Use of scamp3 inhibitors for treating hepatitis b virus infection

Abstract

To provide SCAMP3 inhibitors that reduce cccDNA in HBV infected cells, as well as novel nucleic acid molecules capable of inhibiting the expression of SCAMP3 in vitro and in vivo.SOLUTION: The invention particularly relates to the use of a SCAMP3 inhibitor that destabilizes cccDNA such as HBV cccDNA. The invention also relates to a nucleic acid molecule being complementary to SCAMP3 and capable of reducing the level of a SCAMP3 mRNA, as well as a pharmaceutical composition and its use in the treatment of a HBV infection.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to SCAMP3 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 SCAMP3 inhibitors to destabilize cccDNA, such as HBV cccDNA. The present invention also relates to nucleic acid molecules, such as oligonucleotides, including siRNA, shRNA, and antisense oligonucleotides, that are complementary to SCAMP3 and can reduce the expression of SCAMP3. The present invention also includes pharmaceutical compositions and their use in the treatment and / or prevention of HBV infection. [Background technology]

[0002] Hepatitis B is an infectious disease caused by the hepatitis B virus (HBV), a small, hepatotropic virus that replicates via reverse transcription. Chronic HBV infection is a significant risk factor for severe liver diseases such as cirrhosis and hepatocellular carcinoma. Current treatment for chronic HBV infection is based on the administration of pegylated type 1 interferons or nucleoside(t)ide analogs, such as lamivudine, adefovir, entecavir, tenofovir diisoproxil, and tenofovir alafenamide, which target the viral polymerase, a multifunctional reverse transcriptase. Successful treatment is usually measured as the disappearance of hepatitis B surface antigen (HBsAg). However, complete HBsAg clearance is rarely achieved because hepatitis B viral DNA persists in the body after infection. HBV persistence is mediated by an 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 viral replication intermediate pregenomic RNA (pgRNA). The presence of several copies of cccDNA may be sufficient to reinitiate late-stage HBV infection. Current treatments for HBV do not target cccDNA. However, elimination of cccDNA may 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] SCAMP3( minutes Secretory carriers Membrane Protein 3 ) is an integral membrane protein that belongs to the family of secretory carrier membrane proteins. It functions as a carrier to the cell surface in the post-Golgi recycling pathway. It is also involved in protein trafficking in the endosomal pathway and is regulated by ubiquitination.

[0004] The role of SCAMP3 in endosomes has been studied and it has been shown to interact with ESCRT (endosomal sorting complexes required for transport) and associate with the E3 ubiquitin-protein ligases NEDD4 and Hrs. siRNA-mediated knockdown of SCAMP3 promoted lysosomal degradation of EGFR and EGF but inhibited their recycling (Aoh et al. (2009). Molecular Biology of the Cell. 20(6):1816-32. doi:10.1091 / mbc.E08-09-0894). Knockdown of CAMP3 also reduced Hrs recruitment to enlarged endosomes (Thomas et al., Biochem Biophys Res Commun. 2016 Sep 23;478(3):1028-34. doi:10.1016 / j.bbrc.2016.08.012).

[0005] Furthermore, SCAMP3 mRNA has been shown to be highly expressed in hepatocellular carcinoma (HCC). Specifically, overexpression of SCAMP3 is an indicator of poor prognosis in HCC. It has been reported that knockdown of SCAMP3 by siRNA resulted in suppression of cell proliferation and disruption of the cell cycle in HCC cells, and no correlation was observed between SCAMP3 expression and HBV antigen (Zhang et al., Oncotarget. 2017 Nov 27;8(65):109247-109257. doi:10.18632 / oncotarget.22665).

[0006] To the best of our knowledge, SCAMP3 has never been identified in relation to HBV infection. In particular, SCAMP3 has never been identified as a cccDNA-dependent factor in relation to cccDNA stability and maintenance, and molecules that inhibit SCAMP3 have never been suggested as cccDNA-destabilizing agents for the treatment of HBV infection. Furthermore, to our knowledge, the only disclosures of oligonucleotides potentially relevant to modulating SCAMP3 expression have been made by Aho et al., Thomas et al., and Zhang et al. (see above). However, none of these three publications mention the treatment of HBV infection. Object of the invention

[0007] The present invention demonstrates that there is a relationship between inhibition of SCAMP3 (secretory carrier membrane protein 3) and reduction of cccDNA in HBV-infected cells, which has implications for the treatment of HBV-infected individuals. An object of the present invention is to identify SCAMP3 inhibitors that reduce cccDNA in HBV-infected cells. Such SCAMP3 inhibitors may be used to treat HBV infection. The present invention further identifies novel nucleic acid molecules that can inhibit the expression of SCAMP3 in vitro and in vivo. Summary of the Invention

[0008] The present invention relates to oligonucleotides that target nucleic acids and that regulate the expression of SCAMP3, and that can treat or prevent diseases associated with the function of SCAMP3.

[0009] Thus, in a first aspect, the present invention provides SCAMP3 inhibitors for use in the treatment and / or prevention of hepatitis B virus (HBV) infection. In particular, SCAMP3 inhibitors capable of reducing HBV cccDNA and / or HBV pregenomic RNA (pgRNA) are useful. Such inhibitors are advantageously nucleic acid molecules of 12 to 60 nucleotides in length that can reduce SCAMP3 mRNA. In a further aspect, the present invention relates to a nucleic acid molecule of 12 to 60 nucleotides, e.g., 12 to 30 nucleotides, comprising a contiguous nucleotide sequence of at least 12 nucleotides, particularly 16 to 20 nucleotides, that is at least 90% complementary to mammalian SCAMP3, e.g., human SCAMP3 or mouse SCAMP3. Such a nucleic acid molecule can inhibit SCAMP3 expression in cells that express SCAMP3. Inhibition of SCAMP3 reduces the amount of cccDNA present in the cell. The nucleic acid molecule can be selected from a single-stranded antisense oligonucleotide, a double-stranded siRNA molecule, or an shRNA nucleic acid molecule (particularly a chemically produced shRNA molecule).

[0010] A further aspect of the present invention relates to single-stranded antisense oligonucleotides or siRNAs that inhibit the expression and / or activity of SCAMP3. In particular, modified antisense oligonucleotides or siRNAs containing one or more 2' sugar-modified nucleosides and one or more phosphorothioate linkages that reduce SCAMP3 mRNA are advantageous.

[0011] In a further aspect, the present invention provides a pharmaceutical composition comprising an SCAMP3 inhibitor of the present invention, such as an antisense oligonucleotide or siRNA of the present invention, and a pharmaceutically acceptable excipient.

[0012] In a further aspect, the present invention provides an in vivo or in vitro method for modulating SCAMP3 expression in target cells expressing SCAMP3 by administering to the cells an effective amount of a SCAMP3 inhibitor of the present invention, e.g., an antisense oligonucleotide or composition of the present invention. In some embodiments, SCAMP3 expression is reduced in the target cells by at least 50% or at least 60% compared to levels in the absence of any treatment or treatment with a control. In some embodiments, the target cells are infected with HBV, and cccDNA in the HBV-infected target cells is reduced by at least 50% or at least 60% compared to levels in the absence of any treatment or treatment with a control. In some embodiments, the target cells are infected with HBV, and pgRNA in the HBV-infected target cells is reduced by at least 50%, or at least 60%, or at least 70%, or at least 80% compared to levels in the absence of any treatment or treatment with a control.

[0013] 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 SCAMP3, comprising administering a therapeutically or prophylactically effective amount of a SCAMP3 inhibitor of the present invention, e.g., an antisense oligonucleotide or siRNA of the present invention, to a subject suffering from or susceptible to the disease, disorder or dysfunction.

[0014] Further aspects of the invention are conjugates of the nucleic acid molecules of the invention and pharmaceutical compositions comprising the molecules of the invention, particularly conjugates that target the liver, such as GalNAc clusters. [Brief explanation of the drawings]

[0015] [Figure 1A]Figures 1A-1D show exemplary antisense oligonucleotide conjugates in which the oligonucleotide is represented by the term "oligonucleotide" and the asialoglycoprotein receptor-targeting conjugate moiety is a trivalent N-acetylgalactosamine moiety. The compounds in Figures 1A-1D contain a dilysine brancher molecule, a PEG spacer, and three terminal GalNAc carbohydrate moieties. In the compound in Figure 1A (Figures 1A-1 and 1A-2 show two different diastereomers of the same compound), the oligonucleotide is directly attached to the asialoglycoprotein receptor-targeting conjugate moiety without a linker. [Figure 1B] In the compound of Figure 1B (Figures 1B-1 and 1B-2 show two different diastereomers of the same compound), the oligonucleotide is directly attached to the asialoglycoprotein receptor-targeting conjugate moiety without a linker. [Figure 1C] In the compound of Figure 1C (Figures 1C-1 and 1C-2 show two different diastereomers of the same compound), the oligonucleotide is conjugated to the asialoglycoprotein receptor-targeting conjugate moiety via a C6 linker. [Figure 1D] In the compound of Figure 1D (Figures 1D-1 and 1D-2 show two different diastereomers of the same compound), the oligonucleotide is conjugated to the asialoglycoprotein receptor targeting conjugate moiety via a C6 linker. [Figure 1E] The compounds in Figure IE contain commercially available trebler brancher molecules and spacers of various lengths and structures, as well as three terminal GalNAc carbohydrate moieties. [Figure 1F] The compounds in Figure 1F contain commercially available trebler brancher molecules and spacers of various lengths and structures, as well as three terminal GalNAc carbohydrate moieties. [Figure 1G] The compounds in Figure 1G contain commercially available trebler brancher molecules and spacers of various lengths and structures, as well as three terminal GalNAc carbohydrate moieties. [Figure 1H] The compounds in Figure 1H contain commercially available trebler brancher molecules and spacers of various lengths and structures, as well as three terminal GalNAc carbohydrate moieties. [Figure 1I] The compounds in Figure 1I contain commercially available trebler brancher molecules and spacers of various lengths and structures, as well as three terminal GalNAc carbohydrate moieties. [Figure 1J] The compounds in Figure 1J contain commercially available trebler brancher molecules and spacers of various lengths and structures, as well as three terminal GalNAc carbohydrate moieties. [Figure 1K] The compounds in Figure 1K contain commercially available trebler brancher molecules and spacers of various lengths and structures, as well as three terminal GalNAc carbohydrate moieties. [Figure 1L] The compound in Figure 1L is composed of a monomeric GalNAc phosphoramidite (where X = S or O, independently Y = S or O, and n = 1-3) that was added to the oligonucleotide while it was still on the solid support as part of its synthesis (see International Publication No. WO 2017 / 178656). Figures 1B and 1D are also referred to herein as GalNAc2 and GN2, respectively, without and with the C6 linker. The two different diastereoisomers shown in each of Figures 1A-1D are the results of the conjugation reaction. Thus, a particular pool of antisense oligonucleotide conjugates may contain only one of the two different diastereoisomers, or a particular pool of antisense oligonucleotide conjugates may contain a mixture of the two different diastereoisomers.

[0016] definition HBV infection The term "hepatitis B virus infection" or "HBV infection" is commonly known in the art and refers to an infectious disease caused by hepatitis B virus (HBV) and affecting the liver. HBV infection can be acute or chronic. Chronic hepatitis B virus (CHB) infection is a global disease burden affecting 248 million people worldwide. Approximately 686,000 deaths per year are attributed 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 predicted that without further intervention, the number of CHB-infected individuals will remain at their current high level for the next 40 to 50 years, with a cumulative 20 million deaths between 2015 and 2030 (WHO, 2016). CHB infection is not a homogeneous disease with distinct clinical manifestations. Infected individuals progress through several stages of CHB-related liver disease over their lifetime. These stages also form the basis for treatment with standard of care (SOC). Current guidelines recommend treating only select individuals infected with CHB based on three criteria: serum ALT level, HBV DNA level, and liver disease severity (EASL, 2017). This recommendation is due to the fact that SOC, namely, nucleos(t)ide analogs (NA) and pegylated interferon-α (PEG-IFN), are not curative and must be administered for extended periods, thereby increasing safety risks. NA effectively suppresses HBV DNA replication; however, it has very limited or no effect on other viral markers. Two hallmarks of HBV infection, hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA), are the primary targets of new drugs aimed at curing HBV. In the plasma of CHB individuals, the number of HBsAg subviral (empty) particles is 103- to 105-fold higher than the number of HBV virions (Ganem and Prince, N Engl J Med. 2004 Mar 11;350(11):1118-29), an excess observed after individuals have resolved acute HBV infection. It is thought to contribute to the immunopathogenesis of the disease, including in individuals who fail to develop neutralizing anti-HBs antibodies, a serological marker of HBs immunity.

[0017] In some embodiments, the term "HBV infection" refers to "chronic HBV infection."

[0018] Furthermore, the term encompasses infection with any genotype of HBV.

[0019] In some embodiments, the patient being treated is infected with HBV genotype A.

[0020] In some embodiments, the patient being treated is infected with HBV genotype B.

[0021] In some embodiments, the patient being treated is infected with HBV genotype C.

[0022] In some embodiments, the patient being treated is infected with HBV genotype D.

[0023] In some embodiments, the patient being treated is infected with HBV genotype E.

[0024] In some embodiments, the patient being treated is infected with HBV genotype F.

[0025] In some embodiments, the patient being treated is infected with HBV genotype G.

[0026] In some embodiments, the patient being treated is infected with HBV genotype H.

[0027] In some embodiments, the patient being treated is infected with HBV genotype I.

[0028] In some embodiments, the patient being treated is infected with HBV genotype J.

[0029] cccDNA (covalently closed circular DNA) cccDNA is the viral gene template of HBV present in the nucleus of infected hepatocytes, gives rise to all HBV RNA transcripts required for productive infection, and is involved in viral persistence during the natural history of chronic HBV infection (Locarnini and Zoulim, Antiviral 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 cessation, necessitating long-term, sometimes lifelong, treatment. PEG-IFN can only be administered to a small subset of CHB patients due to its various side effects.

[0030] Therefore, there is a great need for novel therapies that can bring about complete cure, defined by the degradation or elimination of HBV cccDNA, in the majority of CHB patients.

[0031] compound As used herein, the term "compound" refers to any molecule that can inhibit the expression or activity of SCAMP3. A specific compound of the present invention is a nucleic acid molecule, such as an RNAi molecule or an antisense oligonucleotide, according to the present invention, or any conjugate containing such a nucleic acid molecule. For example, in the present specification, the compound may be a nucleic acid molecule, particularly an antisense oligonucleotide or siRNA, that targets SCAMP3.

[0032] Oligonucleotides The term "oligonucleotide" as used herein is defined as a molecule containing two or more covalently linked nucleosides, as commonly understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers.

[0033] The oligonucleotides referred to in the specification and claims are generally therapeutic oligonucleotides less than 70 nucleotides in length. Oligonucleotides can be or include single-stranded antisense oligonucleotides, or can be other oligomeric nucleic acid molecules, such as CRISPR RNA, siRNA, shRNA, aptamers, or ribozymes. Therapeutic oligonucleotide molecules are usually produced in laboratories by solid-phase chemical synthesis followed by purification and isolation. However, shRNAs are often delivered to cells using lentiviral vectors, 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 mechanism (including RNA-induced silencing complex (RISC)). In one embodiment of the present invention, the shRNA is a chemically produced shRNA molecule (not dependent on cell-based expression from a plasmid or virus). When referring to the sequence of an oligonucleotide, it refers to the sequence or order of the nucleobase moieties of covalently linked nucleotides or nucleosides, or their modification. Generally, the oligonucleotide of the present invention is artificial, is chemically synthesized, and is typically purified or isolated.However, in some embodiments, the oligonucleotide of the present invention is the shRNA that is transcribed from a vector when entering target cell.The oligonucleotide of the present invention can comprise one or more modified nucleosides or nucleotides.

[0034] In some embodiments, the oligonucleotides of the present invention comprise or consist of 10 to 70 nucleotides in length, 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, or such as 16 to 20 consecutive nucleotides. Thus, in some embodiments, the oligonucleotides of the present invention may have a length of 12 to 25 nucleotides. Alternatively, in some embodiments, the oligonucleotides of the present invention may have a length of 15 to 22 nucleotides.

[0035] In some embodiments, the oligonucleotide or its contiguous nucleotide sequence comprises or consists of 24 or fewer nucleotides, e.g., 22, e.g., 20 or fewer nucleotides, e.g., 18 or fewer nucleotides, e.g., 14, 15, 16, or 17 nucleotides. Any range provided herein should be understood to include 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.

[0036] In some embodiments, the contiguous nucleotide sequence comprises or consists of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides in length.

[0037] The oligonucleotides are for regulating the expression of target nucleic acids in mammals. In some embodiments, nucleic acid molecules such as siRNA, shRNA, and antisense oligonucleotides are typically for inhibiting the expression of target nucleic acid(s).

[0038] In one embodiment of the invention, the oligonucleotide is selected from an RNAi agent, e.g., an 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.

[0039] In some embodiments, oligonucleotides of the invention may contain one or more modified nucleosides or nucleotides, such as, for example, 2' sugar-modified nucleosides.

[0040] In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages.

[0041] In some embodiments, the oligonucleotide may be linked to a non-nucleoside moiety (conjugate moiety).

[0042] An oligonucleotide library should be understood as a collection of variant oligonucleotides. The purpose of an oligonucleotide library can be varied. In some embodiments, an oligonucleotide library is composed of oligonucleotides having overlapping nucleobase sequences that target one or more mammalian SCAMP3 target nucleic acids, with the goal of identifying the most potent sequences within the oligonucleotide library. In some embodiments, an oligonucleotide library is a library of oligonucleotide design variants (child nucleic acid molecules) of parent or ancestor oligonucleotides, where the oligonucleotide design variants retain the core nucleobase sequence of the parent nucleic acid molecule.

[0043] antisense oligonucleotides The term " antisense oligonucleotide " or " ASO " used herein is defined as an oligonucleotide that can regulate the expression of target gene by hybridizing with target nucleic acid, particularly the continuous sequence on target nucleic acid.Antisense oligonucleotide is not essentially double-stranded, and therefore is not siRNA or shRNA.Preferably, the antisense oligonucleotide of the present invention is single-stranded.It is understood that the single-stranded oligonucleotide of the present invention can form hairpin or intermolecular duplex structure (duplex between two molecules of the same oligonucleotide), as long as the degree of complementarity between itself or itself is less than 50% over the entire length of the oligonucleotide.

[0044] Advantageously, the single-stranded antisense oligonucleotides of the present invention do not contain RNA nucleosides to reduce nuclease resistance.

[0045] Advantageously, the oligonucleotides of the invention comprise one or more modified nucleosides or nucleotides, such as, for example, 2' sugar-modified nucleosides. Furthermore, it is advantageous for the unmodified nucleosides to be DNA nucleosides.

[0046] RNAi molecule As used herein, the term "RNA interference (RNAi) molecule" refers to a short double-stranded oligonucleotide that contains RNA nucleosides and mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC), where it interacts with the catalytic RISC component Argonaute. RNAi molecules regulate, for example, inhibit the expression of target nucleic acids in cells, for example, cells in a subject, such as a mammalian subject. RNAi molecules include single-stranded RNAi molecules (Lima et al., 2012 Cell 150:883) and double-stranded siRNA, as well as short hairpin RNA (shRNA). In some embodiments of the present invention, the oligonucleotide of the present invention, or its continuous nucleotide sequence, is an RNAi agent, such as siRNA.

[0047] siRNA The term "small interfering ribonucleic acid" or "siRNA" refers to a small interfering ribonucleic acid (RNAi) molecule. This is a class of double-stranded RNA molecule, also known in the art as short interfering RNA or silencing RNA. siRNA typically comprises a sense strand (also called a passenger strand) and an antisense strand (also called a guide strand), each strand being 17-30 nucleotides long, typically 19-25 nucleosides long. The antisense strand is complementary to the target nucleic acid (suitably the mature mRNA sequence), e.g., at least 95% complementary, e.g., completely complementary, and the sense strand is complementary to the antisense strand, so that the sense strand and the antisense strand form a double-stranded or double-stranded region. The siRNA strands can form a blunt-end duplex, or advantageously, the 3' ends of the sense strand and the antisense strand can form, for example, 1, 2, or 3 nucleoside 3' overhangs, which resemble the products generated by Dicer to form RISC substrates in vivo. Effective extended forms of Dicer substrates are described in U.S. Patent Nos. 8,349,809 and 8,513,207, which are incorporated herein by reference. In some embodiments, both the sense strand and the antisense strand have a 2-nt 3' overhang. Thus, the double-stranded region can be, for example, 17 to 25 nucleotides in length, e.g., 21 to 23 nucleotides in length.

[0048] Once inside the cell, the antisense strand is incorporated into the RISC complex, which mediates the targeted degradation or target inhibition of the target nucleic acid. siRNA typically contains modified nucleosides in addition to RNA nucleosides. In one embodiment, siRNA molecules can be chemically modified using modified internucleotide linkages and 2' sugar-modified nucleosides, such as 2'-4' bicyclic ribose-modified nucleosides, including LNA and cET, or 2' substitution modifications such as 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, etc. In particular, 2' fluoro, 2'-O-methyl, or 2'-O-methoxyethyl can be incorporated into siRNA.

[0049] In some embodiments, all of the nucleotides of the siRNA sense (passenger) strand may 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 may be discontinuous (see, e.g., WO 2007 / 107162). Incorporation of thermolabile nucleotides in the seed region of the antisense strand of the siRNA has been reported to be useful for reducing the off-target activity of the siRNA (see, e.g., WO 2018 / 098328). Preferably, the siRNA comprises 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.

[0050] In one embodiment, the siRNA molecule further comprises at least one phosphorothioate or methylphosphonate internucleoside bond.The phosphorothioate or methylphosphonate internucleoside bond can be at the 3'-end of one or both strands (for example, antisense strand; or sense strand); or the phosphorothioate or methylphosphonate internucleoside bond can be at the 5'-end of one or both strands (for example, antisense strand; or sense strand); or the phosphorothioate or methylphosphonate internucleoside bond can be at both the 5'-end and 3'-end of one or both strands (for example, antisense strand; or sense strand).In some embodiments, the remaining internucleoside bond is a phosphodiester bond.In some embodiments, the siRNA molecule comprises one or more phosphorothioate internucleoside bond. In siRNA molecules, phosphorothioate internucleoside linkages can reduce nuclease cleavage in RICS, and therefore it is advantageous that not all internucleoside linkages in the antisense strand are modified.

[0051] The siRNA molecule may further comprise a ligand. In some embodiments, the ligand is attached to the 3' end of the sense strand.

[0052] For biodistribution, the siRNA may be conjugated to a targeting ligand and / or formulated into lipid nanoparticles.

[0053] Other aspects of the invention relate to pharmaceutical compositions comprising these dsRNA, such as siRNA molecules, suitable for therapeutic use, and methods of inhibiting target gene expression by administering dsRNA, such as siRNA, molecules of the invention, for the treatment of various disease states, e.g., as disclosed herein.

[0054] shRNA The term "short hairpin RNA" or "shRNA" refers to a molecule that is generally 40-70 nucleotides long, e.g., 45-65 nucleotides long, e.g., 50-60 nucleotides long, and forms a stem-loop (hairpin) RNA structure that interacts with an endonuclease known as Dicer, which is thought to process dsRNA into 19-23 base pair short interfering RNAs with characteristic two-base 3' overhangs, which are then incorporated into the RNA-induced silencing complex (RISC). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing. shRNA oligonucleotides can be chemically modified using modified internucleotide linkages and 2' sugar-modified nucleosides, such as 2'-4' bicyclic ribose-modified nucleosides, including LNA and cET, or 2' substitution modifications such as 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, etc.

[0055] In some embodiments, shRNA molecules contain one or more phosphorothioate internucleoside linkages. In RNAi molecules, phosphorothioate internucleoside linkages can reduce nuclease cleavage in RICS, so it is advantageous not to modify all internucleoside linkages in the stem loop of shRNA molecules. Phosphorothioate internucleoside linkages can be advantageously placed at the 3' and / or 5' ends of the stem loop of shRNA molecules, particularly in parts of the molecule that are not complementary to the target nucleic acid. However, the region of the shRNA molecule that is complementary to the target nucleic acid can also be modified in the first 2-3 internucleoside linkages in the parts that are predicted to be the 3' and / or 5' ends after Dicer cleavage.

[0056] Contiguous nucleotide sequence The term "contiguous 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 "contiguous nucleobase sequence" and "oligonucleotide motif sequence." In some embodiments, all nucleotides of an oligonucleotide constitute a contiguous nucleotide sequence. In some aspects, the contiguous nucleotide sequence is included in the guide strand of an siRNA molecule. In some embodiments, the contiguous nucleotide sequence is a portion of an shRNA molecule that is 100% complementary to a target nucleic acid. In some embodiments, an oligonucleotide comprises a contiguous nucleotide sequence such as an FG-F' gapmer region, and may optionally include a nucleotide linker region that can be used to attach additional nucleotide(s), such as a functional group (e.g., a conjugate group for targeting), to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. In some embodiments, the nucleobase sequence of an antisense oligonucleotide constitutes a contiguous nucleotide sequence. In some embodiments, the contiguous nucleotide sequence is 100% complementary to the target nucleic acid.

[0057] 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 and non-naturally occurring nucleotides and nucleosides.Naturally, nucleotides, such as DNA nucleotides and RNA nucleotides, contain 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."

[0058] Modified Nucleosides As used herein, the term "modified nucleoside" or "nucleoside modification" refers to a nucleoside that has been modified by introducing one or more modifications to the sugar moiety or (nucleic acid) base moiety, compared to an equivalent DNA or RNA nucleoside. Advantageously, one or more of the modified nucleosides contain a modified sugar moiety. The term modified nucleoside may also be used interchangeably herein with the term "nucleoside analog" or modified "unit" or modified "monomer." Nucleosides with unmodified DNA or RNA sugar moieties are referred to herein as DNA or RNA nucleosides. Nucleosides with modifications in the base region of DNA or RNA nucleosides are still generally referred to as DNA or RNA if Watson-Crick base pairing is possible.

[0059] Modified internucleoside linkages The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) linkage that covalently links two nucleosides together, as commonly understood by those skilled in the art. Thus, the oligonucleotides of the present invention can contain one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages or one or more phosphorodithioate internucleoside linkages.

[0060] The oligonucleotides of the invention advantageously use phosphorothioate internucleoside linkages.

[0061] Phosphorothioate internucleoside bond is particularly useful due to its nuclease resistance, favorable pharmacokinetics and ease of manufacture.In some embodiments, the internucleoside bond of at least 50% of oligonucleotide or its consecutive nucleotide sequence is phosphorothioate, and the internucleoside bond of at least 60%, for example at least 70%, for example at least 75%, for example at least 80% or for example at least 90% of oligonucleotide or its consecutive nucleotide sequence is phosphorothioate.

[0062] In some embodiments, all internucleoside linkages of the oligonucleotide or contiguous nucleotide sequence thereof are phosphorothioate. In some advantageous embodiments, all internucleoside linkages of the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate linkages, or all internucleoside linkages of the oligonucleotide are phosphorothioate linkages.

[0063] As disclosed in EP 2742135, antisense oligonucleotides may contain other internucleoside linkages (other than phosphodiester and phosphorothioate), such as alkylphosphonate / methylphosphonate internucleoside linkages, and it is recognized that EP 2742135 allows for other DNA phosphorothioate gap regions.

[0064] Nucleic acid bases The term "nucleobase" includes purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization.In the context of the present invention, the term "nucleobase" also encompasses modified nucleobases that may differ from naturally occurring nucleobases but function during nucleic acid hybridization.In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as non-naturally occurring variants.Such variants are described, for example, in Hirao et al. (2012) Accounts of Chemical Research, Vol. 45, p. 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry, Appendix 37, 1.4.1.

[0065] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolocytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolauracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0066] Nucleobase moieties can be represented by the letter code for each corresponding nucleobase, for example, A, T, G, C, or U, where each letter can optionally include a modified nucleobase of equivalent function. For example, in exemplary oligonucleotides, the nucleobase moieties can be selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides can be used.

[0067] Modified Oligonucleotides The term " modified oligonucleotide " refers to the oligonucleotide that comprises one or more sugar-modified nucleosides and / or modified internucleoside linkages.The term " chimeric " oligonucleotide is used in the literature to describe the oligonucleotide that comprises modified nucleosides and DNA nucleosides.The antisense oligonucleotide of the present invention is preferably a chimeric oligonucleotide.

[0068] Complementarity The term "complementarity" or "complementary" refers to the Watson-Crick base pairing ability of nucleosides / nucleotides. 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; therefore, the term "complementarity" is understood to encompass Watson-Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al. (2012) Accounts of Chemical Research, vol. 45, p. 2055; and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry, Suppl. 37, 1.4.1).

[0069] The term "% complementary," as used herein, refers to the percentage of nucleotides in a contiguous nucleotide sequence of a nucleic acid molecule (e.g., an oligonucleotide) that are complementary to a reference sequence (e.g., a target sequence or sequence motif) over the contiguous nucleotide sequence. Thus, the percentage of complementarity is the percentage of complementary sequences between two sequences (when aligned from 5'-3' to the target sequence and from 3'-5' to the oligonucleotide sequence) that are complementary (Watson, 2002). Complementarity is calculated by counting the number of aligned nucleobases (from Watson-Crick base pairing), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such comparisons, nucleobases / nucleotides that do not align (form base pairs) are referred to as mismatches. Insertions and deletions are not allowed in calculating the percent complementarity of contiguous nucleotide sequences. It will be understood that in determining complementarity, chemical modifications of nucleobases are disregarded so long as the nucleobase retains its functional ability to form Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating percent identity).

[0070] The term "fully complementary" refers to 100% complementarity.

[0071] identity The term "identity" as used herein refers to the percentage (expressed as a percentage) of nucleotides in a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an oligonucleotide) that is identical to a reference sequence (e.g., a sequence motif) across the contiguous nucleotide sequence. Thus, the percentage of identity is calculated by counting the number of identical (matching) aligned nucleobases between two sequences (in the contiguous nucleotide sequence of the compound of the present invention and the reference sequence), dividing this number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, the percentage of identity = (number of matches x 100) / length of the aligned region (e.g., contiguous nucleotide sequence). Insertions and deletions are not allowed in calculating the percentage identity of a contiguous nucleotide sequence. It should be understood that in determining identity, chemical modifications of nucleobases are ignored as long as the nucleobase retains its functional ability to form Watson-Crick base pairs (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating identity percentage).

[0072] Hybridization As used herein, the term "hybridize" or "hybridizing" should 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 binding between two nucleic acid strands is the strength of hybridization. This is determined by the melting temperature (T), which is defined as the temperature at which half of the oligonucleotide forms a duplex with the target nucleic acid. m ) is often explained by the following: 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 binding affinity, ΔG° = -RTln(K d ) to calculate the dissociation constant (K d) where R is the gas constant and T is the absolute temperature. Therefore, a very low ΔG° of the reaction between an oligonucleotide and a target nucleic acid reflects strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the energy associated with a reaction at an aqueous concentration of 1 M, pH 7, and temperature of 37°C. The hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and in the case of a spontaneous reaction, ΔG° is less than zero. ΔG° can be experimentally measured, for example, by isothermal titration calorimetry (ITC), as described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discovery Today. Those skilled in the art will be aware that commercially available devices are available for measuring ΔG°. ΔG° can also be numerically estimated by using the nearest neighbor model described in Santa Lucia, 1998, Proc Natl Acad Sci USA. 95:1460-1465, using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405. To ensure the potential for modulation by hybridization to its intended nucleic acid target, In general, oligonucleotides of the present invention hybridize to target nucleic acids with estimated ΔG° values ​​of less than -10 kcal for oligonucleotides 10 to 30 nucleotides in length. In some embodiments, the degree or strength of hybridization is measured by the standard state Gibbs free energy ΔG°. Oligonucleotides may hybridize to target nucleic acids with estimated ΔG° values ​​of less than -10 kcal, e.g., less than -15 kcal, e.g., less than -20 kcal, and e.g., less than -25 kcal for oligonucleotides 8 to 30 nucleotides in length. In some embodiments, oligonucleotides hybridize to target nucleic acids with estimated ΔG° values ​​in the range of -10 to -60 kcal, e.g., -12 to -40, e.g., -15 to -30 kcal, or 16 to -27 kcal, e.g., -18 to -25 kcal.

[0073] target nucleic acid According to the present invention, the target nucleic acid is a nucleic acid encoding a mammalian SCAMP3, and may be, for example, a gene, RNA, mRNA, pre-mRNA, mature mRNA, or cDNA sequence. Thus, the target may be referred to as a SCAMP3 target nucleic acid.

[0074] Suitably, the target nucleic acid encodes a mammalian SCAMP3, such as a human SCAMP3 gene, that encodes a SCAMP3 protein, in particular the pre-mRNA or mRNA sequences provided herein as SEQ ID NOs: 1, 3, 4 and / or 5.

[0075] The therapeutic oligonucleotides of the present invention can target, for example, exon regions of mammalian SCAMP3 (particularly siRNAs and shRNAs, and antisense oligonucleotides), or can target, for example, any intron region in the SCAMP3 pre-mRNA (particularly antisense oligonucleotides). The human SCAMP3 gene encodes nine transcripts, two of which are protein-coding (SEQ ID NOs: 3 and 4), and are therefore potential nucleic acid targets.

[0076] Table 1 lists the predicted exon and intron regions of SEQ ID NO: 1, the human SCAMP3 pre-mRNA sequence.

[0077] [Table 1]

[0078] Suitably, the target nucleic acid encodes a SCAMP3 protein, in particular a mammalian SCAMP3 such as human SCAMP3 (see, for example, Tables 2 and 3), and the genomic sequences of human, porcine and mouse SCAMP3 (Table 2) and the pre- A summary of the mRNA sequence and mature mRNA of human SCAMP3 (Table 3) is provided.

[0079] In some embodiments, the target nucleic acid is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4 and / or 5, or naturally occurring variants thereof (eg, sequences encoding mammalian SCAMP3).

[0080] [Table 2]

[0081] When the nucleic acid molecules of the invention are used for research or diagnostic purposes, the target nucleic acid can be cDNA or a synthetic nucleic acid derived from DNA or RNA. For in vivo or in vitro applications, therapeutic nucleic acid molecules of the present invention typically inhibit expression of a SCAMP3 target nucleic acid in cells expressing the SCAMP3 target nucleic acid. In some embodiments, the cells contain HBV cccDNA. The contiguous sequence of nucleobases of the nucleic acid molecules of the present invention, when measured over the length of the nucleic acid molecule, is typically complementary to a conserved region of a SCAMP3 target nucleic acid, except for one or two mismatches and a nucleotide-based linker region that may link the oligonucleotide to any functional group, such as a conjugate or other non-complementary terminal nucleotide. The target nucleic acid is a messenger RNA, e.g., a pre-mRNA encoding a mammalian SCAMP3 protein, such as human SCAMP3, e.g., a human SCAMP3 pre-mRNA sequence such as that disclosed as SEQ ID NO: 1, or a mouse SCAMP3 pre-mRNA sequence such as that disclosed as SEQ ID NO: 2, a SCAMP3 pre-mRNA sequence such as that disclosed as SEQ ID NO: 5, or a mature SCAMP3 mRNA, such as the human mature mRNA disclosed as SEQ ID NO: 3 or 4. SEQ ID NOs: 1-5 are DNA sequences. It will be appreciated that the target RNA sequence has uracil (U) bases in place of thymidine (T) bases.

[0082] Further information regarding exemplary target nucleic acids is provided in Tables 2 and 3.

[0083] [Table 3]

[0084] In some embodiments, the target nucleic acid is SEQ ID NO:1.

[0085] In some embodiments, the target nucleic acid is SEQ ID NO:2.

[0086] In some embodiments, the target nucleic acid is SEQ ID NO:3.

[0087] In some embodiments, the target nucleic acid is SEQ ID NO:4.

[0088] In some embodiments, the target nucleic acid is SEQ ID NO:5.

[0089] In some embodiments, the target nucleic acid is SEQ ID NO: 1 and / or SEQ ID NO: 3.

[0090] In some embodiments, the target nucleic acid is SEQ ID NO: 1 and / or 4.

[0091] In some embodiments, the target nucleic acid is SEQ ID NO: 1, 3 and / or 4.

[0092] Target sequence The term "target sequence" as used herein refers to a sequence of nucleotides present in a target nucleic acid that contains a nucleobase sequence complementary to the oligonucleotide or nucleic acid molecule of the present invention. In some embodiments, the target sequence consists of a region on the target nucleic acid that has a nucleobase sequence complementary to the continuous nucleotide sequence of the oligonucleotide of the present invention. This region of the target nucleic acid can be interchangeably referred to as a target nucleotide sequence, a target sequence, or a target region. In some embodiments, the target sequence is longer than the complementary sequence of the nucleic acid molecule of the present invention, and can represent, for example, a preferred region of the target nucleic acid that can be targeted by some nucleic acid molecules of the present invention.

[0093] In some embodiments, the target sequence is a sequence selected from the group consisting of human SCAMP3 mRNA exons, such as human SCAMP3 mRNA exons selected from the group consisting of e1, e2, e3, e4, e5, e6, e7, e8, and e9 (see, e.g., Table 1 above).

[0094] Thus, the present invention provides an oligonucleotide comprising a contiguous sequence that is at least 90% complementary, for example, completely complementary, to an exon region of SEQ ID NO: 1 selected from the group consisting of e1 to e9 (see Table 1).

[0095] In some embodiments, the target sequence is a sequence selected from the group consisting of a human SCAMP3 mRNA intron, such as a human SCAMP3 mRNA intron selected from the group consisting of i1, i2, i3, i4, i5, i6, i7, and i8 (see, e.g., Table 1 above).

[0096] Thus, the present invention provides an oligonucleotide comprising a contiguous sequence that is at least 90% complementary, for example, completely complementary, to an intron region of SEQ ID NO: 1 selected from the group consisting of i1 to i8 (see Table 1).

[0097] In some embodiments, the target sequence is selected from the group consisting of SEQ ID NOs: 6, 7, 8, and 9. In some embodiments, the contiguous nucleotide sequence referred to herein is at least 90% complementary, such as at least 95% complementary, to a target sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, and 9. In some embodiments, the contiguous nucleotide sequence is fully complementary to a target sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8, and 9.

[0098] The oligonucleotides of the invention comprise a contiguous nucleotide sequence that is complementary to or hybridizes to a region on a target nucleic acid, eg, a target sequence described herein.

[0099] The target nucleic acid sequence to which the therapeutic oligonucleotide is complementary or hybridizes generally comprises a stretch of contiguous nucleobases of at least 10 nucleotides, the contiguous nucleotide sequence being 12 to 70 nucleotides, such as 12 to 50, for example 13 to 30, for example 14 to 25, for example 15 to 20, for example 16 to 18 contiguous nucleotides.

[0100] In some embodiments, the oligonucleotides of the invention target the regions shown in Table 4.

[0101] [Table 4]

[0102] In some embodiments, the target sequence is selected from the group consisting of target regions 1A-23A as shown in Table 4 above.

[0103] In some embodiments, the oligonucleotides of the invention target the regions shown in Table 5.

[0104] [Table 5]

[0105] In some embodiments, the target sequence is selected from the group consisting of target regions 1B-44B as shown in Table 5 above.

[0106] target cell As used herein, the term "target cell" refers to a cell expressing a target nucleic acid. For therapeutic use 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 aspects, the target cell is a mammalian cell, such as a rodent cell, such as a mouse cell or a rat cell, or a marmot cell, or a pig cell, or a primate cell, such as a monkey cell (e.g., a cynomolgus monkey cell) or a human cell.

[0107] In a preferred embodiment, the target cell expresses SCAMP3 mRNA, such as SCAMP3 pre-mRNA or SCAMP3 mature mRNA. The polyA tail of SCAMP3 mRNA is typically ignored for antisense oligonucleotide targeting.

[0108] Furthermore, the target cells may be hepatocytes. In one embodiment, the target cells are HBV-infected primary human hepatocytes derived from either an HBV-infected individual or an HBV-infected mouse with a humanized liver (PhoenixBio, PXB mouse). According to the present invention, the target cell may be infected with HBV. Furthermore, the target cell may contain HBV cccDNA. Therefore, the target cell preferably contains SCAMP3 mRNA, such as SCAMP3 pre-mRNA or SCAMP3 mature mRNA, and HBV cccDNA.

[0109] Naturally occurring variants The term "naturally occurring variant" refers to a variant that originates from the same genetic locus as the target nucleic acid, but that does not, for example, have the degeneracy of the genetic code, or protease activity, which results in multiple codons that encode the same amino acid. "Reverse" refers to variants of the SCAMP3 gene or transcript that may differ due to alternative splicing of mRNA or the presence of polymorphisms, such as single nucleotide polymorphisms (SNPs), and allelic variants. Based on the presence of a sufficiently complementary sequence to the oligonucleotide, the oligonucleotides of the invention can therefore target the target nucleic acid and its naturally occurring variants.

[0110] In some embodiments, the naturally occurring variant has at least 95%, e.g., at least 98%, or at least 99% homology to a mammalian SCAMP3 target nucleic acid, e.g., the target nucleic acid of SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the naturally occurring variant has at least 99% homology to the human SCAMP3 target nucleic acid of SEQ ID NO: 1. In some embodiments, the naturally occurring variant is a known polymorphism.

[0111] Inhibition of expression The term "inhibition of expression" as used herein should be understood as a general term for the ability of a SCAMP3 (COP9 signalosome subunit 3) inhibitor to inhibit, i.e., reduce, the amount or activity of SCAMP3 in target cells. Inhibition of expression or activity can be measured by measuring the level of SCAMP3 pre-mRNA or SCAMP3 mRNA, or by measuring the level of SCAMP3 protein or activity in cells. Inhibition of expression can be measured in vitro or in vivo. Advantageously, inhibition is assessed relative to the amount of SCAMP3 before administration of the SCAMP3 inhibitor. Alternatively, inhibition can be measured by reference to a control. A control is generally understood to be an individual or target cells treated with a saline composition or an individual or target cells treated with a non-targeting oligonucleotide (mock).

[0112] The terms "inhibition" or "inhibiting" may also be referred to as downregulating, decreasing, suppressing, reducing, lowering or decreasing the expression or activity of SCAMP3.

[0113] Inhibition of SCAMP3 expression can occur, for example, by degradation of pre-mRNA or mRNA, using, for example, RNase H-recruiting oligonucleotides such as gapmers, or nucleic acid molecules that function via RNA interference pathways such as siRNA or shRNA. Alternatively, inhibitors of the invention can bind to the MCM4 polypeptide and inhibit the activity of MCM4 or prevent its binding to other molecules.

[0114] In some embodiments, inhibiting the expression of SCAMP3 target nucleic acids or the activity of SCAMP3 protein results in a reduction in the amount of HBV cccDNA in target cells. Preferably, the amount of HBV cccDNA is reduced compared to a control. In some embodiments, the reduction in the amount of HBV cccDNA is at least 20%, or at least 30%, compared to a control. In some embodiments, the amount of cccDNA in HBV-infected cells is reduced by at least 50%, e.g., 60%, when compared to a control. In some aspects, the target cells are infected with HBV, and the amount of cccDNA in HBV-infected cells is reduced by at least 25%, e.g., at least 40%, compared to the level in HBV-infected target cells without any treatment or treated with a control.

[0115] In some embodiments, inhibiting the expression of SCAMP3 target nucleic acid or the activity of SCAMP3 protein results in a decrease in the amount of HBV pgRNA in target cells. Preferably, the amount of HBV pgRNA is reduced compared to a control. In some embodiments, HBV The amount of pgRNA is reduced by at least 20%, at least 30% compared to a control, hi some embodiments, the amount of pgRNA in HBV-infected cells is reduced by at least 50%, e.g., 60%, compared to a control.

[0116] sugar modification Oligonucleotides of the invention may contain one or more nucleosides having modified sugar moieties, ie, modifications in the sugar moiety as compared to the ribose sugar moiety found in DNA and RNA.

[0117] Numerous nucleosides with modifications in the ribose sugar moiety have been created primarily with the goal of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0118] Such modifications include those in which the ribose ring structure has been modified, for example, by replacing it with a hexose ring (HNA), or a bicyclic ring typically having a biradical bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring (e.g., UNA), typically lacking a bond between the C2 and C3 carbons. Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclic nucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which the sugar moiety has been replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0119] Sugar modifications also include modifications made by changing the substituent on the ribose ring to a group other than hydrogen or to the 2'-OH group naturally occurring in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' position.

[0120] High-affinity modified nucleosides High affinity modified nucleosides are modified nucleotides that, when incorporated into an oligonucleotide, e.g., increase the melting temperature (T m The high affinity modified nucleosides of the present invention preferably provide an increase in melting temperature in the range of +0.5 to +12°C per modified nucleoside, more preferably in the range of +1.5 to +10°C, and most preferably in the range of +3 to +8°C. Numerous high affinity modified nucleosides are known in the art, including, for example, many 2'-substituted nucleosides and locked nucleic acids (LNAs) (see, e.g., Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).

[0121] 2' sugar-modified nucleosides 2' sugar modified nucleosides are nucleosides that either have a substituent other than H or -OH at the 2' position (2' substituted nucleosides) or contain a 2' linked biradical that can form a bridge between the 2' carbon and a second carbon on the ribose ring, e.g., LNA (2'-4' biradical bridged) nucleosides.

[0122] Indeed, much attention has been focused on the development of 2' sugar-substituted nucleosides, and many 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 Dr. See, Ug Development, 2000, 3(2), 293-213, and Deleavy and Damha, Chemistry and Biology 2012, 19, 937. Below are examples of some 2'-substituted modified nucleosides. [ka] In the context of the present invention, 2'-substituted sugar modified nucleosides do not include 2'-bridged nucleosides such as LNA.

[0123] Locked nucleic acid nucleosides (LNA nucleosides) An "LNA nucleoside" is a 2' sugar-modified nucleoside containing a biradical (also referred to as a "2'-4' bridge") linking the C2' and C4' of the ribose sugar ring of the nucleoside, which restricts or fixes the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNA). Fixation of the ribose conformation is associated with improved hybridization affinity (duplex stabilization) when LNA is incorporated into oligonucleotides of complementary RNA or DNA molecules. This can usually be determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0124] Non-limiting exemplary LNA nucleosides are those 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.

[0125] Specific examples of LNA nucleosides of the present invention are shown in Scheme 1, where B is as defined above. Scheme 1 [ka]

[0126] Particular LNA nucleosides are beta-D-oxy-LNA, 6'-methyl-beta-D-oxyLNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET) and ENA.

[0127] RNase H activity and recruitment The RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when duplexed with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for measuring RNase H activity, which can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is considered capable of recruiting RNase H when provided with a complementary target nucleic acid if it has at least 5%, e.g., at least 10% or more than 20%, of the initial rate, measured in pmol / l / min, determined using an oligonucleotide 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 using the methodology provided in Examples 91-95 of WO 01 / 23613 (incorporated herein by reference). For use in measuring RNase H activity, recombinant human R-antisense oligonucleotides are used. RNASE H1 is available from Creative Biomart® (recombinant human RNASE H1 fused to a His tag expressed in E. coli).

[0128] Gapma The antisense oligonucleotide or its contiguous nucleotide sequence of the present invention may be a gapmer, also referred to as a gapmer oligonucleotide or gapmer design. Antisense gapmers are typically used to inhibit target nucleic acids via RNase H-mediated degradation. Gapmer-type oligonucleotides contain at least three distinct structural regions: a 5'-flank, a gap, and a 3'-flank, FG-F', in the 5'→3' direction. The "gap" region (G) contains a stretch of contiguous DNA nucleotides that allows the oligonucleotide to recruit RNase H. The gap region is flanked by a 5'-flanking region (F) containing one or more sugar-modified nucleosides, preferably high-affinity sugar-modified nucleosides, and a 3'-flanking region (F') containing one or more sugar-modified nucleosides, preferably high-affinity sugar-modified nucleosides. One or more sugar-modified nucleosides in regions F and F' improve the affinity of the oligonucleotide for the target nucleic acid (i.e., it is an affinity-enhancing sugar-modified nucleoside). In some embodiments, one or more sugar-modified nucleosides of regions F and F' are 2' sugar-modified nucleosides, such as high affinity 2' sugar modifications, independently selected from, for example, LNA and 2'-MOE.

[0129] In a gapmer design, the 5'- and 3'-most nucleosides of the gap region are DNA nucleosides, positioned adjacent to sugar-modified nucleosides in the 5' (F) or 3' (F') regions, respectively. Flanks may be further defined by having at least one sugar-modified nucleoside at the ends furthest from the gap region, i.e., at the 5'-end of the 5' flank and at the 3'-end of the 3' flank. The region FG-F' forms a contiguous nucleotide sequence. The antisense oligonucleotide of the present invention or its contiguous nucleotide sequence may comprise a gapmer region of the formula FG-F'.

[0130] The total length of the gapmer designed FG-F' can be, for example, 12 to 32 nucleosides, for example, 13 to 24, for example, 14 to 22 nucleosides, for example, 15 to 20, for example, 16 to 18 nucleosides. By way of example, a gapmer-type 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 FG-F' is at least 12, for example at least 14, nucleotides in length.

[0131] In one aspect of the invention, the antisense oligonucleotide or contiguous nucleotide sequence thereof consists of or comprises a gapmer of the formula 5'-FG-F'-3', where regions F and F' independently comprise or consist of 1 to 8 nucleosides, 1 to 4 of which are 2' sugar modified and define the 5' and 3' ends of the F and F' regions, and G is a region of between 6 and 18 nucleosides capable of recruiting RNase H. In some embodiments, the G region consists of DNA nucleosides.

[0132] In some embodiments, regions F and F' independently consist of or comprise a contiguous sequence of sugar-modified nucleosides. In some embodiments, the sugar-modified nucleosides of region F independently consist of 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. It may be selected from the following positions.

[0133] In some embodiments, regions F and F' independently comprise both LNA and 2'-substituted sugar modified nucleotides (mixed wing design). In some embodiments, the 2'-substituted sugar modified nucleotides are 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. In some embodiments, all modified nucleosides in regions F and F' are independently selected from LNA nucleosides, e.g., beta-D-oxyLNA, ENA, or ScET nucleosides, and regions F or F', or F and F', can comprise DNA nucleosides. In some embodiments, all modified nucleosides in regions F and F' are beta-D-oxyLNA nucleosides, and regions F or F', or F and F', can comprise DNA nucleosides. In such embodiments, flanking regions F or F', or both F and F', comprise at least three nucleosides, and the 5'- and 3'-most nucleosides of the F and / or F' regions are LNA nucleosides.

[0134] LNA Gapma An LNA gapmer is a gapmer in which either or both of regions F and F' comprise or consist of LNA nucleosides. A beta-D-oxy gapmer is a gapmer in which either or both of regions F and F' comprise or consist of beta-D-oxy LNA nucleosides. In some embodiments, the LNA gapmer has the formula: [LNA] 1-5 -[Area G] 6-18 -[LNA] 1-5 and region G is as defined in the definition of gapma region G.

[0135] MOE Gapma An MOE gapmer is a gapmer in which regions F and F' consist of MOE nucleosides. In some embodiments, an MOE gapmer is [MOE] 1-8 -[Area G] 5-16 -[MOE] 1-8 , e.g. [MOE] 2-7 -[Area G] 6-14 -[MOE] 2-7 , e.g. [MOE] 3-6 -[Area G] 8-12 -[MOE] 3-6 , e.g., [MOE]5-[Region G] 10 -[MOE]5 design, where region G is as defined in the gapmer definition. MOE gapmers with a 5-10-5 design (MOE-DNA-MOE) are widely used in the art.

[0136] Region D' or D'' within the oligonucleotide Oligonucleotides of the invention, in some embodiments, can comprise or consist of a contiguous nucleotide sequence of the oligonucleotide that is complementary to a target nucleic acid, such as a gapmer region FG-F', as well as additional 5' and / or 3' nucleosides. The additional 5' and / or 3' nucleosides may or may not be fully complementary to the target nucleic acid. Such additional 5' and / or 3' nucleosides may be referred to herein as regions D' and D''.

[0137] The addition of region D' or D" can be used for the purpose of linking a contiguous nucleotide sequence, such as a gapmer, to a conjugate moiety or another functional group. When used to link a conjugate moiety to a conjugate moiety, it can serve as a biocleavable linker. Alternatively, it can be used to provide exonuclease protection or to facilitate synthesis or manufacturing.

[0138] Regions D' and D'' are linked to the 5' end of region F or the 3' end of region F', respectively, and have the following formula: D'-FG-F', FG-F'-D'' or A D'-FG-F'-D'' design can be generated, where FG-F' is the gapmer portion of the oligonucleotide and regions D' or D'' constitute separate portions of the oligonucleotide.

[0139] Region D' or D" independently comprises or consists of 1, 2, 3, 4, or 5 additional nucleotides and 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 but are, for example, DNA or RNA or base-modified versions thereof. The D' or D" region can serve as a nuclease-sensitive biocleavable 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 biocleavable linkers suitable for use as region D' or D" are disclosed in WO 2014 / 076195, including, by way of example, phosphodiester-linked DNA dinucleotides. The use of biocleavable linkers in polyoligonucleotide constructs is disclosed in WO 2015 / 113922, where they have been used to join multiple antisense constructs (e.g., gapmer regions) within a single oligonucleotide.

[0140] In one embodiment, the oligonucleotide of the present invention comprises regions D' and / or D'' in addition to the contiguous nucleotide sequence that constitutes the gapmer.

[0141] In some embodiments, the oligonucleotides of the invention can be represented by the following formula: FG-F'; especially F 1-8 -G 5-18 -F' 2-8 D'-FG-F', especially D' 1-3 -F 1-8 -G 5-18 -F' 2-8 FG-F'-D'', especially F1-8 -G 5-18 -F' 2-8 -D'' 1-3 D'-FG-F'-D'', especially D' 1-3 -F 1-8 -G 5-18 -F' 2-8 -D'' 1-3 .

[0142] In some embodiments, the internucleoside linkage located between region D' and region F is a phosphodiester bond. In some embodiments, the internucleoside linkage located between region F' and region D'' is a phosphodiester bond.

[0143] Conjugates The term conjugate, as used herein, refers to an oligonucleotide covalently linked to a non-nucleotide moiety (conjugate moiety or region C or third region). The conjugate moiety may be covalently linked to the antisense oligonucleotide, optionally via a linker group such as region D' or D".

[0144] Oligonucleotide conjugates and their synthesis are reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001, and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, each of which is incorporated herein by reference.

[0145] In some embodiments, the non-nucleotide moiety (conjugate moiety) is a carbohydrate ( For example, the compound is selected from the group consisting of a galactose or N-acetylgalactosamine (GalNAc), a cell surface receptor ligand, a drug substance, 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.

[0146] Exemplary conjugate moiety is the conjugate moiety that can be bound to asialoglycoprotein receptor (ASGPR).In particular, the conjugate moiety of trivalent N-acetylgalactosamine is suitable for binding to ASGPR, for example, see International Publication No. 2014 / 076196, International Publication No. 2014 / 207232 and International Publication No. 2014 / 179620 (incorporated herein by reference).This conjugate is useful for enhancing the uptake of oligonucleotide into liver.

[0147] Linker A bond or linker is a connection between two atoms that connects a desired chemical group or segment to another desired chemical group or segment through one or more covalent bonds. The conjugate moiety can be directly or via a linking moiety (e.g., a linker or tether) to the oligonucleotide. The linker functions to covalently link a third region (region C), such as a conjugate moiety, to a first region (region A), such as an oligonucleotide or a continuous nucleotide sequence complementary to a target nucleic acid.

[0148] In some embodiments of the present invention, the conjugate or oligonucleotide conjugate of the present invention may optionally comprise a linker region (second region or region B and / or region Y) located between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid (region A or first region) and the conjugate moiety (region C or third region).

[0149] Region B refers to a biocleavable linker that contains or consists of a physiologically labile bond that is cleavable under conditions normally found in, or similar to, those found in, a mammalian body. Conditions under which a physiologically labile linker undergoes chemical transformation (e.g., cleavage) include chemical conditions such as pH, temperature, oxidizing or reducing conditions or agents, and salt concentration found in, or similar to, mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activity normally present in mammalian cells, such as from proteolytic or hydrolytic enzymes or nucleases. In one embodiment, the biocleavable linker is susceptible to S1 nuclease cleavage. In a preferred embodiment, the nuclease-sensitive linker contains 1 to 5 nucleosides, e.g., 1, 2, 3, 4, or 5 nucleosides, more preferably 2 to 4 nucleosides, and most preferably 2 or 3 nucleosides, which contain at least two consecutive phosphodiester bonds, e.g., at least 3, 4, or 5 consecutive phosphodiester bonds. Preferably, the nucleoside is DNA or RNA. Biocleavable linkers containing phosphodiesters are described in more detail in WO 2014 / 076195, which is incorporated herein by reference.

[0150] Region Y refers to a linker that is not necessarily biocleavable, but primarily serves to covalently attach the conjugate moiety (region C or third region) to the oligonucleotide (region A or first region). Region Y linkers may comprise chain structures or oligomers of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. Oligonucleotide conjugates of the invention may be constructed from the following local elements AC, ABC, ABYC, AYBC, or AYC. In some embodiments, the linker (region Y) is an aminoalkyl, e.g., a C2-C36 aminoalkyl group, including a C6-C12 aminoalkyl group. In some embodiments, the linker (region Y) is a C6 amino It is an alkyl group.

[0151] treatment As used herein, the term "treatment" refers to both the treatment of an existing disease (e.g., a disease or disorder referred to herein) or the prevention of disease, i.e., prophylaxis. Accordingly, it will be recognized that the treatment referred to herein may, in some embodiments, be prophylactic. Prevention can be understood as preventing HBV infection from converting to chronic HBV infection, or preventing serious liver diseases, such as cirrhosis and hepatocellular carcinoma, due to chronic HBV infection.

[0152] patient For the purposes of the present invention, a "subject" (or "patient") may 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 applications. Preferably, the subject is a mammal. More preferably, the subject is a human. As described elsewhere herein, the patient to be treated may be suffering from HBV infection, such as chronic HBV infection. In some embodiments, the patient suffering from HBV infection may be suffering from hepatocellular carcinoma (HCC). In some embodiments, the patient suffering from HBV infection does not suffer from hepatocellular carcinoma. DETAILED DESCRIPTION OF THE INVENTION

[0153] HBV cccDNA in infected hepatocytes is involved in persistent chronic infection and reactivation, serving 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, SCAMP3 has been shown for the first time to be involved in cccDNA stability. This knowledge provides an opportunity to destabilize cccDNA in HBV-infected subjects, potentially opening up the possibility of a complete cure for chronically infected HBV patients.

[0154] One aspect of the present invention is a SCAMP3 inhibitor for use in the treatment and / or prevention of hepatitis B virus (HBV) infection, particularly chronic HBV infection. A SCAMP3 inhibitor can be, for example, a small molecule that specifically binds to the SCAMP3 protein, and the inhibitor prevents or reduces binding of the SCAMP3 protein to cccDNA. One embodiment of the present invention is a SCAMP3 inhibitor that can reduce cccDNA and / or pgRNA in infected cells, such as HBV-infected cells. In a further embodiment, SCAMP3 inhibitors may reduce HBsAg and / or HBeAg in vivo in HBV-infected individuals.

[0155] SCAMP3 inhibitors for use in the treatment of HBV - Patent Application 20070122997 Without being bound by theory, it is believed that SCAMP3 is involved in stabilizing cccDNA in the cell nucleus through direct or indirect binding to cccDNA, and that by blocking the binding / association of SCAMP3 with cccDNA, the cccDNA is destabilized and becomes more susceptible to degradation. Thus, one embodiment of the present invention is a SCAMP3 inhibitor that interacts with the SCAMP3 protein and prevents or reduces its binding / association to cccDNA.

[0156] In some embodiments of the invention, the inhibitor is an antibody, an antibody fragment, or a small molecule compound. In some embodiments, the inhibitor is encoded by SEQ ID NO: 1, 3, or 4. It may be an antibody, antibody fragment or small molecule that specifically binds to a SCAMP3 protein, such as a SCAMP3 protein.

[0157] Nucleic Acid Molecules of the Invention Therapeutic nucleic acid molecules target SCAMP3 transcripts and inhibit the RNA interference pathway or RNase These oligonucleotides are potentially excellent SCAMP3 inhibitors because they can promote its degradation via either H cleavage or H cleavage. Alternatively, oligonucleotides such as aptamers can also act as inhibitors of SCAMP3 protein interactions.

[0158] One aspect of the present invention is a nucleic acid molecule targeting SCAMP3 for use in the treatment and / or prevention of Hepatitis B virus (HBV) infection. Such a nucleic acid molecule may be selected from the group consisting of a single-stranded antisense oligonucleotide, an siRNA molecule, and an shRNA molecule.

[0159] This section discloses novel nucleic acid molecules suitable for use in the treatment and / or prevention of Hepatitis B virus (HBV) infection.

[0160] The nucleic acid molecules of the present invention can inhibit SCAMP3 expression in vitro and in vivo. Inhibition is achieved by hybridizing an oligonucleotide to a target nucleic acid encoding SCAMP3 or involved in the regulation of SCAMP3. The target nucleic acid can be a mammalian SCAMP3 sequence. In some embodiments, the target nucleic acid can be a human SCAMP3 pre-mRNA sequence, such as the sequence of SEQ ID NO: 1, or a human SCAMP3 mRNA sequence, such as SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the target nucleic acid can be a porcine SCAMP3 sequence, such as the sequence of SEQ ID NO: 5.

[0161] In some embodiments, nucleic acid molecules of the present invention can modulate target expression by inhibiting or downregulating the expression of the target. Preferably, such modulation results in at least 20% inhibition of expression compared to the target's normal expression level, more preferably at least 30%, at least 40%, at least 50%, or at least 60% inhibition compared to the target's normal expression level. In some embodiments, nucleic acid molecules of the present invention can inhibit SCAMP3 mRNA expression levels by at least 60% or 70% in vitro by transfecting 25 nM of the nucleic acid molecule into PXB-PHH cells. This range of target reduction is advantageous in selecting nucleic acid molecules that correlate well with cccDNA reduction. Suitably, the Examples provide assays that can be used to measure SCAMP3 RNA or protein inhibition (e.g., Example 1 and the "Materials and Methods" section). Target inhibition is caused by hybridization between a contiguous 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, nucleic acid molecules of the present invention contain mismatches between the oligonucleotide and the target nucleic acid. Despite the mismatch, hybridization to the target nucleic acid may still be sufficient to exhibit the desired inhibition of SCAMP3 expression. The reduced binding affinity due to the mismatch may be advantageously compensated for by increasing the number of nucleotides in the oligonucleotide complementary to the target nucleic acid and / or by increasing the number of modified nucleosides, such as 2' sugar-modified nucleosides containing LNA, present within the oligonucleotide sequence, which may increase binding affinity to the target.

[0162] One aspect of the present invention relates to nucleic acid molecules of 12 to 60 nucleotides in length, which comprise a contiguous nucleotide sequence of at least 12 nucleotides in length, e.g., at least 12 to 30 nucleotides in length, and which are at least 95% complementary, e.g., fully complementary, to a mammalian SCAMP3 target nucleic acid, particularly a human SCAMP3 nucleic acid. These nucleic acid molecules are useful for detecting SCAMP3. The expression of the gene may be inhibited.

[0163] One aspect of the present invention relates to a nucleic acid molecule of 12 to 30 nucleotides in length, comprising a contiguous nucleotide sequence of at least 10 nucleotides, e.g., 12 to 30 nucleotides in length, that is at least 90% complementary, e.g., completely complementary, to a mammalian SCAMP3 target sequence.

[0164] A further aspect of the invention relates to a nucleic acid molecule according to the invention comprising a contiguous nucleotide sequence of 14 to 22 nucleotides in length that is at least 90% complementary, such as completely complementary, to the target sequence of SEQ ID NO:1.

[0165] In some embodiments, the nucleic acid molecule comprises a contiguous sequence of 12 to 30 nucleotides in length that is at least 90% complementary to a region of the target nucleic acid or target sequence, such as at least 91%, for example at least 92%, for example at least 93%, for example at least 94%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, or 100% complementary.

[0166] It is advantageous if the oligonucleotide or its contiguous nucleotide sequence is fully complementary (100% complementary) to a region of the target sequence, or in some embodiments, may contain one or two mismatches between the oligonucleotide and the target sequence.

[0167] In some embodiments, the oligonucleotide sequence is 100% complementary to a region of the target sequence of SEQ ID NO:1 and / or SEQ ID NO:3 and / or SEQ ID NO:4.

[0168] In some embodiments, the nucleic acid molecules or contiguous nucleotide sequences of the invention are at least 90% or 95% complementary, eg, completely (or 100%) complementary, to the target nucleic acids of SEQ ID NOs: 1 and 2.

[0169] In some embodiments, the oligonucleotide or contiguous nucleotide sequence of the invention is at least 90% or 95% complementary, such as completely (or 100%) complementary, to the target nucleic acids of SEQ ID NO:2 and SEQ ID NO:3 or 4.

[0170] In some embodiments, the contiguous sequence of a nucleic acid molecule of the invention is at least 90% complementary, e.g., fully complementary, to a region of SEQ ID NO: 1 and is selected from the group consisting of target regions 1A-23A shown in Table 4.

[0171] In some embodiments, the contiguous sequence of a nucleic acid molecule of the invention is at least 90% complementary, e.g., fully complementary, to a region of SEQ ID NO: 1 and is selected from the group consisting of target regions 1B to 44B shown in Table 5.

[0172] In some embodiments, the nucleic acid molecule of the invention comprises or consists of a length of 12 to 60 nucleotides, such as 13 to 50, such as 14 to 35, such as 15 to 30, such as 16 to 22 contiguous nucleotides, In preferred embodiments, the nucleic acid molecule comprises or consists of a length of 15, 16, 17, 18, 19, 20, 21, or 22 nucleotides.

[0173] In some embodiments, the contiguous nucleotide sequence of the nucleic acid molecule complementary to the target nucleic acid comprises or consists of a length of 12 to 30, such as 13 to 25, such as 15 to 23, such as 16 to 22 contiguous nucleotides.

[0174] In some embodiments, the oligonucleotide is an antisense oligonucleotide, It is selected from the group consisting of siRNA and shRNA.

[0175] In some embodiments, the contiguous nucleotide sequence of the siRNA or shRNA complementary to the target sequence comprises or consists of a length of 18-28, such as 19-26, such as 20-24, such as 21-23 contiguous nucleotides.

[0176] In some embodiments, the contiguous nucleotide sequence of the antisense oligonucleotide complementary to the target nucleic acid comprises or consists of a length of 12 to 22, such as 14 to 20, for example 16 to 20, for example 15 to 18, for example 16 to 18, such as 16, 17, 18, 19 or 20 contiguous nucleotides.

[0177] It is understood that the contiguous oligonucleotide sequence (motif sequence) can be modified, for example, to increase nuclease resistance and / or binding affinity to the target nucleic acid.

[0178] The pattern in which modified nucleosides (such as high affinity modified nucleosides) are incorporated into an oligonucleotide sequence is commonly referred to as the oligonucleotide design.

[0179] The nucleic acid molecules of the present invention can be designed using modified nucleosides and RNA nucleosides (particularly for siRNA and shRNA molecules) or DNA nucleosides (particularly for single-stranded antisense oligonucleotides). It is advantageous to use high-affinity modified nucleosides.

[0180] In advantageous embodiments, the nucleic acid molecule or contiguous nucleotide sequence comprises one or more sugar-modified nucleosides, such as 2'-sugar-modified nucleosides, such as one or more 2'-sugar-modified nucleosides 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. It is advantageous if one or more of the modified nucleoside(s) is / are locked nucleic acid (LNA).

[0181] In some embodiments, the contiguous nucleotide sequence comprises LNA nucleosides.

[0182] In some embodiments, the contiguous nucleotide sequence comprises LNA nucleosides and DNA nucleosides.

[0183] In some embodiments, the contiguous nucleotide sequence comprises 2'-O-methoxyethyl (2'MOE) nucleosides.

[0184] In some embodiments, the contiguous nucleotide sequence comprises 2'-O-methoxyethyl (2'MOE) nucleosides and DNA nucleosides.

[0185] Advantageously, the 3'-most nucleoside of the antisense oligonucleotide, or the contiguous nucleotide sequence thereof, is a 2'-sugar modified nucleoside.

[0186] In a further embodiment, the nucleic acid molecule comprises at least one modified internucleoside linkage. Suitable internucleoside modifications are described in the "Definitions" section under "Modified Internucleoside Linkages."

[0187] Advantageously, the oligonucleotide contains at least one modified internucleoside linkage, such as phosphorothioate or phosphorodithioate.

[0188] In some embodiments, at least one internucleoside linkage in the contiguous nucleotide sequence is a phosphodiester internucleoside linkage.

[0189] It is advantageous if at least 2-3 internucleoside linkages at the 5' or 3' end of the oligonucleotide are phosphorothioate internucleoside linkages.

[0190] For single-stranded antisense oligonucleotides, 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 internucleoside linkages within the continuous sequence of the single-stranded antisense oligonucleotide are phosphorothioate linkages.

[0191] In a preferred embodiment of the present invention, the antisense oligonucleotide of the present invention can recruit RNase H, such as RNase H1. Advantageous structural designs are gapmer designs, such as those described in the "Definitions" section of "Gapmer," "LNA Gapmer," and "MOE Gapmer." In the present invention, it is advantageous if the antisense oligonucleotide of the present invention is a gapmer of FG-F' design.

[0192] In all cases, the FG-F' design may further comprise regions D' and / or D'' as described in the "Definitions" section of "Region D' or D'' in an Oligonucleotide."

[0193] The present invention provides antisense oligonucleotides according to the invention, for example antisense oligonucleotides of 12 to 24, for example 12 to 18 in length, wherein the antisense nucleotide comprises a contiguous nucleotide sequence comprising at least 14, for example at least 15, for example 16 contiguous nucleotides present in SEQ ID NO: 19.

[0194] The present invention provides antisense oligonucleotides according to the invention, for example antisense oligonucleotides of 12 to 24, for example 12 to 18 in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 14, for example at least 15, for example 16 contiguous nucleotides present in SEQ ID NO: 20.

[0195] The present invention provides antisense oligonucleotides according to the invention, for example antisense oligonucleotides of 12 to 24 in length, for example 12 to 18 in length, wherein the antisense oligonucleotide comprises a contiguous nucleotide sequence comprising at least 14, for example at least 15, for example 16 contiguous nucleotides present in SEQ ID NO: 21.

[0196] The present invention provides an LNA gapmer according to the invention comprising or consisting of the contiguous nucleotide sequence set forth in SEQ ID NO: 19, 20 or 21. In some embodiments, the LNA gapmer is an LNA gapmer having CMP ID NO: 19_1, 20_1 or 21_1 of Table 6.

[0197] In a further embodiment of the present invention, nucleic acid molecules such as antisense oligonucleotides, siRNAs or shRNAs of the present invention may be targeted directly to the liver by covalently linking them to a conjugate moiety capable of binding to the asialoglycoprotein receptor (ASGPr), such as a bivalent or trivalent GalNAc cluster.

[0198] Conjugates Because HBV infection primarily affects hepatocytes in the liver, conjugating SCAMP3 inhibitors to a conjugate moiety increases delivery of the inhibitor to the liver compared to unconjugated inhibitors. In one embodiment, the liver-targeting moiety is selected from a moiety that includes cholesterol or other lipids, or a conjugate moiety that can bind to the asialoglycoprotein receptor (ASGPR).

[0199] In some embodiments, the invention provides a conjugate comprising a nucleic acid molecule of the invention covalently attached to a conjugate moiety.

[0200] The asialoglycoprotein receptor (ASGPR) conjugate moiety comprises one or more carbohydrate moieties capable of binding to the asialoglycoprotein receptor (ASPGR targeting moiety) with an affinity equal to or greater than that of galactose. The affinity of numerous galactose derivatives for the asialoglycoprotein receptor has been studied (see, e.g., Jobst, S.T. and Drickamer, K.J.B.C. 1996, 271, 6686) or is readily determined using methods typical in the art.

[0201] 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, Nn-butanoyl-galactosamine, and N-isobutanoylgalactosamine. Advantageously, the asialoglycoprotein receptor targeting moiety is N-acetylgalactosamine (GalNAc).

[0202] To generate an ASGPR conjugate moiety, an ASPGR targeting moiety (preferably GalNAc) can be attached to the conjugate scaffold. Generally, the ASPGR targeting moiety can be at the same end of the scaffold.

[0203] In one embodiment, the conjugate moiety consists of two to four terminal GalNAc moieties attached to a spacer that connects each GalNAc moiety to a brancher molecule that can be attached to an antisense oligonucleotide. In further embodiments, the conjugate moiety is monovalent, bivalent, trivalent, or tetravalent with respect to the asialoglycoprotein receptor targeting moiety. Advantageously, the asialoglycoprotein receptor targeting moiety comprises an N-acetylgalactosamine (GalNAc) moiety.

[0204] GalNAc conjugate moieties include, for example, those described in WO 2014 / 179620 and WO 2016 / 055601 and PCT / EP2017 / 059080 (incorporated herein by reference), as well as small peptides with GalNAc moieties attached, such as Tyr-Glu-Glu-(aminohexylGalNAc)3 (YEE(ahGalNAc)3); glycotripeptides that bind to the asialoglycoprotein receptor on hepatocytes (see, e.g., Duff, et al., Methods Enzymol, 2000, 313, 297); lysine-based galactose clusters (e.g., L3G4; Biessen, et al., Cardovasc. Med., 1999, 214); and cholan-based galactose clusters (e.g., carbohydrate recognition motifs for the asialoglycoprotein receptor).

[0205] 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.

[0206] In one embodiment, the ASGPR conjugate moiety is linked to the 5' end of the oligonucleotide. In one embodiment, the conjugate moiety is a trivalent N-acetylgalactosidase as shown in FIG. In one embodiment, the conjugate moiety is a trivalent N-acetylgalactosamine (GalNAc) of Figure 1A-1 or Figure 1A-2, or a mixture of both. In one embodiment, the conjugate moiety is a trivalent N-acetylgalactosamine (GalNAc) of Figure 1B-1 or Figure 1B-2, or a mixture of both. In one embodiment, the conjugate moiety is a trivalent N-acetylgalactosamine (GalNAc) of Figure 1C-1 or Figure 1C-2, or a mixture of both. In one embodiment, the conjugate moiety is a trivalent N-acetylgalactosamine (GalNAc) of Figure 1D-1 or Figure 1D-2, or a mixture of both.

[0207] Manufacturing method In a further aspect, the present invention provides a method for producing an oligonucleotide of the present invention, comprising reacting nucleotide units to thereby form covalently linked consecutive nucleotide units comprising the oligonucleotide. Preferably, the method uses phosphoramidite chemistry (see, e.g., Caruthers et al., 1987, Methods in (See Enzymology, Vol. 154, pp. 287-313.) In a further embodiment, the method further comprises reacting the contiguous nucleotide sequence with a conjugate moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide. In a further aspect, there is provided a method for producing a composition of the invention, comprising mixing an oligonucleotide or conjugated oligonucleotide of the invention with a pharmaceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0208] Pharmaceutical salts The compounds according to the present invention can exist in the form of their pharmaceutically acceptable salts. The term "pharmaceutically acceptable salts" refers to conventional acid or base addition salts that retain the biological effectiveness and properties of the compounds of the present invention. In a further aspect, the present invention provides a pharmaceutically acceptable salt of the nucleic acid molecule or a conjugate thereof, such as a pharmaceutically acceptable sodium, ammonium or potassium salt.

[0209] Pharmaceutical Composition In a further aspect, the present invention provides pharmaceutical compositions comprising any of the compounds of the present invention, particularly the aforementioned nucleic acid molecules and / or nucleic acid molecule conjugates, or salts 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 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 the pharmaceutically acceptable diluent at a concentration of 50-300 μM solution.

[0210] 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 review of drug delivery methods, see, for example, Langer (Science 249:1527-1533, 1990). WO 2007 / 031091 provides additional suitable and preferred examples of pharmaceutically acceptable diluents, carriers and adjuvants (incorporated herein by reference). Appropriate dosages, formulations, administration routes, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations are also provided in WO 2007 / 031091.

[0211] In some embodiments, a nucleic acid molecule or nucleic acid molecule conjugate of the invention, or The pharmaceutically acceptable salt is in a solid form, for example a powder, for example a lyophilized powder.

[0212] The compounds, nucleic acid molecules, or nucleic acid molecule conjugates of the present invention can be mixed with pharmaceutically acceptable active or inactive substances to prepare pharmaceutical compositions or formulations. The composition and method for preparing pharmaceutical compositions depend on many criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0213] These compositions may be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solutions may be packaged for immediate use or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation will typically be 3 to 11, more preferably 5 to 9 or 6 to 8, and most preferably 7 to 8, e.g., 7 to 7.5. The resulting solid form compositions may be packaged in multiple single-dose units, such as a sealed package of tablets or capsules, each containing a fixed amount of the agent or agents. The solid form compositions may also be packaged in flexible volume containers, such as squeezable tubes designed for topical creams or ointments.

[0214] In some embodiments, the nucleic acid molecules or nucleic acid molecule conjugates of the invention are prodrugs. Particularly with respect to nucleic acid molecule conjugates, once the prodrug is delivered to the site of action, e.g., a target cell, the conjugate moiety is cleaved from the nucleic acid molecule.

[0215] Administration The compounds, nucleic acid molecules or nucleic acid molecule conjugates or pharmaceutical compositions of the invention may be administered topically (e.g., to the skin, inhalation, eye or ear) or enterally (e.g., orally or through the digestive tract) or parenterally (e.g., intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly or intrathecally).

[0216] In a preferred embodiment, the oligonucleotide or pharmaceutical composition of the present invention is administered parenterally, 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.

[0217] In some embodiments, the nucleic acid molecule, nucleic acid molecule conjugate, or pharmaceutical composition of the present invention is administered at a dose of 0.1 to 15 mg / kg, e.g., 0.2 to 10 mg / kg, e.g., 0.25 to 5 mg / kg, and may be administered once a week, once every two weeks, once every three weeks, or even once a month.

[0218] The present invention also provides the use of the nucleic acid molecule or nucleic acid molecule conjugate of the present invention as described for the manufacture of a medicament, wherein the medicament is in a dosage form for subcutaneous administration.

[0219] Combination therapy In some embodiments, the inhibitors of the invention, such as nucleic acid molecules, nucleic acid molecule conjugates, or pharmaceutical compositions of the invention, are for use in combination treatment with another therapeutic agent, which may be, for example, a standard treatment for the disease or disorder described above.

[0220] By way of example, SCAMP3 inhibitors, such as nucleic acid molecules or nucleic acid molecule conjugates of the present invention, may be used in combination with other active agents, such as oligonucleotide-based antivirals - sequence-specific oligonucleotide-based antivirals that act either via antisense (including other LNA oligomers), siRNA (such as ARC520), aptamers, morpholinos, or any other antiviral nucleotide sequence-dependent mode of action.

[0221] As a further example, SCAMP3 inhibitors, such as nucleic acid molecules or nucleic acid molecule conjugates of the present invention, may be used in combination with other active agents, such as immunostimulatory antiviral compounds, such as interferons (e.g., pegylated interferon alpha), TLR7 agonists (e.g., GS-9620), or therapeutic vaccines.

[0222] As a further example, SCAMP3 inhibitors, such as nucleic acid molecules or nucleic acid molecule conjugates of the present invention, can be used in combination with other active agents, such as small molecules with antiviral activity, such as nucleoside / nucleotide inhibitors (e.g., entecavir or tenofovir disoproxil fumarate), inclusion inhibitors, or entry inhibitors (e.g., Myrcludex B).

[0223] In certain embodiments, the additional therapeutic agent may be an HBV agent, a hepatitis C virus (HCV) agent, a chemotherapeutic agent, an antibiotic, an analgesic, a nonsteroidal anti-inflammatory (NSAID) agent, an antifungal agent, an antiparasitic agent, an antinausea agent, an antidiarrheal agent, or an immunosuppressant.

[0224] In particular, in related embodiments, the additional HBV agent may be interferon alpha-2b, interferon alpha-2a, and interferon alfacon-1 (pegylated and non-pegylated), ribavirin; an HBV RNA replication inhibitor; a second antisense oligomer; an HBV therapeutic vaccine; an HBV prophylactic vaccine; lamivudine (3TC); entecavir (ETV); tenofovir diisoproxil fumarate (TDF); telbivudine (LdT); adefovir; or HBV antibody therapy (monoclonal or polyclonal).

[0225] In certain other related embodiments, the additional HCV agent may be interferon alpha-2b, interferon alpha-2a, and interferon alphacon-1 (pegylated and non-pegylated); ribavirin; Pegasys; HCV RNA replication inhibitors (e.g., ViroPharma VP50406 series); HCV antisense agents; HCV therapeutic vaccines; HCV protease inhibitors; HCV helicase inhibitors; or HCV monoclonal or polyclonal antibody therapy.

[0226] Purpose

[0227] The nucleic acid molecules of the present invention can be utilized, for example, as research reagents for diagnostics, therapeutics, and prophylaxis. In research, such nucleic acid molecules can be used to specifically regulate the synthesis of SCAMP3 protein in cells (e.g., in vitro cell cultures) and experimental animals, thereby facilitating functional analysis of the target or 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 modulators of the gene or mRNA that produces the protein.

[0228] When the nucleic acid molecules of the invention are used for research or diagnostic purposes, the target nucleic acid can be cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0229] The present invention also encompasses an in vivo or in vitro method for regulating SCAMP3 expression in a target cell expressing SCAMP3, which comprises administering to the cell an effective amount of a nucleic acid molecule, conjugated compound or pharmaceutical composition of the present invention.

[0230] In some embodiments, the target cells are mammalian cells, particularly human cells. The target cells may be in vitro cell cultures or in vivo cells that form part of mammalian tissue. In a preferred embodiment, the target cells are present in the liver. The target cells may be hepatocytes.

[0231] One aspect of the present invention relates to a SCAMP3 inhibitor, such as a nucleic acid molecule, a conjugated compound or a pharmaceutical composition of the present invention for use as a medicament.

[0232] In one embodiment of the present invention, SCAMP3 inhibitors, such as nucleic acid molecules, conjugated compounds, or pharmaceutical compositions of the present invention, can reduce cccDNA levels in infected cells, thereby inhibiting HBV infection. In particular, the nucleic acid molecules can affect one or more of the following parameters: i) reducing cccDNA, and / or ii) reducing pgRNA, and / or iii) reducing HBV DNA, and / or iv) reducing HBV viral antigens in infected cells.

[0233] For example, a nucleic acid molecule that inhibits HBV infection may (i) reduce cccDNA levels in infected cells by at least 40%, for example, 50% or 60%, compared to a control, or (ii) reduce pgRNA levels by at least 40%, for example, 50% or 60%, compared to a subject. The control may be untreated cells or animals, or cells or animals treated with an appropriate negative control.

[0234] Inhibition of HBV infection can be measured in vitro using HBV-infected primary human hepatocytes or in vivo using the humanized hepatocyte PXB mouse model (available from PhoenixBio; see also Kakuni et al. (2014) Int. J. Mol. Sci. 15:58-74). Inhibition of HBsAg and / or HBeAg secretion can be measured using, for example, a CLIA ELISA kit (Autobio) according to the manufacturer's instructions. The reduction of intracellular cccDNA or HBV mRNA and pgRNA can be measured by ELISA using a ELISA kit (Diagnostic). The reduction of intracellular cccDNA or HBV mRNA and pgRNA can be measured by qPCR, for example, as described in the Materials and Methods section. Another method for evaluating whether a test compound inhibits HBV infection is to measure the secretion of HBV DNA by qPCR, for example, as described in WO 2015 / 173208, or using Northern blot, in situ hybridization, or immunofluorescence.

[0235] By reducing SCAMP3 levels, SCAMP3 inhibitors such as 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, by destabilizing and reducing cccDNA, the nucleic acid molecules, conjugate compounds, or pharmaceutical compositions of the present invention more efficiently inhibit the onset of chronic HBV infection or more efficiently treat chronic HBV infection than compounds that only reduce HBsAg secretion.

[0236] Thus, one aspect of the present invention relates to the use of a SCAMP3 inhibitor, such as a nucleic acid molecule, conjugated compound or pharmaceutical composition of the present invention, to reduce cccDNA and / or pgRNA in an HBV-infected individual.

[0237] A further aspect of the present invention relates to the use of a SCAMP3 inhibitor, such as a nucleic acid molecule, a conjugated compound or a pharmaceutical composition of the present invention, to inhibit the development of or treat chronic HBV infection.

[0238] A further aspect of the present invention relates to the use of SCAMP3 inhibitors, such as the nucleic acid molecules, conjugated compounds or pharmaceutical compositions of the present invention, to reduce infectivity in HBV-infected individuals. In a specific aspect of the present invention, the nucleic acid molecules, conjugated compounds or pharmaceutical compositions of the present invention inhibit the development of chronic HBV infection.

[0239] The subject to be treated with a SCAMP3 inhibitor, such as a nucleic acid molecule, conjugated compound or pharmaceutical composition of the present invention (or receive a nucleic acid, conjugated compound or pharmaceutical composition of the present invention prophylactically) is preferably a human, more preferably an HBsAg-positive and / or HBeAg-positive human patient, and even more preferably an HBsAg-positive and HBeAg-positive human patient.

[0240] Thus, the present invention relates to a method for treating HBV infection, comprising administering an effective amount of a SCAMP3 inhibitor, such as a nucleic acid molecule, conjugated compound or pharmaceutical composition of the present invention.

[0241] The present invention further relates to methods for preventing cirrhosis and hepatocellular carcinoma resulting from chronic HBV infection.

[0242] The present invention also provides the use of a nucleic acid molecule, conjugate compound or pharmaceutical composition of the present invention for the manufacture of a medicament, particularly a medicament for use in treating HBV infection or chronic HBV infection, or reducing the infectivity of an HBV-infected individual. In a preferred embodiment, the medicament is prepared in a dosage form for subcutaneous administration.

[0243] The present invention also provides the use of a SCAMP3 inhibitor, such as a nucleic acid molecule or conjugated compound, in the manufacture of a medicament, wherein the medicament is in a dosage form for intravenous administration, and the pharmaceutical composition of the present invention.

[0244] SCAMP3 inhibitors, such as the nucleic acid molecules, conjugates, or pharmaceutical compositions of the present invention, can be used in combination therapy. For example, the nucleic acid molecules, conjugates, or pharmaceutical compositions of the present invention can be used in combination therapy with other anti-HBV agents, such as 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 inhibitors, for the treatment and / or prevention of HBV. RNA replication inhibitors, HBsAg secretion inhibitors, HBV capsid inhibitors, antisense oligomers (e.g., as described in WO 2012 / 145697, WO 2014 / 179629, and WO 2017 / 216390), siRNA (e.g., as described in WO 2005 / 014806, WO 2012 / 024170, WO 2012 / 2055362), No. WO 2013 / 003520, WO 2013 / 159109, WO 2017 / 027350, and WO 2017 / 015175), an HBV therapeutic vaccine, an HBV prophylactic vaccine, an HBV antibody therapy (monoclonal or polyclonal), or a TLR2, 3, 7, 8, or 9 agonist.

[0245] Embodiments of the present invention The following embodiments of the invention may be used in combination with any other embodiment described herein. The definitions and explanations provided above, particularly in the "Summary of the Invention", "Definitions" and "Detailed Description of the Invention" sections, apply mutatis mutandis below.

[0246] 1. SCAMP3 (secretory carrier membrane protein 3) inhibitors for use in the treatment and / or prevention of Hepatitis B virus (HBV) infection.

[0247] 2. The SCAMP3 inhibitor for use according to embodiment 1, administered in an effective amount.

[0248] 3. The SCAMP3 inhibitor for use according to embodiment 1 or 2, wherein the HBV infection is a chronic infection.

[0249] 4. A SCAMP3 inhibitor for use according to any one of embodiments 1 to 3, which is capable of reducing cccDNA and / or pgRNA in infected cells.

[0250] 5. A SCAMP3 inhibitor for use according to any one of embodiments 1 to 4, which prevents or reduces the association of SCAMP3 with cccDNA.

[0251] 6. The SCAMP3 inhibitor for use according to embodiment 5, wherein the inhibitor is a small molecule that specifically binds to the SCAMP3 protein, and the inhibitor prevents or reduces the association of the SCAMP3 protein with cccDNA.

[0252] 7. The SCAMP3 inhibitor for use according to embodiment 6, wherein the SCAMP3 protein is encoded by SEQ ID NO: 3 or 4.

[0253] 8. A SCAMP3 inhibitor for use according to any one of embodiments 1 to 7, wherein the inhibitor is a nucleic acid molecule of 12 to 60 nucleotides in length comprising or consisting of a contiguous nucleotide sequence of at least 12 nucleotides in length that is at least 90% complementary to a mammalian SCAMP3 target nucleic acid.

[0254] 9. The SCAMP3 inhibitor for use according to embodiment 8, which is capable of reducing the level of a SCAMP3 target nucleic acid in a mammal.

[0255] 10. The SCAMP3 inhibitor for use according to embodiment 8 or 9, wherein the mammalian SCAMP3 target nucleic acid is RNA.

[0256] 11. The SCAMP3 inhibitor for use according to embodiment 10, wherein the RNA is pre-mRNA.

[0257] 12. The SCAMP3 inhibitor for use according to any one of embodiments 8 to 11, wherein the nucleic acid molecule is selected from the group consisting of antisense oligonucleotides, siRNA and shRNA.

[0258] 13. The SCAMP3 inhibitor for use according to embodiment 12, wherein the nucleic acid molecule is a single-stranded antisense oligonucleotide or a double-stranded siRNA.

[0259] 14. A SCAMP3 inhibitor for use according to any one of embodiments 8 to 13, wherein the mammalian SCAMP3 target nucleic acid is selected from the group consisting of SEQ ID NOs: 1, 3 and 4.

[0260] 15. A SCAMP3 inhibitor for use according to any one of embodiments 8 to 13, wherein the contiguous 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.

[0261] 16. The SCAM for use according to any one of embodiments 8 to 13, wherein the contiguous nucleotide sequence of the nucleic acid molecule is completely complementary to the target nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 2. P3 inhibitors.

[0262] 17. A SCAMP3 inhibitor for use according to any one of embodiments 1 to 16, wherein cccDNA in HBV-infected cells is reduced by at least 50%, such as 60%, when compared to a control.

[0263] 18. A SCAMP3 inhibitor for use according to any one of embodiments 1 to 16, wherein pgRNA in HBV-infected cells is reduced by at least 50%, for example 60%, compared to a control.

[0264] 19. A SCAMP3 inhibitor for use according to any one of embodiments 8 to 18, in which mammalian SCAMP3 target nucleic acids are reduced by at least 50%, for example 60%, compared to a control.

[0265] 20. A nucleic acid molecule of 12 to 60 nucleotides in length comprising or consisting of a contiguous nucleotide sequence of 12 to 30 nucleotides in length, wherein the contiguous nucleotide sequence is at least 90% complementary, such as 95%, for example 98%, for example fully complementary, to a mammalian SCAMP3 target nucleic acid.

[0266] 21. The nucleic acid molecule of embodiment 20, wherein the nucleic acid molecule is chemically produced.

[0267] 22. The nucleic acid molecule of embodiment 20 or 21, wherein the mammalian SCAMP3 target nucleic acid is selected from the group consisting of SEQ ID NOs: 1, 3 and 4.

[0268] 23. The nucleic acid molecule of embodiment 20 or 21, wherein the contiguous nucleotide sequence is at least 98% complementary to the target nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 2.

[0269] 24. The nucleic acid molecule of embodiment 20 or 21, wherein the contiguous nucleotide sequence is perfectly complementary to the target nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 2.

[0270] 25. The nucleic acid molecule of any one of embodiments 20 to 23, which is 12 to 30 nucleotides in length.

[0271] 26. The nucleic acid molecule according to any one of embodiments 20 to 25, which is an RNAi molecule, such as a double-stranded siRNA or shRNA.

[0272] 27. The nucleic acid molecule of any one of embodiments 20 to 25, which is a single-stranded antisense oligonucleotide.

[0273] 28. The nucleic acid molecule of any one of embodiments 20 to 27, wherein the contiguous nucleotide sequence is perfectly complementary to a target nucleic acid sequence selected from Table 4.

[0274] 29. The nucleic acid molecule according to any one of embodiments 20 to 28, which is capable of hybridizing to the target nucleic acids of SEQ ID NO: 1 and SEQ ID NO: 2 with a ΔG° of less than −15 kcal.

[0275] 30. The nucleic acid molecule according to any one of embodiments 20 to 29, wherein the contiguous nucleotide sequence comprises or consists of at least 14 contiguous nucleotides, in particular 15, 16, 17, 18, 19, 20, 21 or 22 contiguous nucleotides.

[0276] 31. The contiguous nucleotide sequence contains or consists of 14 to 22 nucleotides. The nucleic acid molecule according to any one of embodiments 20 to 29,

[0277] 32. The nucleic acid molecule of embodiment 31, wherein the contiguous nucleotide sequence comprises or consists of 16 to 20 nucleotides.

[0278] 33. The nucleic acid molecule according to any one of embodiments 20 to 32, comprising or consisting of a length of 14 to 25 nucleotides.

[0279] 34. The nucleic acid molecule according to embodiment 33, comprising or consisting of at least one oligonucleotide strand of 16 to 22 nucleotides in length.

[0280] 35. The nucleic acid molecule of any one of embodiments 19 to 34, wherein the contiguous nucleotide sequence is perfectly complementary to a target sequence selected from the group consisting of SEQ ID NOs: 6, 7, 8 and 9.

[0281] 36. A nucleic acid molecule according to any one of embodiments 20 to 35, wherein the contiguous nucleotide sequence has 0 to 3 mismatches compared to the mammalian SCAMP3 target nucleic acid to which it is complementary.

[0282] 37. The nucleic acid molecule of embodiment 36, wherein the contiguous nucleotide sequence has one mismatch compared to the mammalian SCAMP3 target nucleic acid.

[0283] 38. The nucleic acid molecule of embodiment 36, wherein the contiguous nucleotide sequence has two mismatches compared to the mammalian SCAMP3 target nucleic acid.

[0284] 39. The nucleic acid molecule of embodiment 36, wherein the contiguous nucleotide sequence is perfectly complementary to a mammalian SCAMP3 target nucleic acid.

[0285] 40. The nucleic acid molecule of any one of embodiments 20 to 39, comprising one or more modified nucleosides.

[0286] 41. The nucleic acid molecule of embodiment 40, wherein one or more modified nucleosides are high-affinity modified nucleosides.

[0287] 42. The nucleic acid molecule of embodiment 40 or 41, wherein one or more modified nucleosides are 2' sugar-modified nucleosides.

[0288] 43. The nucleic acid molecule of 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.

[0289] 44. The nucleic acid molecule of any one of embodiments 40 to 43, wherein one or more modified nucleosides is an LNA nucleoside.

[0290] 45. The nucleic acid molecule of embodiment 44, wherein the modified LNA nucleoside is selected from the group consisting of oxy-LNA, amino-LNA, thio-LNA, cET and ENA.

[0291] 46. ​​The nucleic acid molecule of embodiment 44 or 45, wherein the modified LNA nucleoside is oxy-LNA having the following 2'-4' bridge: -O-CH2-.

[0292] 47. The nucleic acid molecule of embodiment 46, wherein the oxy-LNA is beta-D-oxy-LNA.

[0293] 48. The nucleic acid molecule of embodiment 44 or 45, wherein the modified LNA nucleoside is cET having the following 2'-4' bridge: -O-CH(CH3)-.

[0294] 49. The nucleic acid molecule of embodiment 48, wherein the cET is (S)cET, i.e. 6'(S)methyl-beta-D-oxy-LNA.

[0295] 50. The nucleic acid molecule of embodiment 44 or 45, wherein the LNA is an ENA having the following 2'-4' bridge: -O-CH2-CH2-.

[0296] 51. The nucleic acid molecule according to any one of embodiments 20 to 50, comprising at least one modified internucleoside linkage.

[0297] 52. The nucleic acid molecule of embodiment 51, wherein at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[0298] 53. The nucleic acid molecule according to any one of embodiments 20 to 52, which is an antisense oligonucleotide capable of recruiting RNase H.

[0299] 54. The nucleic acid molecule of embodiment 53, wherein the antisense oligonucleotide or the consecutive nucleotide sequence is a gapmer.

[0300] 55. The nucleic acid molecule of embodiment 54, wherein the antisense oligonucleotide or its consecutive nucleotide sequence comprises or consists of a gapmer of the formula 5'FG-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 between 6 and 18 nucleosides capable of recruiting RNase H.

[0301] 56. The nucleic acid molecule of embodiment 55, wherein the one to four 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.

[0302] 57. The nucleic acid molecule of embodiment 55 or 56, wherein one or more of the 1 to 4 2' sugar-modified nucleosides in regions F and F' is an LNA nucleoside.

[0303] 58. The nucleic acid molecule of embodiment 57, wherein all 2' sugar-modified nucleosides in regions F and F' are LNA nucleosides.

[0304] 59. The nucleic acid molecule of any one of embodiments 56 to 58, wherein the LNA nucleoside is selected from 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.

[0305] 60. The nucleic acid of any one of embodiments 56 to 59, wherein regions F and F' consist of identical LNA nucleosides. A nucleic acid molecule described in any one of claims 1 to 4.

[0306] 61. The nucleic acid molecule of any one of embodiments 56-60, wherein all 2' sugar-modified nucleosides in regions F and F' are oxy-LNA nucleosides.

[0307] 62. The nucleic acid molecule of any one of embodiments 55-61, wherein the nucleosides in region G are DNA nucleosides.

[0308] 63. The nucleic acid molecule of embodiment 62, wherein region G consists of at least 75% DNA nucleosides.

[0309] 64. The nucleic acid molecule of embodiment 63, wherein all nucleosides in region G are DNA nucleosides.

[0310] 65. A conjugate compound comprising a nucleic acid molecule according to any one of embodiments 20 to 64 and at least one conjugate moiety covalently attached to said nucleic acid molecule.

[0311] 66. The conjugate compound according to embodiment 65, wherein the nucleic acid molecule is a double-stranded siRNA and the conjugate moiety is covalently linked to the sense strand of the siRNA.

[0312] 67. The conjugated compound according to embodiment 65 or 66, wherein the conjugate moiety is selected from a carbohydrate, a cell surface receptor ligand, a drug substance, a hormone, a lipophilic substance, a polymer, a protein, a peptide, a toxin, a vitamin, a viral protein or a combination thereof.

[0313] 68. The conjugated compound according to any one of embodiments 65-67, wherein the conjugated moiety is capable of binding to an asialoglycoprotein receptor.

[0314] 69. The conjugate compound of 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, Nn-butanoyl-galactosamine, and N-isobutanoylgalactosamine.

[0315] 70. The conjugated compound according to embodiment 69, wherein the asialoglycoprotein receptor targeting moiety is N-acetylgalactosamine (GalNAc).

[0316] 71. The conjugated compound of embodiment 69 or 70, wherein the conjugated moiety is monovalent, bivalent, trivalent, or tetravalent with respect to the asialoglycoprotein receptor targeting moiety.

[0317] 72. The conjugate compound of embodiment 71, wherein the conjugate moiety consists of two to four terminal GalNAc moieties and a spacer linking each GalNAc moiety to a brancher molecule capable of conjugating to an antisense compound.

[0318] 73. The conjugate compound according to embodiment 72, wherein the spacer is a PEG spacer.

[0319] 74. The conjugate compound according to any one of embodiments 68-73, wherein the conjugate moiety is a trivalent N-acetylgalactosamine (GalNAc) moiety.

[0320] 75. The conjugate compound according to any one of embodiments 68-74, wherein the conjugate moiety is selected from one of the trivalent GalNAc moieties in Figure 1.

[0321] 76. The conjugate compound of embodiment 75, wherein the conjugate moiety is a trivalent GalNAc moiety of Figure 1D-1 or Figure 1D-2, or a mixture of both.

[0322] 77. The conjugate compound according to any of embodiments 65-76, comprising a linker disposed between the nucleic acid molecule and the conjugate moiety.

[0323] 78. The conjugate compound according to embodiment 77, wherein the linker is a physiologically labile linker.

[0324] 79. The conjugated compound according to embodiment 78, wherein the physiologically labile linker is a nuclease-sensitive linker.

[0325] 80. The conjugate compound according to embodiment 78 or 79, wherein the physiologically labile linker consists of 2 to 5 consecutive phosphodiester bonds.

[0326] 81. A conjugated compound according to any one of embodiments 68 to 80, which exhibits improved cellular distribution between the liver and kidney or improved cellular uptake of the conjugated compound into the liver compared to unconjugated nucleic acid.

[0327] 82. A pharmaceutical composition comprising a nucleic acid molecule according to any one of embodiments 20 to 64, a conjugated compound according to any one of embodiments 65 to 81, or an acceptable salt thereof, and a pharmaceutically acceptable diluent, carrier, salt and / or adjuvant.

[0328] 83. A method for identifying a compound that prevents, ameliorates, and / or inhibits Hepatitis B Virus (HBV) infection, comprising: a. Test compound i. a SCAMP3 polypeptide; or ii. contacting with cells expressing SCAMP3; b. measuring the expression and / or activity of SCAMP3 in the presence and absence of the test compound; and c. A method comprising identifying a compound that reduces the expression and / or activity of SCAMP3 and reduces cccDNA.

[0329] 84. An in vivo or in vitro method for modulating SCAMP3 expression in a target cell expressing SCAMP3, comprising administering to said cell an effective amount of a nucleic acid molecule according to any one of embodiments 20 to 64, a conjugated compound according to any one of embodiments 65 to 81 or a pharmaceutical composition according to embodiment 82.

[0330] 85. The method of embodiment 84, wherein SCAMP3 expression is reduced in the target cells by at least 50%, or at least 60%, compared to levels without any treatment or treatment with a control.

[0331] 86. The method of embodiment 84, wherein the target cells are 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 cells compared to the level without any treatment or treated with a control.

[0332] 87. A method for treating or preventing a disease such as HBV infection, comprising administering a therapeutically effective amount or a prophylactic Administering a prophylactically effective amount of the nucleic acid molecule of any one of embodiments 20 to 64, the conjugate compound of any one of embodiments 65 to 81, or the pharmaceutical composition of embodiment 82 to a subject suffering from or susceptible to a disease.

[0333] 88. A nucleic acid molecule according to any one of embodiments 20 to 64, or a conjugated compound according to any one of embodiments 65 to 81, or a pharmaceutical composition according to embodiment 82, for use as a medicament for treating or preventing a disease, such as an HBV infection, in a subject.

[0334] 89. Use of a nucleic acid molecule according to any one of embodiments 20 to 64, or a conjugate compound according to any one of embodiments 65 to 81, for the preparation of a medicament for treating or preventing a disease, such as an HBV infection, in a subject.

[0335] 90. The method, nucleic acid molecule, conjugated compound or use according to any one of embodiments 87 to 89, wherein the subject is a mammal.

[0336] 91. The method, nucleic acid molecule, conjugated compound or use according to embodiment 90, wherein the mammal is a human.

[0337] 92. The conjugate compound of embodiment 75, wherein the conjugate moiety is a trivalent GalNAc moiety of Figure 1B-1 or Figure 1B-2, or a mixture of both.

[0338] The invention will now be illustrated by the following examples, which have no limiting character. [Example]

[0339] material and method siRNA sequences and compounds

[0340] [Table 6A]

[0341] The pool of siRNA (ON-TARGETplus SMARTpool siRNA Cat. No. LU-013442-00-0005, Dharmacon) contains four individual siRNA molecules targeting the sequences listed in the table above.

[0342] [Table 6B]

[0343] Oligonucleotide synthesis Oligonucleotide synthesis is generally known in the art. Below are applicable protocols. The oligonucleotides of the present invention may be produced by methods that differ slightly in terms of the equipment, supports, and concentrations used.

[0344] Oligonucleotides are synthesized on a uridine universal support using the phosphoramidite approach in Oligomaker 48 on a 1 μmol scale. At the end of synthesis, oligonucleotides are cleaved from the solid support using aqueous ammonia at 60 °C for 5–16 h. Oligonucleotides are purified by reverse-phase HPLC (RP-HPLC) or solid-phase extraction, characterized by UPLC, and molecular weights are further confirmed by ESI-MS.

[0345] Oligonucleotide extension: 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 carried out using 0.1 M of the 5'-O-DMT-protected amidite in acetonitrile and a solution of DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as the activator. In the final cycle, a phosphoramidite bearing the desired modification, such as a C6 linker for attaching a conjugate group, or such a conjugate group, can be used. Thiolation to introduce a phosphorothioate bond is carried out using hydrogenated xanthan gum (0.01 M in acetonitrile / pyridine 9:1). Phosphodiester bonds can be introduced using 0.02 M iodine in THF / pyridine / water 7:2:1. The remaining reagents are those commonly used in oligonucleotide synthesis.

[0346] For post-solid-phase synthesis conjugation, commercially available C6 amino linker phosphoramidites can be used in the final 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 functional group activation using standard synthetic methods.

[0347] Purification by RP-HPLC: The crude compound is purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10 μm 150 × 10 mm column. 0.1 M ammonium acetate pH 8 and acetonitrile are used as buffers at a flow rate of 5 mL / min. The collected fractions are lyophilized to obtain the purified compound, typically as a white solid.

[0348] Abbreviation: DCI: 4,5-dicyanoimidazole DCM: dichloromethane DMF: dimethylformamide DMT: 4,4'-dimethoxytrityl THF: tetrahydrofuran Bz: benzoyl Ibu: Isobutyryl RP-HPLC: reversed-phase high-performance liquid chromatography

[0349] T m Assay: Dilute the oligonucleotide and RNA target (phosphate-linked, PO) duplex to 3 mM in 500 mL of RNase-free water and 500 mL of 2x T m Mix with 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 ) is measured on a Lambda 40 UV / VIS spectrophotometer equipped with a Peltier temperature programmer PTP6 using PE Templab software (Perkin Elmer). The temperature is increased from 20°C to 95°C and then decreased to 25°C, and the absorbance is recorded at 260 nm. The first derivative and both the melting and annealing maxima are used to calculate the duplex T m Evaluate.

[0350] Clonal growth medium (dHCGM). dHCGM is DMEM medium containing 100 U / ml penicillin, 100 μg / ml streptomycin, 20 mM Hepes, 44 mM NaCO, 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 a humidified atmosphere containing 5% CO in an incubator at 37°C. Culture medium was changed 24 h after seeding and every 2 days until harvest.

[0351] ASO sequences and compounds

[0352] [Table 7]

[0353] HBV infected PHH cells Fresh primary human hepatocytes (PHH) were provided by PhoenixBio, Higashihiroshima, Japan, at 70,000 cells / well in a 96-well plate format (PXB cells are also described in Ishida et al., 2015 Am J Pathol. 185(5):1275-85).

[0354] Upon arrival, PHH cells were infected with HepG2 2.2.15-derived HBV (batch Z12) at an MOI of 2GE by incubating PHH cells with HBV in 4% (v / v) PEG in PHH medium for 16 hours. Cells were then washed three times with PBS and resuspended in 10% (v / v) heat-inactivated fetal bovine serum (GIBCO, catalog no. 10082). ), 2% (v / v) DMSO, 1% (v / v) penicillin / streptomycin (GIBCO, Cat. No. 15140-148), 20 mM HEPES (GIBCO, Cat. No. 15630-080), 44 mM NaHCO3 (Wako, Cat. No. 195-14515), 15 μg / ml L-proline (MP-Biomedicals, Cat. No. 0219472825), 0.25 μg / ml insulin (Sigma, Cat. No. I1882), 50 nM dexamethasone (Sigma, Cat. No. D8893), 5 ng / ml EGF (Sigma, Cat. No. E9644), and 0.1 mM L-ascorbic acid 2-phosphate (Wako, Cat. No. 013-12061), 5% The cells were cultured in a humidified atmosphere containing 5% CO2 in an incubator at 37°C. The culture medium was changed 24 hours after seeding and every 2 days until harvest.

[0355] siRNA transfection Four days after infection, cells were transfected in triplicate with SCAMP3 siRNA pools. No drug control (NDC), negative control siRNA, and HBx siRNA were included as controls (see Table 6B).

[0356] Transfection mixes were prepared per well with 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) containing 2 μl of either negative control siRNA (stock concentration 1 μM), SCAMP3 siRNA pool (stock concentration 1 μM), HBx control siRNA (stock concentration 0.1 μM), or HO (NDC). The transfection mixes were mixed and incubated at room temperature for 5 minutes before transfection. Prior to transfection, media was removed from PHH cells and replaced with 100 μl / well of William's E Medium + GlutaMAX (Gibco, No. 32551) supplemented with P / S-free HepaRG supplement (Biopredic International, No. ADD711C). 20 μl of transfection mix was added to each well to achieve a final concentration of 1 μl of negative control siRNA or SCAMP3 siRNA pool. The siRNA pool was 16 nM, or the HBx control siRNA was 1.92 nM. The plates were gently rocked and then placed in an incubator. After 6 hours, the medium was replaced with PHH medium. The siRNA treatment was repeated on day 6 post-infection as described above. Supernatants were collected on day 8 post-infection and stored at -20°C. HBsAg and HBeAg could be measured from the supernatants if desired.

[0357] LNA Processing Two LNA master mix plates were prepared from 500 μM stock LNAs. For LNA treatments at a final concentration of 25 μM, 200 μL of 500 μM stock LNA was prepared in the first master mix plate. For LNA treatments at a final concentration of 5 μM, a second master mix plate containing 100 μM SCAMP3 LNA was prepared by mixing 40 μL of each 500 μM SCAMP3 LNA with 160 μL of PBS.

[0358] On day 4 post-infection, cells were treated with SCAMP3 LNA (see Table 7) at a final concentration of 25 μM in either duplicate or triplicate, or with PBS without drug control (NDC). Prior to LNA treatment, old medium was removed from cells and replaced with 114 μl / well of fresh PHH medium. Per well, 6 μL of each SCAMP3 LNA was added to 114 μL of PHH medium at either 500 μM or PBS as NDC. The same treatment was repeated three times on days 4, 11, and 18 post-infection. Cell culture medium was changed every 3 days at days 7, 14, and 2 post-infection. On day 1, the medium was replaced with fresh medium. For cccDNA quantification, infected cells were treated with entecavir (ETV) at a final concentration of 10 nM from day 7 to day 21 postinfection. Fresh ETV treatment was repeated five times on days 7, 11, 14, 18, and 21 postinfection. This ETV treatment inhibited the synthesis of new viral DNA intermediates and allowed specific detection of HBV cccDNA sequences.

[0359] Measurement of HBV antigen expression HBV antigen expression and secretion can be measured in the collected supernatants, if necessary. HBV proliferation parameters, HBsAg and HBeAg levels, were measured using CLIA ELISA kits (Autobio Diagnostic, CL0310-2, CL0312-2) according to the manufacturer's protocol. Briefly, 25 μL of supernatant per well was transferred to each antibody-coated microtiter plate, and 25 μL of enzyme conjugate reagent was added. The plate was incubated on a shaker at room temperature for 60 minutes, after which the wells were washed five times with wash buffer using an automated washer. 25 μL of substrates A and B were added to each well. The plate was incubated on a shaker at room temperature for 10 minutes, after which luminescence was measured using an EnVision® luminescence reader (Perkin Elmer).

[0360] Cell viability measurement Cell viability was measured in supernatant-free cells using Cell Counting Kit-8 (CCK8, product number 96992, Sigma-Aldrich). For measurements, CCK8 reagent was diluted 1:10 in normal culture medium and 100 μl / well was added to the cells. After 1 hour of incubation in an incubator, 80 μl of the supernatant was transferred to a clear, flat-bottom 96-well plate, and the absorbance was read at 450 nm using a microplate reader (Tecan). Absorbance values ​​were normalized to NDC, which was set to 100% to calculate relative cell viability.

[0361] Cell viability measurements were used to confirm that the reduction in viral parameters was not the cause of cell death; values ​​closer to 100% indicate lower toxicity. LNA treatments showing cell viability values ​​below 20% relative to the NDC were excluded from further analysis.

[0362] Real-time PCR to measure SCAMP3 mRNA expression and quantification of viral parameters pgRNA, cccDNA and HBV DNA After measuring cell viability, cells were washed once with PBS. For siRNA treatment, cells were lysed with 50 μl / well lysis solution from the TaqMan® Gene Expression Cells-to-CT™ Kit (Thermo Fisher Scientific, no. AM1729) and stored at -80°C. For LNA-treated cells, total RNA was extracted using the MagNA Pure robot and the MagNA Pure 96 Cellular RNA Large Volume Kit (Roche, no. 05467535001) according to the manufacturer's protocol. For quantification of SCAMP3 RNA and viral pgRNA levels and normalization controls, including GUS B, the TaqMan® RNA-to-Ct™ 1-Step Kit (Life Technologies, no. 4392656) was used. For each reaction, 2 or 4 μl of cell lysate, 0.5 μl of 20× SCAMP3 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 are 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 the quantification was based on the DNA present. A minus RT control is included to assess potential amplification.

[0363] Target mRNA expression levels, as well as viral pgRNA, were quantified by RT-qPCR using a QuantStudio 12K Flex (Applied Biosystems) with the following protocol: 48°C for 15 min, 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 60 s in technical duplicates.

[0364] SCAMP3 mRNA and pgRNA expression levels were analyzed using the comparative cycle threshold 2-ΔΔCt method, normalized to the reference gene GUS B and untransfected cells. Expression levels in siRNA-treated cells are shown as a percentage of the mean no-drug control sample (i.e., the lower the value, the greater the inhibition / reduction). In LNA-treated cells, expression levels are shown as the inhibitory effect compared to non-treated cells (NDC), which was set as 100%, and are expressed as a percentage of the mean + SD 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, a fraction of the siRNA-treated cell lysate was digested with T5 enzyme (10 U / 500 ng DNA; New England Biolabs, no. M0363L) to remove viral DNA intermediates, and only cccDNA molecules were quantified. T5 digestion was performed at 37°C for 30 min. To avoid qPCR interference in the assay, T5 digestion was not applied to LNA-treated cell lysates. To remove HBV DNA intermediates and quantify cccDNA levels in LNA-treated cells, cells were treated with entecavir (10 nM) for 3 weeks as described in the LNA treatment section.

[0365] 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 number AI1RW7N, FAM dye listed in the table below), 5 μl of TaqMan® Fast Advanced Master Mix (Applied Biosystems, number 4444557), and 2.5 μl of DEPC-treated water. Technical triplicates were performed for each sample.

[0366] [Table 7A]

[0367] For quantification of cccDNA in LNA-treated cells by qPCR, 10 ul of 2x FastSYBR™ Green Master Mix (Appli Prepare 16 uL / well of a master mix containing 10 ul of 2x FastSYBR™ Green Master Mix (Applied Biosystems, No. 4385614), 2 ul of cccDNA Primer Mix (1 uM each forward and reverse), and 4 ul of nuclease-free water per well. A master mix containing 10 ul per well of 2x FastSYBR™ Green Master Mix (Applied Biosystems, No. 4385614), 2 ul of mitochondrial genome primer mix (1 uM each forward and reverse), and 4 ul of nuclease-free water is also prepared for cccDNA standardization.

[0368] For quantification of intracellular HBV DNA and the normalization control, human hemoglobin beta (HBV), each reaction mixture contained 2 μl of undigested cell lysate, 0.5 μl of 20× HBV Taqman primer / probe (Life Technologies, no. Pa03453406_s1, FAM dye), 0.5 μl of 20× HBB Taqman primer / probe (Life Technologies, no. Hs00758889_s1, VIC dye), 5 μl of TaqMan® Fast Advanced Master Mix (Applied Biosystems, no. 4444557), and 2 μl of DEPC-treated water. Technical triplicates were performed for each sample.

[0369] qPCR was performed on a QuantStudio™ K12 flex using standard settings for a rapid heating block (95°C for 20 seconds, followed by 40 cycles of 95°C for 1 second and 60°C for 20 seconds).

[0370] Outliers were removed from the dataset by excluding values ​​with a difference of more than 0.9 from 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 calculated using a 2 -ddCT Expression levels were determined from Ct values ​​via the ≈ ≈ method and normalized to HBB or mitochondrial DNA as housekeeping genes. For siRNA-treated cells, expression levels are shown as % of the mean of drug-free samples (i.e., lower values ​​indicate greater inhibition / reduction). For LNA-treated cells, the inhibitory effect on cccDNA was expressed as a percentage of the mean + / - SD from three independent biological replicates compared to non-treated cells (NDC), which were set at 100%.

[0371] [Table 8]

[0372] Example 1: Measurement of reduction of SCAMP3 mRNA, intracellular HBV DNA, and cccDNA in HBV-infected PHH cells due to siRNA treatment In the following experiments, the effect of SCAMP3 knockdown on HBV parameters, HBV DNA and cccDNA, was examined.

[0373] HBV-infected PHH cells were treated with a pool of siRNA from Dharmacon (LU-013442-00-0005) as described in the Materials and Methods section “siRNA transfection.”

[0374] After 4 days of treatment, SCAMP3 mRNA, cccDNA, and intracellular HBV DNA were measured by qPCR as described in the Materials and Methods section “Real-time PCR to measure the expression of SCAMP3 mRNA and viral parameters pgRNA, cccDNA, and HBV DNA.”

[0375] The results are shown in Table 9 as a % of the mean of the no drug control samples (ie, the lower the value, the greater the inhibition / reduction).

[0376] Table 9: Effect on HBV parameters after knockdown of SCAMP3 by a pool of siRNAs. Values ​​are given as the mean of biological and technical triplicates.

[0377] [Table 9]

[0378] This indicates that the SCAMP3 siRNA pool can highly efficiently reduce SCAMP3 mRNA, cccDNA, and HBV DNA. The positive control reduced intracellular HBV DNA as expected, but had no effect on cccDNA when compared with the negative control.

[0379] Example 2: Measurement of reduction of SCAMP3 mRNA, intracellular HBV pgRNA and cccDNA in HBV-infected PHH cells due to LNA treatment In the following experiments, the effect of SCAMP3 knockdown on HBV parameters, HBV DNA and cccDNA, was examined.

[0380] HBV-infected PHH cells were treated with SCAMP3 naked LNA (see Table 7) as described in the Materials and Methods section "LNA Treatment."

[0381] After 21 days of treatment, SCAMP3 mRNA, cccDNA, and intracellular HBV pgRNA were measured by qPCR as described in the Materials and Methods section "Real-time PCR for measuring SCAMP3 mRNA expression and viral parameters pgRNA, cccDNA, and HBV DNA." Results are shown in Table 9 as inhibitory effects compared to untreated cells (NDC), which were set at 100%, and are expressed as percentages of the mean + SD from two independent biological replicates.

[0382] Table 10: Effect on HBV parameters after knockdown of SCAMP3 by naked LNA. Values ​​are given as the average of either two or three biological replicates. Data show the effect of LNA at a final concentration of 25 mM.

[0383] [Table 10]

[0384] This indicates that SCAMP3 LNA significantly reduces SCAMP3 mRNA expression and can very efficiently reduce the expression levels of both pgRNA and cccDNA.

Claims

1. SCAMP3 (secretory carrier membrane protein 3) inhibitors for use in the treatment of hepatitis B virus (HBV) infection.

2. The SCAMP3 inhibitor for use according to claim 1, wherein the HBV infection is a chronic infection.

3. 3. The SCAMP3 inhibitor for use according to claim 1 or 2, which is capable of reducing the amount of cccDNA (covalently closed circular DNA) in HBV-infected cells.

4. The SCAMP3 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 that is at least 95% complementary to a mammalian SCAMP3 target sequence, particularly a human SCAMP3 target sequence, and is capable of reducing expression of SCAMP3 mRNA in cells that express the SCAMP3 mRNA.

5. The SCAMP3 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 SCAMP3 inhibitor for use according to any one of claims 1 to 5, wherein the mammalian SCAMP3 target sequence is selected from the group consisting of SEQ ID NOs: 1, 3, and 4.

7. The SCAMP3 inhibitor for use according to any one of claims 4 to 6, wherein the contiguous nucleotide sequence is at least 98% complementary, e.g., completely complementary, to the target sequences of SEQ ID NO: 1 and SEQ ID NO:

2.

8. The SCAMP3 inhibitor for use according to any one of claims 3 to 7, wherein the amount of cccDNA in HBV-infected cells is reduced by at least 60%.

9. The SCAMP3 inhibitor for use according to any one of claims 4 to 7, wherein the amount of SCAMP3 mRNA is reduced by at least 60%.

10. A nucleic acid molecule of 12 to 30 nucleotides in length comprising a continuous nucleotide sequence of at least 12 nucleotides that is 90% complementary, for example, completely complementary, to a mammalian SCAMP3 target sequence, particularly a human SCAMP3 target sequence, wherein the nucleic acid molecule is capable of inhibiting the expression of SCAMP3.

11. 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, 3 and 4.

12. 12. The nucleic acid molecule according to claim 10 or 11, comprising a contiguous nucleotide sequence of 12 to 25, in particular 16 to 20, nucleotides in length.

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

15. The nucleic acid molecule according to any one of claims 10 to 14, comprising 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. The nucleic acid molecule of claim 15 or 16, wherein the one or more 2' sugar modified nucleosides are LNA nucleosides.

18. 18. The nucleic acid molecule of claim 10, wherein the contiguous nucleotide sequence comprises at least one phosphorothioate internucleoside linkage.

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

20. The nucleic acid molecule according to any one of claims 10 to 19, which 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', wherein regions F and F' independently comprise 1 to 4 2' sugar-modified nucleosides, and G is a region of between 6 and 18 nucleosides capable of recruiting RNase H, such as a region comprising between 6 and 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, such as a trivalent GalNAc moiety, e.g., a GalNAc moiety selected from one of the trivalent GalNAc moieties of Figure 1.

24. 24. The conjugate compound of claim 22 or 23, comprising a physiologically labile linker composed of 2 to 5 linked nucleosides comprising at least two consecutive phosphodiester bonds, wherein the physiologically labile linker is 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 pharmaceutically acceptable salt of the conjugate compound according to any one of claims 22 to 24.

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

27. An in vivo or in vitro method for inhibiting SCAMP3 expression in a target cell expressing SCAMP3, comprising administering to the 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 pharmaceutically acceptable salt according to claim 25, or a pharmaceutical composition according to claim 26. The method includes providing.

28. 26. A method for the treatment or prevention of a disease, comprising administering a therapeutically or prophylactically effective amount of the nucleic acid molecule of any one of claims 10 to 21, the conjugate compound of any one of claims 22 to 24, the pharmaceutically acceptable salt of claim 25, or the pharmaceutical composition of claim 26 to a subject suffering from or susceptible to said disease.

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

30. 27. 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 pharmaceutically acceptable salt according to claim 25, or a pharmaceutical composition according to claim 26, for use in medicine.

31. 27. The nucleic acid molecule of any one of claims 10 to 21, the conjugate compound of any one of claims 22 to 24, the pharmaceutically acceptable salt of claim 25, or the pharmaceutical composition of claim 26, for use in the treatment of hepatitis B virus (HBV) infection, such as chronic HBV infection.

32. 27. Use of a nucleic acid molecule according to any one of claims 10 to 21, a conjugated compound according to any one of claims 22 to 24, a pharmaceutically 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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