the nucleic acid molecule, nucleic acid molecule conjugate or pharmaceutical composition of the present invention, is for use in combination treatment with another therapeutic agent. The therapeutic agent can be, for example, a standard therapeutic agent for the above-mentioned diseases or disorders.

[0237] By way of example, a COPS3 inhibitor such as the nucleic acid molecule or nucleic acid molecule conjugate of the present invention can be used in combination with other active agents, such as oligonucleotide-based antiviral agents that act via any of antisense (including other LNA oligomers), siRNA (such as ARC520), aptamers, morpholinos, or any other antiviral, nucleotide sequence-dependent mode of action, for example sequence-specific oligonucleotide-based antiviral agents.

[0238] As a further example, a COPS3 inhibitor such as the nucleic acid molecule or nucleic acid molecule conjugate of the present invention can be used in combination with other active substances, such as immunostimulatory antiviral compounds such as interferon (for example, pegylated interferon alpha), TLR7 agonists (for example, GS-9620), or therapeutic vaccines.

[0239] As a further example, a COPS3 inhibitor such as the nucleic acid molecule or nucleic acid molecule conjugate of the present invention can be used in combination with other active substances having antiviral activity, such as small molecules. These other active substances can be, for example, nucleoside / nucleotide inhibitors (for example, entecavir or tenofovir disoproxil fumarate), envelope inhibitors, entry inhibitors (for example, Myrcludex B).

[0240] In certain embodiments, additional therapeutic agents can be HBV agents, hepatitis C virus (HCV) agents, chemotherapeutic agents, antibiotics, analgesics, non-steroidal anti-inflammatory (NSAID) agents, antifungal agents, anti-parasitic agents, anti-emetic agents, anti-diarrheal agents, or immunosuppressive agents.

[0241] In particular, in related embodiments, additional HBV agents can be interferon alpha-2b, interferon alpha-2a, and interferon alphacon-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).

[0242] In other specific related embodiments, additional HCV agents can be interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and non-pegylated); ribavirin; Pegasis; HCV RNA replication inhibitors (e.g., VP50406 series from ViroPharma); HCV antisense agents; HCV therapeutic vaccines; HCV protease inhibitors; HCV helicase inhibitors; or HCV monoclonal antibody therapy or HCV polyclonal antibody therapy.

[0243] Use The nucleic acid molecules of the present invention can be utilized, for example, as research reagents for diagnosis, treatment, and prevention.

[0244] In the research, such nucleic acid molecules can be used to specifically regulate the synthesis of COPS3 protein in cells (e.g., in vitro cell cultures) and experimental animals, thereby facilitating the functional analysis of the target or the evaluation of its usefulness as a target for therapeutic intervention. Typically, target regulation is achieved by degrading or inhibiting the mRNA that produces the protein, thereby preventing protein formation, or by degrading or inhibiting the modulator of the gene or mRNA that produces the protein.

[0245] When the nucleic acid molecules of the present invention are used for research or diagnosis, the target nucleic acid can be cDNA, or a synthetic nucleic acid derived from DNA or RNA.

[0246] The present invention also encompasses in vivo or in vitro methods for regulating COPS3 expression in target cells expressing COPS3, which methods include administering to the cells an effective amount of the nucleic acid molecules, conjugate compounds, or pharmaceutical compositions of the present invention.

[0247] In some embodiments, the target cells are mammalian cells, particularly human cells. The target cells can be in vitro cell cultures or in vivo cells that form part of a mammalian tissue. In a preferred embodiment, the target cells are present in the liver. The target cells can be hepatocytes.

[0248] One aspect of the present invention relates to COPS3 inhibitors such as the nucleic acid molecules, conjugate compounds, or pharmaceutical compositions of the present invention for use as a medicament.

[0249] In one aspect of the present invention, the COPS3 inhibitor such as the nucleic acid molecule, conjugate compound or pharmaceutical composition of the present invention can reduce the cccDNA level in infected cells and thus inhibit HBV infection. In particular, the nucleic acid molecule can affect one or more of the following parameters in infected cells: (i) reduction of cccDNA, and / or (ii) reduction of pgRNA, and / or (iii) reduction of HBV DNA, and / or (iv) reduction of HBV viral antigen.

[0250] For example, a nucleic acid molecule that inhibits HBV infection can (i) reduce the cccDNA level in infected cells by at least 40%, such as 50% or 60% reduction compared to the control, or (ii) reduce the pgRNA level by at least 40%, such as 50% or 60% reduction compared to the control. The control can be untreated cells or animals, or cells or animals treated with an appropriate negative control.

[0251] Inhibition of HBV infection can be measured in vitro using primary human hepatocytes infected with HBV, or in vivo using a humanized liver cell PXB mouse model (available from PhoenixBio, see also Kakuni et al 2014 Int.J.Mol.Sci.15:58-74). Inhibition of the secretion of HBsAg and / or HBeAg can be measured by ELISA using, for example, a 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. Further methods for evaluating whether a test compound inhibits HBV infection are, for example, by measuring the secretion of HBV DNA by qPCR as described in International Publication No. 2015 / 173208, or using Northern blot, in situ hybridization, or immunofluorescence.

[0252] Due to the reduction of COPS3 levels, the nucleic acid molecules, conjugate compounds or pharmaceutical compositions of the present invention can be used to inhibit the onset of HBV infection or in the treatment of HBV infection. In particular, due to the destabilization and reduction of cccDNA, the nucleic acid molecules, conjugate compounds or pharmaceutical compositions of the present invention can more efficiently inhibit the onset of chronic HBV infection or treat chronic HBV infection compared to compounds that only reduce the secretion of HBsAg.

[0253] Therefore, one aspect of the present invention relates to the use of a COPS3 inhibitor such as the nucleic acid molecule, conjugate compound or pharmaceutical composition of the present invention to reduce cccDNA and / or pgRNA in HBV-infected individuals.

[0254] A further aspect of the present invention relates to the use of a COPS3 inhibitor such as the nucleic acid molecule, conjugate compound or pharmaceutical composition of the present invention to inhibit or treat the onset of chronic HBV infection.

[0255] A further aspect of the present invention relates to the use of a COPS3 inhibitor such as the nucleic acid molecule, conjugate compound or pharmaceutical composition of the present invention to reduce the infectivity of HBV-infected subjects. In a particular aspect of the present invention, a COPS3 inhibitor such as the nucleic acid molecule, conjugate compound or pharmaceutical composition of the present invention inhibits the onset of chronic HBV infection.

[0256] Subjects treated with a COPS3 inhibitor such as the nucleic acid molecule, conjugate compound or pharmaceutical composition of the present invention (or who prophylactically receive the nucleic acid molecule, conjugate compound or pharmaceutical composition of the present invention) are preferably humans, more preferably human patients who are HBsAg positive and / or HBeAg positive, and even more preferably human patients who are HBsAg positive and HBeAg positive.

[0257] Accordingly, the present invention relates to a method for treating HBV infection, which comprises administering an effective amount of a COPS3 inhibitor such as a nucleic acid molecule, a conjugate compound or a 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. In one embodiment, the COPS3 inhibitor of the present invention is not intended for the treatment of hepatocellular carcinoma, but only for its prevention.

[0258] The present invention also provides the use of a COPS3 inhibitor such as a nucleic acid molecule, a conjugate compound or a pharmaceutical composition of the present invention for the manufacture of a pharmaceutical, particularly a pharmaceutical for use in the treatment of HBV infection or chronic HBV infection or for reducing the infectivity of HBV-infected individuals. In a preferred embodiment, the pharmaceutical is manufactured in a dosage form for subcutaneous administration.

[0259] The present invention also provides a pharmaceutical composition of the present invention for the manufacture of a pharmaceutical, wherein the pharmaceutical is in a dosage form for intravenous administration, which is the use of a COPS3 inhibitor such as a nucleic acid molecule, a conjugate compound.

[0260] The COPS3 inhibitor such as the nucleic acid molecule, conjugate or pharmaceutical composition of the present invention can be used in combination therapy. For example, the COPS3 inhibitor such as the nucleic acid molecule, conjugate or pharmaceutical composition of the present invention can be used in combination with interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and non-pegylated), ribavirin, lamivudine (3TC), entecavir, tenofovir, telbivudine (LdT), adefovir, or other anti-HBV agents such as HBV RNA replication inhibitors, HBsAg secretion inhibitors, HBV capsid inhibitors, antisense oligomers (for example, described in International Publication Nos. 2012 / 145697, 2014 / 179629, and 2017 / 216390), siRNA (for example, described in International Publication Nos. 2005 / 014806, 2012 / 024170, 2012 / 2055362, 2013 / 003520, 2013 / 159109, 2017 / 027350, and 2017 / 015175), HBV therapeutic vaccines, HBV prophylactic vaccines, HBV antibody therapies (monoclonal or polyclonal), or other anti-HBV agents such as TLR2, 3, 7, 8, or 9 agonists for the treatment and / or prevention of HBV.

[0261] Embodiments of the present invention The following embodiments of the present invention can be used in combination with any other embodiments described herein. The definitions and explanations provided above, particularly in the sections of "Summary of the Invention", "Definitions", and "Detailed Description of the Invention", shall apply mutatis mutandis hereinafter.

[0262] 1. A COPS3 inhibitor for the treatment and / or prevention of hepatitis B virus (HBV) infection.

[0263] 2. The COPS3 inhibitor according to embodiment 1, wherein the COPS3 inhibitor is administered in an effective amount.

[0264] 3. The COPS3 inhibitor according to embodiment 1 or 2, wherein the HBV infection is a chronic infection.

[0265] 4. The COPS3 inhibitor according to any one of Embodiments 1 to 3, which is capable of reducing cccDNA and / or pgRNA in infected cells.

[0266] 5. The COPS3 inhibitor according to any one of Embodiments 1 to 4, which is capable of preventing or reducing the association of COPS3 with cccDNA.

[0267] 6. The COPS3 inhibitor according to Embodiment 5, which is a small molecule that specifically binds to the COPS3 protein and is capable of preventing or reducing the association of the COPS3 protein with cccDNA.

[0268] 7. The COPS3 inhibitor according to Embodiment 6, wherein the COPS3 protein is encoded by SEQ ID NO: 4, 5, 6, 7, 8 or 9.

[0269] 8. The COPS3 inhibitor according to any one of Embodiments 1 to 7, which is a nucleic acid molecule having a length of 12 to 60 nucleotides and containing or consisting of a continuous nucleotide sequence of at least 12 nucleotides that is at least 90% complementary to the mammalian COPS3 target nucleic acid.

[0270] 9. The COPS3 inhibitor according to Embodiment 8, which is capable of reducing the level of the mammalian COPS3 target nucleic acid.

[0271] 10. The COPS3 inhibitor according to Embodiment 8 or 9, wherein the mammalian COPS3 target nucleic acid is RNA.

[0272] 11. The COPS3 inhibitor according to Embodiment 10, wherein the RNA is pre-mRNA.

[0273] 12. The COPS3 inhibitor according to any one of Embodiments 8 to 11, wherein the nucleic acid molecule is selected from the group consisting of antisense oligonucleotides, siRNAs and shRNAs.

[0274] 13. The COPS3 inhibitor according to embodiment 12, wherein the nucleic acid molecule is a single-stranded antisense oligonucleotide or a double-stranded siRNA.

[0275] 14. The COPS3 inhibitor according to any one of embodiments 8 to 13, wherein the mammalian COPS3 target nucleic acid is selected from the group consisting of SEQ ID NOs: 1, 4, 5, 6, 7, 8, and 9.

[0276] 15. The COPS3 inhibitor according to any one of embodiments 8 to 13, wherein the continuous nucleotide sequence of the nucleic acid molecule is at least 98% complementary to the target nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 2.

[0277] 16. The continuous nucleotide sequence of the nucleic acid molecule is at least 98% complementary to the target nucleic acids of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, according to any one of embodiments 8 to 13 The COPS3 inhibitor described for use in any one of the above.

[0278] 17. The COPS3 inhibitor according to any one of embodiments 1 to 16, wherein the cccDNA in HBV-infected cells is reduced by at least 50%, such as 60%, when compared to the control.

[0279] 18. The COPS3 inhibitor according to any one of embodiments 1 to 16, wherein the pgRNA in HBV-infected cells is reduced by at least 50%, such as 60%, when compared to the control.

[0280] 19. The COPS3 inhibitor according to any one of embodiments 8 to 18, wherein the mammalian COPS3 target nucleic acid is reduced by at least 50%, such as 60%, when compared to the control.

[0281] 20. A nucleic acid molecule having a nucleotide sequence of 12 to 60 nucleotides in length, comprising or consisting of a nucleotide sequence of 12 to 30 nucleotides in length, wherein the continuous nucleotide sequence is at least 90% complementary, such as 95%, such as 98%, such as completely complementary, to the mammalian COPS3 target nucleic acid.

[0282] 21. The nucleic acid molecule according to embodiment 20, wherein the nucleic acid molecule is chemically produced.

[0283] 22. The nucleic acid molecule according to embodiment 20 or 21, wherein the mammalian COPS3 target nucleic acid is selected from the group consisting of SEQ ID NOs: 1, 4, 5, 6, 7, 8, and 9.

[0284] 23. The nucleic acid molecule according to embodiment 20 or 21, wherein the continuous nucleotide sequence is at least 98% complementary to the target nucleic acids of SEQ ID NOs: 1 and 2.

[0285] 24. The nucleic acid molecule according to embodiment 20 or 21, wherein the continuous nucleotide sequence is completely complementary to the target nucleic acids of SEQ ID NOs: 1, 2, and 3.

[0286] 25. The nucleic acid molecule according to any one of embodiments 20 to 23, wherein the nucleic acid molecule is 12 to 30 nucleotides in length.

[0287] 26. The nucleic acid molecule according to any one of embodiments 20 to 25, wherein the nucleic acid molecule is an RNAi molecule such as double-stranded siRNA or shRNA.

[0288] 27. The nucleic acid molecule according to any one of embodiments 20 to 25, wherein the nucleic acid molecule is a single-stranded antisense oligonucleotide.

[0289] 28. The nucleic acid molecule according to any one of embodiments 20 to 27, wherein the continuous nucleotide sequence is completely complementary to a target nucleic acid sequence selected from Table 4 or Table 5.

[0290] 29. The nucleic acid molecule according to any one of embodiments 20 to 28, which can hybridize with the target nucleic acids of SEQ ID NOs: 1 and 2 with a ΔG° of less than -15 kcal.

[0291] 30. The nucleic acid molecule according to any one of embodiments 20 to 29, wherein the continuous nucleotide sequence comprises or consists of at least 14 consecutive nucleotides, particularly 15, 16, 17, 18, 19, 20, 21, or 22 consecutive nucleotides.

[0292] 31. The nucleic acid molecule according to any one of embodiments 20 to 29, wherein the continuous nucleotide sequence comprises or consists of 14 to 22 nucleotides. from.

[0293] 32. The nucleic acid molecule according to embodiment 31, wherein the continuous nucleotide sequence comprises or consists of 16 to 20 nucleotides.

[0294] 33. The nucleic acid molecule according to any one of embodiments 20 to 32, wherein the nucleic acid molecule comprises or consists of 14 to 25 nucleotides in length.

[0295] 34. The nucleic acid molecule according to embodiment 33, wherein the nucleic acid molecule comprises or consists of at least one oligonucleotide strand 16 to 22 nucleotides in length.

[0296] 35. The nucleic acid molecule according to any one of embodiments 20 to 34, wherein the continuous nucleotide sequence is completely complementary to a target sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, and 13.

[0297] 36. The nucleic acid molecule according to any one of embodiments 20 to 35, wherein the continuous nucleotide sequence has 0 to 3 mismatches compared to the mammalian COPS3 target nucleic acid to which it is complementary.

[0298] 37. The nucleic acid molecule according to embodiment 36, wherein the continuous nucleotide sequence has 1 mismatch compared to the mammalian COPS3 target nucleic acid.

[0299] 38. The nucleic acid molecule according to embodiment 36, wherein the continuous nucleotide sequence has 2 mismatches compared to the mammalian COPS3 target nucleic acid.

[0300] 39. The nucleic acid molecule according to embodiment 36, wherein the continuous nucleotide sequence is completely complementary as compared with a mammalian COPS3 target nucleic acid.

[0301] 40. The nucleic acid molecule according to any one of embodiments 20 to 39, comprising one or more modified nucleosides.

[0302] 41. The nucleic acid molecule according to embodiment 40, wherein the one or more modified nucleosides are high-affinity modified nucleosides.

[0303] 42. The nucleic acid molecule according to embodiment 40 or 41, wherein the one or more modified nucleosides are 2'-sugar modified nucleosides.

[0304] 43. The nucleic acid molecule according to embodiment 42, wherein the 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, 2'-fluoro-ANA, and LNA nucleosides.

[0305] 44. The nucleic acid molecule according to any one of embodiments 40 to 43, wherein the one or more modified nucleosides are LNA.

[0306] 45. The nucleic acid molecule according to embodiment 44, wherein the modified LNA nucleoside is selected from the group consisting of oxy-LNA, amino-LNA, thio-LNA, cET, and ENA.

[0307] 46. The modified LNA nucleoside has the following 2'-4' bridge -O-CH2- oxy-LNA, which is the nucleic acid molecule according to embodiment 44 or 45.

[0308] 47. The nucleic acid molecule according to embodiment 46, wherein the oxy-LNA is beta-D-oxy-LNA.

[0309] 48. The modified LNA nucleoside has the following 2'-4' bridge -O-CH(CH3)- The nucleic acid molecule according to embodiment 44 or 45, which is a cET having the above structure.

[0310] 49. The nucleic acid molecule according to embodiment 48, wherein the cET is (S)cET, that is, 6'(S)-methyl-beta-D-oxy-LNA.

[0311] 50. The LNA is ENA and has the following 2'-4' bridge -O-CH2-CH2- The nucleic acid molecule according to embodiment 44 or 45.

[0312] 51. The nucleic acid molecule according to any one of embodiments 20 to 50, wherein the nucleic acid molecule comprises at least one modified nucleoside internucleoside linkage.

[0313] 52. The nucleic acid molecule according to embodiment 51, wherein at least one modified nucleoside internucleoside linkage is a phosphorothioate nucleoside internucleoside linkage.

[0314] 53. The nucleic acid molecule according to any one of embodiments 20 to 52, wherein the nucleic acid molecule is an antisense oligonucleotide capable of recruiting RNase H.

[0315] 54. The nucleic acid molecule according to embodiment 53, wherein the antisense oligonucleotide or the continuous nucleotide sequence is a gapmer.

[0316] 55. The nucleic acid molecule according to embodiment 54, wherein the antisense oligonucleotide or its continuous nucleotide sequence consists of or contains a gapmer of the formula 5'-F-G-F'-3', wherein regions F and F' independently comprise or consist of 1 to 4 2'-sugar modified nucleosides, and G is a region of 6 to 18 nucleosides capable of recruiting RNase H.

[0317] 56. The nucleic acid molecule according to embodiment 55, wherein the 1 to 4 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, arabinonucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleosides.

[0318] 57. The nucleic acid molecule according to embodiment 55 or 56, wherein one or more of the 1 to 4 2'-sugar-modified nucleosides in regions F and F' are LNA nucleosides.

[0319] 58. The nucleic acid molecule according to embodiment 57, wherein all of the 2'-sugar-modified nucleosides in regions F and F' are LNA nucleosides.

[0320] 59. The nucleic acid molecule according to embodiments 56 to 58, wherein the LNA nucleoside is selected from the group consisting of beta-D-oxy-LNA, alpha-L-oxy-LNA, beta-D-amino-LNA, alpha-L-amino-LNA, beta-D-thio-LNA, alpha-L-thio-LNA, (S)cET, (R)cET beta-D-ENA, and alpha-L-ENA.

[0321] 60. The nucleic acid molecule according to any one of embodiments 56 to 59, wherein regions F and F' consist of the same LNA nucleoside. 61. The nucleic acid molecule according to any one of embodiments 56 to 60, wherein all of the 2'-sugar-modified nucleosides in regions F and F' are oxy-LNA nucleosides.

[0322] 62. The nucleic acid molecule according to any one of embodiments 55 to 61, wherein the nucleosides in region G are DNA nucleosides.

[0323] 63. The nucleic acid molecule according to embodiment 62, wherein region G consists of at least 75% DNA nucleosides.

[0324] 63. The nucleic acid molecule according to embodiment 62, wherein region G consists of at least 75% DNA nucleosides.

[0325] The nucleic acid molecule according to embodiment 63, wherein all nucleosides in region G are DNA nucleosides.

[0326] A conjugate compound comprising the nucleic acid molecule according to any one of embodiments 20 to 64 and at least one conjugate moiety covalently bound to the nucleic acid molecule.

[0327] The conjugate compound according to embodiment 65, wherein the nucleic acid molecule is a double-stranded siRNA and the conjugate moiety is covalently bound to the sense strand of the siRNA.

[0328] The conjugate compound according to embodiment 65 or 66, wherein the conjugate moiety is selected from carbohydrates, cell surface receptor ligands, prodrugs, hormones, lipophilic substances, polymers, proteins, peptides, toxins, vitamins, viral proteins, or combinations thereof.

[0329] The conjugate compound according to any one of embodiments 65 to 67, wherein the conjugate moiety is capable of binding to an asialoglycoprotein receptor.

[0330] The conjugate compound according to embodiment 68, wherein the conjugate moiety comprises at least one asialoglycoprotein receptor targeting moiety selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, N-n-butanoyl-galactosamine, and N-isobutanoylgalactosamine.

[0331] The conjugate compound according to embodiment 69, wherein the asialoglycoprotein receptor targeting moiety is N-acetylgalactosamine (GalNAc).

[0332] The conjugate compound according to embodiment 69 or 70, wherein the conjugate moiety is monovalent, divalent, trivalent, or tetravalent with respect to the asialoglycoprotein receptor targeting moiety.

[0333] 72. The conjugate compound according to embodiment 71, wherein the conjugate moiety consists of 2 to 4 terminal GalNAc moieties and a spacer that binds each GalNAc moiety to a branching molecule that can be conjugated to the antisense compound.

[0334] 73. The conjugate compound according to embodiment 72, wherein the spacer is a PEG spacer.

[0335] 74. The conjugate compound according to any one of embodiments 68 to 73, wherein the conjugate moiety is a trivalent N-acetylgalactosamine (GalNAc) moiety.

[0336] 75. The conjugate compound according to any one of embodiments 68 to 74, wherein the conjugate moiety is selected from one of the trivalent GalNAc moieties in FIG. 1.

[0337] 76. The conjugate compound according to embodiment 75, wherein the conjugate moiety is the trivalent GalNAc moiety of FIG. 1D-1 or FIG. 1D-2, or a mixture of both.

[0338] 77. The conjugate compound according to any one of embodiments 65 to 76, comprising a linker disposed between the nucleic acid molecule and the conjugate moiety.

[0339] 78. The conjugate compound according to embodiment 77, wherein the linker is a physiologically labile linker.

[0340] 79. The conjugate compound according to embodiment 78, wherein the physiologically labile linker is a nuclease-sensitive linker.

[0341] 80. The conjugate compound according to embodiment 78 or 79, wherein the physiologically labile linker is composed of 2 to 5 consecutive phosphodiester bonds.

[0342] 81. A conjugate compound according to any one of embodiments 68 to 80, which exhibits improved cell distribution between the liver and the kidney or improved cellular uptake of the conjugate compound into the liver as compared to non-conjugated nucleic acids.

[0343] 82. A pharmaceutical composition comprising a nucleic acid molecule according to any one of embodiments 20 to 64, a conjugate compound according to embodiments 65 to 81 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier, salt and / or adjuvant.

[0344] 83. A method for identifying a compound for preventing, ameliorating, and / or inhibiting hepatitis B virus (HBV) infection, comprising: a. contacting a test compound with i. a COPS3 polypeptide, or ii. a cell expressing COPS3; b. measuring the expression and / or activity of COPS3 in the presence or absence of the test compound; and c. identifying a compound that reduces the expression and / or activity of COPS3 and decreases cccDNA.

[0345] 84. An in vivo or in vitro method for regulating COPS3 expression in a target cell expressing COPS3, comprising administering to the cell an effective amount of a nucleic acid molecule according to any one of embodiments 20 to 64, a conjugate compound according to any one of embodiments 65 to 81, or a pharmaceutical composition according to embodiment 82.

[0346] 85. The method according to embodiment 84, wherein the COPS3 expression is reduced by at least 50% or at least 60% in the target cell as compared to the level without any treatment or treatment with a control.

[0347] 86. The method according to embodiment 84, wherein the target cells are infected with HBV, and the cccDNA of the HBV-infected cells is reduced by at least 50% or at least 60% in the HBV-infected target cells as compared to the level without treatment or treated with a control.

[0348] 87. A method for treating or preventing a disease such as HBV infection, comprising administering to a subject suffering from or susceptible to the disease a therapeutically effective amount or a prophylactically effective amount of a nucleic acid molecule according to any one of embodiments 20-64, a conjugate compound according to any one of embodiments 65-81, or a pharmaceutical composition according to embodiment 82. 87. A method for treating or preventing a disease such as HBV infection, comprising administering to a subject suffering from or susceptible to the disease a therapeutically effective amount or a prophylactically effective amount of a nucleic acid molecule according to any one of embodiments 20-64, a conjugate compound according to any one of embodiments 65-81, or a pharmaceutical composition according to embodiment 82.

[0349] 88. A nucleic acid molecule according to any one of embodiments 20-64, or a conjugate compound according to any one of embodiments 65-81, or a pharmaceutical composition according to embodiment 82, for use as a medicament for treating or preventing a disease such as HBV infection in a subject.

[0350] 89. Use of a nucleic acid molecule according to any one of embodiments 20-64, or a conjugate compound according to any one of embodiments 65-81, for the preparation of a medicament for treating or preventing a disease such as HBV infection in a subject.

[0351] 90. The method, nucleic acid molecule, antisense oligonucleotide, conjugate compound or use according to any one of embodiments 87-89, wherein the subject is a mammal.

[0352] 91. The method, nucleic acid molecule, conjugate compound or use thereof according to embodiment 90, wherein the mammal is a human.

[0353] 92. The conjugate compound according to embodiment 75, wherein the conjugate moiety is the trivalent GalNAc moiety of Figure 1B-1 or Figure 1B-2, or a mixture of both.

[0354] Hereinafter, the present invention will be described by the following examples without limiting features.

Example

[0355] Materials and Methods siRNA Sequences and Compounds

Table 6

[0356] The siRNA pool (ON-TARGETplus SMART pool siRNA catalog number LU-011494-00-0005, Dharmacon) contains four individual siRNA molecules targeting the sequences listed in the table above.

Table 7

[0357] Oligonucleotide Synthesis Oligonucleotide synthesis is generally known in the art. The following is an applicable protocol. The oligonucleotides of the present invention may be generated by slightly different methods with respect to the apparatus, support, and concentration used.

[0358] Oligonucleotides are synthesized on a uridine universal support using the phosphoramidite approach of Oligomaker 48 on a 1 μmol scale. At the end of the synthesis, the oligonucleotides are cleaved from the solid support using aqueous ammonia at 60 °C for 5 - 16 hours. The oligonucleotides are purified by reverse-phase HPLC (RP-HPLC) or solid-phase extraction, characterized by UPLC, and the molecular weight is further confirmed by ESI-MS.

[0359] Extension of Oligonucleotides: Coupling of β-cyanoethyl-phosphoramidites (DNA-A(Bz), DNA-G(ibu), DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), or LNA-T) is performed using a solution of 0.1 M 5'-O-DMT protected amidite in acetonitrile and DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. In the final cycle, phosphoramidites with the desired modification, such as a C6 linker for attaching a conjugate group, or such a conjugate group itself can be used. Thiolation to introduce phosphorothioate linkages is performed by using xanthane hydride (0.01 M in acetonitrile / pyridine 9:1). Phosphodiester linkages can be introduced using 0.02 M iodine in THF / pyridine / water 7:2:1. The remaining reagents are those typically used in oligonucleotide synthesis.

[0360] For conjugation after solid-phase synthesis, commercially available C6 amino linker phosphoramidite can be used in the last cycle of solid-phase synthesis, and after deprotection and cleavage from the solid support, the amino-linked deprotected oligonucleotide is isolated. The conjugate is introduced by activation of functional groups using standard synthetic methods.

[0361] Purification by RP-HPLC: The crude compound is purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10μm 150×10mm column. 0.1 M ammonium acetate pH8 and acetonitrile are used as buffer at a flow rate of 5 mL / min. The collected fractions are lyophilized to obtain the purified compound typically as a white solid. Abbreviations: DCI: 4,5-dicyanoimidazole DCM: dichloromethane DMF: dimethylformamide DMT: 4,4'-dimethoxytrityl THF: tetrahydrofuran Bz: benzoyl Parent: Isobutyryl RP-HPLC: Reversed-phase high performance liquid chromatography

[0362] T m Assay: Dilute the oligonucleotide and RNA target (phosphate binding, PO) duplex to 3 mM with 500 mL of RNase-free water and mix with 500 mL of 2-fold T m buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM phosphate, pH 7.0). Heat this solution at 95 °C for 3 minutes and then anneal at room temperature for 30 minutes. The melting temperature (T m ) of the duplex is measured using PE Templab software (Perkin Elmer) with a Lambda 40 UV / VIS spectrophotometer equipped with a Peltier temperature programmer PTP6. Raise the temperature from 20 °C to 95 °C and then lower it to 25 °C, and record the absorption at 260 nm. Use the first derivative and the maxima of both melting and annealing to evaluate the duplex T m .

[0363] Clone growth medium (dHCGM). dHCGM is DMEM medium containing 100 U / ml penicillin, 100 μg / mL streptomycin, 20 mM HEPES, 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). Cells were cultured in an incubator at 37 °C under a humidified atmosphere containing 5% CO2. The culture medium was changed every 2 days from 24 hours after seeding until collection.

[0364] ASO sequences and compounds

Table 8

[0365] HBV-infected PHH cells Fresh primary human hepatocytes (PHH) were provided by PhoenixBio (PXB cells are also described in Ishida et al 2015 Am J Pathol.185(5):1275-85) in 96-well plate format at 70,000 cells / well.

[0366] Upon arrival, PHH were infected with a purified inoculum (genotype C) from a chronic patient at an MOI of 7E08 GE / mL using HBV from HepG2 2.2.15 (batch Z12) at an MOI of 2 GE / mL or by incubating PHH cells with HBV in 4% (v / v) PEG in PHH medium for 16 hours. The cells were then washed three times with PBS and resuspended in 10% (v / v) heat-inactivated fetal bovine serum (GIBCO, catalog number 10082), 2% (v / v) DMSO, 1% (v / v) penicillin / streptomycin (GIBCO, catalog number 15140-148), 20 mM HEPES (GIBCO, catalog number 15630-080), 44 mM NaHCO3 (Wako, catalog number 195-14515), 15 μg / ml L-proline (MP-Biomedicals, catalog number 0219472825), 0.25 μg / ml insulin (Sigma, catalog number I1882), 50 nM dexamethasone (Sigma, catalog number D8893), 5 ng / ml EGF (Sigma, catalog number E9644) and 0.1 mM L-ascorbic acid 2-phosphate (Wako, catalog number 013-12061) It was cultured in fresh PHH medium consisting of DMEM (GIBCO, catalog number 21885) supplemented with 5% CO₂ in a humidified atmosphere. The cells were cultured in an incubator at 37 °C in a humidified atmosphere containing 5% CO₂. The culture medium was changed three times a week 24 hours after plating and until harvest.

[0367] siRNA transfection Four days after infection, the cells were transfected in triplicate with the COPS3 siRNA pool. As controls, vehicle control (NDC), negative control siRNA, and HBx siRNA were included (see Table 6A).

[0368] For each well, a transfection mixture was prepared with 2 μl of negative control siRNA (stock concentration 1 μM), COPS3 siRNA pool (stock concentration 1 μM), HBx control siRNA (stock concentration 0.12 μM), or H2O (NDC), 18.2 μl of OptiMEM (Thermo Fisher Scientific Reduced Serum media), and 0.6 μl of Lipofectamine® RNAiMAX Transfection Reagent (Thermofisher Scientific catalog No. 13778). The transfection mixture was mixed and incubated at room temperature 5 minutes prior to transfection. Prior to transfection, the medium was removed from the PHH cells and replaced with 100 μl / well of William's E Medium + GlutaMAX (Gibco, #32551) supplemented with HepaRG supplement without P / S (Biopredic International, #ADD711C). 20 μl of the transfection mix was added to each well to a final concentration of 16 nM for the negative control siRNA or COPS3 siRNA pool, or 1.92 nM for the HBx control siRNA, and the plate was gently rocked before placing in the incubator. The medium was replaced with PHH medium 6 hours later. The siRNA treatment was repeated on day 6 post-infection as described above. The supernatant was collected on day 8 post-infection and stored at -20 °C. If desired, HBsAg and HBeAg can be determined from the supernatant.

[0369] LNA treatment Two LNA master mix plates were prepared from a 500 μM stock. For LNA treatment at a final concentration of 25 μM, 200 μL of the 500 μM stock LNA was prepared in the first master mix plate. A second master mix plate containing 100 μM COPS3 LNA was prepared for LNA treatment at a final concentration of 5 μM by mixing 40 μL of each 500 μM COPS3 LNA with 160 μL of PBS.

[0370] Four days after infection, cells were treated either in duplicate or triplicate with COPS3 LNA at a final concentration of 25 μM (see Table 7), or with PBS (NDC) as a drug-free control. Prior to LNA treatment, old medium was removed from the cells and replaced with 114 μl / well of fresh PHH medium. For each well, 6 μL of each COPS3 LNA was added to 114 μL of PHH medium either with 500 uM or with PBS as NDC. The same treatment was repeated three times on days 4, 11, and 18 after infection. The cell culture medium was replaced with fresh medium every three days on days 7, 14, and 21 after infection.

[0371] For quantification of cccDNA, infected cells were treated with entecavir (ETV) at a final concentration of 10 nM from day 7 to day 21 after infection. Fresh ETV treatment was repeated five times on days 7, 11, 14, 18, and 21 after infection. This ETV treatment was used to inhibit the synthesis of new viral DNA intermediates and specifically detect the HBV cccDNA sequence.

[0372] Measurement of HBV antigen expression The expression and secretion of HBV antigens can be measured, if desired, in the collected supernatant. HBsAg and HBeAg levels, which are HBV growth parameters, are measured using CLIA ELISA kits (Autobio Diagnostic, CL0310-2, CL0312-2) according to the manufacturer's protocol. Briefly, 25 μL / well of the supernatant is transferred to each antibody-coated microtiter plate, and 25 μL of the enzyme conjugate reagent is added. After incubating the plate on a shaker at room temperature for 60 minutes, the wells are washed five times with wash buffer using an automatic washer. 25 μL of substrate A and B are added to each well. After incubating the plate on a shaker at room temperature for 10 minutes, luminescence is measured using an EnVision® luminescence reader (Perkin Elmer).

[0373] Measurement of cell viability Cell viability was measured in cells without supernatant using Cell Counting Kit-8 (CCK8, Sigma Aldrich #96992). For the measurement, the CCK8 reagent was diluted 1:10 in normal culture medium and 100 μl / well was added to the cells. After incubating for 1 hour in the incubator, 80 μl of the supernatant was transferred to a clear flat-bottom 96-well plate and the absorbance at 450 nm was read using a microplate reader (Tecan). The absorbance values were normalized against NDC, which was set to 100% to calculate the relative cell viability.

[0374] Cell viability measurements were used to confirm that the reduction in viral parameters was not the cause of cell death, and the closer the value was to 100%, the lower the toxicity. LNA treatments that gave cell viability values of 20% or less relative to NDC were excluded from further analysis.

[0375] Real-time PCR for measuring COPS3 mRNA expression and quantification of viral parameters pgRNA, cccDNA, and HBV DNA After determining cell viability, the cells were washed once with PBS. For siRNA treatment, the cells were lysed with 50 μl / well lysis solution from the TaqMan® Gene Expression Cells-to-CT™ Kit (Thermo Fisher Scientific, #AM1729) and stored at -80 °C. For cells treated with LNA, total RNA was extracted using a MagNA Pure robot and MagNA Pure 96 Cellular RNA Large Volume Kit (Roche, #05467535001) according to the manufacturer's protocol. Quantification of COPS3 RNA and viral pgRNA levels and normalization controls, GUS B, were performed using the TaqMan® RNA-to-Ct™ 1-Step Kit (Life Technologies, number 4392656). For each reaction, 2 or 4 μl of cell lysate, 0.5 μl of 20× COPS3 Taqman primer / probe, 0.5 μl of 20× GUS B Taqman primer / probe, 5 μl of 2× TaqMan® RT-PCR Mix, 0.25 μl of 40× TaqMan® RT Enzyme Mix, and 1.75 μl of DEPC-treated water were used. Primers used for quantification of GUS B RNA and target mRNA are listed in Table 8. Technical replicates were performed for each sample, and minus RT controls were included to assess potential amplification by contaminating DNA.

[0376] Target mRNA expression levels as well as viral pgRNA were quantified by RT-qPCR using the QuantStudio 12 K Flex (Applied Biosystems) using the following protocol: 15 min at 48 °C, 10 min at 95 °C, then 40 cycles of 15 s at 95 °C and 60 s at 60 °C for technical replicates.

[0377] The COPS3 mRNA and pgRNA expression levels were analyzed using the comparative cycle threshold 2-ΔΔCt method normalized to the reference gene GUS B and non-transfected cells. The expression levels in siRNA-treated cells are shown as a percentage of the mean drug-free control sample (i.e., the lower the value, the greater the inhibition / decrease). In LNA-treated cells, the expression levels are presented as the inhibitory effect compared to non-treated cells (NDC) set as 100%, and are represented as the mean + SD percentage from two independent biological replicates. For cccDNA quantification, total DNA was extracted from HBV-infected primary human hepatocytes treated with siRNA or LNA. Prior to cccDNA qPCR analysis, fractions of siRNA-treated cell lysates were digested with T5 enzyme (10 U / 500 ng DNA; New England Biolabs, #M0363L) to remove viral DNA intermediates and only cccDNA molecules were quantified. T5 digestion was performed at 37 °C for 30 minutes. T5 digestion was not applied to LNA-treated cell lysates to avoid qPCR interference in the assay. To remove HBV DNA intermediates and quantify the cccDNA levels in LNA-treated cells, cells were treated with entecavir (10 nM) for 3 weeks as described in the LNA-treated section.

[0378] For quantification of cccDNA in siRNA-treated cells, each reaction mixture / well contained 2 μl of T5-digested cell lysate, 0.5 μl of 20× cccDNA_DANDRI Taqman primer / probe (Life Technologies, custom #AI1RW7N, FAM dye listed in the table below), 5 μl of TaqMan® Fast Advanced Master Mix (Applied Biosystems, #4444557) and 2.5 μl of DEPC-treated water. Technical triplicates were performed for each sample.

Table 9

[0379] For quantification of cccDNA in LNA-treated cells by qPCR, prepare a 16 μL / well master mix containing 10 μL of 2× Fast SYBR® Green Master Mix (Applied Biosystems, #4385614), 2 μL of cccDNA Primer Mix (1 μM each of forward and reverse), and 4 μL of nuclease-free water per well. Also prepare a master mix containing 10 μL of 2× Fast SYBR® Green Master Mix (Applied Biosystems, #4385614), 2 μL of mitochondrial genome primer mix (1 μM each of forward and reverse), and 4 μL of nuclease-free water per well for normalization of cccDNA.

[0380] For quantification of intracellular HBV DNA and the normalization control human hemoglobin beta (HBB), use each reaction mixture containing 2 μL of undigested cell lysate, 0.5 μL of 20× HBV Taqman primer / probe (Life Technologies, #Pa 03453406_s1, FAM dye), 0.5 μL of 20× HBB Taqman primer / probe (L ife Technologies, #Hs00758889_s1, VIC dye), 5 μL of TaqMan® Fast Advanced Master Mix (Applied Biosystems, #4444557), and 2 μL of DEPC-treated water. Technical triplicates were performed for each sample.

[0381] Perform qPCR on a QuantStudio™ 12 Flex using the standard settings of a rapid heating block (20 seconds at 95 °C, then 40 cycles of 1 second at 95 °C and 20 seconds at 60 °C).

[0382] Outliers were removed from the dataset by excluding values having a difference greater than 0.9 for the median Ct of all three biological replicates for each treatment condition. Fold changes in cccDNA (siRNA and LNA-treated cells) and total HBV DNA (siRNA-treated cells only) were determined from Ct values via the 2 -ddCT method and normalized to HBB or mitochondrial DNA as the housekeeping gene. For siRNA-treated cells, the expression levels are shown as % of the mean drug-free control sample (i.e., the lower the value, the greater the inhibition / decrease). In LNA-treated cells, the inhibitory effect on cccDNA was represented as the percentage of mean + / −SD from three independent biological replicates, compared to non-treated cells (NDC) set to 100%.

Table 10

[0383] Example 1: Measurement of the decrease in COPS3 mRNA, HBV intracellular DNA, and cccDNA in HBV-infected PHH cells resulting from siRNA treatment In the following experiment, the effect of COPS3 knockdown on HBV parameters, HBV DNA and cccDNA, was tested.

[0384] HBV-infected PHH cells were treated with a pool of siRNAs from Dharmacon (LU-011494-00-0005, see Table 6A) as described in the “siRNA transfection” section of the Materials and Methods. After 4 days of treatment, COPS3 mRNA, cccDNA, and intracellular HBV DNA were measured by qPCR as described in the “COPS3 mRNA expression and real-time PCR for measuring viral parameters pgRNA, cccDNA, and HBV DNA” section of the Materials and Methods.

[0385] The results are shown in Table 9 as % of the mean drug-free control sample (i.e., the lower the value, the greater the inhibition / decrease).

Table 11

[0386] From this, it can be seen that the COPS3 siRNA pool can very efficiently reduce COPS3 mRNA, cccDNA, and HBV DNA. The positive control reduced intracellular HBV DNA as expected, but had no effect on cccDNA when compared to the negative control.

[0387] Example 2: Measurement of the reduction of COPS3 mRNA, HBV intracellular pgRNA, and cccDNA in HBV-infected PHH cells resulting from LNA treatment In the following experiment, the effect of COPS3 knockdown on HBV parameters, HBV DNA, and cccDNA was tested.

[0388] HBV-infected PHH cells were treated with COPS3 naked LNA (see Table 7) as described in the "LNA treatment" section of the Materials and Methods.

[0389] After 21 days of treatment, COPS3 mRNA, cccDNA, and intracellular HBV pgRNA were measured by qPCR as described in the "Real-time PCR for measuring COPS3 mRNA expression and viral parameters pgRNA, cccDNA, and HBV DNA" section of the Materials and Methods. The results are shown in Table 10 as the inhibitory effect compared to untreated cells (NDC) set as 100%, and are presented as the mean + SD percentage from two independent biological replicates. [Table 12]

[0390] From this, it can be seen that SCAMP3 LNA can significantly reduce SCAMP3 mRNA expression, and as a result, the expression levels of both pgRNA and cccDNA are very efficiently reduced.

Claims

1. A COPS3 (COP9 signalosome subunit 3) inhibitor for use in the treatment of Hepatitis B virus (HBV) infection.

2. The COPS3 inhibitor for use according to claim 1, wherein the HBV infection is a chronic infection.

3. The COPS3 inhibitor for use according to claim 1 or 2, wherein the COPS3 inhibitor is capable of reducing the amount of cccDNA (covalently closed circular DNA) in HBV infected cells.

4. The COPS3 inhibitor for use according to any one of claims 1 to 3, wherein the inhibitor is a nucleic acid molecule of 12 to 60 nucleotides in length comprising a contiguous nucleotide sequence of at least 12 nucleotides in length that is at least 95% complementary, e.g. completely complementary, to a mammalian COPS3 target nucleic acid, in particular a human COPS3 target sequence, and is capable of reducing the expression of COPS3 mRNA.

5. The COPS3 inhibitor for use according to any one of claims 1 to 4, wherein the inhibitor is selected from the group consisting of single-stranded antisense oligonucleotides, siRNA and shRNA.

6. The COPS3 inhibitor for use according to any one of claims 1 to 5, wherein the mammalian COPS3 target sequence is selected from the group consisting of SEQ ID NOs: 1, 4, 5, 6, 7, 8 and 9.

7. The COPS3 inhibitor for use according to any one of claims 4 to 6, wherein the contiguous nucleotide sequence is at least 98% complementary, such as completely complementary, to the target sequences of SEQ ID NO:1 and SEQ ID NO:

2.

8. The COPS3 inhibitor for use according to any one of claims 3 to 7, wherein the amount of cccDNA in the HBV infected cells is reduced by at least 60%.

9. The COPS3 inhibitor for use according to any one of claims 4 to 7, wherein the amount of COPS3 mRNA is reduced by at least 60%.

10. A nucleic acid molecule of 12 to 30 nucleotides in length comprising a contiguous nucleotide sequence of at least 12 nucleotides that is 90% complementary, e.g., completely complementary, to a mammalian COPS3 target sequence, particularly a human COPS3 target sequence, wherein the nucleic acid molecule is capable of inhibiting expression of COPS3 mRNA.

11. The nucleic acid molecule of claim 10, wherein the contiguous nucleotide sequence is completely complementary to a sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 6, 7, 8 and 9.

12. The nucleic acid molecule according to claim 10 or 11, wherein the nucleic acid molecule comprises a contiguous nucleotide sequence of 12 to 25, such as 16 to 20, nucleotides in length.

13. The nucleic acid molecule according to any one of claims 10 to 12, wherein the nucleic acid molecule is an RNAi molecule such as a double-stranded siRNA or shRNA.

14. The nucleic acid molecule according to any one of claims 10 to 12, wherein the nucleic acid molecule is a single-stranded antisense oligonucleotide.

15. The nucleic acid molecule according to any one of claims 10 to 14, wherein the nucleic acid molecule comprises one or more 2' sugar modified nucleosides.

16. 16. The nucleic acid molecule of claim 15, wherein the 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, arabinonucleic acid (ANA), 2'-fluoro-ANA and LNA nucleosides.

17. 17. The nucleic acid molecule of claim 15, wherein the one or more 2' sugar modified nucleosides are LNA nucleosides.

18. 18. The nucleic acid molecule according to any one of claims 10 to 17, wherein the contiguous nucleotide sequence comprises at least one phosphorothioate internucleoside linkage.

19. 20. The nucleic acid molecule of claim 18, wherein all internucleoside linkages in the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

20. The nucleic acid molecule according to any one of claims 10 to 19, wherein the nucleic acid molecule is capable of recruiting RNase H.

21. 21. The nucleic acid molecule of any one of claims 10 to 20, wherein the nucleic acid molecule or contiguous nucleotide sequence thereof comprises a gapmer of the formula 5'-F-G-F'-3', where regions F and F' independently comprise 1 to 4 2' sugar modified nucleosides and G is a region of 6 to 18 nucleosides capable of recruiting RNase H, such as a region comprising 6 to 18 DNA nucleosides.

22. A conjugate compound comprising a nucleic acid molecule according to any one of claims 10 to 21 and at least one conjugate moiety covalently attached to said nucleic acid molecule.

23. 23. The conjugate compound of claim 22, wherein the conjugate moiety is or comprises a GalNAc moiety, e.g., a trivalent GalNAc moiety, e.g., a GalNAc moiety selected from one of the trivalent GalNAc moieties of FIG.

24. 24. The conjugate compound of claim 22 or 23, wherein the conjugate compound comprises a physiologically labile linker composed of 2 to 5 linked nucleosides containing at least two consecutive phosphodiester bonds, the physiologically labile linker being covalently attached to the 5' or 3' end of the nucleic acid molecule.

25. A nucleic acid molecule according to any one of claims 10 to 21, or a pharma- ceutically acceptable salt of the conjugate compound according to any one of claims 22 to 24.

26. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 10 to 21, a conjugate compound according to any one of claims 22 to 24, or a pharma- ceutically acceptable salt according to claim 25, and a pharma- ceutically acceptable excipient.

27. In vivo use to inhibit COPS3 expression in target cells expressing COPS3 27. An in vivo or in vitro method, said method comprising administering to said cell an effective amount of a nucleic acid molecule according to any one of claims 10 to 21, a conjugate compound according to any one of claims 22 to 24, a pharma- ceutically acceptable salt according to claim 25, or a pharmaceutical composition according to claim 26.

28. 27. A method for treating or preventing a disease, comprising administering to a subject suffering from or susceptible to said disease a therapeutically or prophylactically effective amount of a nucleic acid molecule according to any one of claims 10 to 21, a conjugate compound according to any one of claims 22 to 24, a pharma- ceutically acceptable salt according to claim 25, or a pharmaceutical composition according to claim 26.

29. 29. The method of claim 28, wherein the disease is a Hepatitis B virus (HBV) infection, such as a chronic HBV infection.

30. A nucleic acid molecule according to any one of claims 10 to 21, a conjugate compound according to any one of claims 22 to 24, a pharma- ceutically acceptable salt according to claim 25, or a pharmaceutical composition according to claim 26, for use in medicine.

31. A nucleic acid molecule according to any one of claims 10 to 21, a conjugate compound according to any one of claims 22 to 24, a pharma- ceutically acceptable salt according to claim 25, or a pharmaceutical composition according to claim 26, for use in the treatment of Hepatitis B Virus (HBV) infection, such as chronic HBV infection.

32. Use of a nucleic acid molecule according to any one of claims 10 to 21, a conjugate compound according to any one of claims 22 to 24, a pharma- ceutically acceptable salt according to claim 25, or a pharmaceutical composition according to claim 26, for the preparation of a medicament for treating Hepatitis B Virus (HBV) infection, such as chronic HBV infection.

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