Oligonucleotides for modulating RTEL1 expression

Abstract

To provide a RTEL1 inhibitor for use in treatment of an HBV infection, in particular a chronic HBV infection.SOLUTION: The invention in particular relates to the use of RTEL1 inhibitors for destabilizing cccDNA, such as HBV cccDNA. The invention also relates to antisense oligonucleotides which are complementary to RTEL1 and capable of reducing a RTEL1 mRNA. Also comprised in the present invention is a pharmaceutical composition and its use in the treatment and / or prevention of a HBV infection.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to RTEL1 inhibitors, such as oligonucleotides (oligomers) complementary to RTEL1, which result in the modulation of RTEL1 expression or modulation of RTEL1 activity. The present invention particularly relates to the use of RTEL1 to target nucleic acid molecules for the treatment and / or prevention of Hepatitis B virus (HBV) infection, especially chronic HBV infection. The present invention particularly relates to the use of RTEL1 inhibitors to destabilize cccDNA, such as HBV cccDNA. 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 an important 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 analogues, such as lamivudine, adefovir, entecavir, tenofovir diisoproxil, and tenofovir alafenamide, that 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 that is stably maintained in the nucleus. This episomal form is called "covalently closed circular DNA" (cccDNA). cccDNA serves as the template for all HBV transcripts, including the viral replication intermediate, pregenomic RNA (pgRNA). The presence of several copies of cccDNA would be sufficient to reinitiate a late-stage HBV infection. Current treatments for HBV do not target cccDNA. Removal of cccDNA is required for the cure of chronic HBV infection (reviewed in Nassal, Gut. (December 2015) 64, no. 12, 1972-84, doi:10.1136 / gutjnl-2015-309809).

[0003] Regulator of telomere elongation helicase 1 (RTEL1) encodes a DNA helicase that functions in telomere stability, protection, and elongation and interacts with proteins in the shelterin complex, known to protect telomeres during DNA replication. Mutations in this gene are associated with dyskeratosis congenita and Hoyerall-Hreidarsson syndrome (see, e.g., review by Vannier et al. (2014) Trends Cell Biol. 24:416).

[0004] Located in the nucleus, RTEL1 functions as an ATP-dependent DNA helicase involved in telomere length regulation, DNA repair, and maintaining genomic stability. RTEL1 acts as an anti-recombinase to counter toxic recombination, restrict crossovers during meiosis, and regulate meiotic recombination and crossover homeostasis by physically dissociating strand invasion events, thereby promoting meiotic synthesis dependent strand annealing (SDSA) and non-crossover repair by resolution of D-loop recombination intermediates. Moreover, RTEL1 prevents telomere fragility by resolving T-loops and antagonizing telomeric G4-DNA structures, ensuring both telomere dynamics and stability.

[0005] RTEL1 has been identified in siRNA screens as a stabilizer of HPV episomes: (Edwards et al. (2013) PLoS One 8:e75406). siRNA targeting RTEL1 has also been used to identify interactors with RTEL1 in Hoyeraal-Hreidarsson syndrome (Schertzer et al. (2015) Nucleic Acid Res 43:1834). In addition, RTEL1 was identified as an HIV host-dependence factor from an siRNA screen against essential host proteins providing a target to inhibit HIV infection (WO 2007 / 094818).

[0006] To the best of our knowledge, RTEL1 has not been identified as a cccDNA-dependent factor for cccDNA stability and maintenance, nor have molecules that inhibit RTEL1 been suggested as cccDNA-destabilizing agents for the treatment of HBV infection.

[0007] Object of the invention The present invention shows that there is a correlation between inhibition of RTEL1 and reduction of cccDNA in HBV-infected cells, which is relevant for the treatment of HBV-infected individuals.The object of the present invention is to identify RTEL1 inhibitors that reduce cccDNA in HBV-infected cells.Such RTEL1 inhibitors can be used to treat HBV infection.

[0008] The present invention further identifies novel nucleic acid molecules capable of inhibiting the expression of RTEL1 in vitro and in vivo. Summary of the Invention

[0009] The present invention relates to nucleic acid targeted oligonucleotides capable of regulating the expression of RTEL1 and for treating or preventing diseases associated with the function of RTEL1.

[0010] Thus, in a first aspect, the present invention provides an RTEL1 inhibitor for the treatment and / or prevention of Hepatitis B virus (HBV) infection. In particular, RTEL1 inhibitors capable of reducing cccDNA and / or pregenomic RNA (pgRNA) are useful. Such inhibitors are advantageously selected from nucleic acid molecules of 12-60 nucleotides in length, which are capable of reducing RTEL1 mRNA, such as single-stranded antisense oligonucleotides, siRNAs, or shRNAs complementary to mammalian RTEL1.

[0011] In a further aspect, the invention relates to an oligonucleotide of 12 to 60 nucleotides, e.g. 12 to 30 nucleotides, comprising a contiguous nucleotide sequence of at least 10 nucleotides, in particular 16 to 20 nucleotides, that is complementary to a mammalian RTEL1. Such an oligonucleotide is capable of inhibiting expression of RTEL1. The oligonucleotide may be a single-stranded antisense oligonucleotide or an shRNA nucleic acid molecule.

[0012] The antisense oligonucleotide may have a gapmer binding design.Preferably, the antisense oligonucleotide can inhibit the expression of RTEL1 by cleaving the target nucleic acid.Cleavage is preferably achieved via nuclease recruitment.

[0013] In a further aspect, the present invention provides a pharmaceutical composition comprising an antisense oligonucleotide of the invention and a pharmaceutical excipient.

[0014] In a further aspect, the present invention provides an in vivo or in vitro method for modulating RTEL1 expression in a target cell expressing RTEL1 by administering to the cell an effective amount of an antisense oligonucleotide or composition of the present invention.

[0015] 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 RTEL1, comprising administering to a subject suffering from or susceptible to the disease, disorder, or dysfunction a therapeutically or prophylactically effective amount of an antisense oligonucleotide of the present invention.

[0016] Further aspects of the invention are conjugates of the nucleic acid molecules of the invention and pharmaceutical compositions comprising the molecules of the invention, in particular conjugates that are liver targeted, such as GalNAc clusters. [Brief description of the drawings]

[0017] [Figure 1]Illustrated are exemplary antisense oligonucleotide conjugates, where the oligonucleotides are represented as either wavy lines (A-D) or "oligonucleotides" (E-H) or T2 (I), and the asialoglycoprotein receptor-targeted conjugate moiety is a trivalent N-acetylgalactosamine moiety. Compounds A-D contain a dilysine brancher molecule, a PEG3 spacer, and three terminal GalNAc carbohydrate moieties. In compounds A and B, the oligonucleotide is directly attached to the asialoglycoprotein receptor-targeted conjugate moiety without a linker. In compounds C and D, the oligonucleotide is attached to the asialoglycoprotein receptor-targeted conjugate moiety via a C6 linker. Compounds E-I contain commercially available trebler brancher molecules and spacers of various lengths and structures, and three terminal GalNAc carbohydrate moieties.

[0018] 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 an acute infection or a chronic infection. Chronic hepatitis B virus (CHB) infection is a global disease burden affecting 248 million people worldwide. Approximately 686,000 deaths per year are due to HBV-related end-stage liver disease and hepatocellular carcinoma (HCC) (GBD, 2013; Schweitzer et al., 2015). WHO has predicted that without further intervention, the number of CHB-infected individuals will remain at their current high levels for the next 40-50 years, with a cumulative 20 million deaths between 2015 and 2030 (WHO, 2016). CHB infection is not a homogenous disease with distinct clinical manifestations. Infected individuals progress through several stages of CHB-related liver disease over their lifetime. These stages are also the basis for treatment with standard of care (SOC). Current guidelines recommend treating only selected individuals infected with CHB based on three criteria: serum ALT level, HBV DNA level, and severity of liver disease (EASL, 2017). This recommendation is due to the fact that SOC, namely nucleos(t)ide analogs (NA) and pegylated interferon-α (PEG-IFN), are not curative and must be administered for a long period of time, thereby increasing safety risks. NA effectively suppresses HBV DNA replication; however, it has very limited / no effect on other viral markers. Two hallmarks of HBV infection, hepatitis B surface antigen (HBsAg) and covalently closed circular DNA (cccDNA), are the main targets of new drugs aimed at HBV cure. In the plasma of CHB patients, HBsAg subviral (empty) particles outnumber HBV virions by 103-105 times (Ganem & Prince, 2014). This excess is thought to contribute to the immunopathogenesis of the disease, including in individuals who fail to develop neutralizing anti-HBs antibodies, a serological marker observed after resolution of acute HBV infection.

[0019] cccDNA (covalently closed circular DNA) cccDNA is the viral genetic template present in the nucleus of infected hepatocytes, gives rise to all HBV RNA transcripts required for productive infection, and is responsible for viral persistence during the natural history of chronic HBV infection (Locarnini & Zoulim (2010) Antivir Ther. vol. 15, suppl. no. 3, pp. 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.

[0020] Therefore, there is a strong need for novel therapies that can bring about a complete cure, defined by the degradation or elimination of HBV cccDNA, in the majority of CHB patients.

[0021] compound As used herein, the term "compound" refers to any molecule capable of inhibiting the expression or activity of RTEL1. A particular 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, as used herein, a compound may be a nucleic acid molecule, particularly an antisense oligonucleotide or siRNA, that targets RTEL1.

[0022] Oligonucleotides The term "oligonucleotide," as used herein, is defined as a molecule that contains two or more covalently linked nucleosides, as it is commonly understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as a nucleic acid molecule or oligomer, which may be used interchangeably.

[0023] The oligonucleotides described in the specification and claims are generally therapeutic oligonucleotides less than 70 nucleotides in length. The oligonucleotides may be or include single-stranded antisense oligonucleotides, or may be other oligomeric nucleic acid molecules, such as CRISPR RNA, siRNA, shRNA, aptamers, or ribozymes. Therapeutic oligonucleotide molecules are usually produced in the laboratory by solid-phase chemical synthesis, followed by purification and isolation. However, shRNAs are often delivered to cells using lentiviral vectors, which are then transcribed to generate single-stranded RNA, which will form an RNA stem-loop (hairpin) RNA structure that can interact with the RNA interference machinery (including RNA-induced silencing complex (RISC)). In one embodiment of the present invention, the shRNA is a chemically generated shRNA molecule (not relying on cell-based expression from a plasmid or virus).

[0024] When referring to the sequence of an oligonucleotide, it refers to the sequence or order of the nucleic acid base moieties of covalently linked nucleotides or nucleosides, or their modification.Generally, the oligonucleotide of the present invention is artificial, chemically synthesized, and typically purified or isolated.However, in some embodiments, the oligonucleotide of the present invention is shRNA that is transcribed from a vector upon entry into target cell.The oligonucleotide of the present invention may comprise one or more modified nucleosides or nucleotides.

[0025] In some embodiments, the oligonucleotide of the present invention comprises or consists 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 12 to 25, such as 16 to 22, such as 16 to 20 consecutive nucleotides in length. Thus, the oligonucleotide of the present invention may have a length of 12 to 25 nucleotides in some embodiments. Alternatively, the oligonucleotide of the present invention may have a length of 15 to 22 nucleotides in some embodiments.

[0026] In some embodiments, the oligonucleotide or contiguous nucleotide sequence thereof comprises or consists of 24 or fewer nucleotides, such as 22, such as 20 or fewer nucleotides, such as 18 or fewer nucleotides, such as 14, 15, 16, or 17 nucleotides. 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.

[0027] 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.

[0028] The oligonucleotide(s) are for modulating expression of a target nucleic acid in a mammal. In some embodiments, the nucleic acid molecules, such as siRNA, shRNA, and antisense oligonucleotides, are typically for inhibiting expression of the target nucleic acid(s).

[0029] In one embodiment of the invention, the oligonucleotide is selected from an RNAi agent, such as 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 RNaseH.

[0030] In some embodiments, oligonucleotides of the invention may contain one or more modified nucleosides or nucleotides, such as, for example, 2' sugar modified nucleosides.

[0031] In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages.

[0032] In some embodiments, the oligonucleotide may be linked to a non-nucleoside moiety (a conjugate moiety).

[0033] 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 with overlapping nucleobase sequences that target one or more mammalian RTEL1 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.

[0034] Antisense oligonucleotides The term "antisense oligonucleotide" as used herein is defined as an oligonucleotide that can regulate the expression of a target gene by hybridizing to a target nucleic acid, particularly a continuous sequence on the target nucleic acid. The antisense oligonucleotide herein is not double-stranded in nature, 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 a hairpin or intermolecular duplex structure (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self-complementarity over the entire length of the oligonucleotide is less than 50%.

[0035] Advantageously, the single-stranded antisense oligonucleotides of the present invention are free of RNA nucleosides to reduce nuclease resistance.

[0036] 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.

[0037] RNAi molecule As used herein, the term "RNA interference (RNAi) molecule" refers to a short double-stranded RNA-based oligonucleotide that can induce RNA-dependent gene silencing via the RNA-induced silencing complex (RISC) in the cytoplasm of a cell and that interacts with the catalytic RISC component Argonaute. RNAi molecules regulate, e.g., inhibit, the expression of a target nucleic acid in a cell, e.g., a cell in a subject, such as a mammalian subject. One type of RNAi molecule is small interfering RNA (siRNA), which is a double-stranded RNA molecule composed of two complementary oligonucleotides, where one strand binds to the complementary mRNA after transcription, resulting in its degradation and loss of translation. Small hairpin RNA (shRNA) is a single-stranded RNA-based oligonucleotide that forms a stem-loop (hairpin) structure, which can reduce mRNA via DICER and the RNA-reducing silencing complex (RISC). RNAi molecules can be designed based on the sequence of a gene of interest (target nucleic acid). The corresponding RNAi can then be synthesized chemically or by in vitro transcription, or expressed from a vector or PCR product.

[0038] siRNA The term "siRNA" refers to a small interfering ribonucleic acid RNAi molecule. It is a class of double-stranded RNA molecules, also known in the art as small interfering RNA or silencing RNA. siRNA typically comprises a sense strand (also called passenger strand) and an antisense strand (also called guide strand), where each strand is 17-30 nucleotides long, typically 19-25 nucleosides long, the antisense strand is complementary, e.g. at least 95% complementary, e.g. fully complementary, to a target nucleic acid (preferably a mature mRNA sequence), and the sense strand is complementary to the antisense strand such that the sense strand and the antisense strand form a duplex or duplex region. The siRNA strands may form a blunt-ended duplex, or advantageously, the sense strand and the antisense strand 3'-end may form a 3'-overhang of, e.g., 1, 2, or 3 nucleosides, similar to the product generated by Dicer to form a RISC substrate 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 and antisense strands have a 2 nt 3' overhang. Thus, the duplex region can be, for example, 17-25 nucleotides long, for example, 21-23 nucleotides long.

[0039] Once inside the cell, the antisense strand is incorporated into the RISC complex, which mediates the targeted degradation or targeted inhibition of the target nucleic acid. siRNA typically includes modified nucleosides in addition to RNA nucleosides. In one embodiment, siRNA molecules may also be chemically modified with 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). In particular, 2'fluoro, 2'-O-methyl, or 2'-O-methoxyethyl can be incorporated into the siRNA.

[0040] 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). It has been reported that the incorporation of thermally destabilized nucleotides occurring in the seed region of the antisense strand of the siRNA is useful for reducing the off-target activity of the siRNA (see, e.g., WO 2018 / 098328). Suitably, 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.

[0041] 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 (e.g., antisense strand or sense strand), or the phosphorothioate or methylphosphonate internucleoside bond can be at the 5'-end of one or both strands (e.g., 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 (e.g., antisense strand or sense strand). In some embodiments, the remaining internucleoside bonds are phosphodiester bonds. In some embodiments, the siRNA molecule comprises one or more phosphorothioate internucleoside bonds. In siRNA molecules, phosphorothioate internucleoside linkages can reduce nuclease cleavage in RICS, therefore it is advantageous that not all internucleoside linkages in the antisense strand are modified.

[0042] The siRNA molecule can further comprise a ligand, hi some embodiments, the ligand is attached to the 3' end of the sense strand.

[0043] For biodistribution, the siRNA can be conjugated to a targeting ligand and / or formulated in, for example, lipid nanoparticles.

[0044] Other aspects of the invention relate to pharmaceutical compositions comprising these dsRNA, such as siRNA molecules suitable for therapeutic use, and methods of inhibiting expression of target genes by administering dsRNA molecules, such as siRNA molecules, of the invention, for the treatment of various disease conditions, e.g., as disclosed herein.

[0045] shRNA Short hairpin RNA or shRNA molecules are generally 40-70 nucleotides long, e.g., 45-65 nucleotides long, e.g., 50-60 nucleotides long, and form a stem-loop (hairpin) RNA structure that interacts with an endonuclease known as Dicer, which is believed to process dsRNA into 19-23 base pair short interfering RNAs with characteristic 2-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, inducing silencing. RNAi oligonucleotides may be chemically modified with 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).

[0046] In some embodiments, the shRNA nucleic acid molecule comprises one or more phosphorothioate internucleoside bonds. In RNAi molecules, phosphorothioate internucleoside bonds can reduce nuclease cleavage in RICS, so it is advantageous not to modify all internucleoside bonds in the stem loop of the shRNA molecule. Phosphorothioate internucleoside bonds can be advantageously located at the 3' and / or 5' ends of the stem loop of the shRNA molecule, especially in parts of the molecule that are not complementary to the target nucleic acid (e.g., the sense strand or passenger strand of an siRNA molecule). The region of the shRNA molecule that is complementary to the target nucleic acid can, however, also be modified in the first 2-3 internucleoside bonds in the parts predicted to be the 3' and / or 5' ends after cleavage by Dicer.

[0047] Contiguous nucleotide sequence The term "contiguous nucleotide sequence" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. This term is used interchangeably herein with the terms "contiguous nucleobase sequence" and "oligonucleotide motif sequence." In some embodiments, all nucleotides of an oligonucleotide constitute a contiguous nucleotide sequence. In some embodiments, 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 may 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 a target nucleic acid.

[0048] 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 and RNA nucleotides, contain a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (not present in nucleosides).Nucleosides and nucleotides may also be referred to interchangeably as "units" or "monomers."

[0049] Modified Nucleosides The term "modified nucleoside" or "nucleoside modification," as used herein, refers to a nucleoside that has been modified by the introduction of one or more modifications in the sugar moiety or (nucleic acid) base moiety, as compared to an equivalent DNA or RNA nucleoside. In a preferred embodiment, the modified nucleoside comprises a modified sugar moiety. The term modified nucleoside may also be used interchangeably 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 they are capable of Watson-Crick base pairing.

[0050] Modified Internucleoside Linkages The term "modified internucleoside linkage" is defined as understood by those skilled in the art as a linkage other than a phosphodiester (PO) linkage that covalently links two nucleosides together. Thus, the oligonucleotide of the present invention can include one or more modified internucleoside linkages, such as one or more phosphorothioate internucleoside linkages or one or more phosphorodithioate internucleoside linkages. In some embodiments, the modified internucleoside linkage increases the nuclease resistance of the oligonucleotide compared to a phosphodiester linkage. In the case of naturally occurring oligonucleotides, the internucleoside linkage comprises a phosphate group that forms a phosphodiester bond between adjacent nucleosides. The modified internucleoside linkage is particularly useful for stabilizing oligonucleotides for in vivo applications and can serve to protect against nuclease cleavage in regions of DNA or RNA nucleosides of the oligonucleotide of the present invention, such as in the gap region G of a gapmer oligonucleotide, and in regions F and F' of modified nucleosides.

[0051] In one embodiment, the oligonucleotide comprises one or more internucleoside linkages modified from natural phosphodiester, e.g., such that the one or more modified internucleoside linkages are more resistant to nuclease attack. Nuclease resistance can be determined by incubating the oligonucleotide in serum or by using a nuclease resistance assay (e.g., snake venom phosphodiesterase (SVPD)), both of which are well known in the art. An internucleoside linkage that can improve the nuclease resistance of an oligonucleotide is referred to as a nuclease-resistant internucleoside linkage. In some embodiments, at least 50% of the internucleoside linkages of the oligonucleotide, or its contiguous nucleotide sequence, are modified, e.g., at least 60%, e.g., at least 70%, e.g., at least 75%, e.g., at least 80%, or e.g., at least 90% of the internucleoside linkages of the oligonucleotide, or its contiguous nucleotide sequence, are modified. In some embodiments, all of the internucleoside bonds of the oligonucleotide, or the consecutive nucleotide sequence thereof, are modified. It will be appreciated that in some embodiments, the nucleoside that connects the oligonucleotide of the present invention to a non-nucleotidic functional group, such as a conjugate, can be phosphodiester. In some embodiments, all of the internucleoside bonds of the oligonucleotide, or the consecutive nucleotide sequence thereof, are nuclease-resistant internucleoside bonds.

[0052] It is advantageous to use phosphorothioate internucleoside linkages in the oligonucleotides of the invention.

[0053] Phosphorothioate internucleoside linkages are particularly useful due to their nuclease resistance, favorable pharmacokinetics, and ease of manufacture.In some embodiments, at least 50% of the internucleoside linkages of the oligonucleotide, or its consecutive nucleotide sequence, are phosphorothioate, for example at least 60%, such as at least 70%, for example at least 75%, for example at least 80% or for example at least 90% of the internucleoside linkages of the oligonucleotide, or its consecutive nucleotide sequence, are phosphorothioate.In some embodiments, all of the internucleoside linkages of the oligonucleotide, or its consecutive nucleotide sequence, are phosphorothioate.

[0054] Nuclease-resistant linkages such as phosphorothioate linkages are particularly useful in regions of oligonucleotides that can recruit nucleases when duplexed with a target nucleic acid, such as region G of a gapmer. However, phosphorothioate linkages may also be useful in non-nuclease recruiting and / or affinity enhancing regions, such as regions F and F' of a gapmer. A gapmer oligonucleotide may, in some embodiments, contain one or more phosphodiester linkages in region F or F', or both regions F and F', where all of the internucleoside linkages in region G may be phosphorothioate.

[0055] Advantageously, all of the internucleoside linkages of the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate or all of the internucleoside linkages of the oligonucleotide are phosphorothioate linkages.

[0056] As disclosed in EP 2 742 135, it will be appreciated that antisense oligonucleotides may contain other internucleoside linkages (other than phosphodiester and phosphorothioate), e.g., alkylphosphonate / methylphosphonate internucleoside linkages, which according to EP 2 742 135 may be tolerated, e.g., within the gap region of another DNA phosphorothioate.

[0057] Nucleic acid bases The term nucleobase includes the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses modified nucleobases that may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, and non-naturally occurring variants. Such variants are described, for example, in Hirao et al. (2012) "Accounts of Chemical Research" Vol. 45, p. 2055, and Bergstrom (2009) "Current Protocols in Nucleic Acid Chemistry Suppl." Vol. 37, p. 1.4.1.

[0058] 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, e.g., a nucleobase selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0059] The nucleobase moieties are represented by the letter code of each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter can optionally include a modified nucleobase of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides can be used.

[0060] Modified Oligonucleotides The term modified oligonucleotide describes an oligonucleotide that contains one or more sugar-modified nucleosides and / or modified internucleoside linkages. The term "chimeric" oligonucleotide is a term used in the literature to describe oligonucleotides that have modified nucleosides and DNA nucleosides. The antisense oligonucleotide of the present invention is preferably a chimeric oligonucleotide.

[0061] Complementarity The term "complementarity" describes the ability of nucleosides / nucleotides to undergo Watson-Crick base pairing. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). It will be understood that oligonucleotides may contain nucleosides with modified nucleobases, e.g., 5-methylcytosine is often used in place of cytosine, and thus the term complementarity encompasses Watson-Crick base pairing between unmodified and modified nucleobases (see, e.g., Hirao et al. (2012) Accounts of Chemical Research 45:2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37:1.4.1).

[0062] The term "% complementarity" as used herein refers to the percentage of nucleotides of 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) across the contiguous nucleotide sequence. Thus, the percentage of complementarity is calculated by counting the number of aligned (by Watson-Crick base pairing) nucleobases that are complementary between two sequences (when the target sequence 5'-3' and the oligonucleotide sequence 3'-5' are aligned), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. In such a comparison, nucleobases / nucleotides that do not align (form base pairs) are referred to as mismatches. Insertions and deletions are not allowed in the calculation of the % complementarity of a contiguous nucleotide sequence. It will be understood that in determining complementarity, chemical modifications of nucleobases are disregarded so long as the functional ability of the nucleobase to form Watson-Crick base pairs is retained (e.g., 5-methylcytosine is considered identical to cytosine for purposes of calculating % complementarity).

[0063] The term "fully complementary" refers to 100% complementarity.

[0064] Below is an example of an oligonucleotide motif (SEQ ID NO:33) that is perfectly complementary to a target nucleic acid (SEQ ID NO:11).

[0065] 5'-CTTTGACCAGAGTATGTAAAATTCTC-3' (SEQ ID NO: 11) 3'-AAACTGGTCTCATACATTTT-5' (SEQ ID NO: 33)

[0066] identity The term "identity", as used herein, refers to the proportion (expressed as a percentage) of nucleotides of a contiguous nucleotide sequence of a nucleic acid molecule (e.g., an oligonucleotide) that are identical to a reference sequence (e.g., a sequence motif) over the contiguous nucleotide sequence. Thus, the percentage of identity is calculated by counting the number of aligned nucleobases that are identical (matching) between two sequences (in the contiguous nucleotide sequence of the compound of the invention and the reference sequence), dividing that number by the total number of nucleotides in the oligonucleotide, and multiplying by 100. Thus, the percentage of identity = (matching 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 will be understood that in determining identity, chemical modifications of nucleobases are disregarded so long as the functional ability of the nucleobase to form Watson-Crick base pairs is retained (e.g., 5-methylcytosine is considered to be identical to cytosine for purposes of calculating % identity).

[0067] Hybridization The term "hybridizing" or "hybridizing" as used herein should be understood as two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) forming 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 often measured 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 ) Under physiological conditions, T m is not strictly proportional to affinity (Mergny and Lacroix (2003) Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° more accurately represents the binding affinity, and ΔG° =-RTln(K d) determines the dissociation constant (K d), where R is the gas constant and T is the absolute temperature. Thus, a very low ΔG° for the reaction of an oligonucleotide with 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 1M, pH of 7, and temperature of 37°C. Hybridization of an oligonucleotide to a target nucleic acid is a spontaneous reaction, and for spontaneous reactions, ΔG° is less than zero. ΔG° can be experimentally measured using, for example, isothermal titration calorimetry (ITC), as described in Hansen et al. (1965) Chem. Comm. 36-38 and Holdgate et al. (2005) Drug Discov Today. Those skilled in the art will know that commercially available equipment is available for measuring ΔG°. ΔG° can also be estimated numerically by using the nearest neighbor model, as described in SantaLucia (1998) Proc Natl Acad Sci USA. 95, 1460-1465, with appropriately derived thermodynamic parameters as described in Sugimoto et al. (1995) Biochemistry 34, 11211-11216 and McTigue et al. (2004) Biochemistry 43, 5388-5405. To have the potential to modulate its intended nucleic acid target by hybridization, the oligonucleotides of the invention hybridize to the target nucleic acid with an estimated ΔG° value of less than -10 kcal for oligonucleotides of 10-30 nucleotides in length. In some embodiments, the degree or strength of hybridization is measured by the standard state Gibbs free energy ΔG°. The oligonucleotides may hybridize to the target nucleic acid with estimated ΔG° values ​​in the range of less than −10 kcal, such as less than −15 kcal, such as less than −20 kcal, and such as less than −25 kcal for oligonucleotides 8 to 30 nucleotides in length.In some embodiments the oligonucleotide hybridizes to the target nucleic acid with an estimated ΔG° value of -10 to -60 kcal, such as -12 to -40, such as -15 to -30 kcal or -16 to -27 kcal, such as -18 to -25 kcal.

[0068] target nucleic acid In accordance with the present invention, a target nucleic acid is a nucleic acid encoding a mammalian RTEL1, and may be, for example, a gene, an RNA, an mRNA, and a pre-mRNA, a mature mRNA, or a cDNA sequence. Thus, the target may be referred to as an RTEL1 target nucleic acid.

[0069] The oligonucleotides of the invention can target, for example, exonic regions of mammalian RTEL1 (particularly siRNA and shRNA target exonic regions, but also antisense oligonucleotides) or can target, for example, intronic regions of the RTEL1 pre-mRNA (particularly antisense oligonucleotides targeting intronic regions). The human RTEL1 gene encodes 15 transcripts of these seven transcripts that code for proteins and are therefore potential nucleic acid targets. Table 1 lists the predicted exonic and intronic regions of the seven transcripts located on the human RTEL1 pre-mRNA of SEQ ID NO:1. It will be understood that the oligonucleotides of the invention can target one or more of the mature mRNA sequences of the transcripts listed in Table 1.

[0070] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0071] Suitably, the target nucleic acid encodes an RTEL1 protein, in particular a mammalian RTEL1, such as human RTEL1 (see, for example, Tables 2 and 3), which provide pre-mRNA sequences for human and monkey RTEL1.

[0072] In some embodiments, the target nucleic acid is selected from SEQ ID NO: 1 and / or 2, or a naturally occurring variant thereof (eg, a sequence encoding a mammalian RTEL1 protein in Table 1).

[0073] For research or diagnostic uses of the oligonucleotides of the invention, the target nucleic acid can be cDNA or synthetic nucleic acid derived from DNA or RNA.

[0074] For in vivo or in vitro applications, the oligonucleotides of the invention are typically capable of inhibiting expression of a RTEL1 target nucleic acid in a cell expressing the RTEL1 target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotides of the invention is typically complementary to the RTEL1 target nucleic acid, measured over the length of the oligonucleotide, optionally except for one or two mismatches, and optionally except for a nucleotide-based linker region or other non-complementary terminal nucleotides (e.g., region D' or D'') that may attach the oligonucleotide to any functional group, such as a conjugate. The target nucleic acid may in some embodiments be RNA, such as a messenger RNA, such as a mature mRNA (e.g., an exon region of a transcript listed in Table 1) or a pre-mRNA, or DNA.

[0075] In some embodiments, the target nucleic acid is RNA or DNA encoding a mammalian RTEL1 protein, such as human RTEL1, e.g., the human RTEL1 mRNA sequence as disclosed as SEQ ID NO: 1. Further information regarding exemplary target nucleic acids is provided in Tables 2 and 3.

[0076] [Table 2]

[0077] [Table 3]

[0078] NOTE: SEQ ID NO:2 contains multiple NNNN regions where sequencing cannot precisely refine the sequence and therefore degenerate sequences are included. For the avoidance of doubt, the compounds of the present invention are complementary to the actual target sequence and are therefore not degenerate compounds.

[0079] In some embodiments, the target nucleic acid is SEQ ID NO:1.

[0080] In some embodiments, the target nucleic acid is SEQ ID NO:2.

[0081] Target sequence The term "target sequence" as used herein refers to a sequence of nucleotides present in a target nucleic acid, which comprises a nucleobase sequence that is complementary to an oligonucleotide of the present invention. In some embodiments, the target sequence is comprised of a region on the target nucleic acid that has a nucleobase sequence that is complementary to the continuous nucleotide sequence of an 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 a single oligonucleotide, and can represent, for example, a preferred region of the target nucleic acid that can be targeted by several oligonucleotides of the present invention.

[0082] In some embodiments, the target sequence is a sequence selected from the group consisting of human RTEL1 mRNA exons, such as an RTEL1 human mRNA exon selected from the list in Table 1 above.

[0083] In some embodiments, the target sequence is a sequence selected from the group consisting of a human RTEL1 mRNA intron, such as a RTEL1 human mRNA intron selected from the list in Table 1 above.

[0084] The oligonucleotides of the invention comprise a contiguous nucleotide sequence that is complementary to or hybridizes to a target nucleic acid, such as a target sequence described herein.

[0085] The target sequence to which the oligonucleotide is complementary or hybridizes generally comprises a contiguous nucleic acid base sequence of at least 10 nucleotides. The contiguous nucleotide sequence is 10-35 nucleotides, such as 12-30, such as 14-20, such as 16-20 contiguous nucleotides. In one embodiment of the present invention, the target sequence is selected from the group consisting of SEQ ID NOs: 3-21 as shown in Table 4.

[0086] [Table 4]

[0087] In some embodiments, the target sequence is selected from a region shown in Table 5A or Table 5B.

[0088] target cell The term "target cell" as used herein 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 embodiments, the target cell is a mammalian cell, such as a rodent cell, such as a mouse cell or a rat cell, or a woodchuck cell, or a primate cell, such as a monkey cell (e.g., a cynomolgus monkey cell) or a human cell.

[0089] In a preferred embodiment, the target cell expresses RTEL1 mRNA, such as RTEL1 pre-mRNA or RTEL1 mature mRNA. The polyA tail of RTEL1 mRNA is typically ignored for antisense oligonucleotide targeting.

[0090] Furthermore, the target cells may be hepatocytes. In one embodiment, the target cells are primary human hepatocytes infected with HBV, either from HBV-infected individuals or from HBV-infected mice with humanized livers (PhoenixBio, PXB mice).

[0091] According to the present invention, the target cell may be infected with HBV. Furthermore, the target cell may contain HBV cccDNA. Thus, it is preferred that the target cell contains RTEL1 mRNA, such as RTEL1 pre-mRNA or RTEL1 mature mRNA, and HBV cccDNA.

[0092] Naturally occurring variants The term "naturally occurring variant" refers to variants of the RTEL1 gene or transcript that originate from the same locus as the target nucleic acid, but may differ, for example, due to the degeneracy of the genetic code resulting in multiple codons encoding the same amino acid, or due to alternative splicing of pre-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 oligonucleotide of the present invention can thus target the target nucleic acid and its naturally occurring variants.

[0093] In some embodiments, a naturally occurring variant has at least 95%, such as at least 98% or at least 99% homology to a mammalian RTEL1 target nucleic acid, such as SEQ ID NO: 1 and / or 2. In some embodiments, a naturally occurring variant has at least 99% homology to a human RTEL1 target nucleic acid of SEQ ID NO: 1. In some embodiments, a naturally occurring variant is a known polymorphism.

[0094] Regulation of expression The term "modulation of expression" as used herein should be understood as a general term for the ability of an oligonucleotide to modify the amount of RTEL1 when compared to the amount of RTEL1 before administration of the oligonucleotide. Alternatively, modulation of expression can be determined by reference to a control experiment. A control is generally understood to be an individual or target cell treated with a saline composition or an individual or target cell treated with a non-targeting oligonucleotide (mock).

[0095] One type of modulation is the ability of the oligonucleotide to inhibit, downregulate, reduce, suppress, eliminate, stop, block, prevent, decrease, reduce, avoid, or terminate expression of RTEL1, for example, by degradation of mRNA or blocking transcription. Another type of modulation is the ability of the oligonucleotide to restore, increase, or enhance expression of RTEL1, for example, by repairing a splice site or preventing splicing, or by removing or blocking an inhibitory mechanism such as microRNA suppression.

[0096] 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.

[0097] A large number of nucleosides with modifications of the ribose sugar moiety have been produced with the primary goal of improving certain properties of oligonucleotides, such as affinity and / or nuclease resistance.

[0098] Such modifications include those in which the ribose ring structure has been modified, for example by replacing it with a hexose ring (HNA) or bicyclic ring (typically having a biradical bridge between the C2 and C4 carbons of the ribose ring (LNA)), or an unlinked ribose ring (e.g., UNA), which typically lacks 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.

[0099] Sugar modifications also include modifications by changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group that occurs naturally in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4', or 5' positions.

[0100] 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 invention preferably provide an increase in melting temperature of +0.5 to +12° C., more preferably +1.5 to +10° C., and most preferably +3 to +8° C. per modified nucleoside. A large number of 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, vol. 25, pp. 4429-4443, and Uhlmann; Curr. Opinion in Drug Development, 2000, vol. 3, no. 2, pp. 293-213).

[0101] 2' sugar modified nucleosides A 2' sugar modified nucleoside is a nucleoside that has a substituent other than H or -OH at the 2' position (2' substituted nucleoside) or that contains a 2' linked biradical that can form a bridge between the 2' carbon and the second carbon of the ribose ring, e.g., LNA (2'-4' biradical bridge).

[0102] In fact, much attention has been paid to 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 provide oligonucleotides with improved binding affinity and / or increased nuclease resistance. 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, e.g., Freier & Altmann; Nucl. Acid Res. (1997) vol. 25, pp. 4429-4443, and Uhlmann; Curr. Opinion in Drug Development (2000) vol. 3, no. 2, pp. 293-213, and Deleavey and Damha, Chemistry and Biology (2012) vol. 19, pp. 937. Below is a description of some 2'-substituted modified nucleosides. [ka]

[0103] In the context of the present invention, 2' substituted sugar modified nucleosides do not include 2' bridged nucleosides such as LNA.

[0104] Locked Nucleoside (LNA) Nucleoside An "LNA nucleoside" is a 2' sugar modified nucleoside that contains 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). Fixing the conformation of the ribose is associated with improved hybridization affinity (duplex stabilization) when LNA is incorporated into an oligonucleotide of a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complementary duplex.

[0105] Non-limiting exemplary LNA nucleosides are described in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, WO 2008 / 150729, Morita et al., Bioorganic & Med. Chem. Lett. Vol. 12, pp. 73-76; Seth et al., J. Org. Chem. (2010) Vol. 75, No. 5, pp. 1569-81; Mitsuoka et al., Nucleic Acids Research (2009) Vol. 37, No. 4, pp. 1225-1238; and Wan and Seth, J. Medical Chemistry (2016) Vol. 59, pp. 9645-9667.

[0106] Specific examples of LNA nucleosides of the present invention are shown in Scheme 1, where B is as defined above.

[0107] Scheme 1 [ka]

[0108] Particular LNA nucleosides are β-D-oxy-LNA, 6'-methyl-β-D-oxy LNA, such as (S)-6'-methyl-β-D-oxy-LNA (ScET) and ENA.

[0109] Pharmaceutically acceptable salts The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid, without being biologically or otherwise undesirable. Salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, especially hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcysteine, etc. In addition, these salts can be prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins. Compounds of formula (I) can also exist in zwitterionic form. Particularly preferred pharmaceutically acceptable salts of compounds of formula (I) are salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and methanesulfonic acid.

[0110] RNase H activity and recruitment RNase H activity of an antisense oligonucleotide refers to its ability to recruit RNase H when it forms a duplex with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for determining RNase H activity, which can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is considered to be 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 determined when using an oligonucleotide having the same base sequence as the modified oligonucleotide being tested, measured in pmol / l / min, but containing only DNA monomers and having phosphorothioate linkages between all monomers of the oligonucleotide, and using the methodology provided by Examples 91-95 of WO 01 / 23613 (herein incorporated by reference). For use in determining RNase H activity, recombinant human RNase H1 is available from Creative Biomart® (recombinant human RNASEH1 fused to a His tag expressed in E. coli).

[0111] 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 oligonucleotides contain at least three distinct structural regions, 5'-flank, gap and 3'-flank, FG-F', in a 5'->3' orientation. 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) that contains one or more sugar-modified nucleosides, advantageously high affinity sugar-modified nucleosides, and a 3' flanking region (F') that contains one or more sugar-modified nucleosides, advantageously high affinity sugar-modified nucleosides. The one or more sugar-modified nucleosides of regions F and F' enhance the affinity of the oligonucleotide to 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, e.g., high affinity 2' sugar modifications, independently selected from LNA and 2'-MOE.

[0112] In a gapmer design, the 5' and 3' most nucleosides of the gap region are DNA nucleosides, each positioned adjacent to a 5' (F) or 3' (F') sugar-modified nucleoside. The flanks may be further defined by having at least one sugar-modified nucleoside at the end furthest from the gap region, i.e., at the 5' end of the 5' flank and at the 3' end of the 3' flank.

[0113] The regions FG-F' form a contiguous nucleotide sequence. The antisense oligonucleotide of the invention, or the contiguous nucleotide sequence thereof, may comprise a gapmer region of the formula FG-F'.

[0114] The total length of the gapmer designed FG-F' may be, for example, 12 to 32 nucleosides, for example, 13 to 24, for example, 14 to 22 nucleosides, for example, 14 to 17, for example, 16 to 18 nucleosides.

[0115] By way of example, a gapmer oligonucleotide of the present invention can be represented by the following formula: F 1-8 -G 5-18 -F' 1-8 ,for example F 1-8 -G 5-16 -F' 1-8 ,for example F 1-8 -G 7-16 -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.

[0116] 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 1-8 nucleosides, 1-4 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 6-18 nucleosides, e.g., a region of 6-16 nucleosides, capable of recruiting RNase H. In some embodiments, the G region consists of DNA nucleosides.

[0117] Regions F, G, and F' are further defined below and can be incorporated into the formula FG-F'.

[0118] Gapmer region G The region G (gap region) of the gapmer is a region of nucleosides, typically DNA nucleosides, that allows the oligonucleotide to recruit RNase H, e.g. human RNase H1. RNase H is a cellular enzyme that recognizes a duplex between DNA and RNA and enzymatically cleaves the RNA molecule. Suitably, the gapmer may have a gap region (G) of at least 5 or 6 consecutive DNA nucleosides, such as 5-18 consecutive DNA nucleosides, such as 5-17 consecutive DNA nucleosides, such as 5-16 consecutive DNA nucleosides, such as 6-15 consecutive DNA nucleosides, such as 7-14 consecutive DNA nucleosides, such as 8-12 consecutive DNA nucleotides, such as 8-12 consecutive DNA nucleotides in length. The gap region G may in some embodiments consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 consecutive DNA nucleosides. Cytosine (C) DNA in the gap region may be methylated in some cases, and such residues include 5'-methyl-cytosine ( me C, or e instead of c). Methylation of cytosine DNA in the gap is advantageous when a cg dinucleotide is present in the gap to reduce potential toxicity, and the modification does not significantly affect the potency of the oligonucleotide. 5'-substituted DNA nucleosides, such as 5'-methyl DNA nucleosides, have been reported for use in DNA gap regions (European Patent Application Publication No. EP2742136).

[0119] In some embodiments, the gap region G can consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 consecutive phosphorothioate-linked DNA nucleosides. In some embodiments, all internucleoside linkages within the gap are phosphorothioate linkages.

[0120] A traditional gapmer has a DNA gap region, but there are numerous examples of modified nucleosides that allow for RNase H recruitment when used within the gap region. Modified nucleosides that have been reported as capable of recruiting RNase H when contained within the gap region include, for example, α-L-LNA, C4'-alkylated DNA (as described in International Application No. PCT / EP2009 / 050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) pp. 2296-2300, both of which are incorporated herein by reference), arabinose-derived nucleosides such as ANA and 2'F-ANA (Mangos et al., (2003) J. AM. CHEM. SOC. 125: 654-661), UNA (unlocked nucleic acid) (as described in Fluiter et al., Mol. Biosyst. (2009) vol. 10: 1039, which is incorporated herein by reference). UNAs are typically unlocked nucleic acids in which the bond between C2 and C3 of the ribose has been removed to form an unlocked "sugar" residue. The modified nucleosides used in such gapmers can be nucleosides that adopt a 2'endo (DNA-like) structure when introduced into the gap region (i.e., modifications that allow RNase H recruitment). In some embodiments, the DNA gap region (G) described herein may optionally contain 1-3 sugar modified nucleosides that adopt a 2'endo (DNA-like) structure when introduced into the gap region.

[0121] Gappa flanking regions, F and F' Region F is positioned immediately adjacent to the 5' DNA nucleoside of region G. The 3'-most nucleoside of region F is a sugar-modified nucleoside, such as a high affinity sugar-modified nucleoside, such as a 2'-substituted nucleoside, such as an MOE nucleoside, or an LNA nucleoside.

[0122] Region F' is positioned immediately adjacent to the 3' DNA nucleoside of region G. The 5'-most nucleoside of region F' is a sugar-modified nucleoside, such as a high affinity sugar-modified nucleoside, such as a 2'-substituted nucleoside, such as an MOE nucleoside, or an LNA nucleoside.

[0123] Region F is 1 to 8 contiguous nucleotides in length, such as 2 to 6, such as 3 to 4 contiguous nucleotides in length. Advantageously, the 5'-most nucleoside of region F is a sugar-modified nucleoside. In some embodiments, the two 5'-most nucleosides of region F are sugar-modified nucleosides. In some embodiments, the two 5'-most nucleosides of region F are LNA nucleosides. In some embodiments, the two 5'-most nucleosides of region F are LNA nucleosides. In some embodiments, the two 5'-most nucleosides of region F are 2'-substituted nucleosides, such as two 3'MOE nucleosides. In some embodiments, the 5'-most nucleosides of region F are 2'-substituted nucleosides, such as MOE nucleosides.

[0124] Region F' is 2 to 8 contiguous nucleotides in length, such as 3 to 6, such as 4 to 5 contiguous nucleotides in length. Advantageously, the 3'-most nucleosides of region F' are sugar-modified nucleosides. In some embodiments, the two 3'-most nucleosides of region F' are sugar-modified nucleosides. In some embodiments, the two 3'-most nucleosides of region F' are LNA nucleosides. In some embodiments, the 3'-most nucleosides of region F' are LNA nucleosides. In some embodiments, the two 3'-most nucleosides of region F' are 2'-substituted nucleosides, such as two 3' MOE nucleosides. In some embodiments, the 3'-most nucleosides of region F' are 2'-substituted nucleosides, such as MOE nucleosides.

[0125] It should be noted that when the length of region F or region F' is 1, it is an LNA nucleoside.

[0126] In some embodiments, region F and region F' independently consist of or comprise a contiguous sequence of sugar-modified nucleosides. In some embodiments, the sugar-modified nucleosides of region F may be independently selected from 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, LNA units, arabinonucleic acid (ANA) units, and 2'-fluoro-ANA units.

[0127] In some embodiments, region F and region F' independently comprise both LNA and 2'-substituted modified nucleosides (mixed wing designs).

[0128] In some embodiments, region F and region F' are composed of only one type of sugar modified nucleoside, for example only MOE, or only β-D-oxy LNA, or only ScET. Such designs are also referred to as uniform flank or uniform gapmer designs.

[0129] In some embodiments, all nucleosides of regions F or F', or F and F', are LNA nucleosides, e.g., independently selected from β-D-oxy LNA, ENA, or ScET nucleosides. In some embodiments, region F consists of 1-5, e.g., 2-4, e.g., 3-4, e.g., 1, 2, 3, 4 or 5 contiguous LNA nucleosides. In some embodiments, all nucleosides of regions F and F' are β-D-oxy LNA nucleosides.

[0130] In some embodiments, all nucleosides of region F or F', or F and F', are 2'-substituted nucleosides, such as OMe or MOE. In some embodiments, region F consists of 1, 2, 3, 4, 5, 6, 7, or 8 contiguous OMe or MOE nucleosides. In some embodiments, only one of the flanking regions can consist of a 2'-substituted nucleoside, such as an OMe or MOE nucleoside. In some embodiments, it is the 5' (F) flanking region that consists of a 2'-substituted nucleoside, such as an OMe or MOE nucleoside, while the 3' (F') flanking region comprises at least one LNA nucleoside, such as a β-D-oxyLNA nucleoside or a cET nucleoside. In some embodiments, it is the 3' (F') flanking region that consists of a 2' substituted nucleoside, such as OMe or MOE, while the 5' (F) flanking region comprises at least one LNA nucleoside, such as a β-D-oxyLNA nucleoside or a cET nucleoside.

[0131] In some embodiments, all modified nucleosides in regions F and F' are LNA nucleosides, independently selected from, for example, β-D-oxy LNA, ENA, or ScET nucleosides, and regions F or F', or F and F', may optionally comprise DNA nucleosides (alternating flanks, see these definitions for details). In some embodiments, all modified nucleosides in regions F and F' are β-D-oxy LNA nucleosides, and regions F or F', or F and F', may optionally comprise DNA nucleosides (alternating flanks, see these definitions for details).

[0132] In some embodiments, the 5'-most and 3'-most nucleosides of regions F and F' are LNA nucleosides, such as β-D-oxyLNA nucleosides or ScET nucleosides.

[0133] In some embodiments, the internucleoside linkage between region F and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between region F' and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between regions F or F', F and F' is a phosphorothioate internucleoside linkage.

[0134] LNA GapMa An LNA gapmer is a gapmer in which either one or both of regions F and F' comprises or consists of LNA nucleosides. A β-D-oxy gapmer is a gapmer in which either one or both of regions F and F' comprises or consists of β-D-oxy LNA nucleosides.

[0135] In some embodiments, the LNA gapmer has the formula: [LNA] 1-5 -[Area G]-[LNA] 1-5 and region G is as defined in the definition of gapmer region G.

[0136] MOE Gapma An MOE gapmer is a gapmer in which regions F and F' consist of MOE nucleosides. In some embodiments, an MOE gapmer has the design [MOE] 1-8 -[Area G]-[MOE] 1-8 , e.g. [MOE] 2-7 -[Area G] 5-16 -[MOE] 2-7 , e.g. [MOE] 3-6 -[Area G]-[MOE] 3-6 and region G is as defined in the gapmer definition. MOE gapmers with the 5-10-5 design (MOE-DNA-MOE) are widely used in the art.

[0137] Region D' or D'' in the oligonucleotide The oligonucleotides of the invention may, in some embodiments, comprise or consist of a contiguous nucleotide sequence of the oligonucleotide that is complementary to a target nucleic acid, such as a gapmer FG-F', and further 5' and / or 3' nucleosides. The further 5' and / or 3' nucleosides may or may not be fully complementary to the target nucleic acid. Such further 5' and / or 3' nucleosides may be referred to herein as regions D' and D''.

[0138] The addition of region D' or D'' may be used to link a contiguous nucleotide sequence, such as a gapmer, to a conjugate moiety or other functional group. When used to link a contiguous nucleotide sequence to a conjugate moiety, it may serve as a biocleavable linker. Alternatively, it may be used to provide exonuclease protection or to facilitate synthesis or manufacture.

[0139] The regions D' and D'' are linked to the 5' end of the region F or the 3' end of the region F', respectively, to the following formula: D'-FG-F', FG-F'-D'', or

[0140] A D'-FG-F'-D'' design can be generated, where FG-F' is the gapmer portion of the oligonucleotide and the regions D' or D'' constitute separate portions of the oligonucleotide.

[0141] Regions D' or D'' independently comprise or consist 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' regions are not sugar-modified nucleotides, but are, for example, DNA or RNA or base-modified versions thereof. The D' and D'' regions may serve as nuclease-sensitive biocleavable linkers (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 regions D' and D'' are disclosed in WO 2014 / 076195, which includes, as examples, 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.

[0142] 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.

[0143] In some embodiments, the oligonucleotides of the invention can be represented by the following formula: FG-F'; especially F 1-8 -G 5-16 -F' 2-8 D'-FG-F', especially D' 1-3 -F 1-8 -G 5-16 -F' 2-8 FG-F'-D'', especially F 1-8 -G 5-16 -F' 2-8 -D'' 1-3 D'-FG-F'-D'', especially D' 1-3 -F 1-8 -G 5-16 -F' 2-8 -D''1-3

[0144] In some embodiments, the internucleoside linkage located between region D' and region F is a phosphodiester linkage. In some embodiments, the internucleoside linkage located between region F' and region D'' is a phosphodiester linkage.

[0145] Conjugates The term conjugate, as used herein, refers to an oligonucleotide covalently attached to a non-nucleotide moiety (the conjugate moiety or region C or the third region).

[0146] Conjugation of the oligonucleotide of the present invention to one or more non-nucleotide moieties can improve the pharmacology of the oligonucleotide, for example, by affecting the activity, cellular distribution, cellular uptake, or stability of the oligonucleotide. In some embodiments, the conjugate moiety regulates or improves the pharmacokinetic properties of the oligonucleotide by improving the cellular distribution, bioavailability, metabolism, excretion, permeability, and / or cellular uptake of the oligonucleotide. In particular, the conjugate can target the oligonucleotide to a specific organ, tissue, or cell type, thereby enhancing the efficacy of the oligonucleotide in that organ, tissue, or cell type. At the same time, the conjugate can help to reduce the activity of the oligonucleotide in non-target cell types, tissues, or organs (e.g., off-target activity or activity in non-target cell types, tissues, or organs).

[0147] WO 93 / 07883 and WO 2013 / 033230 provide suitable conjugate moieties, which are incorporated herein by reference.More suitable conjugate moieties are those that can bind to asialoglycoprotein receptor (ASGPR).In particular, trivalent N-acetylgalactosamine conjugate moieties are suitable for binding to ASGPR, see, for example, WO 2014 / 076196, WO 2014 / 207232 and WO 2014 / 179620 (which are incorporated herein by reference).Such conjugates enhance the uptake of oligonucleotides into the liver while reducing their presence in the kidney, thereby increasing the liver / kidney ratio of conjugated oligonucleotides compared to the unconjugated version of the same oligonucleotide.

[0148] Oligonucleotide conjugates and their synthesis are also reported in comprehensive reviews by Manoharan, Antisense Drug Technology, Principles, Strategies, and Applications, edited by S. T. Crooke, Chapter 16, Marcel Dekker, Inc. (2001) and Manoharan, Antisense and Nucleic Acid Drug Development (2002) Vol. 12, p. 103, each of which is incorporated herein by reference in its entirety.

[0149] In one embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophiles, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0150] linker Linkage or linker is a connection between two atoms that connects a chemical group or segment of interest to another chemical group or segment through one or more covalent bonds. The conjugate moiety can be attached to the oligonucleotide directly or through a linking moiety (e.g., linker or tether). The linker serves to covalently link a third region, e.g., the conjugate moiety (region C), to a first region, e.g., an oligonucleotide or a continuous nucleotide sequence that is complementary to the target nucleic acid (region A).

[0151] In some embodiments of the invention, the conjugate or oligonucleotide conjugate of the 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).

[0152] Region B refers to a biocleavable linker that comprises or consists of a physiologically labile bond that is cleavable under conditions normally or similar to those encountered 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 drugs, and salt concentrations similar to those found or encountered in mammalian cells. Mammalian intracellular conditions also include the presence of enzymatic activities normally present in mammalian cells, such as 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 comprises 1-10 nucleosides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleosides, more preferably 2-6 nucleosides, and most preferably 2-4 linked nucleosides, which comprise at least two consecutive phosphodiester bonds, such as at least 3 or 4 or 5 consecutive phosphodiester bonds. Preferably, the nucleosides are DNA or RNA. Phosphodiester-containing biocleavable linkers are described in more detail in WO 2014 / 076195, which is incorporated herein by reference.

[0153] Region Y refers to a linker that is not necessarily biocleavable, but serves primarily to covalently link the conjugate moiety (region C or third region) to the oligonucleotide (region A or first region). Region Y linkers may include chain structures or oligomers of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. The oligonucleotide conjugates of the present invention can be constructed from the following region elements AC, ABC, ABYC, AYBC, or AYC. In some embodiments, the linker (region Y) is an aminoalkyl, such as, for example, a C2-C36 aminoalkyl group, including, for example, a C6-C12 aminoalkyl group. In a preferred embodiment, the linker (region Y) is a C6 aminoalkyl group.

[0154] treatment The term "treatment" as used herein 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. It will therefore be appreciated that the treatment referred to herein may, in some embodiments, be prophylactic. Prevention may be understood as preventing HBV infection from transforming into chronic HBV infection, or preventing serious liver diseases, such as cirrhosis and hepatocellular carcinoma, due to chronic HBV infection.

[0155] prevention As used herein, the terms "preventing", "prevention" or "preventing" refer to prophylactic treatment, i.e., measures or means whose purpose is to prevent rather than cure a disease. Prevention means that the desired pharmacological and / or physiological effect is obtained prophylactically, in terms of completely or partially preventing a disease or its symptoms. Thus, "prevention of HBV infection" as used herein includes preventing the occurrence of HBV infection in a subject, and preventing the occurrence of symptoms of HBV infection. In particular, the present invention contemplates the prevention of HBV infection in children from mothers infected with HBV. It is also contemplated to prevent acute HBV infection from turning into chronic HBV infection.

[0156] 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. Thus, as used herein, a subject may be an animal, such as a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bovine species, horse, camel, or primate. Preferably, the subject is a mammal. More preferably, the subject is a human. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0157] HBV cccDNA in infected hepatocytes is involved in persistent chronic infection and reactivation, and is the template for all viral subgenomic transcripts and pregenomic RNA (pgRNA), ensuring both newly synthesized viral progeny and cccDNA pool replenishment via intracellular nucleocapsid recycling. In the context of the present invention, it has been shown for the first time that RTEL1 is associated with cccDNA stability. This knowledge provides an opportunity to destabilize cccDNA in HBV-infected subjects, opening the door to a complete cure for chronically infected HBV patients.

[0158] One aspect of the present invention is an RTEL1 inhibitor for the treatment and / or prevention of Hepatitis B virus (HBV) infection, particularly chronic HBV infection.

[0159] An RTEL1 inhibitor can be, for example, a small molecule that specifically binds to an RTEL1 protein, where the inhibitor prevents or reduces binding of the RTEL1 protein to cccDNA.

[0160] An embodiment of the present invention is an RTEL1 inhibitor that can reduce cccDNA and / or pgRNA in infected cells, such as HBV-infected cells.

[0161] In a further embodiment, RTEL1 inhibitors are capable of reducing HBsAg and / or HBeAg in vivo in HBV-infected individuals.

[0162] The oligonucleotides of the present invention Therapeutic oligonucleotides are potentially good RTEL1 inhibitors because they can target the RTEL1 transcript and promote its degradation either via the RNA interference pathway or RNaseH cleavage. Alternatively, oligonucleotides such as aptamers can also act as inhibitors of RTEL1 protein interactions.

[0163] One aspect of the present invention is an RTEL1-targeting oligonucleotide for the treatment and / or prevention of Hepatitis B Virus (HBV) infection. Such an oligonucleotide may be selected from the group consisting of a single-stranded antisense oligonucleotide, a siRNA molecule, or a shRNA molecule.

[0164] The present invention section describes novel oligonucleotides suitable for the treatment and / or prevention of Hepatitis B virus (HBV) infection.

[0165] The oligonucleotides of the invention are capable of inhibiting expression of RTEL1 in vitro and in vivo. Inhibition is achieved by hybridizing the oligonucleotide to a target nucleic acid that encodes RTEL1 or is involved in the regulation of RTEL1. The target nucleic acid can be a mammalian RTEL1 sequence, such as the sequences of SEQ ID NO: 1 and / or 2.

[0166] In some embodiments, the oligonucleotides of the invention can modulate the expression of a target by inhibiting or downregulating it. Preferably, such modulation results in at least 20% inhibition compared to the normal expression level of the target, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition compared to the normal expression level of the target. In some embodiments, the oligonucleotides of the invention can inhibit the expression level of RTEL1 mRNA by at least 60% or 70% in vitro using 10 μM in PXB-PHH cells. In some embodiments, the oligonucleotides of the invention can inhibit the expression level of RTEL1 protein by at least 50% in vitro using 10 μM PXB-PHH cells, this range of target reduction being advantageous in terms of selecting nucleic acid molecules that have a good correlation with cccDNA reduction. Advantageously, the examples provide assays that can be used to measure RTEL1 RNA or protein inhibition (e.g., Example 1). Target inhibition is caused by hybridization between a continuous nucleotide sequence of the oligonucleotide and the target nucleic acid. In some embodiments, the oligonucleotide of the present invention comprises a mismatch between the oligonucleotide and the target nucleic acid. Despite the mismatch, hybridization to the target nucleic acid may still be sufficient to show the desired inhibition of RTEL1 expression. The decrease in binding affinity resulting from the mismatch may be advantageously compensated for by increasing the number of nucleotides in the oligonucleotide and / or the number of modified nucleosides, including LNA, present in the oligonucleotide sequence, which can increase the binding affinity to the target.

[0167] One aspect of the invention relates to oligonucleotides, 12-60 nucleotides in length, that comprise a contiguous nucleotide sequence at least 10 nucleotides in length, e.g., at least 12-30 nucleotides in length, and that are at least 95% complementary, e.g., fully complementary, to a mammalian RTEL1 target nucleic acid, in particular a human RTEL1 nucleic acid. These oligonucleotides are capable of inhibiting expression of RTEL1.

[0168] One aspect of the present invention relates to an oligonucleotide according to the present invention, which is an antisense oligonucleotide of 12 to 30 nucleotides in length and comprises a continuous nucleotide sequence of at least 10 nucleotides in length, for example 10 to 30 nucleotides in length, that is at least 90% complementary, for example completely complementary, to mammalian RTEL1.

[0169] A further aspect of the present invention relates to an oligonucleotide according to the present invention comprising a contiguous nucleotide sequence of 12 to 20, such as 15 to 22 nucleotides in length, which has at least 90% complementarity, such as being completely complementary, to a target nucleic acid of SEQ ID NO:1.

[0170] In some embodiments the oligonucleotide comprises a contiguous sequence of 10-30 nucleotides in length which 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%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, for example at least 97%, such as at least 98%, or 100% complementary.

[0171] It may be advantageous if the oligonucleotide of the invention, or its contiguous nucleotide sequence, is fully complementary (100% complementary) to a region of the target nucleic acid, or in some embodiments may contain one or two mismatches between the oligonucleotide and the target nucleic acid.

[0172] In some embodiments, the antisense oligonucleotide sequence is 100% complementary to the corresponding target nucleic acid region of SEQ ID NO:1.

[0173] In some embodiments, the oligonucleotide or contiguous nucleotide sequence of the invention is at least 95% complementary, such as completely (or 100%) complementary, to the target nucleic acids of SEQ ID NO:1 and SEQ ID NO:2.

[0174] In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence of 15-22 nucleotides in length having at least 90% complementarity, e.g., 100% complementarity, to a corresponding target sequence present in SEQ ID NO:1, where the target sequence is selected from the group consisting of SEQ ID NOs:3-21 (Table 4) or regions 1A-959A of Table 5A.

[0175] [Table 5A-1] [Table 5A-2] [Table 5A-3] [Table 5A-4] [Table 5A-5] [Table 5A-6] [Table 5A-7] [Table 5A-8]

[0176] In some embodiments, the oligonucleotide comprises a contiguous nucleotide sequence of 16-20, e.g., 15-22 nucleotides in length having at least 90% complementarity, e.g., 100% complementarity, to a corresponding target sequence present in SEQ ID NO:1, wherein the target sequence is selected from the group consisting of SEQ ID NOs:3-21 (Table 4) or regions B1-B28 of Table 5B.

[0177] [Table 5B]

[0178] In some embodiments, the oligonucleotide 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 20 contiguous nucleotides, in preferred embodiments, the oligonucleotide comprises or consists of a length of 15, 16, 17, 18, 19, or 20 nucleotides.

[0179] In some embodiments the contiguous nucleotide sequence of the oligonucleotide that is complementary to the target nucleic acid comprises or consists of a length of 12-30, such as 13-25, such as 15-23, such as 16-22 contiguous nucleotides.

[0180] In some embodiments, the contiguous nucleotide sequence of the siRNA or shRNA that is complementary to the target nucleic acid comprises or consists of a length of 18-28, such as 19-26, such as 20-24, such as 21-23 contiguous nucleotides.

[0181] In some embodiments, the contiguous nucleotide sequence of a single-stranded antisense oligonucleotide complementary to a target nucleic acid comprises or consists of a length of 12-22, such as 14-20, for example 16-20, such as 15-21, for example 15-18, such as 16-18, for example 16-17 contiguous nucleotides.

[0182] In some embodiments, the oligonucleotide or contiguous nucleotide sequence comprises or consists of a sequence selected from the group consisting of the sequences listed in Table 6 (Materials and Methods section).

[0183] In some embodiments, the oligonucleotide or contiguous nucleotide sequence comprises or consists of 10-30 nucleotides in length having at least 90%, preferably 100%, identity to a sequence selected from the group consisting of SEQ ID NOs: 22-237 (see motif sequences listed in Table 6). In certain embodiments, the oligonucleotide or contiguous nucleotide sequence is selected from SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 32, 35, 36, 37, 38, 39, 40, 41, 42, 42, 42, 43, 43, 46, 49, 83, 109, 130, 203, and 232.

[0184] 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.

[0185] 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.

[0186] The oligonucleotides of the invention can be designed using modified nucleosides and RNA nucleosides (particularly siRNA and shRNA molecules) or DNA nucleosides (particularly single-stranded antisense oligonucleotides). It is advantageous to use high affinity modified nucleosides.

[0187] In one embodiment, the oligonucleotide comprises at least one modified nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 modified nucleosides. In one embodiment, the oligonucleotide comprises 1-10 modified nucleosides, such as 2-9 modified nucleosides, such as 3-8 modified nucleosides, such as 4-7 modified nucleosides, such as 6 or 7 modified nucleosides. Suitable modifications are described in the "Definitions" sections of "Modified Nucleosides", "High Affinity Modified Nucleosides", "Sugar Modifications", "2' Sugar Modifications", and Locked Nucleic Acid (LNA).

[0188] In one embodiment, the oligonucleotide comprises one or more sugar-modified nucleosides, such as 2' sugar-modified nucleosides.Preferably, the oligonucleotide of the present invention comprises 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 nucleosides is a locked nucleic acid (LNA).

[0189] In further embodiments, the oligonucleotide comprises at least one modified internucleoside bond. Suitable internucleoside bonds are described in the "Definitions" section under "Modified Internucleoside Bonds". It is advantageous if at least 2-3 internucleoside bonds at the 5'-end or 3'-end of the oligonucleotide are phosphorothioate internucleoside bonds. For single-stranded antisense oligonucleotides, it is advantageous if at least 75%, such as all, of the internucleoside bonds in the contiguous nucleotide sequence are phosphorothioate bonds. In some embodiments, all internucleoside bonds in the contiguous sequence of the single-stranded antisense oligonucleotide are phosphorothioate bonds.

[0190] In some embodiments, the oligonucleotide of the invention comprises at least one LNA nucleoside, such as 1, 2, 3, 4, 5, 6, 7 or 8 LNA nucleosides, such as 2-6 LNA nucleosides, such as 3-7 LNA nucleosides, 4-8 LNA nucleosides, or 3, 4, 5, 6, 7 or 8 LNA nucleosides. In some embodiments, at least 75% of the modified nucleosides of the oligonucleotide are LNA nucleosides, such as 80%, such as 85%, such as 90% of the modified nucleosides. In still further embodiments, all modified nucleosides of the oligonucleotide are LNA nucleosides. In further embodiments, the oligonucleotide may comprise both β-D-oxy-LNA and one or more of the following LNA nucleosides: thio-LNA in either β-D or α-L conformation, amino-LNA, oxy-LNA, ScET and / or ENA, or a combination thereof. In a further embodiment, all LNA cytosine units are 5-methyl-cytosine. For nuclease stability of an oligonucleotide or a contiguous nucleotide sequence, it is advantageous to have at least one LNA nucleoside at the 5'-end and at least two LNA nucleosides at the 3'-end of the nucleotide sequence.

[0191] In one embodiment of the invention, the oligonucleotide of the invention is capable of recruiting RNase H.

[0192] In the present invention, advantageous structural designs are the gapmer designs described in the "Definitions" section, for example "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. In some embodiments, the gapmer is an LNA gapmer with uniform flanks.

[0193] In some embodiments of the invention, the LNA gapmers are selected from the following uniform flank designs: 2-12-3, 4-14-2, 3-10-3, 3-9-3, 2-15-2, 2-12-4, 1-13-2, 3-13-2, 4-13-2, 2-12-2, 3-12-2, 3-15-2, 3-14-2, 3-13-3, 2-14-4, 3-12-3, 1-14-3, 3-14-3, 2-14-3, 2-15-3, 3-11-3, 1-12-3, 1-11-4, 1-13-2, 2-13-2, 2-16-2, 1-14-2, 1-17-3, and 1-18-2.

[0194] Table 6 (Materials and Methods section) lists the preferred designs for each motif sequence.

[0195] In all cases, the FG-F' design may further comprise a region D' and / or a region D'' as described in the "Definitions" section under "Region D' or Region D'' in an Oligonucleotide". In some embodiments, the oligonucleotides of the invention have one, two, or three phosphodiester-linked nucleoside units, e.g., DNA units, at the 5' or 3' end of the gapmer region. In some embodiments, the oligonucleotides of the invention consist of two 5' phosphodiester-linked DNA nucleosides followed by an FG-F' gapmer region as defined in the "Definitions" section. Oligonucleotides comprising phosphodiester-linked DNA units at the 5' or 3' end are suitable for conjugation and may further comprise a conjugate moiety as described herein. For delivery to the liver, ASGPR targeting moieties are particularly advantageous as conjugate moieties.

[0196] For some embodiments of the invention, the oligonucleotide is selected from the group of oligonucleotide compounds having CMP numbers 22_1, 23_1, 24_1, 25_1, 26_1, 27_1, 28_1, 29_1, 30_1, 31_1, 32_1, 33_1, 34_1, 35_1, 36_1, 37_1, 38_1, 39_1, 40_1, 41_1, 42_1, 42_2, 42_3, 43_1, 43_2, 44_1, 45_1, 46_1, 47_1, 48_1, 49_1, 130_1, 109_1, 83_1, 203_1, and 232_1 (see Table 6).

[0197] Conjugates Since HBV infection primarily affects hepatocytes in the liver, it is advantageous to conjugate RTEL1 inhibitors to increase delivery of the inhibitor to the liver compared to unconjugated inhibitors. In one embodiment, the liver targeting moiety is selected from a moiety that contains cholesterol or other lipids, or a conjugate moiety that can bind to the asialoglycoprotein receptor (ASGPR).

[0198] In some embodiments, the invention provides a conjugate comprising a nucleic acid molecule of the invention covalently attached to a conjugate moiety.

[0199] Asialoglycoprotein receptor (ASGPR) conjugate moieties include one or more carbohydrate moieties capable of binding to the asialoglycoprotein receptor (ASGPR targeting moiety) with an affinity equal to or greater than that of galactose. The affinity of a number of galactose derivatives for the asialoglycoprotein receptor has been studied (see, e.g., Jobst, "ST and Drickamer", K. J. B. C. (1996) 271:6686) or is readily determined using methods typical in the art.

[0200] 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).

[0201] To generate the ASGPR conjugate moiety, an ASGPR targeting moiety (preferably GalNAc) can be attached to the conjugate scaffold. In general, the ASGPR targeting moiety can be at the same end of the scaffold. In one embodiment, the conjugate moiety consists of 2-4 terminal GalNAc moieties attached to a spacer that connects each GalNAc moiety to a brancher molecule that can be attached to an antisense oligonucleotide.

[0202] 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.

[0203] GalNAc conjugate moieties can include, for example, those described in WO 2014 / 179620 and WO 2016 / 055601 and PCT / EP2017 / 059080, which are incorporated by reference herein, 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, vol. 313, p. 297); lysine-based galactose clusters (e.g., L3G4; Biessen et al., Cardovasc. Med., 1999, vol. 214); and cholan-based galactose clusters (e.g., carbohydrate recognition motifs for the asialoglycoprotein receptor).

[0204] The ASGPR conjugate moiety, particularly the trivalent GalNAc conjugate moiety, can be attached to the 3' or 5' end of the oligonucleotide using methods known in the art. In one embodiment, the ASGPR conjugate moiety is attached to the 5' end of the oligonucleotide.

[0205] In one embodiment, the conjugate moiety is a trivalent N-acetylgalactosamine (GalNAc), as shown in FIG. 1, and particularly FIG. 1D.

[0206] Manufacturing method In a further aspect, the invention provides a method for making an oligonucleotide of the invention, comprising reacting nucleotide units to thereby form covalently linked contiguous nucleotide units comprising an oligonucleotide. Preferably, the method uses phosphoramidite chemistry (see, e.g., Caruthers et al. (1987) Methods in Enzymology vol. 154, pp. 287-313). In a further embodiment, the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently link the conjugated moiety to the oligonucleotide. In a further aspect, a method for making a composition of the invention is provided, comprising mixing an oligonucleotide or conjugated oligonucleotide of the invention with a pharma- ceutically acceptable diluent, solvent, carrier, salt, and / or adjuvant.

[0207] Pharmaceutical Salts The compounds of the present invention may exist in the form of their pharma- ceutically acceptable salts. The term "pharma- ceutically acceptable salts" refers to conventional acid or base addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid addition salts include, for example, those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, and fumaric acid. Base addition salts include those derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides, such as tetramethylammonium hydroxide. Chemical modification of pharmaceutical compounds into salts is a technique well known to pharmacists to improve the physical and chemical stability, hygroscopicity, flowability, and solubility of the compounds. This is described, for example, in Bastin, "Organic Process Research & Development" (2000) Vol. 4, pp. 427-435, or Ansel, "In: Pharmaceutical Dosage Forms and Drug Delivery Systems" 6th Edition (1995) pp. 196 and 1456-1457. For example, a pharma- ceutically acceptable salt of the compounds provided herein can be a sodium salt.

[0208] In a further aspect, the present invention provides a pharma- ceutically acceptable salt of the antisense oligonucleotide or conjugate thereof. In a preferred embodiment, the pharma- ceutically acceptable salt is a sodium or potassium salt.

[0209] Pharmaceutical Compositions In a further aspect, the present invention provides a pharmaceutical composition comprising any of the aforementioned oligonucleotides and / or oligonucleotide conjugates or salts thereof, and a pharma- ceutically acceptable diluent, carrier, salt and / or adjuvant. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS) and pharma- ceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharma- ceutically acceptable diluent is sterile phosphate-buffered saline. In some embodiments, the oligonucleotide is used in the pharma- ceutically acceptable diluent at a concentration of 50-300 μM solution.

[0210] Suitable formulations for use in the present invention can be found in "Remington's Pharmaceutical Sciences", Mack Publishing Company, Philadelphia, PA, 17th Edition (1985). For a brief review of methods of drug delivery, see, for example, Langer (Science, vol. 249, pp. 1527-1533 (1990)). WO 2007 / 031091 provides further suitable and preferred examples of pharma-ceutically acceptable diluents, carriers and adjuvants (herein incorporated by reference). Suitable dosages, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, and prodrug formulations are also provided in WO 2007 / 031091.

[0211] In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention, or a pharma- ceutically acceptable salt thereof, is in a solid form, such as a powder, e.g., a lyophilized powder.

[0212] The compounds, oligonucleotides, or oligonucleotide conjugates of the present invention may be mixed with pharma- ceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. The composition and method for the formulation of 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 may be sterile and filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation will typically be 3-11, more preferably 5-9 or 6-8, most preferably 7-8, e.g., 7-7.5. The resulting solid form compositions may be packaged in a plurality of single dose units, each containing a fixed amount of the agent or agents, such as a sealed package of tablets or capsules. The solid form compositions may also be packaged in flexible volume containers, such as squeezable tubes designed for topically applicable creams or ointments.

[0214] In some embodiments, the oligonucleotide or oligonucleotide conjugate of the invention is a prodrug. Particularly with respect to oligonucleotide conjugates, when the prodrug is delivered to the site of action, e.g., a target cell, the conjugate moiety is cleaved from the oligonucleotide.

[0215] Administration The compounds, oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions of the invention may be administered topically (such as to the skin, inhalation, eye, or ear), enterally (orally or through the digestive tract), or parenterally (such as intravenously, subcutaneously, intramuscularly, intracerebrally, intraventricularly, or intrathecally).

[0216] In a preferred embodiment, the oligonucleotide or pharmaceutical composition of the present invention is administered by parenteral routes, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intracerebral or intraventricular, intravitreal administration. In one embodiment, the active oligonucleotide or oligonucleotide conjugate is administered intravenously. In another embodiment, the active oligonucleotide or oligonucleotide conjugate is administered subcutaneously.

[0217] In some embodiments, the oligonucleotide, oligonucleotide conjugate, or pharmaceutical composition of the invention is administered at a dose of 0.1-15 mg / kg, such as 0.2-10 mg / kg, such as 0.25-5 mg / kg, which may be administered weekly, biweekly, triweekly, or monthly.

[0218] The present invention also provides the use of an oligonucleotide or oligonucleotide conjugate of the 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, such as compounds, oligonucleotides, oligonucleotide conjugates, or pharmaceutical compositions of the invention are for use in combination therapy with another therapeutic agent, which may be, for example, a standard treatment for the disease or disorder described above.

[0220] By way of example, the oligomers or oligomer conjugates of the invention can be used in combination with other active agents, such as oligonucleotide-based antiviral agents, e.g., sequence-specific oligonucleotide-based antiviral agents, 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, the oligomer or oligomer conjugate of the invention can be used in combination with other active agents, such as interferons (e.g., pegylated interferon alpha), TLR7 agonists (e.g., GS-9620), or immunostimulatory antiviral compounds, such as therapeutic vaccines.

[0222] As a further example, the oligomers or oligomer conjugates of the invention can be used in combination with other active agents, such as small molecules, that have antiviral activity. These other active agents can be, for example, nucleoside / nucleotide inhibitors (e.g., entecavir or tenofovir disoproxil fumarate), inclusion inhibitors, 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, an antidiarrheal agent, or an immunosuppressant agent.

[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 antibody therapy or HCV polyclonal antibody therapy.

[0226] Purpose The oligonucleotides of the invention can be utilized, for example, as research reagents for diagnosis, therapy and prophylaxis.

[0227] In research, such oligonucleotides can be used to specifically regulate the synthesis of RTEL1 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 a modulator of the gene or mRNA that produces the protein.

[0228] For research or diagnostic uses of the oligonucleotides of the invention, the target nucleic acid can be cDNA or synthetic nucleic acid derived from DNA or RNA.

[0229] The present invention also includes an in vivo or in vitro method for modulating RTEL1 expression in a target cell expressing RTEL1, comprising administering to the cell an effective amount of an oligonucleotide, 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 a 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 the oligonucleotide, the conjugated compound, or the pharmaceutical composition of the present invention for use as a medicament.

[0232] In one aspect of the present invention, the oligonucleotide, conjugate compound or pharmaceutical composition of the present invention can reduce cccDNA levels in infected cells and thus inhibit HBV infection. In particular, the antisense oligonucleotide can affect one or more of the following parameters in infected cells: (i) reduction of cccDNA, and / or (ii) reduction of pgRNA, and / or (iii) reduction of HBV DNA, and / or (iv) reduction of HBV viral antigens.

[0233] For example, a nucleic acid molecule that inhibits HBV infection can (i) reduce cccDNA levels in infected cells by at least 40%, e.g., 50%, 60%, 70%, 80%, or 90%, as compared to a control, or (ii) reduce pgRNA levels by at least 40%, e.g., 50%, 60%, 70%, 80%, or 90%, as compared to a control. The control can be untreated cells or animals, or cells or animals treated with an appropriate control.

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

[0235] By reducing RTEL1 levels, the onset or treatment of HBV infection can be inhibited using the oligonucleotides, conjugated compounds, or pharmaceutical compositions of the present invention. In particular, the destabilization and reduction of cccDNA, oligonucleotides, conjugated compounds, or pharmaceutical compositions of the present invention more efficiently inhibits or treats the onset of chronic HBV infection compared to compounds that only reduce the secretion of HBsAg.

[0236] Thus, one aspect of the present invention relates to the use of an oligonucleotide, a conjugated compound, or a pharmaceutical composition of the present invention for reducing cccDNA and / or pgRNA in an HBV-infected individual.

[0237] A further aspect of the invention relates to the use of an oligonucleotide, a conjugated compound or a pharmaceutical composition of the invention for inhibiting or treating the development of chronic HBV infection.

[0238] A further aspect of the invention relates to the use of the oligonucleotide, conjugated compound or pharmaceutical composition of the invention to reduce infectivity in an HBV-infected individual. In a particular aspect of the invention, the oligonucleotide, conjugated compound or pharmaceutical composition of the invention inhibits the development of chronic HBV infection.

[0239] The subject to be treated with the oligonucleotide, conjugate compound, or pharmaceutical composition of the invention (or which prophylactically receives the antisense oligonucleotide, conjugate compound, or pharmaceutical composition of the invention) is preferably a human, more preferably an HBsAg-positive and / or HBeAg-positive human patient, more preferably an HBsAg-positive and HBeAg-positive human patient.

[0240] Therefore, the present invention relates to a method for treating HBV infection comprising administering an effective amount of an oligonucleotide, a conjugated compound, or a pharmaceutical composition of the present invention.The present invention further relates to a method for preventing cirrhosis and hepatocellular carcinoma resulting from chronic HBV infection.

[0241] The present invention also provides the use of an oligonucleotide, a conjugated compound or a pharmaceutical composition for the manufacture of a medicament, in particular a medicament for use in the treatment of HBV infection or chronic HBV infection or for reducing the infectivity of an HBV-infected individual. In a preferred embodiment, the medicament is prepared in a dosage form for subcutaneous administration.

[0242] The present invention also provides the use of the oligonucleotide, conjugate compound, pharmaceutical composition of the present invention for the manufacture of a medicament, wherein the medicament is in a dosage form for intravenous administration.

[0243] The oligonucleotide, conjugate, or pharmaceutical composition of the present invention may be used in combination therapy. For example, the oligonucleotide, conjugate, or pharmaceutical composition of the present invention may be used in combination therapy with other anti-HBV agents, such as interferon alpha-2b, interferon alpha-2a, and interferon alfacon-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. It may also be combined with other anti-HBV agents such as 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, WO 2013 / 003520, WO 2013 / 159109, WO 2017 / 027350, and WO 2017 / 015175), HBV therapeutic vaccines, HBV prophylactic vaccines, HBV antibody therapy (monoclonal or polyclonal), or TLR2, 3, 7, 8, or 9 agonists.

[0244] EMBODIMENTS OF THE PRESENT DISCLOSURE The following embodiments of the invention may be used in combination with any other embodiments described herein.

[0245] 1. RTEL1 inhibitors for the treatment and / or prevention of Hepatitis B virus (HBV) infection.

[0246] 2. An RTEL1 inhibitor as described for use in embodiment 1, wherein the RTEL1 inhibitor is administered in an effective amount.

[0247] 3. An RTEL1 inhibitor as described for use in embodiment 1 or 2, wherein the HBV infection is a chronic infection.

[0248] 4. An RTEL1 inhibitor whose use is described in embodiments 1 to 3, wherein the RTEL1 inhibitor is capable of reducing cccDNA and / or pgRNA in infected cells.

[0249] 5. An RTEL1 inhibitor whose use is described in any one of embodiments 1 to 4, wherein the RTEL1 inhibitor prevents or reduces the binding of RTEL1 to DNA, such as cccDNA.

[0250] 6. An RTEL1 inhibitor whose use is described in embodiment 5, wherein the inhibitor is a small molecule that specifically binds to the RTEL1 protein, wherein the inhibitor prevents or reduces binding of the RTEL1 protein to cccDNA.

[0251] 7. An RTEL1 inhibitor whose use is described in any one of embodiments 1 to 5, wherein the inhibitor is an oligonucleotide of 12 to 60 nucleotides in length comprising or consisting of a contiguous nucleotide sequence of at least 10 nucleotides in length that is at least 90% complementary to a mammalian RTEL1 target nucleic acid.

[0252] 8. An RTEL1 inhibitor whose use is described in embodiment 7, capable of reducing the level of said RTEL1 target nucleic acid.

[0253] 9. An RTEL1 inhibitor as described for use in embodiment 7 or 8, wherein the target nucleic acid is RNA.

[0254] 10. The RTEL1 inhibitor as described for use in embodiment 9, wherein the RNA is pre-mRNA.

[0255] 11. An RTEL1 inhibitor according to any one of embodiments 7 to 10, wherein the oligonucleotide is selected from an antisense oligonucleotide, an siRNA, or an shRNA.

[0256] 12. The RTEL1 inhibitor as described in embodiment 11 for use, wherein the oligonucleotide is a single-stranded antisense oligonucleotide or a double-stranded siRNA.

[0257] 13. An RTEL1 inhibitor whose use is described in any one of embodiments 7 to 12, wherein the mammalian RTEL1 target nucleic acid is selected from SEQ ID NO: 1 or 2.

[0258] 14. An RTEL1 inhibitor for use as described in any one of embodiments 7 to 12, wherein the contiguous nucleotide sequence of the oligonucleotide is at least 98% complementary to the target nucleic acids of SEQ ID NO:1 and SEQ ID NO:2.

[0259] 15. An RTEL1 inhibitor as described in any one of embodiments 1 to 14 for use, wherein the cccDNA in HBV-infected cells is reduced by at least 50%, such as 60%, for example 70%, such as 80%, for example 90%, for example 95%, for example 100% compared to a control.

[0260] 16. An oligonucleotide for use as described in any one of embodiments 7 to 15, wherein the RTEL1 mRNA is reduced by at least 50%, such as 60%, for example 70%, for example 80%, such as 90%, for example 95%, for example 100% compared to a control.

[0261] 17. An oligonucleotide 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, e.g., 95%, e.g., 98% completely complementary, to a mammalian RTEL1 target nucleic acid.

[0262] 18. The oligonucleotide of embodiment 17, wherein the oligonucleotide is chemically produced.

[0263] 19. The oligonucleotide of embodiments 17-18, wherein the mammalian RTEL1 target nucleic acid is selected from SEQ ID NO: 1 or 2.

[0264] 20. The oligonucleotide according to embodiments 17-18, wherein the contiguous nucleotide sequence is at least 98% complementary to the target nucleic acid of SEQ ID NO:1 and SEQ ID NO:2.

[0265] 21. The oligonucleotide according to any one of embodiments 17 to 20, wherein the oligonucleotide is 12 to 30 nucleotides in length.

[0266] 22. The oligonucleotide according to any one of embodiments 17 to 21, wherein the oligonucleotide is an RNAi molecule, such as a double-stranded siRNA or shRNA.

[0267] 23. The oligonucleotide according to any one of embodiments 17 to 21, wherein the oligonucleotide is a single-stranded antisense oligonucleotide.

[0268] 24. The oligonucleotide according to any one of embodiments 17 to 23, wherein the contiguous nucleotide sequence is complementary to a target sequence selected from SEQ ID NOs: 3 to 21 (Table 4).

[0269] 25. The oligonucleotide according to any one of embodiments 17 to 24, 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.

[0270] 26. The oligonucleotide according to any one of embodiments 17 to 25, 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.

[0271] 27. The oligonucleotide according to any one of embodiments 17 to 25, wherein the contiguous nucleotide sequence comprises or consists of 14 to 22 nucleotides.

[0272] 28. The oligonucleotide according to embodiment 27, wherein the contiguous nucleotide sequence comprises or consists of 16 to 20 nucleotides.

[0273] 29. The oligonucleotide according to any one of embodiments 17 to 28, wherein the oligonucleotide comprises or consists of a length of 14 to 25 nucleotides.

[0274] 30. The oligonucleotide according to embodiment 29, wherein the oligonucleotide comprises or consists of a length of 16 to 22 nucleotides.

[0275] 31. The nucleotide according to any one of embodiments 17 to 30, wherein the oligonucleotide comprises a sequence selected from SEQ ID NOs: 22 to 237.

[0276] 32. The oligonucleotide according to any one of embodiments 17 to 31, wherein the contiguous nucleotide sequence has 0 to 3 mismatches compared to the target nucleic acid to which it is complementary.

[0277] 33. The oligonucleotide according to embodiment 32, wherein the contiguous nucleotide sequence has one mismatch compared to the target nucleic acid.

[0278] 34. The oligonucleotide according to embodiment 32, wherein the contiguous nucleotide sequence has two mismatches compared to the target nucleic acid.

[0279] 35. The oligonucleotide according to embodiment 32, wherein the contiguous nucleotide sequences are perfectly complementary to both of the target nucleic acid sequences.

[0280] 36. The oligonucleotide according to any one of embodiments 17 to 35, comprising one or more modified nucleosides.

[0281] 37. The oligonucleotide of embodiment 36, wherein the one or more modified nucleosides are high-affinity modified nucleosides.

[0282] 38. The oligonucleotide according to embodiment 36 or 37, wherein the one or more modified nucleosides are 2' sugar modified nucleosides.

[0283] 39. The oligonucleotide of embodiment 38, 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.

[0284] 40. The oligonucleotide of embodiments 36-39, wherein the one or more modified nucleosides are LNA nucleosides.

[0285] 41. The oligonucleotide of embodiment 40, wherein the modified LNA nucleoside is selected from oxy-LNA, amino-LNA, thio-LNA, cET, and ENA.

[0286] 42. The oligonucleotide of embodiment 40 or 41, wherein the modified LNA nucleoside is oxy-LNA having the following 2'-4' bridge -O-CH2-.

[0287] 43. The oligonucleotide according to embodiment 42, wherein said oxy-LNA is β-D-oxy-LNA.

[0288] 44. The oligonucleotide of embodiment 40 or 41, wherein the modified LNA nucleoside is cET having the following 2'-4' bridge: -O-CH(CH3)-.

[0289] 45. The oligonucleotide of embodiment 44, wherein said cET is (S)cET, i.e. 6'(S)methyl-β-D-oxy-LNA.

[0290] 46. ​​The oligonucleotide according to embodiment 40 or 41, wherein said LNA is an ENA having the following 2'-4' bridge -O-CH2-CH2-.

[0291] 47. The oligonucleotide according to any one of embodiments 17 to 46, wherein the nucleotide comprises at least one modified internucleoside bond.

[0292] 48. The oligonucleotide according to embodiment 47, wherein the modified internucleoside linkages are nuclease resistant.

[0293] 49. The oligonucleotide according to embodiment 47 or 48, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[0294] 50. The oligonucleotide according to any one of embodiments 17 to 49, wherein the oligonucleotide is an antisense oligonucleotide capable of recruiting RNase H.

[0295] 51. The antisense oligonucleotide according to embodiment 50, wherein the antisense oligonucleotide or the contiguous nucleotide sequence is a gapmer.

[0296] 52. The antisense oligonucleotide according to embodiment 51, wherein the antisense oligonucleotide or its contiguous nucleotide sequence consists of or comprises a gapmer of the formula 5'-FG-F'-3', in which regions F and F' independently comprise or consist of 1 to 4 2' sugar modified nucleosides and G is a region of 6 to 18 nucleosides capable of recruiting RNase H.

[0297] 53. The antisense oligonucleotide of embodiment 52, wherein the 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.

[0298] 54. The antisense oligonucleotide of embodiment 52 or 53, wherein one or more of the 2' sugar modified nucleosides within region F and region F' is an LNA nucleoside.

[0299] 55. The antisense oligonucleotide of embodiment 54, wherein all of the 2' sugar modified nucleosides within region F and region F' are LNA nucleosides.

[0300] 56. The oligonucleotide of embodiment 53 or 55, wherein the LNA nucleoside is selected from β-D-oxy-LNA, α-L-oxy-LNA, β-D-amino-LNA, α-L-amino-LNA, β-D-thio-LNA, α-L-thio-LNA, (S)cET, (R)cET, β-D-ENA, and α-L-ENA.

[0301] 57. The antisense oligonucleotide according to embodiments 53 to 56, wherein region F and region F' consist of the same LNA nucleosides.

[0302] 58. The antisense oligonucleotide according to embodiments 53 to 57, wherein all of the 2' sugar modified nucleosides within region F and region F' are oxy-LNA nucleosides.

[0303] 59. The antisense oligonucleotide according to any one of embodiments 52 to 58, wherein the nucleosides within region G are DNA and / or α-L-LNA nucleosides.

[0304] 60. The antisense oligonucleotide of embodiment 59, wherein region G consists of at least 75% DNA nucleosides.

[0305] 61. The antisense oligonucleotide of embodiment 60, wherein all of the nucleosides within region G are DNA nucleosides.

[0306] 62. The oligonucleotide according to any one of embodiments 17 to 62, wherein the oligonucleotide is selected from CMP numbers 22_1, 23_1, 24_1, 25_1, 26_1, 27_1, 28_1, 29_1, 30_1, 31_1, 32_1, 33_1, 34_1, 35_1, 36_1, 37_1, 38_1, 39_1, 40_1, 41_1, 42_1, 42_2, 42_3, 43_1, 43_2, 44_1, 45_1, 46_1, 49_1, 130_1, 109_1, 83_1, 203_1, and 232_1, or a pharma- ceutically acceptable salt thereof.

[0307] 63. A conjugate compound comprising an oligonucleotide according to any one of embodiments 17 to 50 or an antisense oligonucleotide according to any one of embodiments 51 to 62, and at least one conjugate moiety covalently bonded to the antisense oligonucleotide.

[0308] 64. The conjugate compound according to embodiment 63, wherein the oligonucleotide is a double-stranded siRNA and the conjugate moiety is covalently linked to the sense strand of the siRNA.

[0309] 65. The conjugate compound according to embodiment 63 or 64, 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.

[0310] 66. The conjugate compound according to any one of embodiments 63 to 65, wherein the conjugate moiety is capable of binding to an asialoglycoprotein receptor.

[0311] 67. The conjugate compound according to embodiment 66, 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.

[0312] 68. The conjugated compound according to embodiment 67, wherein the asialoglycoprotein receptor targeting moiety is N-acetylgalactosamine (GalNAc).

[0313] 69. The conjugate compound of embodiment 67 or 68, wherein the conjugate moiety is monovalent, bivalent, trivalent, or tetravalent with respect to the asialoglycoprotein receptor targeting moiety.

[0314] 70. The conjugate compound according to embodiment 69, wherein the conjugate moiety consists of two to four terminal GalNAc moieties and a spacer that connects each GalNAc moiety to a brancher molecule that can be conjugated to the antisense compound.

[0315] 71. The conjugate compound according to embodiment 70, wherein the spacer is a PEG spacer.

[0316] 72. The conjugate compound according to embodiments 66-71, wherein the conjugate moiety is a trivalent N-acetylgalactosamine (GalNAc) moiety.

[0317] 73. The conjugate compound according to embodiments 66-72, wherein the conjugate moiety is selected from one of the trivalent GalNAc moieties in Figure 1.

[0318] 74. The conjugate compound according to embodiment 73, wherein the conjugate moiety is a trivalent GalNAc moiety of FIG. 1D.

[0319] 75. The conjugate compound of embodiments 63-74, comprising a linker located between the oligonucleotide or the antisense oligonucleotide and the conjugate moiety.

[0320] 76. The conjugate compound according to embodiment 75, wherein the linker is a physiologically labile linker.

[0321] 77. The conjugate compound according to embodiment 76, wherein the physiologically labile linker is a nuclease-sensitive linker.

[0322] 78. The oligonucleotide conjugate of embodiment 76 or 77, wherein the physiologically labile linker consists of 2 to 5 consecutive phosphodiester bonds.

[0323] 79. A conjugated compound according to embodiments 66 to 78, which exhibits improved cellular distribution between the liver and the kidney, or improved cellular uptake of the conjugated compound into the liver, compared to an unconjugated oligonucleotide or antisense oligonucleotide.

[0324] 80. A pharmaceutical composition comprising an oligonucleotide according to any one of embodiments 17 to 50 or an antisense oligonucleotide according to any one of embodiments 51 to 61, a conjugate compound according to embodiments 63 to 79 or an acceptable salt thereof, and a pharma- ceutically acceptable diluent, carrier, salt and / or adjuvant. 81. A method for identifying a compound that prevents, ameliorates, and / or inhibits Hepatitis B Virus (HBV) infection, comprising: a. Test compound i. an RTEL1 polypeptide; or ii. contacting with a cell expressing RTEL1; and b. measuring the expression and / or activity of RTEL1 in the presence and absence of the test compound; and c. identifying a compound that reduces the expression and / or activity of RTEL1 and reduces cccDNA.

[0325] 82. An in vitro or in vivo method for regulating RTEL1 expression in a target cell expressing RTEL1, comprising administering to the cell an effective amount of an oligonucleotide described in any one of embodiments 17 to 50, or an antisense oligonucleotide described in any one of embodiments 51 to 61, a conjugate compound described in embodiments 63 to 79, or a pharmaceutical composition described in embodiment 80.

[0326] 83. The method of embodiment 82, wherein the RTEL1 expression is reduced in the target cells by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, compared to levels in the target cells treated with no treatment or a control.

[0327] 84. The method of embodiment 82, wherein the target cells are infected with HBV and cccDNA in the HBV-infected cells is reduced by at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95% in the HBV-infected target cells compared to levels in the HBV-infected target cells treated with no treatment or a control.

[0328] 85. A method for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of an oligonucleotide according to any one of embodiments 17 to 50, or an antisense oligonucleotide according to any one of examples 51 to 61, a conjugate compound according to embodiments 63 to 79, or a pharmaceutical composition according to embodiment 80 to a subject suffering from or susceptible to the disease.

[0329] 86. An oligonucleotide according to any one of embodiments 17 to 50, or an antisense oligonucleotide according to any one of embodiments 51 to 61, or a conjugate compound according to any one of embodiments 63 to 79, or a pharmaceutical composition according to embodiment 80, for use as a medicament for treating or preventing a disease in a subject.

[0330] 87. Use of an oligonucleotide according to any one of embodiments 17 to 50, or an antisense oligonucleotide according to any one of embodiments 51 to 61, or a conjugate compound according to any one of embodiments 63 to 79 for the preparation of a medicament for treating or preventing a disease in a subject.

[0331] 88. The method, oligonucleotide, antisense oligonucleotide, conjugate or use according to embodiments 85 to 87, wherein the subject is a mammal.

[0332] 89. The method, oligonucleotide, antisense oligonucleotide, conjugate or use according to embodiment 88, wherein the mammal is a human.

[0333] The invention will now be illustrated by the following examples, which have no limiting character. EXAMPLES

[0334] Materials and Methods Oligonucleotide motif sequences and oligonucleotide compounds

[0335] [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8]

[0336] A motif sequence represents a contiguous sequence of nucleobases present in an oligonucleotide.

[0337] Design refers to gapmer design FG-F', where each number represents the number of consecutive modified nucleosides, e.g., a 2' modified nucleoside (first number=5' flank), followed by the number of DNA nucleosides (second number=gap region), followed by the number of modified nucleosides, e.g., a 2' modified nucleoside (third number=3' flank), optionally preceded or followed by further repeated regions of DNA and LNA that are not necessarily part of the contiguous sequence that is complementary to the target nucleic acid.

[0338] The oligonucleotide compounds represent the specific design of the motif sequence. The capital letters represent β-D-oxy LNA nucleosides, the lower case letters represent DNA nucleosides, all LNA C's are 5-methylcytosines, 5-methyl DNA cytosines are represented by "e", and all internucleoside linkages are phosphorothioate internucleoside linkages.

[0339] Oligonucleotide synthesis Oligonucleotide synthesis is generally known in the art. Below are applicable protocols. The oligonucleotides of the invention may be produced by methods that differ slightly in terms of the equipment, supports and concentrations used.

[0340] Oligonucleotides are synthesized on a uridine universal support using the phosphoramidite approach in Oligomaker 48 on a 1 μmol scale. At the end of the synthesis, oligonucleotides are cleaved from the solid support with aqueous ammonia at 60 °C for 5-16 h. Oligonucleotides are purified by reversed-phase HPLC (RP-HPLC) or solid-phase extraction, characterized by UPLC, and molecular weights are further confirmed by ESI-MS.

[0341] 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 5'-O-DMT protected amidite in acetonitrile and a solution of DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. In the final cycle, a phosphoramidite with the desired modification can be used, such as a C6 linker for attaching a conjugate group, or such a conjugate group. Thiolation to introduce a phosphorothioate bond is carried out by using hydrogenated xanthan (0.01 M in acetonitrile / pyridine 9:1). A phosphodiester bond 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.

[0342] For conjugation after solid-phase synthesis, commercially available C6 amino linkerphoramidites can be used in the last cycle of solid-phase synthesis, and after deprotection and cleavage from the solid support, the amino-linked deprotected oligonucleotide is isolated. The conjugates are introduced by activation of functional groups using standard synthetic methods.

[0343] Purification by RP-HPLC: The crude compound is purified by preparative RP-HPLC on a Phenomenex Jupiter C18 10μ 150x10mm column. 0.1M ammonium acetate pH 8 and acetonitrile are used as buffers at a flow rate of 5mL / min. The collected fractions are lyophilized to give the purified compound, typically as a white solid.

[0344] Abbreviations: 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

[0345] T m Assay: Dilute the oligonucleotide and RNA target (phosphate-linked, PO) duplex to 3 mM in 500 mL of RNase-free water and suspend in 500 mL of 2x T m Mix with buffer (200 mM NaCl, 0.2 mM EDTA, 20 mM phosphate, pH 7.0). Heat the solution at 95° C. for 3 min and then anneal at room temperature for 30 min. The melting temperature of the duplex (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 Rate the following.

[0346] Clonal growth medium (dHCGM). dHCGM is DMEM medium containing 100 U / mL penicillin, 100 μg / mL streptomycin, 20 mM Hepes, 44 mM NaCO3, 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 37 °C incubator in a humidified atmosphere containing 5% CO2. Culture medium was changed 24 h after seeding and every 2 days until harvest.

[0347] Primary human hepatocytes (PXB-PHH) Fresh primary human hepatocytes (PXB-PHH) (herein referred to as PHH) harvested from humanized mice (uPA / SCID mice) were obtained from Phoenix Bio Co., Ltd. (Japan). Cells were seeded onto collagen I-coated plates at the following cell densities: 35,000 cells / well (384 wells), 70,000 cells / well (96 wells), or 400,000 cells / well (24 wells) in modified hepatocyte clonal growth medium (dHCGM). dHCGM is DMEM medium containing 100 U / mL penicillin, 100 μg / mL streptomycin, 20 mM Hepes, 44 mM NaCO3, 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 37 °C incubator in a humidified atmosphere containing 5% CO2. Culture medium was changed 24 h after seeding and every 2 days until harvest.

[0348] HBV infection and oligonucleotide therapy PHH were incubated with HBV (purified from CHB individuals) at a multiplicity of infection (MOI) of 40 with 4% PEG for 24 hours. The virus inoculum was removed the next day. cccDNA establishment compound treatment in PHH was initiated on day 3 after HBV infection. Fresh oligonucleotides dissolved in the medium were replenished every 2 days (example 1) or fresh oligonucleotides were replenished on days 3, 5, 7, and 9, and then the medium was replenished every 2 days (example 2), and cells were harvested on day 19.

[0349] HBV antigen measurement Supernatants were collected on day 19 to assess the effect on HBV antigen expression and secretion. HBV proliferation parameters HBsAg and HBeAg levels were measured using CLIA ELISA kits (Autobio Diagnostic, no. CL0310-2, no. CL0312-2) according to the manufacturer's protocol. Briefly, 25 μL / well of supernatant was transferred to each antibody-coated microtiter plate and 25 μL of enzyme conjugate reagent was added. After incubating the plates on a shaker at room temperature for 60 min, 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. After incubating the plates on a shaker at room temperature for 10 min, luminescence was measured using an Envision luminescence reader (Perkin Elmer).

[0350] CCK8 cytotoxicity measurement To evaluate the effect of cytotoxicity on oligonucleotide treatment, cells were treated as described for HBV infection and oligonucleotide treatment, and on day 18, PHH were preincubated for 24 h in a 37 °C incubator in a humidified atmosphere of 5% CO2. 10 μL of CCK-8 solution was added to each well of a 96-well plate in 100 μL of solution and incubated for 1-4 h. The absorbance at 450 nM was measured for each plate using a microplate reader (Tecan) and values ​​were calculated as % of control (untreated cells).

[0351] Real-time PCR of intracellular HBV pgRNA and RTEL1 RNA mRNA was extracted from cells using the Qiagen BioRobot Universal System and RNeasy 96-well extraction plates (RNeasy 96 BioRobot 8000 Kit(12) / Cat. No. / ID:967152) according to the manufacturer's protocol. Relative HBV and cellular mRNA expression levels were analyzed using real-time PCR on an ABI QuantStudio 12k Flex.

[0352] β-actin (ACT B) and HBV pgRNA were quantified by qPCR using TaqMan Fast Advanced Master Mix (Life Technologies, Cat. No. 4444558) in technical triplicates. Results were normalized to human ACT B endogenous control. mRNA expression was analyzed using the comparative cycle threshold2-ΔΔCt method normalized to the reference gene ACT B and non-transfected cells. Primers used for quantification of ACTB RNA and HBV pgRNA are listed in Table 7.

[0353] [Table 7]

[0354] HBV cccDNA quantification DNA was extracted from HBV-infected primary human hepatocytes using SDS lysis buffer and purified using the ZymoResearch Genomic DNA Clean&Concentrator kit (ZymoResearch, Cat. No. D4067) protocol. cccDNA levels were determined after digestion with T5 exonuclease (New England Biolabs, MA, USA) using 10U T5 for 500ng DNA for 1 hour at 37°C in a total volume of 20uL. After digestion, samples were diluted to 50μL, of which 4μL was used for qPCR reaction. mRNA expression was analyzed using the comparative cycle threshold2-ΔΔCt method normalized against the reference gene mitochondrial DNA and non-transfected cells. Quantitative real-time polymerase chain reaction measurements were performed on a QuantStudio 12K Flex PCR system (Applied Biosystems). qPCR was performed using Fast SYBR™ Green Master Mix (Life Technologies, Cat. No. 4385612). The primers are shown in Table 8.

[0355] [Table 8]

[0356] Example 1: Effects of antisense oligonucleotides targeting RTEL1 on HBV parameters in HBV-infected PHH In the following experiments, the effect of RTEL1 knockdown on the HBV parameters HBsAg, HBeAg, HBV pgRNA, and cccDNA was examined using the oligonucleotide compounds in Table 6.

[0357] PHH were cultured as described in the Materials and Methods section. Cells were treated 3 days after HBV infection with a final oligonucleotide concentration of 10 μM dissolved in dHCGM medium in a final culture volume of 100 μL / well. Experiments were performed in biological triplicates, with oligonucleotides replenished every 2 days and cells harvested 19 days after HBV infection. Cytotoxicity was determined using CCK8 according to Materials and Methods, supernatants were collected to measure HBsAg and HBeAg, and cells were collected in two fractions: one for RTEL1 mRNA and pgRNA measurements using one lysis buffer and one for cccDNA measurements using another lysis buffer as described in Materials and Methods. All values ​​are shown in Table 9 as % of control (untreated cells). That is, for RTEL1 mRNA, cccDNA, and pgRNA, the greater the inhibition, the lower the value.

[0358] CCK8 cytotoxicity was measured as described in the Materials and Methods section to ensure that reduction in any of the viral parameters was not the cause of cell death, although values ​​closer to 100% indicate lower toxicity. The results are shown in Table 9.

[0359] [Table 9]

[0360] All tested antisense oligonucleotides targeting RTEL1 demonstrate target knockdown and HBV antiviral efficacy at a single measurement time point for at least one of the parameters cccDNA, pgRNA, HBsAg, and HBeAg with no observed cytotoxicity as measured by CCK8.

[0361] Example 2: Further oligonucleotide libraries targeting RTEL1 tested for their effect on cccDNA in infected PHH In the following experiments, an additional library of 236 oligonucleotides targeting across the RTEL1 transcript was generated and shown in Table 6 as CMP numbers 50_1 through 237_1, and tested for their ability to reduce RTEL1 as well as cccDNA.

[0362] PHH were cultured as described in the Materials and Methods section. Cells were dosed on days 3, 5, 7, and 9 post-HBV infection with a final oligonucleotide concentration of 10 μM dissolved in dHCGM medium in a final culture volume of 100 μL / well. Experiments were performed in biological triplicates and cells were harvested on day 19 post-HBV infection with supplemented medium every 2 days until day 19.

[0363] According to Materials and Methods, cells were collected in two fractions, one for RTEL1 mRNA using one lysis buffer and one for cccDNA measurement using another lysis buffer as described in Materials and Methods. All values ​​are shown in Table 10 as % of control (untreated cells). That is, for RTEL1 mRNA and cccDNA, the greater the inhibition / reduction, the lower the value.

[0364] [Table 10-1] [Table 10-2] [Table 10-3]

[0365] CCK8 cytotoxicity was measured as described in the Materials and Methods section to assess whether the reduction in viral parameters could be caused by cell death. Values ​​closer to 100% indicate lower toxicity. Results are shown in Table 11. For CCK8 values ​​above 80% of the control, cell death is unlikely to affect the reduction in RTEL1 and cccDNA shown in Table 10.

[0366] [Table 11-1] [Table 11-2] [Table 11-3]

[0367] The results show that only 5% of the 236 oligonucleotides in the library were unable to reduce either RTEL1 or cccDNA to at least 80% of the control, indicating that it is possible to generate oligonucleotides targeting the entire RTEL1 transcript that can reduce RTEL1 and / or cccDNA, and that in general, the reduction in RTEL1 and cccDNA is not due to cell death, although for some compounds this may be the case.

Claims

1. RTEL1 inhibitors for the treatment and / or prevention of Hepatitis B virus (HBV) infection.

2. The RTEL1 inhibitor for use according to claim 1, wherein the HBV infection is a chronic infection.

3. The RTEL1 inhibitor according to claim 1 or 2, wherein the RTEL1 inhibitor is capable of reducing cccDNA in infected cells.

4. The RTEL1 inhibitor according to any one of claims 1 to 3, wherein said inhibitor is an oligonucleotide of 12 to 60 nucleotides in length comprising a contiguous nucleotide sequence of at least 10 nucleotides in length, which is at least 95% complementary to a mammalian RTEL1 target nucleic acid, in particular a human RTEL1 nucleic acid, and capable of reducing RTEL1 mRNA.

5. The RTEL1 inhibitor according to any one of claims 1 to 4, which is selected from a single-stranded antisense oligonucleotide, an siRNA, or an shRNA molecule.

6. The RTEL1 inhibitor according to any one of claims 1 to 5, wherein the mammalian RTEL1 target nucleic acid is selected from SEQ ID NO: 1 or 2.

7. The RTEL1 inhibitor for use according to any one of claims 4 to 6, 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.

8. The RTEL1 inhibitor for use according to any one of claims 3 to 7, wherein said cccDNA in HBV infected cells is reduced by at least 60% compared to a control.

9. The RTEL1 inhibitor according to any one of claims 4 to 7, wherein the RTEL1 mRNA is reduced by at least 60% compared to a control.

10. A single-stranded antisense oligonucleotide having a length of 12 to 30 nucleotides, comprising a contiguous nucleotide sequence of at least 10 nucleotides complementary to mammalian RTEL1, particularly human RTEL1, wherein said oligonucleotide is capable of inhibiting expression of RTEL1.

11. The antisense oligonucleotide of claim 10, wherein the contiguous nucleotide sequence is at least 90% complementary, e.g., completely complementary, to SEQ ID NO:

1.

12. The antisense oligonucleotide according to claim 10 or 11, which comprises a contiguous nucleotide sequence of 12 to 25, in particular 15 to 21 nucleotides in length.

13. The oligonucleotide according to any one of claims 11 to 12, wherein the contiguous nucleotide sequence is 100% complementary to a target sequence selected from SEQ ID NOs: 3 to 21.

14. The oligonucleotide of any one of claims 10 to 13, wherein the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 22 to 237.

15. The antisense oligonucleotide of any one of claims 10 to 14, comprising one or more 2' sugar modified nucleosides.

16. 16. The antisense oligonucleotide 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 antisense oligonucleotide of any one of claims 15 to 16, wherein the one or more 2' sugar modified nucleosides are LNA nucleosides.

18. The antisense oligonucleotide of any one of claims 10 to 17, wherein the oligonucleotide comprises at least one phosphorothioate internucleoside linkage.

19. 19. The antisense oligonucleotide of claim 18, wherein all of the internucleoside linkages between the nucleosides in the contiguous nucleotide sequence are phosphorothioate internucleoside linkages.

20. 20. The antisense oligonucleotide of any one of embodiments 10 to 19, wherein said oligonucleotide is capable of recruiting RNase H.

21. The antisense oligonucleotide according to any one of claims 10 to 20, wherein the antisense oligonucleotide or its contiguous nucleotide sequence consists of or comprises a gapmer of the formula 5'-F-G-F'-3', where regions F and F' independently comprise 1 to 4 2' sugar modified nucleosides and G is a region of 6 to 16 nucleosides capable of recruiting RNase H, such as a region comprising 6 to 18 DNA nucleosides.

22. A conjugate comprising an oligonucleotide or antisense oligonucleotide according to any one of claims 10 to 21 and at least one conjugate moiety covalently attached to said oligonucleotide.

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

24. 24. The conjugate compound of claim 22 or 23, comprising a physiologically labile linker consisting of 2 to 5 linked nucleosides containing at least two consecutive phosphodiester bonds, wherein said physiologically labile linker is covalently attached to the 5' or 3' end of an oligonucleotide component.

25. A pharma- ceutically acceptable salt of the oligonucleotide according to any one of claims 10 to 21, or the conjugate according to claims 22 to 24.

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

27. An in vivo or in vitro method for modulating RTEL1 expression in a target cell expressing RTEL1, comprising administering to said cell an effective amount of an oligonucleotide according to any one of claims 10 to 21, or a conjugate according to any one of claims 22 to 24, a pharma- ceutically acceptable salt according to claim 25, or a pharmaceutical composition according to claim 26.

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

29. 29. The method of claim 28, wherein the disease is Hepatitis B virus (HBV).

30. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 10 to 21, or a conjugate according to claims 22 to 24, or a pharma- ceutically acceptable salt according to claim 25, or a pharmaceutical composition according to claim 26, for use in medicine.

31. Use of the oligonucleotide according to any one of claims 10 to 21, or the conjugate according to claims 22 to 24, or the pharma- ceutically acceptable salt according to claim 25, or the pharmaceutical composition according to claim 26, for the preparation of a medicament for treating or preventing the Hepatitis B virus HBV.

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