Compositions and methods for treating hepatitis b virus infection

Abstract

The present disclosure provides compositions and methods for inhibiting hepatitis B virus (HBV) in infected cells. Exemplary methods include contacting an infected cell with one or more agents that induce interferon regulatory factor 3 (IRF3) activation in the infected cell. In some embodiments, the one or more agents include a nucleic acid molecule containing a pathogen-associated molecular pattern (PAMP), a small molecule agent (e.g., a benzothiazole derivative molecule), or a combination thereof. In some embodiments, the methods further include contacting the infected cell with an NRTI. The methods can be in vivo methods of treating a subject having an HBV infection, which include administering a therapeutically relevant amount of one or more agents formulated in one or more therapeutically effective compositions. Exemplary compositions are formulated for treating a hepatitis B virus (HBV) infection in a subject, which include: a RIG-I agonist, a vehicle for intracellular delivery, and a pharmaceutically acceptable carrier.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 909,321, filed October 2, 2019, the entire contents of which are incorporated herein by reference.

[0003] Declaration of sequence list

[0004] The sequence list relating to this application is provided in text format instead of a paper copy and is incorporated herein by reference. The text file containing the sequence list is named 72750_Sequence_Listing_final_2020-09-28.txt. The text file is 31KB; created on September 28, 2020; and is being submitted via EFS-Web along with the specification.

[0005] Government License Rights Statement

[0006] This invention was completed with government support under grant numbers R01 AI118916 and R01 AI127463 granted by the National Institutes of Health in the United States. The government holds certain rights to this invention. Background Technology

[0007] Hepatitis B virus (HBV) is a global public health problem, with more than 250 million people worldwide chronically infected. Chronic HBV infection is a leading cause of liver diseases, including cirrhosis, hepatocellular carcinoma (HCC), and liver failure, and therefore causes more than 700,000 deaths annually [WHO, 2017].

[0008] HBV is a small, hepatotropic DNA virus that replicates partly via reverse transcription. HBV specifically enters hepatocytes via the sodium taurocholate cotransport polypeptide (NTCP) receptor to replicate and produce viral particles. Upon entering the hepatocyte cytosol, the viral nucleocapsid translocates to the nucleus to break down and release the relaxed circular (RC) DNA. In the nucleus, the RC DNA is converted into covalently closed circular DNA (cccDNA), a long-lived viral miniature chromosome that serves as the primary template for the synthesis of all HBV RNA transcripts, including pregenomic (pg) RNA, pre-S, S, and X viral RNA. After synthesis, the mature nucleocapsid-containing RC DNA acquires an envelope by budding in the endoplasmic reticulum (ER) and then produces progeny viral particles. A portion of the mature HBV nucleocapsid library is used to promote further cccDNA synthesis in a process known as the intracellular amplification pathway. This process helps maintain a homeostatic population of 3–50 molecules per cell, marking chronic infection. Removing cccDNA from the liver by eradicating cccDNA from infected cells or consuming infected cells is considered a necessary condition for HBV cure.

[0009] Current treatment for chronic HBV relies on two classes of therapies: (i) nucleoside analogues (NAs), which inhibit viral reverse transcriptase and DNA polymerase function, and (ii) pegylated interferon-alpha (peg-IFNα) therapy, which induces innate immune defense to suppress HBV antigen production. While these therapies can suppress active viral replication, reduce cccDNA levels, and slow disease progression, they do not eliminate the cccDNA nuclear reservoir and are associated with significant side effects in treated patients. The establishment of persistent cccDNA in the body takes 6–22 weeks, with most patients requiring lifelong antiviral therapy to maintain viral replication. Problematically, IFN-based therapies for chronic HBV are poorly tolerable, and only a low frequency of treatment shows complete loss of HBsAg, defining clinical HBV cure.

[0010] Therefore, despite the development of suppressive HPV therapies, there remains a need for effective therapies and treatment strategies capable of specifically eradicating cccDNA, leading to a more complete and functional cure and avoiding the harmful effects of prolonged suppressive therapy. This disclosure addresses these and related needs. Summary of the Invention

[0011] This overview is provided to present the selection of concepts in a simplified form, which will be further described in the detailed description of the invention below. This overview is not intended to identify key features of the claimed subject matter, nor is it intended to serve as an aid in determining the scope of the claimed subject matter.

[0012] In one aspect, this disclosure provides a method for inhibiting the level of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in infected cells. The method includes contacting the infected cells with an agent that induces activation of interferon regulatory factor 3 (IRF3) in the infected cells. "Inhibiting cccDNA" may include inhibiting cccDNA formation in the infected cells or reducing the stability of existing cccDNA in the infected cells.

[0013] In some embodiments, the reagent induces IRF3 activation by inducing the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. In some embodiments, the reagent is or comprises a nucleic acid molecule containing a pathogen-associated molecular pattern (PAMP), wherein the PAMP comprises: a 5' arm containing a terminal triphosphate; a polyuracil core containing at least eight consecutive uracil residues; and a 3' arm containing at least eight nucleic acid residues, wherein the 5' terminal nucleic acid residue of the 3' arm is not uracil, and wherein the 3' arm contains at least 30% uracil residues. In some embodiments, the reagent is a small molecule reagent. In some embodiments, the small molecule reagent is or comprises a benzothiazole derivative molecule, such as a small molecule reagent containing the chemical formula N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthalene-2-carboxamide. In some embodiments, the method includes contacting cells with a combination of nucleic acid molecules containing pathogen-associated molecular patterns (PAMPs) and small molecule reagents (e.g., benzothiazole derivative molecules, such as N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthyl-2-carboxamide).

[0014] In some embodiments, the method further includes contacting the cells with a nucleoside reverse transcriptase inhibitor (NRTI). In some embodiments, the NRTI is selected from lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, tenofovir alafenamide (TAF), clevudine, besivo, zadaxin, remdesivir, etc.

[0015] In some implementations, the cells are liver cells.

[0016] In another aspect, this disclosure provides a method for treating or preventing hepatitis B virus (HBV) infection in a subject in need. The method includes administering a therapeutically effective amount of a composition to the subject, the composition inducing activation of interferon regulatory factor 3 (IRF3) in the subject's infected cells.

[0017] In some embodiments, the composition comprises a nucleic acid molecule containing a pathogen-associated molecular pattern (PAMP), wherein the PAMP comprises: a 5' arm containing a terminal triphosphate; a polyuracil core containing at least eight consecutive uracil residues; and a 3' arm containing at least eight nucleic acid residues, wherein the terminal nucleic acid residue of the 5' arm is not uracil, and wherein the 3' arm comprises at least 30% uracil residues. In some embodiments, the reagent is a small molecule reagent that induces RIG-I signaling. In some embodiments, the small molecule reagent is or comprises a benzothiazole derivative molecule, such as a small molecule reagent comprising the chemical formula N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthalene-2-carboxamide. In some embodiments, the method comprises administering to a subject a combination or coordinated dose of a therapeutically effective amount of the nucleic acid molecule containing the pathogen-associated molecular pattern (PAMP) and the small molecule reagent.

[0018] In some embodiments, the method further includes administering a nucleoside reverse transcriptase inhibitor (NRTI) to the subject. In some embodiments, the NRTI is selected from lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, tenofovir alafenamide (TAF), clavidine, Besivo, thymosin alpha 1, remdesivir, etc. The NRTI may be administered in combination with or in coordination with one or more agents that induce interferon regulatory factor 3 (IRF3) activation in the subject's infected cells.

[0019] In another aspect, this disclosure provides compositions for treating hepatitis B virus (HBV) infection in subjects, comprising: a RIG-I agonist, a medium for intracellular delivery, and a pharmaceutically acceptable carrier.

[0020] In some embodiments, the RIG-I agonist is or comprises a nucleic acid molecule containing a pathogen-associated molecular pattern (PAMP), wherein the PAMP comprises: a 5' arm containing a terminal triphosphate; a polyuracil core containing at least eight consecutive uracil residues; and a 3' arm containing at least eight nucleic acid residues, wherein the 5' terminal nucleic acid residue of the 3' arm is not uracil, and wherein the 3' arm comprises at least 30% uracil residues. In some embodiments, the RIG-I agonist is or comprises a small molecule reagent. In some embodiments, the small molecule reagent is or comprises a benzothiazole derivative molecule, such as a small molecule reagent containing the chemical formula N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthyl-2-carboxamide. In some embodiments, the composition comprises a nucleic acid molecule containing a pathogen-associated molecular pattern (PAMP) and a small molecule reagent (e.g., a benzothiazole derivative molecule, such as one containing the chemical formula N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthyl-2-carboxamide). In some embodiments, the composition further comprises a nucleoside reverse transcriptase inhibitor (NRTI). In some embodiments, the NRTI is selected from lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, tenofovir alafenamide (TAF), clavone, Besivo, thymosin alpha 1, remdesivir, etc. In some embodiments, the mediator is a liposome, nanocapsule, nanoparticle, exosome, microparticle, microsphere, lipid particle, vesicle, etc., configured to introduce a RIG-I agonist into HBV-infected target host cells.

[0021] In another aspect, this disclosure provides a method for treating a subject suffering from hepatitis B virus (HBV) infection, comprising administering to the subject a therapeutically effective amount of the composition disclosed herein. Attached Figure Description

[0022] The foregoing aspects and many accompanying advantages of the invention will become more readily understood as they are better understood by referring to the following detailed description taken in conjunction with the accompanying drawings, wherein:

[0023] Figure 1A-1DThis study demonstrates the differential induction of innate immune genes by F7 small molecules and polyU / UC PAMP. (1A) Structure of F7 and sequence of representative polyU / UC PAMP-RNA (SEQ ID NO: 1). (1B) Immunofluorescence analysis of IRF3 translocation. HepG2-hNTCP cells were cultured in medium containing 2.5% DMSO and infected with Sendai virus (SenV; 10 HAU / ml), or treated with F7 (10 μM), X-RNA (200 ng / ml in liposomes), or polyU / UC PAMP (200 ng / ml in liposomes) for 24 h. Cells were fixed with 3% paraformaldehyde, stained with mouse anti-hNTCP (green) and rabbit anti-IRF3 (red) antibodies, and counterstained with DAPI (blue). Scale bar represents 20 μm. (1C) F7 and polyU / UC induce IRF3 activation and expression of innate immune genes. As shown, Sendai virus, F7, X-RNA, and polyU / UC PAMP were administered to HepG2-hNTCP and dHepaRG cells for 24 and 48 hours. Cell lysates were analyzed by SDS-PAGE followed by Western blotting. Protein expression levels of p-IRF3 (the S386 phosphorylated active form of IRF3), IRF3 (total IRF3), and IFIT1 relative to tubulin expression levels were determined using the corresponding antibodies. (1D) Gene expression analysis. As described above, HepG2-hNTCP cells were administered SenV, F7, X-RNA, and polyU / UC-RNA for 24 and 72 hours, and total cellular RNA was then purified from the harvested cells. The expression levels of innate immune genes IFIT1, CXCL10, IFITM1, RSAD2, RIG-I, MDA5, SAMHD1, APOBEC3A, APOBEC3G, IFN-α, IFN-β, and IFN-λ3 were measured by qRT-PCR and normalized to GAPDH expression levels. Each is shown as the mean fold induction from three independent experiments relative to results obtained with 2.5% DMSO treatment.

[0024] Figures 2A-2EThe therapeutic inhibition of cccDNA formation is illustrated. (2A) Infection and treatment protocol (top) and Southern blot analysis (bottom) to measure protein-free DNA, including cccDNA. The protocol illustrates the timeline of HBV inoculation (1000 Geq / cell) and administration of cyclosporine A (CsA), F7, X-RNA, and poly-U / UC PAMP. 2.5% DMSO was added to the culture medium 1 day post-infection (Dpi). cccDNA and PF-RC DNA were harvested from HepG2-hNTCP cells using the Hirt extraction method and analyzed by Southern blot analysis using HBV-specific DNA probes. Protein-free DNA (protein-free relaxed circular DNA [PF-RC DNA] and cccDNA) were labeled. (2B and 2C) HepG2-hNTCP 2 (B) or differentiated HepaRG cells (2C) were infected with HBV at 1000 Geq / cell moi and administered with the specified concentration of CsA (10 μM) or F7. For the upper plots of (2B) and (2C), DNA was isolated after Hirt extraction at 3 dpi and Southern blot analysis was performed using an HBV-DNA probe. For the lower plots of (2B) and (2C), the inhibitory effect of F7 on cccDNA formation was measured in HepG2-hNTCP and dHepaRG cells, respectively, using RT-qPCR analysis. (2D and 2E) HepG2-hNTCP (2D) or dHepaRG cells (2E) were infected with HBV at 1000 Geq / cell moi and administered CsA (10 μM), X-RNA (100 ng / ml), or a specified concentration of polyU / UC PAMP. For the upper plots of (2D) and (2E), DNA was analyzed using Southern blot analysis. For the lower plots of (2D) and (2E), DNA was analyzed by RT-qPCR. IC50 was calculated based on the decrease in cccDNA relative to the DMSO-treated control. 90 and IC 50 Value. IC 90 and IC 50 The value is the mean of the three experiments ± one standard deviation. This was achieved using CellTiter-Glo TM Reagents are used to measure cellular ATP content as a measure of cell viability to determine cytotoxicity. The CC shown... 50 The value is the mean of the three experiments ± one standard deviation. The location of the quality marker is shown on each Southern blobs.

[0025] Figures 3A-3FThe inhibition of de novo HBV cccDNA synthesis by F7 and polyU / UC PAMP was demonstrated. (3A) Top: Infection and polyU / UC PAMP treatment timeline. Cells were treated with 100 ng / ml polyU / UC PAMP for 24 h before HBV infection, 48 h after HBV infection, or 72 h after infection (pre / post). Cells were harvested at 3 dpi and Hirt extracts were prepared. (3B) HepG2-hNTCP cells and (3C) dHepaRG cells were analyzed by Southern blotting (top) and RT-qPCR (bottom). For RT-qPCR analysis, values ​​from DMSO-treated controls were set as 100%, and data were shown as mean ± standard deviation (SD) percentage of cccDNA in control samples. ***P < 0.005, and ns = not significant. (3D) HBV infection and F7 treatment timeline. Cells were treated with F7 (10 μM) for 24 h before infection (Pre), 24 h during infection (Co), 48 h after infection (Post), or 96 h after infection (pre / co / post). Cells were harvested at 3 Dpi and Hirt extracts were prepared for HepG2-hNTCP (3E) and dHepaRG cells (3F). The top figure shows Southern blot analysis. The bottom figure shows RT-qPCR analysis and the percentage of residual cccDNA in treated cells compared to control DMS-treated cells. Values ​​from control cultures treated with DMSO were set as 100%. Data are expressed as mean ± standard deviation (SD), *P<0.01, **P<0.005, ***P<0.001, ****P<0.0001, and ns = not significant. CsA was used as a treatment control. For Southern blot analysis, the location of the quality markers is indicated.

[0026] Figures 4A-4DSubcellular compartmental analysis illustrating antiviral activity was presented. (4A) and (4C): HBV infection protocols of F7 treatment (4A) or polyU / UC PAMP treatment (4C). HepG2-hNTCP cultures were seeded with HBV at 1000 Geq / cell moi for 24 hours. On day 0 of treatment, cultures received F7 (10 μM) or polyU / UC PAMP (100 ng / ml). Cells were harvested at each time point shown over three days for the production of Hirt extract. Parallel cultures were collected for Western blot analysis to monitor lamin B1 and calcinin as markers of whole-cell lysate and cytosol, respectively. (4B) and (4D) DNA was analyzed by Southern blotting using HBV-specific DNA probes. Virus-free protein DNA (no protein-relaxed circular DNA [PF-RC DNA] and cccDNA) is shown. The location of quality markers is indicated. The following figure shows the Western blot of lamin B1 and calcinin abundance.

[0027] Figures 5A-5FThe F7 and polyU / UC PAMP treatments guide HBV cccDNA decay. (5A) HBV infection and treatment schedule. On day 0, HepG2-hNTCP cells were infected with HBV at 1000 Geq / cell moi, incubated for 24 hours, and the medium was changed. On day 3, cells were harvested or treated with DMSO, ETV (500 nM), F7 (10 μM), polyU / UC (100 ng / ml), or ETC in combination with F7 or polyU / UC PAMP. Cells were harvested at designated points throughout the 20-day time series. (5B), (5C) DNA was isolated by Hirt extract and Southern blot analysis was performed using HBV-specific DNA probes. The percentage values ​​below each lane represent the relative amount of cccDNA compared to the day 3 control before each treatment. The location of the quality markers is indicated. (5D) cccDNA levels were measured by RT-qPCR. The cccDNA values ​​relative to mitochondrial DNA (MT-CO3) are shown. Statistical significance was determined using Student's t-test. Data are expressed as mean ± standard deviation (SD), *P < 0.01, **P < 0.005, ***P < 0.001, and ns = not significant. (E) The half-life of cccDNA from RT-qPCR analysis was estimated by fitting three replicates simultaneously under each treatment strategy. C(t) is the percentage of cccDNA at time “t” after treatment, and C(0) is the percentage of cccDNA at the start of treatment. (5F) HBsAg throughout the time course for each treatment series. Statistical significance was determined using Student's t-test. Data are expressed as mean ± standard deviation (SD), **P < 0.005, and ns = not significant.

[0028] Figures 6A-6DThis demonstrates that F7 and polyU / UC PAMP specifically signal IRF3 activation via RIG-I to inhibit HBV cccDNA. (6A) F7 was administered to HepG2-hNTCP-NT (expressing non-targeted guide RNA), RKO (expressing RIG-I-targeted guide RNA with RIG-I knockout), and MKO (expressing MDA5-targeted guide RNA with MDA5 knockout) cells for 24 hours. Cells were harvested and analyzed by immunoblotting. Protein expression levels of p-IRF3 (S386 phosphorylated, active IRF3), IRF3 (total IRF), IFIT1, RIG-I, and MDA5 relative to tubulin expression levels were determined using the appropriate antibodies. (6B) HepG2-hNTCP-NT, RKO, and MKO cells were treated with 100 ng / ml X-RNA or 100 ng / ml or 200 ng / ml polyU / UC PAMP for 24 hours. Cells were harvested as in (6A) and analyzed by immunoblotting. Cells (6C and 6D) were infected with HBV at 1000 Geq / moi. After 24 hours, cultures were treated with DMSO (negative control), CsA (treatment control), (6C) 10 μM, or F7 or (6D)X RNA, or polyU / UC PAMP. Cells were harvested after three days, Hirt extracts were prepared, and Southern blotting analysis was performed using HBV-DNA probes. The location of quality markers was indicated. Values ​​below each lane show the percentage of residual cccDNA compared to the control treatment.

[0029] Figures 7A-7CThis demonstrates the inhibition of cccDNA in primary human hepatocytes. (7A) PHH cultures were cultured alone or treated with polyU / UC PAMP at 100 ng / ml (X100) or 50 ng / ml (P50), 100 ng / ml (P100), or 200 ng / ml (P200), or infected with SenV (control) and harvested after 24 and 72 hours. Cell lysates were analyzed by immunoblotting using the corresponding antibodies for p-IRF3 (S386 phosphorylated, active IRF3), IRF3 (total IRF3), IFIT1, and actin (control). (7B) PHH cultures were cultured alone or treated with polyU / UC PAMP at 100 ng / ml (X100) or 100 ng / ml (P100), or 200 ng / ml (P200), or infected with SenV (control) and harvested after 24 and 72 hours. Cells were harvested, RNA was extracted, and RT-qPCR analysis was performed to measure the expression levels of specified innate immune genes normalized to GAPDH expression levels. Values ​​are shown in a heatmap as the mean fold induction relative to untreated cells from three independent experiments. (7C)PHH cultures were inoculated with HBV at 200 Geq / moi. After 24 hours, cultures were treated with CsA (treatment control; 10 μM), 100 ng / ml X-RNA (X100), or 100 ng / ml (P100), or 200 ng / ml (P200) polyU / UC PAMP. Cells were harvested after three days, and isolated DNA was extracted via Hirt extract and analyzed by Southern blotting using HBV-specific probes. Virus-free protein DNA (protein-relaxed circular DNA [PF-RC DNA] and cccDNA) was labeled. Values ​​under each lane show the percentage of cccDNA remaining compared to the untreated control. The position of the quality marker is shown on the left.

[0030] Figure 8A-8LThis is a series of graphs illustrating the differential expression of innate immune genes induced by F7 and polyU / UC. HepG2-hNTCP cells were infected with SenV (positive control) or treated with F7 (5 or 10 uM, as shown), X-RNA (100 ng / ml; X-100 and 200 ng / ml; X-200) or polyU / UC PAMP 100 ng / ml or 200 ng / ml as shown for 24 h (black column) and 72 h (grey column). Cells were harvested at each time point. Total cellular RNA was purified and analyzed by RT-qPCR to measure the expression levels of a set of innate immune genes, including IFIT1, CXCL10, IFITM1, RSAD2, RIG-I, MDA5, SAMHD1, APOBEC3A, APOBEC3G, IFN-α, IFN-β, and IFN-λ3. Gene expression levels were normalized to GAPDH expression levels in each sample and expressed as fold induction from three independent experiments relative to results obtained using 2.5% DMSO treatment (negative control). Data are expressed as mean ± standard deviation (SD), *P < 0.01, **P < 0.005, ***P < 0.0005, ****P < 0.0001, and ns = not significant.

[0031] Figures 9A-9EThe kinetics of HBV replication in parallel cell cultures during treatment with F7 or polyU / UC PAMP were illustrated. (9A) HepG2-hNTCP cells were infected with HBV at 1000 Geq / moi per cell. After 24 hours, cells were treated with F7 (10 μM) (top panel) or 100 ng / ml X RNA or 100 ng / ml polyU / UC PAMP (bottom panel). Cells were harvested at each time point, Hirt supernatant was prepared, and Southern blot analysis was performed. (B) HBV pgRNA analysis by RT-qPCR. For RT-PCR analysis, the “HBV only” value from 20 Dpi was set to 100%. Data are expressed as mean ± standard deviation (SD), ***P < 0.001, ****P < 0.0001, and ns = not significant. (9C) Analysis of HBV cell inner shell-associated DNA from cells treated with 10 μM F7 (top panel) or 100 ng / ml XRNA or polyU / UC PAMP (bottom panel) by Southern blotting. (9D) Detection of HBsAg secreted by HBV-infected cells treated with F7 (top panel) or polyU / UC (bottom panel) by ELISA. (9E) Measurement of extracellular HBV-DNA from cells treated with 10 μM (left panel) or 100 ng / ml XRNA or polyU / UC PAMP (right panel) by qPCR. Data are presented as mean ± standard deviation (SD) from three independent experiments. ***P = 0.0002.

[0032] Figures 10A-10E The diagram illustrates the CC treatment of F7 and poly-U / UC PAMP. 50 Analysis. HepG2-NTCP cells (10A and 10C), dHepaRG (10B and 10D), and PHH (10E) were treated with increased doses of F7 or poly-U / UC PAMP for 72 h. Cell viability was determined by measuring ATP content, and values ​​were normalized to simulated treatments. Figures show the mean of triplicate samples in each of the three independent experiments, and error bars show the standard deviation. ns = not significant.

[0033] Figure 11The half-life of cccDNA is described. HepG2-NTCP cells were infected with HBV at 1000 Geq / cell moi. At 3 dpi, cultures were either left untreated or treated with ETV (500 nM) daily by replacing the medium with fresh medium alone or containing ETV over the entire 50-day time series. Cells were harvested at designated time points, DNA was isolated by Hirt extraction, and analyzed by Southern blotting using HBV-specific probes. The percentage values ​​below each lane represent the relative amount of cccDNA present compared to the level at day 3.

[0034] Figure 12 This study describes the immunoblotting analysis of HepG2-hNTCP cells transduced using CRISPR / Cas9-guided RNA constructs targeting RIG-I (RKO) or MDA5 (MKO), or transduced with a non-targeted guide RNA (NT) control. Cells were treated with 100 U / ml IFN-β for 24 hours. Cell lysates were prepared and analyzed by immunoblotting. The levels of RIG-I, MDA5, IFIT1, and tubulin (housekeeping protein control) were determined using the corresponding antibodies. Invention Details

[0036] Hepatitis B virus (HBV) mediates persistent infection, chronic hepatitis, and liver disease. HBV covalently closed circular DNA (cccDNA) is crucial for viral persistence, and its elimination is considered a cornerstone of HBV cure. Inefficient detection by pathogen recognition receptors (PRRs) in infected hepatocytes, through avoidance of innate immune activation and antiviral gene expression induced by interferon regulatory factor 3 (IRF3), contributes to HBV persistence. Given these challenges, the inventors evaluated the ability of RIG-I-induced signaling to inhibit cccDNA, where RIG-I is a PRR that signals the innate immune response. As described in more detail below, two different RIG-I agonists were used for the proof-of-concept: a small molecule compound called “F7”, and a 5'-poly(U / UC) pathogen-associated molecular pattern (PAMP) RNA. Treatment of HBV-infected cells with F7 and poly-U / UC PAMP induced IRF3 activation and RIG-I signaling to induce the inhibition of antiviral genes for cccDNA formation and accelerated decay of established cccDNA, additively with the effects of entecavir. This study demonstrates that activation of IRF3, for example, through induction of the RIG-I pathway, induces innate immune responses, providing a therapeutic benefit for the elimination of cccDNA.

[0037] Based on the foregoing, in one aspect, this disclosure provides a method for inhibiting the level of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) in infected cells. The method includes contacting the infected cells with an agent that induces activation of interferon regulatory factor 3 (IRF3) in the infected cells.

[0038] As described above, hepatitis B virus DNA is released from the viral nucleocapsid in the nucleus of the infected cell. This viral DNA is released from the viral capsid in the form of relaxed circular DNA (RC DNA). RC DNA is converted into covalently closed circular DNA (cccDNA) and used as the primary template for the synthesis of all HBV RNA transcripts, including pregenomic (pg) RNA, pre-S, S, and X viral RNA. cccDNA can replicate intracellularly via intracellular amplification pathways. The lifetime of cccDNA can be relatively long and can serve as the basis for long-term chronic infection, even after treatment. As used herein, the term "inhibition of cccDNA" includes inhibiting the formation of new cccDNA in the infected cell. The term "inhibition of cccDNA" may also include reducing the stability of existing cccDNA in the infected cell prior to the contact step. Reduced stability can result in a shortened half-life of cccDNA. Reduced stability can be observed by a decrease in the level of cccDNA within the infected cell. In some embodiments, the reduction in cccDNA levels is associated with infected cells that are also infected with HBV (e.g., of the same lineage or tissue type). The reduction can be any detectable reduction in the cccDNA level in the infected cells, such as a reduction of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 99%, and complete eradication.

[0039] The cell can be any cell infected with HBV. In some implementations, the infected cell is a hepatocyte.

[0040] A key component of the innate immune response to viral infection is the activation of interferon regulatory factor 3 (IRF3). IRF3 induces the expression of antiviral genes and also induces IFN. Antiviral genes can suppress viral replication in infected cells, while IFN directs the suppression of viral replication in infected cells and neighboring bystander cells by expressing hundreds of interferon-stimulated genes (ISGs) with antiviral and immunomodulatory activities. In addition to IFN suppression in viral infection, programmed cell death in viral-infected cells can be used to prevent viral spread. Therefore, the combination of IRF3 actions, including the resulting IFN action and cell death signaling, provides a synergistic program for viral control of many viruses. This disclosure is based in part on evidence that this pathway can be used to combat HBC, which generally avoids inducing such IRF3 actions.

[0041] In some implementations, agents indirectly induce IRF3 activation via the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling pathway. RLRs are cytoplasmic RNA helicases that act as PRRs to recognize RNA virus infections. RLRs include RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (Laboratory Genetics and Physiology 2). While RIG-I and MDA5 encode tandem N-terminal caspase activation and recruitment domains (CARDs), LGP2 lacks a CARD and is thought to play a regulatory role in signaling initiated by either RIG-I or MDA5. Following recognition and binding of viral PAMPRNAs, RIG-I signals via the adaptor protein mitochondrial antiviral signaling pathway (MAVS, also known as IPS-1 / VISA / Cardif). Downstream signaling of RLRs induces activation of latent transcription factors, including interferon regulator factor (IRF)-3 and NF-κB, leading to the production of type I interferon (IFN) by infected cells. Those skilled in the art will be able to readily determine activation of the RLR pathway, for example, by measuring the transcription of known downstream RLR-regulating genes. For instance, in some embodiments, RLR activation can be established by increasing IFNβ or ISG54 expression. In another embodiment, RLR activation can be established by increasing IRF3 phosphorylation. Therefore, in some embodiments, the RLR signaling pathway comprises RIG-I, melanoma differentiation-associated gene 5 (MDA5), genetics and physiology laboratory 2 (LGP2), and / or mitochondrial antiviral signaling (MAVS) proteins.

[0042] In some embodiments, the reagent that induces RIG-I signaling is or contains a nucleic acid molecule containing a pathogen-associated molecular pattern (PAMP). Exemplary PAMPs and PAMP-containing nucleic acid molecules covered by this disclosure are disclosed in U.S. Publications 2015 / 0017207 and 2018 / 0104325, which relate to PAMP-induced innate immune response signaling and are incorporated herein by reference in their entirety. Elements and exemplary embodiments of PAMP-containing nucleic acids covered by this disclosure are addressed herein.

[0043] As a preliminary consideration, the term "nucleic acid" as used herein refers to a polymer of monomeric units or "residues." Each monomeric subunit or residue of a nucleic acid contains a nitrogenous base (i.e., a nucleobase), a pentose sugar, and a phosphate group. The identity of each residue is generally indicated herein by reference to the identity of the nucleobase (or nitrogenous base) structure of each residue. Typical nucleobases include adenine (A), guanine (G), thymine (T), uracil (U) (in place of the thymine (T) residue in RNA), and cytosine (C). However, nucleic acids of this disclosure may include any modified nucleobases, nucleobase analogs, and / or non-canonical nucleobases known in the art. Modifications to nucleic acid monomers or residues include any chemical changes in the structure of the nucleic acid monomer or residue that result in a non-canonical subunit structure. Such chemical changes may arise from, for example, epigenetic modifications (e.g., genomic DNA or RNA), or damage caused by radiation, chemicals, or other means. Illustrative and non-limiting examples of non-classical subunits that can be generated by modification include uracil (for DNA), 5-methylcytosine, 5-hydroxymethylcytosine, 5-formethylcytosine, 5-carboxycytosine, β-glucosyl-5-hydroxymethylcytosine, 8-oxoguanine, 2-amino-adenosine, 2-amino-deoxyadenosine, 2-thiothymidine, pyrrolopyrimidine, 2-thiocytidine, or abasic lesions. Abasic lesions are positions along the deoxyribose backbone that lack a base. Known analogs of natural nucleotides hybridize with nucleic acids, such as peptide nucleic acids (PNAs) and phosphate-thioester DNA, in a manner similar to naturally occurring nucleotides.

[0044] The pentose sugar attached to a nucleotide base can vary depending on the type of nucleic acid. For example, the sugar is deoxyribose in DNA and ribose in RNA. In some cases, as used herein, nucleic acid residues may also refer to nucleoside structures such as adenosine, guanosine, 5-methyluridine, uridine, and cytidine. Furthermore, alternative nomenclature for nucleosides includes indicating the prefix "ribose" or "deoxyribose" before the nucleotide base to infer the type of pentose sugar. For example, "ribocytosine" used occasionally herein is equivalent to a cytidine residue because it indicates the presence of ribose at that residue in an RNA molecule. Nucleic acid polymers can be or contain deoxyribonucleotide (DNA) polymers, ribonucleotide (RNA) polymers, including mRNA. Nucleic acids can also be or contain PNA polymers, or combinations of any of the polymer types described herein (e.g., containing residues with different sugars).

[0045] In some embodiments, the PAMP-containing nucleic acid is synthetic. In this context, the term "synthetic" refers to a non-natural characteristic of the nucleic acid. Such nucleic acids can be synthesized de novo using standard synthetic techniques. Alternatively, recombinant techniques well known in the art can be used to generate or derive nucleic acid PAMPs from naturally occurring pathogen sequences. In some embodiments, the sequence of the synthetic nucleic acid PAMP construct is not naturally occurring.

[0046] In some embodiments, the PAMP-containing nucleic acid is an RNA construct. In some of these embodiments, the PAMP-containing nucleic acid is derived from or reflects the sequence of the HCV poly-U / UC region, and in this context, it is generally referred to as a poly-U / UCPAMP RNA construct. In some embodiments, the poly-U / UC PAMP RNA construct is synthetic.

[0047] The PAMP-containing nucleic acid of this disclosure typically comprises (a) a 5' arm region containing a terminal triphosphate (“ppp” or “3xp”); (b) a polyuracil core (also referred to as a “poly-U core”); and (c) a 3' arm region. In one embodiment, the three regions (a, b, and c) are covalently linked in a single nucleic acid polymer macromolecule. The covalent linking can be direct (without one or more dispersed adapter sequences) or indirect (with one or more dispersed adapters and / or one or more sequences). In one embodiment, the 5' arm region is covalently linked to the 5' end of the poly-U core. In one embodiment, the 3' arm region is covalently linked to the 3' arm region of the poly-U core. The polymer can be single-stranded or double-stranded, or can appear as a combination of single-stranded and double-stranded portions.

[0048] In one embodiment, the poly-U core comprises at least eight consecutive uracil residues. In a further embodiment, it comprises eight to 60 consecutive uracil residues, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 consecutive uracil residues. In one embodiment, the poly-U core comprises more than eight consecutive uracil residues. In one embodiment, the poly-U core comprises 12 or more consecutive uracil residues. In some implementations, the poly-U core consists of multiple consecutive uracil residues, such as 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 consecutive uracil residues.

[0049] In one embodiment, the 3' arm contains a 5' terminal nucleic acid residue that is not a uracil residue. Instead, the 5' terminal nucleic acid residue of the 3' arm can be an adenine, guanine, or cytosine residue, or any non-classical residue. In one embodiment, the 5' terminal nucleic acid residue of the 3' arm is a cytosine, guanine, or adenine residue.

[0050] In one embodiment, the nucleotide composition of the 3' arm region is at least about 40% uracil residues. In some embodiments, the 3' arm region is at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% uracil residues. In one embodiment, the 3' arm region comprises a plurality of short segments of consecutive uracil residues (e.g., between about 2 and about 15 nucleotides in length) interspersed with one or more cytosine residues. In one embodiment, the 3' arm region comprises a plurality of short segments of consecutive uracil residues (e.g., between about 2 and about 15 nucleotides in length) interspersed with one or more guanine residues. In one embodiment, the 3' arm region comprises a plurality of short segments of consecutive uracil residues (e.g., between about 2 and about 15 nucleotides in length) interspersed with one or more adenine residues. In one embodiment, the 3' arm region comprises a continuous uracil residue segment that does not exceed the length of the poly-U core of the synthesized PAMP-containing nucleic acid molecule. In one embodiment, the 3' arm region does not contain a continuous uracil residue that is equal to and / or exceeds the length of the poly-U core of the synthesized PAMP-containing nucleic acid molecule. In some embodiments, the 3' arm region contains at least seven consecutive uracil residues, such as 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 consecutive uracil residues.

[0051] At least, the 5' arm region consists of a terminal triphosphate (ppp) portion. In such an embodiment, the triphosphate is located at the 5' end of the synthesized PAMP-containing nucleic acid molecule and can be represented as "5'-ppp". In a further embodiment, the terminal triphosphate is directly linked to the 5' end of the poly-U core sequence. In another embodiment, the 5' arm region comprises a 5' terminal triphosphate and one or more additional nucleic acid residues whose sequence terminates at a 3' end. In this embodiment, the one or more additional nucleic acid residues in the 5' arm region are located between the terminal triphosphate and the uracil residue at the 5' end of the poly-U core. Those skilled in the art will readily understand that the one or more additional nucleic acid residues in the 5' arm region can be any number of nucleic acid residues and can present any sequence without limitation. The sequence of the one or more additional nucleic acid residues in the 5' arm region does not affect the function of the PAMP-containing nucleic acid molecule. For example, as described in U.S. Publications 2015 / 0017207 and 2018 / 0104325, adding a poly-U core region to a non-stimulatory nucleic acid containing 5' triphosphate (e.g., the HCV X region) confers stimulatory properties to innate immune system signaling. In one embodiment, the sequence of one or more additional nucleic acid residues in the 5' arm region does not consist of the entire 5' terminal portion of a naturally occurring HCV genome sequence that is naturally present "upstream" or at the 5' end of the poly-U core of the poly-U / UC region of the HCV strain. In other words, in this embodiment, the entire synthetic PAMP-containing nucleic acid molecule is not a naturally occurring HCV genome with the complete 5' triphosphate, the entire coding region, and the untranslated 3' poly-U / UC region. Therefore, in this embodiment, the 5' arm region, one or more nucleic acid residues in the 5' arm region, and the polyuracil core are not naturally present together in the HCV genome. However, in this embodiment, one or more nucleic acid residues in the 5' arm region may comprise or consist of a sub-fragment of the entire naturally occurring sequence present between the 5' arm region and the polyuracil core. Alternatively, in this embodiment, one or more nucleic acid residues in the 5' arm region may comprise sequences other than the portion or the entire naturally occurring HCV genome sequence present between the 5' end and the polyuracil core.

[0052] In some embodiments, the nucleic acid molecule comprises a sequence of at least 16 nucleotides. In some embodiments, the nucleic acid molecule comprises a sequence of at least about 16 nucleotides to about 1000 nucleotides, such as about 20 to about 1000 nucleotides, about 30 to about 1000 nucleotides, about 40 to about 1000 nucleotides, about 50 to about 1000 nucleotides, about 60 to about 1000 nucleotides, about 70 to about 1000 nucleotides, about 80 to about 1000 nucleotides, about 90 to about 1000 nucleotides, about 100 to about 1000 nucleotides, about 150 to about 1000 nucleotides, or about 1000 nucleotides. The nucleic acid contains approximately 200 to 1000 nucleotides, approximately 250 to 1000 nucleotides, approximately 300 to 1000 nucleotides, approximately 350 to 1000 nucleotides, approximately 400 to 1000 nucleotides, approximately 450 to 1000 nucleotides, approximately 500 to 1000 nucleotides, approximately 550 to 1000 nucleotides, approximately 600 to 1000 nucleotides, approximately 650 to 1000 nucleotides, approximately 700 to 1000 nucleotides, and any number or range thereof. In a further embodiment, the nucleic acid contains approximately 20 to 100 nucleotides, approximately 30 to 100 nucleotides, approximately 40 to 100 nucleotides, approximately 50 to 100 nucleotides, approximately 60 to 100 nucleotides, approximately 70 to 100 nucleotides, approximately 80 to 100 nucleotides, and approximately 90 to 100 nucleotides, and any number or range thereof. In some embodiments, the nucleic acid comprises about 16 to 60 nucleotides, for example, about 16 to about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 nucleotides. In some embodiments, the nucleic acid has up to about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60 nucleotides, and the 3' arm comprises a plurality of short segments of 2 to 15 consecutive uracil residues, wherein one or more cytosine or guanine residues are scattered between the plurality of short segments. In some embodiments, the nucleic acid molecule has up to 53 nucleotides and the 3' arm region is at least 60% uracil residues.

[0053] Non-limiting examples of PAMP-containing nucleic acid sequences covered by the disclosed PAMP-containing nucleic acids are disclosed in U.S. Publications 2015 / 0017207 and 2018 / 0104325, and Saito, T. et al. (2008). Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Nature 454, 523-527; and Schnell, G. et al. (2012). Uridine composition of the poly-U / UC tract of HCV RNA defines non-self recognition by RIG-I. PLoSpathogens 8, e1002839 (each of which is incorporated herein by reference) and are shown herein as SEQ ID NO: 34-123. In some embodiments, the PAMP-containing nucleic acid contains a poly-U core region and / or a 3'-arm sequence, which is independently selected from any poly-U core region and / or 3' arm region of any of the disclosed sequences of SEQ ID NO: 34-123. These exemplary non-restrictive sequences are also provided in Table 1 below. The disclosed PAMP-containing nucleic acids may contain any of the sequences listed therein. It should be understood that such exemplary PAMP-containing nucleic acids will conform to the general structural parameters of PAMP-containing molecules as described herein, including having a 5' terminal triphosphate (ppp) motif. In one embodiment, the PAMP-containing molecule contains the sequence: GGCCAUCCUGUUUUUUUCCCUUUUUUUUUUUCUCCUUUUUUUUUCCUUUUUUUCCUUUUUCUUUUCCUU ...

[0054] Table 1: Sequences of poly-U / UC PAMP constructs with exemplary 5' arms, poly-U cores, and 3'-arm domains

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] a Except for the sequence shown, the 5' arm contains a 5'-ppp portion.

[0062] As described in U.S. Publications 2015 / 0017207 and 2018 / 0104325, nucleic acids containing an HCV-derived RNA PAMP with a polyuracil core sequence can trigger retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling. Therefore, in some embodiments, PAMP-containing nucleic acid molecules are capable of inducing retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) activation. In one embodiment, the RLR is RIG-I. Those skilled in the art will be able to readily determine RLR activation, for example, by measuring the transcription of known downstream RLR-regulated genes, as described in more detail below. For example, in some embodiments, RLR activation can be established by increased expression of IFNβ or ISG54. In another embodiment, RLR activation can be established by increased IRF3 phosphorylation.

[0063] In some embodiments, PAMP-containing nucleic acid molecules are contacted with cells at a concentration of about 80 ng / mL or higher to induce RLR activation. Therefore, in some embodiments, the method includes contacting cells with a PAMP-containing nucleic acid molecule reagent at a concentration of at least about 80 ng / mL to about 500 ng / mL, for example, 100 ng / mL to 250 ng / mL. In some embodiments, the method includes reacting cells with a concentration of at least about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 110 ng / mL, about 120 ng / mL, about 130 ng / mL, about 140 ng / mL, about 150 ng / mL, about 160 ng / mL, about 170 ng / mL, about 180 ng / mL, about 190 ng / mL, about 200 ng / mL, about 210 ng / mL, about 220 ng / mL, about 230 ng / mL, about 240 ng / mL, about 250 ng / mL, about 260 ng / mL, about 270 ng / mL, about 280 ng / mL. Contact with PAMP-containing nucleic acid molecules at concentrations of approximately 290 ng / mL, 300 ng / mL, 310 ng / mL, 320 ng / mL, 330 ng / mL, 340 ng / mL, 350 ng / mL, 360 ng / mL, 370 ng / mL, 380 ng / mL, 390 ng / mL, 400 ng / mL, 410 ng / mL, 420 ng / mL, 430 ng / mL, 440 ng / mL, 450 ng / mL, 460 ng / mL, 470 ng / mL, 480 ng / mL, 490 ng / mL, and 500 ng / mL.

[0064] In some embodiments, the reagent is or comprises a small molecule reagent. Those skilled in the art can readily identify small molecule reagents that induce the RLR and / or IRF3 signaling pathways. Exemplary small-molecule agonists that induce the RLR signaling pathway and / or IRF3 signaling pathway as covered in this disclosure are described, for example, in Bedard, KM et al. (2012). Isoflavone agonists of IRF-3 dependent signaling have antiviral activity against RNA viruses. Journal of Virology 86, 7334-7344; Pattabhi, S. et al. (2016). Targeting Innate Immunity for Antiviral Therapy through Small Molecule Agonists of the RLR Pathway. Journal of Virology 90, 2372-2387; Probst, P. et al. (2017). A small-molecule IRF3 agonist functions as an influenza vaccine adjuvant by modulating the antiviral immune response. Vaccine 35, 1964-1971, all of which are incorporated herein by reference in their entirety. In some embodiments, the small-molecule agent is or comprises a benzothiazole derivative molecule. An exemplary molecule is disclosed in US 9884876. In one particular embodiment, used as a proof of concept in the study described below, the small molecule reagent has the chemical formula N-(6-benzoylamino-1,3-benzothiazo-2-yl)naphthalene-2-carboxamide. This small molecule is referred to herein as "F7" and has the following structure:

[0065]

[0066] In some embodiments, the method includes contacting infected cells with two or more agents that induce IRF3 activation in infected cells. The two or more agents (e.g., a first agent, a second agent, a third agent, etc.) may be contacted together with the cells, for example, when formulated as a single mixture, or in separate applications (coordinated such that the effect of each agent is manifested in the cells within overlapping timeframes). The two or more agents may include, for example, nucleic acids and small molecule agents containing PAMP. Each of the PAMP-containing nucleic acids and small molecule agents may encompass the characteristics and embodiments of each agent as described in more detail above. For example, in one illustrative embodiment, the two or more agents comprise a nucleic acid containing a pathogen-associated molecular pattern (PAMP), wherein the PAMP comprises: a 5' arm containing a terminal triphosphate; a polyuracil core containing at least eight consecutive uracil residues; and a 3' arm containing at least eight nucleic acid residues, wherein the 5' terminal nucleic acid residue of the 3' arm is not uracil, and wherein the 3' arm is at least 30% uracil residues. Furthermore, the small molecule reagent in this illustrative embodiment is or contains a benzothiazole derivative molecule, such as a small molecule containing the chemical formula N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthalene-2-carboxamide.

[0067] In some embodiments, the method further includes contacting cells with a reverse transcriptase inhibitor and at least one agent that induces IRF3 activation in infected cells as described above. Exemplary reverse transcriptase inhibitors may include nucleotide or nucleoside reverse transcriptase inhibitors (NTRIs), which are analogs of naturally occurring nucleotides or nucleosides required for the synthesis of viral DNA. NTRIs compete with natural deoxynucleotides for incorporation into growing viral DNA. However, chain elongation is prevented due to structural differences (e.g., lack of a 3' hydroxyl group) because the next nucleotide to enter cannot form the phosphodiester bond required for the extended chain. Exemplary, non-limiting NTRIs covered by this disclosure include lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, tenofovir alafenamide (TAF), clavidine, Besivo, dazometrazine, remdesivir, etc. Those skilled in the art may choose other suitable reverse transcriptase inhibitors to perform the disclosed methods.

[0068] In one specific implementation, the method includes contacting the cells with two or more of the following:

[0069] (1) A nucleic acid molecule containing a pathogen-associated molecular pattern (PAMP), wherein the PAMP comprises:

[0070] The 5' arm region contains the terminal triphosphate;

[0071] A polyuracil core comprising at least eight consecutive uracil residues; and

[0072] A 3' arm region comprising at least 8 nucleic acid residues, wherein the 5' terminal nucleic acid residue of the 3' arm region is not uracil, and wherein the 3' arm region comprises at least 30% uracil residues, as described in more detail above;

[0073] (2) Small molecule reagents, such as benzothiazole derivatives, such as N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthalene-2-carboxamide; and

[0074] (3) NRTI, such as lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, tenofovir alafenamide (TAF), clavidine, Besivo, tadalafil, remdesivir, etc.

[0075] In one specific embodiment, the method includes contacting infected cells with a nucleic acid molecule comprising a pathogen-associated molecular pattern (PAMP) as described above and an NRTI, said NRTI being, for example, selected from lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, tenofovir alafenamide (TAF), clavidine, Besivo, dazometrazine, remdesivir, etc. As described above, multiple reagents (including NRTIs) can be formulated in combination or mixture, or can be contacted individually but in a coordinated manner (such that each reagent exerts its effect in the cell at overlapping timelines). As described in more detail below, the simultaneous administration of said reagents results in a synergistic effect of inhibition of cccDNA in the infected cells.

[0076] The above-described method can be an in vitro method applied to infected cells maintained in a culture. In such an embodiment, the method may include screening for potential antiviral agents to further aid in the inhibition of cccDNA.

[0077] Alternatively, the method may be an in vivo method performed on a subject who has HBC infection, is suspected of having HBV infection, or is at risk of HBV infection. Therefore, in another aspect, this disclosure provides a method for treating or preventing hepatitis B virus (HBV) infection in a subject in need. The method includes administering a therapeutically effective amount of a composition to the subject, the composition inducing activation of interferon regulatory factor 3 (IRF3) in the subject's infected cells.

[0078] As used herein, the term "treatment" refers to the medical management of a disease, symptom, or condition in a subject (e.g., a human or a non-human mammal, such as another primate, horse, dog, mouse, rat, guinea pig, rabbit, etc.). Treatment may include any marker of successful treatment or improvement of a disease or symptom (e.g., HBV infection). In this document, the term "treatment" refers to the prevention or suppression of infection colonization by a pathogen (e.g., hepatitis B virus). Furthermore, the term "treatment" refers to therapeutic uses, such as resolving an already initiated infection. In one embodiment, the term "treatment" refers to curing an infection to the point where no active pathogen (e.g., hepatitis B virus) remains in the host. In another embodiment, the term "treatment" also includes slowing or suppressing the spread of infection within the body, such as slowing or suppressing the replication rate of the pathogen (e.g., hepatitis B virus). The term also includes reducing the pathogenic burden in cells (or host tissues or the body). In some embodiments, this includes reducing cccDNA levels in body cells. The term also includes accelerating the rate of pathogen clearance relative to the time required for the host's endogenous immune response to clear the pathogen without the application of the disclosed reagents. Treatment or improvement of symptoms can be based on objective or subjective parameters, including the results of a physician's examination. Therefore, the term "treatment" includes the application of the reagents or compositions disclosed in this disclosure to reduce, prevent, or inhibit the development of symptoms or conditions associated with a disease or condition (e.g., HBV infection). The term "therapeutic effect" refers to the improvement, reduction, or elimination of the disease or condition, symptoms of the disease or condition, or side effects of the disease or condition in a subject. The term "therapeuticly effective" refers to the amount of composition that produces a therapeutic effect and can be readily determined.

[0079] In one specific embodiment, the composition is or comprises a nucleic acid molecule comprising a pathogen-associated molecular pattern (PAMP). The PAMP may comprise: a 5' arm containing a terminal triphosphate; a polyuracil core containing at least eight consecutive uracil residues; and a 3' arm containing at least eight nucleic acid residues, wherein the terminal nucleic acid residue of the 5' arm is not uracil, and wherein the 3' arm comprises at least 30% uracil residues. Other embodiments and features of the PAMP and / or nucleic acids containing the PAMP included in this aspect have been described in more detail above and will not be repeated here.

[0080] In another embodiment, the composition is or comprises a small molecule agent that induces RIG-I signaling. In some embodiments, the small molecule agent is or comprises a benzothiazole derivative molecule, such as N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthalene-2-carboxamide. Other embodiments and features of the small molecule agent included in this aspect have been described in more detail above and will not be repeated here.

[0081] In some embodiments, the composition is formulated as a mixture of two or more therapeutic agents (e.g., comprising a first agent, a second agent, etc.). Alternatively, the method may include administering individual compositions (e.g., independently comprising a first agent, a second agent, etc.) to a subject. The separate administrations may be simultaneous or coordinated, such that the effects of the individual compositions are achieved in the subject within overlapping timeframes.

[0082] A representative example of a combined implementation scheme is a method comprising administering to a subject a therapeutically effective amount of a first reagent and a second reagent (in the same or different compositions), wherein:

[0083] The first reagent is or contains a nucleic acid molecule comprising: a 5' arm containing a terminal triphosphate; a polyuracil core containing at least eight consecutive uracil residues; and a 3' arm containing at least eight nucleic acid residues, wherein the terminal 5' nucleic acid residue of the 3' arm is not uracil, and wherein the 3' arm is composed of at least 30% uracil residues; and

[0084] The second reagent is or contains a benzothiazole derivative molecule, such as a small molecule containing the chemical formula N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthyl-2-carboxamide.

[0085] The method may also include administering other antiviral therapies to the subject, such as known therapies for treating HBV infection. In some embodiments, the treatment further includes administering a therapeutically effective amount of a reverse transcriptase inhibitor to the subject, in addition to at least one agent that induces IRF3 activation in infected cells as described above. For example, the method may further include administering a therapeutic amount of an NRTI, such as an NRTI selected from lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, tenofovir alafenamide (TAF), clavidine, Besivo, thymosin alpha 1, remdesivir, etc.

[0086] In one specific embodiment, the method includes administering a therapeutically effective amount of a first reagent and a second reagent (in a single composition or in separate compositions) to a subject, wherein the first reagent is or comprises a nucleic acid molecule, the nucleic acid molecule comprising:

[0087] The 5' arm region contains the terminal triphosphate;

[0088] A polyuracil core comprising at least eight consecutive uracil residues; and

[0089] A 3' arm region comprising at least 8 nucleic acid residues, wherein the 5' terminal nucleic acid residue of the 3' arm region is not uracil, and wherein the 3' arm region comprises at least 30% uracil residues; and

[0090] The second reagent is or contains an NRTI, such as lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, tenofovir alafenamide (TAF), clavidine, Besivo, thymosin alpha 1, remdesivir, etc. In a further embodiment, the NRTI is entecavir or remdesivir.

[0091] In some implementations, one or more compositions are administered only once. Alternatively, one or more compositions may be administered multiple times according to a protocol developed by a medical professional. Factors influencing the protocol include observed cccDNA levels, treatment tolerance, etc.

[0092] In another aspect, this disclosure provides therapeutic compositions for treating hepatitis B virus (HBV). This aspect also includes methods of administering the disclosed therapeutic compositions to treat and / or prevent HBV infection in a subject.

[0093] The therapeutic compositions of this aspect comprise a RIG-I agonist, a vehicle for intracellular delivery, and a pharmaceutically acceptable carrier. In some embodiments, the RIG-I agonist is a nucleic acid molecule comprising a pathogen-associated molecular pattern (PAMP). Specific exemplary embodiments of the nucleic acid molecule and PAMP are described in more detail above and are included in this aspect. In other embodiments, the RIG-I agonist is or comprises a benzothiazole derivative molecule. Exemplary embodiments are described in more detail above and are included in this aspect. In one embodiment, the RIG-I agonist comprises N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthyl-2-carboxamide.

[0094] One or more active agents can be incorporated into a delivery medium to facilitate intracellular delivery. Various therapeutic delivery mediators or systems are known and can be used in therapeutic compositions. Delivery mediators or systems may include particulate formulations, such as emulsions, microparticles, immunostimulatory complexes (ISCOM), nanoparticles (e.g., particles and / or matrices), microspheres, liposomes, nanocapsules, etc., which facilitate antigen delivery. Such delivery mediators can be formulated and used using known and conventional techniques. In one embodiment, the disclosed PAMP-containing nucleic acid and any optional additional therapeutic agent are formulated into a liposome delivery medium. Liposomes are vesicle structures characterized by a phospholipid bilayer and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo rearrangement before forming a closed structure and trap solution, including dissolved solutes within and / or between the lipid bilayers. Exemplary applications of liposome formulations are described in Yallapu, U. et al., Liposomal Formulations in Clinical Use: An Updated Review, Pharmaceutics 9(2):12(2017), which is incorporated herein by reference in its entirety.

[0095] In any of the above-described compositions or therapeutic aspects and embodiments, the composition or reagent is suitably formulated according to known methods for the desired therapeutic administration. For example, the composition can be suitably formulated according to known methods for a preferred route of administration. Pharmaceutical compositions can be formulated for delivery via any systemic route of administration (e.g., intramuscular, intradermal, subcutaneous, dermal, transdermal, intravenous, intraperitoneal, intracranial, intranasal, mucosal, anal, vaginal, oral, or oral routes, or they may be inhaled). Certain routes of administration are particularly suitable for pharmaceutical compositions intended to induce at least an inherent immune response. In particular, transdermal, intramuscular, subcutaneous, and intravenous administration are especially suitable.

[0096] Formulations suitable for introducing therapeutic compositions vary depending on the route of administration. Formulations suitable for parenteral administration (e.g., via intra-articular, intravenous, intramuscular, intradermal, intraperitoneal, intranasal, and subcutaneous routes) include aqueous and non-aqueous isotonic sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Formulations may be presented in single-dose or multi-dose sealed containers (e.g., ampoules and vials).

[0097] Solutions of active compounds that are free bases or pharmacologically acceptable salts can be prepared in water appropriately mixed with a surfactant (e.g., hydroxypropyl cellulose). Dispersions can also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and oils. Under normal storage and use conditions, such formulations may contain preservatives to prevent microbial growth. Suitable forms of pharmaceuticals for injection include sterile aqueous solutions or dispersions and sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions (US Patent No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form should be sterile and fluid enough to be easily injected. It should be stable under the conditions of manufacture and storage and should be preserved to prevent contamination by microorganisms (e.g., bacteria and fungi).

[0098] As used herein, "carrier" includes any and all solvents, dispersion media, diluents, antibacterial and antifungal agents, isotonic agents and absorption-delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The carrier can be a solvent or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, mannitol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Suitable flowability can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. Prevention of microbial action can be facilitated by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, are preferred. Extended absorption of injectable compositions can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0099] The phrase “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce allergic reactions or similar adverse reactions when administered to a subject (e.g., a human).

[0100] General definition

[0101] Unless otherwise defined herein, all terms used herein have the same meaning as understood by one of ordinary skill in the art regarding the content of this disclosure. Practitioners of the art shall refer in particular to Ausubel, FM et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, New York (2010); Coligan, JE et al. (eds.), Current Protocols in Immunology, John Wiley & Sons, New York (2010); Mirzaei, H. and Carrasco, M. (eds.), Modern Proteomics–Sample Preparation, Analysis and Practical Applications in Advances in Experimental Medicine and Biology, Springer International Publishing, 2016; and Comai, L et al. (eds.), Proteomic: Methods and Protocols in Methods in Molecular Biology, Springer International Publishing, 2017.

[0102] For convenience, certain terms used herein, in the specification, embodiments, and appended claims are provided. These definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims.

[0103] The term “or” as used in the claims is used to mean “and / or” unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive, although this disclosure supports the definitions of “and / or” and “alternatives only”.

[0104] Unless otherwise specified, when used in conjunction with the word "comprising" in the claims or description, the words "a" and "an" mean one or more / a combination of one or more.

[0105] Unless the context explicitly requires otherwise, throughout the specification and claims, the terms “comprising,” “including,” etc., should be interpreted in an inclusive sense, rather than an exclusive or exhaustive sense, meaning “including but not limited to.” The use of singular or plural terms also includes both singular and plural, respectively. Furthermore, when used in this application, the terms “here,” “above,” and “below,” and terms with similar meanings, should refer to the entire application and not any particular part of it. The term “about” indicates a number within a small range of variation above or below the specified reference number. For example, in some embodiments, the term “about” refers to a number within a range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% above and / or below the specified reference number.

[0106] As used herein, the terms "peptide" or "protein" refer to a polymer in which monomers are linked together by amide bonds of amino acid residues. When the amino acid is an α-amino acid, either the L-optic isomer or the D-optic isomer may be used, with the L-isomer preferred. The terms peptide or protein, as used herein, cover any amino acid sequence and include modified sequences such as glycoproteins. The term peptide is specifically intended to cover naturally occurring proteins, as well as recombinant or synthetically produced proteins.

[0107] Materials, compositions, and components that can be used in, in combination with, or in the preparation of the disclosed methods and compositions, or as products of the disclosed methods and compositions, are disclosed. It should be understood that when combinations, subsets, interactions, groups, etc., of these materials are disclosed, each of the various individual and collective combinations is particularly considered, even if specific references to the individual compounds and each single combination and arrangement may not be explicitly disclosed. This concept applies to all aspects of this disclosure, including but not limited to steps in the methods. Therefore, specific elements of any of the foregoing embodiments may be combined with or substitute for elements in other embodiments. For example, if multiple additional steps are available, it should be understood that each of these additional steps can be performed using any specific method step or combination of method steps of the disclosed method, and each such combination or subset is specifically considered and should be considered disclosed. Furthermore, it should be understood that the embodiments described herein can be implemented using any suitable materials, such as those described elsewhere herein or materials known in the art.

[0108] The publications cited in this article and the topics on which they are cited are incorporated herein in their entirety by reference.

[0109] The following examples are provided to illustrate certain features and / or implementations of this disclosure. These examples should not be construed as limiting the invention to the specific features or implementations described. Example

[0110] Example 1

[0111] This example illustrates how the induction of the RIG-I signaling pathway destabilizes cccDNA and prevents the formation of new cccDNA in hepatocytes, providing a strategy for eradicating cccDNA and the corresponding hepatitis B virus infection.

[0112] introduction

[0113] Acute viral infection typically triggers activation of the intracellular innate immune system, leading to the induction of intracellular antiviral defenses. This process controls viral replication and spread from the site of infection and modulates adaptive immune responses for systemic viral control. Innate immune activation occurs through the host cell's sensing of pathogen-associated molecular patterns (PAMPs) embedded in viral replication products, including viral nucleic acids. PAMPs are sensed by cellular pattern recognition receptors (PRRs). PRRs that sense viral infection include Toll-like receptors (TLRs), NOD-like receptors (NLRs), intracellular DNA sensors such as cGAS, STING, IFI16, and DAI, and RIG-I-like receptors (RLRs), including retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation antigen 5 (MDA5). Each PRR detection originates from a specific PAMP of the ingress virus or viral replication products, while certain host cell nucleic acids produced during viral infection can also trigger PRR signaling. The induction of TLR, RLR, or STING signaling drives downstream activation of latent transcription factors, including interferon regulatory factor (IRF)3 and NF-κB, to promote the expression of antiviral effector genes and immunomodulatory genes, including chemokines, IFN, and other immunomodulatory cytokines. Notably, acute HBV infection in primary human hepatocytes (PHH) neither activates nor suppresses innate immune PRR signaling, thus reinforcing the concept that HBV is a “latent” virus, as previously shown in vivo in non-human primate infection models.

[0114] Previous studies have shown that RIG-I signaling in response to PAMP RNA can guide innate immune activation and antiviral defense, inhibiting hepatitis C virus (HCV) replication, which also causes chronic hepatitis. HCV PAMPs are 100nt polyuridine / cytosine (polyU / UC) motifs containing the 3' untranslated region of the HCV genome. When introduced into cells as 5'ppp synthetic RNA, polyU / UC PAMPs specifically activate RIG-I to drive IRF3 activation and antiviral innate immunity, thereby inhibiting HCV infection in vitro and activating hepatic innate immunity in vivo. Furthermore, small molecule benzothiazoles or 5'ppp RNA ligands that bind to and activate RIG-I or induce IRF3 activation and innate immunity have been identified as therapeutically inhibiting RNA virus infection and enhancing immune responses. In summary, these studies demonstrate that the RIG-I pathway functions in hepatocytes, where targeted activation of RIG-I confers potent antiviral activity that works fully in the liver. However, it remains unclear how the direct targeting and activation of RIG-I and IRF3 affect HBV infection.

[0115] Here, the role of targeted RIG-I activation in the treatment of HBV infection was evaluated in vitro. RIG-I signaling, triggered by poly-U / UCPAMP RNA or small molecule activators of RIG-I, guides robust IRF3 activation and RIG-I-dependent antiviral action to suppress cccDNA levels. The results indicate that the RIG-I response via IRF3 contributes to reducing the half-life (t) of cccDNA. 1 / 2 This approach embodies cccDNA decay kinetics and blocks RC DNA formation, accompanied by inhibition of HBsAg secretion in HepG2 cells (HepG2-hNTCP) expressing ectopic human sodium / taurocholate cotransporter (hNTCP), differentiated HepaRG (dHepaRG) cells, and primary human hepatocytes (PHH). Targeting RIG-I does not promote cytotoxicity. Notably, when used in combination with therapeutic nucleoside reverse transcriptase inhibitors (NRTIs), entecavir, or poly-U / UC PAMP, treatment rapidly depletes the established cccDNA library. Therefore, targeting RIG-I to activate and activate IRF3-guided innate immune activation processes provides a novel and effective treatment option for curing HBV.

[0116] result

[0117] RIG-I and IRF3 agonists trigger innate immune activation in hepatocytes

[0118] Small-molecule agonists of IRF3 have previously been identified that confer innate immune activation, leading to the induction of IRF3 target genes and antiviral activity against a range of RNA viruses (Bedard, KM, et al. (2012). Isoflavone agonists of IRF-3 dependent signaling have antiviral activity against RNAviruses. Journal of Virology 86, 7334-7344; Pattabhi, S., et al. (2016). Targeting Innate Immunity for Antiviral Therapy through Small Molecule Agonists of the RLR Pathway. Journal of Virology 90, 2372-2387; Probst, P., et al. (2017). A small-molecule IRF3 agonist functions as an influenza vaccine adjuvant by modulating the antiviral immune response. Vaccine 35, 1964-1971). Based on the disclosed structure in US 9884876, (N-(6-benzoylamino-1,3-benzothiazo-2-yl)naphthalene-2-carboxamide), referred to herein as F7, was prepared for analysis of anti-HBV activity. Figure 1A Similarly, a PAMP motif of approximately 100 nt was identified in the HCV genome, containing a 5' ppp and a poly-U / UC region of viral RNA. This region is specifically recognized by RIG-I and confers IRF3-activated RIG-I signaling, leading to antiviral gene expression (Saito, T. et al. (2008). Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Nature 454, 523-527; and Schnell, G. et al. (2012). Uridine composition of the poly-U / UC tract of HCV RNA defines non-self recognition by RIG-I. PLoS pathogens 8, e1002839). Figure 1A(See figure below). The therapeutic effects of F7 and polyU / UC PAMP against HBV infection were evaluated. First, HepG2-hNTCP cells were treated with 10 μM MF7 or 200 ng / ml polyU / UC PAMP formulated in liposomes for 24 h. Control cells were treated with DMSO or infected with Sendai virus (SenV; a potent activator of RIG-I-dependent signaling) or transfected with 200 ng / ml X-RNA in liposomes, a non-PAMP / non-signaling 100 nt RNA motif containing 5'ppp with similar mass to polyU / UC PAMP (Saito et al. (2008), ibid.). Similar to the SenV control, F7 and polyU / UC PAMP, but not DMSO and X-RNA treatment, specifically induced innate immune activation, as indicated by IRF3 translocation into the nucleus (…). Figure 1B Immunoblot analysis showed that F7 and poly-U / UC PAMP but not XRNA treatment specifically induced IRF3 phosphorylation and the expression of the IRF3 target gene IFIT1 in HepG2-hNTCP and dHepaRG cells in a dose-dependent manner. (Fensterl, V., and Sen, GC (2011). The ISG56 / IFIT1 gene family. Journal of interferon & cytokineresearch: the official journal of the International Society for Interferon and Cytokine Research 31, 71-78; and Fensterl, V., and Sen, GC (2015). Interferon-induced Ifit proteins: their role in viral pathogenesis. Journal of virology 89, 2462-2468) Figure 1C The mRNA expression of a group of innate immune response genes (including IFN, ISG, and direct IRF3 target genes) in response to F7 or poly-U / UC PAMP was also evaluated. Figure 1D ; Figure 8A-8LWhile poly-U / UC PAMP treatment also induced type I and type III IFN and ISG expression, F7 treatment induced only IRF3 target gene expression. This difference is consistent with the signaling properties of each molecule and the nature of IFN expression, as IFN expression depends on the activation of both IRF3 and NF-κB, while F7 specifically activates IRF3 but not NF-κB (Bedard et al. (2012), ibid.). Conversely, poly-U / UC PAMP triggered RIG-I signaling of both transcription factors to induce IRF3 target genes, IFN, and therefore ISG (Saito, T. et al. (2008), ibid.; Schnell, G. et al. (2012), ibid.). It is noteworthy that type I IFN, SAMHD1, APOBEC3A, and APOBEC3G induced by poly-U / UC PAMP treatment have been shown to have antiviral activity against HBV infection (Bonvin, M., et al. (2006). Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication. Hepatology (Baltimore, Md) 43, 1364-1374; Chen, Z., et al. (2014). Inhibition of Hepatitis B virus replication by SAMHD1. Biochemical and biophysical research communications 450, 1462-1468; Lucifora, J., et al. (2014). Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science (New York, NY) 343, 1221-1228). In summary, these results indicate that F7 and poly-U / UC PAMP induce innate immune activation in treated cells, which begins with IRF3 activation and the induction of IRF3 target genes.

[0119] IRF3 activation inhibits HBV cccDNA formation.

[0120] To determine how IRF3 activation affects cccDNA production in HBV-infected and infected cells, HepG2-hNTCP cells were treated with F7 or poly-U / UC PAMP (100 ng / ml [=2.94 nM] or 200 ng / ml [=5.87 nM]) post-HBV infection. Cyclosporin A (CsA) treatment was used as an antiviral control for HBV entry inhibitors (Watashi, K. et al. (2014). Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholatecotransporting polypeptide (NTCP). Hepatology (Baltimore, Md) 59, 1726-1737). Cells were harvested 3 days post-infection (dpi) and extracts were prepared using the Hirt extraction method for isolating protein-free DNA, such as... Figure 2A As shown in (Guo, H. et al. (2007). Characterization of the Intracellular Deproteinized Relaxed Circular DNA of Hepatitis B Virus: an Intermediate of Covalently Closed Circular DNA Formation. Journal of Virology 81, 12472-12484; Hirt, B. (1967). Selective extraction of polyoma DNA from infected mouse cellcultures. Journal of Molecular Biology 26, 365-369). Southern blot analysis showed that F7 and polyU / UC PAMP treatments inhibited HBV cccDNA formation compared to the expression levels in controls treated with DMSO or X-RNA. Furthermore, the levels of protein-free relaxed circular (PF-RC) DNA (the precursor to cccDNA) were also significantly reduced by F7 or polyU / UC PAMP treatment. Since both F7 and polyU / UC PAMP activate IRF3, these results link the IRF3 response to the inhibition of cccDNA formation in HBV-infected hepatocytes.

[0121] To further determine how IRF3 activation affects the HBV replication cycle, the expression of viral DNA, viral RNA, extracellular HBV DNA, and secreted HBsAg was analyzed during the infection / treatment timeline. Figure 9A As shown, PF-RC DNA was first detected at 1 dpi. In control-treated cells, cccDNA synthesis occurred at 2 dpi, and both PF-RC and cccDNA accumulated over a 20 dpi time series. However, cccDNA production was significantly inhibited in cells treated with F7 or polyU / UC PAMP within 2 dpi. pgRNA production throughout the infection / treatment time series indicated that it accumulated in untreated control cells from 6 to 20 dpi, but was significantly inhibited in cells treated with F7 or polyU / UC PAMP. Figure 9B The levels of capsid-associated intracellular HBV DNA reverse transcription intermediates generated during the HBV infection / treatment timeline were also measured. Relaxed circular (RC) DNA, double-stranded linear (DL) DNA, and single-stranded (SS) DNA products were found to accumulate in control / untreated cells from 6 dpi to 20 dpi. However, F7 or polyU / UC PAMP treatment significantly reduced the levels of these DNA species. Interestingly, the levels of introduced viral capsid-associated HBV DNA (observed from 1–2 dpi) were unaffected by treatment, but their production was suppressed within 6 dpi. Figure 9C Overall, the suppression of HBV DNA and RNA was associated with a significant decrease in the level of secreted HBsAg in the culture supernatant of cells treated with F7 or polyU / UC PAMP. Figure 9D Importantly, both F7 and poly-U / UC PAMP treatments resulted in arrest of de novo HBV production, as revealed by a reduction in extracellular HBV DNA. Figure 9E These results indicate that F7 and poly-U / UCPAMP treatments affect HBV replication in the post-cccDNA synthesis steps, thereby influencing viral RNA transcription, reverse transcription of HBV DNA, and the production of progeny viral particles.

[0122] To evaluate the antiviral activity of F7 and poly-U / UC PAMP against HBV, a dose-response analysis was performed. To determine the 90% maximum inhibitory concentration (IC50), a dose-response analysis was conducted. 90 ) and half-maximal inhibitory concentration (IC50) 50 The values ​​of ) and the cytotoxic concentrations (CC) of F7 and poly-U / UC PAMP. 50Two different cell lines, HepG2-hNTCP and dHepaRG cells, were used. Cultures were treated with increased concentrations of F7 or polyU / UC, followed by HBV infection, and harvested at 3 dpi. Southern blot and qPCR analysis showed that with increasing concentrations of F7 ( Figure 2B and 2C ) and poly-U / UC PAMP ( Figure 2D and 2E With increasing concentration, cccDNA levels decreased linearly. In HepG2-hNTCP cells, the IC50 of F7 cells was [missing information]. 90 and IC 50 The values ​​were defined as approximately 17.38 μM and 8.48 μM, and in differentiated HepaRG cells as 12.84 μM and 3.38 μM. 50% cytotoxic concentration (CC) 50 (Measured by ATP release from treated cells) exceeding 40 μM ( Figure 10A and 10B IC of poly-U / UC PAMP 90 and IC 50 The values ​​were approximately 11.97 nM and 1.94 nM in HepG2-NTCP cells, and 3.3 nM and 1.61 nM in dHepaRG cells, respectively. 50 Exceeding 23.49 nM (=800 ng / ml) Figure 10C and 10D ).

[0123] Inhibition of de novo HBV cccDNA synthesis

[0124] To further determine the inhibitory effects of F7 and poly-U / UC on cccDNA biosynthesis, addition time experiments were performed in HepG2-hNTCP and dHepaRG cells. For poly-U / UC PAMP, treatments were performed 24 hours before infection (pre-treatment), 1 to 3 dpi (post-treatment), and 24 hours before infection up to 3 dpi (pre-treatment / post-treatment). Figure 3A For F7, treatment was administered 24 hours before infection (before treatment), 24 hours after infection (at treatment), from 1 dpi to 3 dpi (after treatment), or starting treatment 24 hours before infection and continuing until 3 dpi (before / at / after treatment). Figure 3DThe study included simultaneous treatment with CsA and viral inoculation as a positive control. Cultures were inoculated with HBV and harvested at the stated time intervals for assessment of cccDNA levels using Southern blotting and qPCR analysis. Notably, cccDNA synthesis was consistently and significantly inhibited in HepG2-hNTCP and HepaRG cells using the poly-U / UC PAMP regimen, reaching levels similar to those observed in CsA treatment. Figure 3B and 3C In contrast, F7 had little effect on HBV cccDNA levels when administered once before or during treatment, but mediated a significant suppression of cccDNA levels when administered post-infection or throughout the entire 3-day period from pre-infection. Figure 3D-3F These results indicate that poly-U / UC PAMP induces an innate immune response that affects immediate and sustained cccDNA synthesis after HBV infection, while F7 directs an innate immune response that influences cccDNA synthesis after viral entry.

[0125] The antiviral activity of IRF3 agonists is distributed to the nucleus to inhibit cccDNA synthesis.

[0126] To determine one or more steps of HBV cccDNA synthesis affected by F7 and polyU / UC PAMP treatment, we assessed cccDNA levels over a 3-day period (dpi). Cells were seeded with HBV at 4°C for 6 hours, then the inoculum was removed, cells were washed, and placed in 37°C medium to begin synchronous infection (time 0), at which point F7 ( Figure 4A ) or poly-U / UCPAMP ( Figure 4C Cells were processed up to 3 days post-infection (dpi). Whole-cell (W), nuclear (N), and cytoplasmic (C) extracts were harvested daily at 3 hours and 6 hours post-infection, and PF-RC and cccDNA abundances were analyzed by Southern blotting. Figure 4B As shown, PF-RC DNA was detected in the cytoplasmic and whole-cell lysate fractions 3 hours post-infection (hpi) and in the nuclear fraction at 6 hpi, but accumulation decreased after 2–3 dpi with F7 treatment. cccDNA was detected at 1 dpi in untreated cells, but accumulation was delayed and decreased in F7-treated cells. Cells were also harvested at similar time intervals for poly-U / UC PAMP treatment (see [link to original text]). Figure 4CEvaluation of HBV DNA in subcellular fractions also showed that PF-RC DNA was present in whole-cell and cytoplasmic extracts of untreated cells at early 3 and 6 hpi, with levels subsequently accumulating in the nuclear fraction. Nuclear PF-RC DNA levels decreased from 1–3 dpi in cells treated with poly-U / UC PAMP, accompanied by a reduction in ccc DNA in the treated cells. Figure 4D These results indicate that the inhibitory effects of F7 and poly-U / UC on HBV cccDNA biosynthesis occur in the cell nucleus during treatment at 1–3 dpi, potentially affecting the early PF-RC DNA to cccDNA conversion step in the HBV replication process.

[0127] IRF3 activation, alone and in combination with ETV, limits the stability of HBV cccDNA.

[0128] To determine how the host response to IRF3 activation suppresses HBV cccDNA levels, the effects of single treatment with F7 or polyU / UCPAMP, as well as combinations of ETV / F7 or ETV / polyU / UCPAMP, on cccDNA decay kinetics were evaluated. To assess cccDNA decay, F7 and polyU / UCPAMP treatment of HBV-infected cells were compared with entecavir (ETV) treatment, starting at 3 dpi and maintained up to 20 dpi by daily medium changes with fresh ETV, F7, or polyU / UCPAMP. Figure 5A ETV is a nucleoside analog that prevents viral reverse transcription and replication of newly synthesized HBV DNA, thereby preventing the de novo formation of supplemental cccDNA. ETV has an IC50 value of 100 times. 50 This technique was applied to HBV-infected cultures (Langley, DR et al. (2007). Inhibition of Hepatitis B Virus Polymerase by Entecavir. Journal of Virology 81, 3992-4001), allowing for the measurement of cccDNA half-life under conditions that maintain only levels from the initial cccDNA library. Cells were then harvested during the treatment time course, and DNA extracts were analyzed by Southern blotting and qPCR. Figure 5B As shown, in untreated control cells, cccDNA levels moderately increased with the progression of infection time. Cells from ETV-treated cultures consistently maintained cccDNA levels similar to those of 3-dpi cultures throughout the entire time course, indicating persistent and stable cccDNA levels beyond 20 dpi in our in vitro culture system, compared to the reported cccDNA half-life exceeding 40 days. 1 / 2 Consistent Figure 11 (Huang, Q. et al. (2020). Rapid Turnover of HBV cccDNA Indicated by Monitoring Emergence and Reversion of Signature-Mutation in Treated Chronic Hepatitis B Patients. Hepatology; Ko, C. et al. (2018). Hepatitis B virus genome recycling and de novo secondary infection events maintains stable cccDNA levels. J Hepatol 69, 1231-1241). However, as measured by Southern blot analysis, F7 single treatment stimulated cccDNA decay ( Figure 5B The enhanced decay kinetics of cccDNA through F7 treatment were also confirmed by RT-PCR analysis. Figure 5D F7 single treatment reduced the t of cccDNA. 1 / 2 The combination of ETV and F7 further enhanced their antiviral activity, reducing cccDNA abundance and persistence to almost undetectable levels at 20 dpi. Figure 5B and 5D It was also found that, as analyzed by Southern blotting, a single application of poly-U / UC PAMP reduced cccDNA abundance, while it continued to accumulate in cells treated with the X RNA control. Figure 5C qPCR assays also confirmed the inhibitory effect of poly-U / UC PAMP on cccDNA levels. Figure 5D It was also found that, compared with ETV or poly-U / UC PAMP alone, the combination of poly-U / UC PAMP and ETV reduced cccDNA abundance and persistence throughout the treatment time course, with cccDNA being almost undetectable after 20 dpi following simultaneous treatment. Figure 5C Mathematical models show that, compared to each individual simultaneous treatment, the combination of F7 or poly-U / UC treatment with ETV resulted in a higher cccDNA t yield per treatment compared to single treatments. 1 / 2 The average duration decreased from 8.7 days to 6.5 days (F7) and from 7.7 days to 6.9 days (poly-U / UC PAMP). Figure 5E (See Table 2). Neither ETV nor X RNA treatment of cells had any effect on HBsAg production and secretion. However, single and combined treatment with poly-U / UC PAMP and F7 inhibited HBsAg secretion (see Table 2). Figure 5F Therefore, F7 and poly-U / UC PAMP confer therapeutic attenuation of established cccDNA. Furthermore, combined treatment with F7 and poly-U / UC PAMP and ETV confers additive antiviral activity to inhibit cccDNA t. 1 / 2 And lasting for several weeks (Huang, Q. et al. (2020), ibid.; Ko, C. et al. (2018), ibid.) to less than 7 days.

[0129] Table 2: Half-life (t) of cccDNA estimated from decay kinetics under treatment 1 / 2 The delay (τ) before cccDNA begins to decrease.

[0130]

[0131] The inhibition of HBV cccDNA by IRF3 activation is RIG-I dependent.

[0132] To determine whether the activation of IRF3 and inhibition of cccDNA in HBV-infected cells via F7 or polyU / UC PAMP treatment are dependent on RIG-I, rather than being an off-target effect of treatment as an IRF3 agonist, HepG2-hNTCP cells expressing non-targeted guide RNA (HepG2-hNTCP-NT) or guide RNA for knocking out (KO) RIG-I expression (HepG2-hNTCP-RKO) or MDA5 (HepG2-hNTCP-MKO) were generated using CRISPR / Cas9 genome editing technology. Immunoblot analysis of different cell populations showed that HepG2-hNTCP-RKO or HepG2-hNTCP-MKO cells did not express detectable levels of RIG-I or MDA5, respectively. Figure 12 Use F7 ( Figure 6A ) or poly-U / UC PAMP ( Figure 6BCell treatment showed that HepG2-hNTCP-RKO cells did not respond to treatment, while HepG2-hNTCP-NT control cells and HepG2-hNTCP-MKO cells fully responded to F7 to accumulate phosphorylated / activated IRF3, accompanied by IFIT1 expression. HepG2-hNTCP-MKO cells were used as an RLR KO control to reveal the specificity of F7 and polyU / UC PAMP for triggering RIG-I-dependent IRF3 activation (Saito et al. (2008), ibid.). Next, each cell population was infected with HBV and then treated with F7 or polyU / UC PAMP once at 1 dpi. Cells were harvested at 3 dpi for Southern blot analysis of cccDNA levels. Parallel cultures of each cell population were treated with DMSO or a single dose of X-RNA (negative control) or CsA as a treatment control. F7 treatment reduced cccDNA levels in HepG2-hNTCP-NT and HepG2-hNTCP-MKO cells but not in non-HepG2-hNTCP-RKO cells. Figure 6C Similarly, poly-U / UC PAMP inhibition of cccDNA is RIG-I dependent, as cccDNA is inhibited by poly-U / UC PAMP treatment in HepG2-hNTCP-NT and HepG2-hNTCP-MKO cells, but not in HepG2-hNTCP-RKO cells. Figure 6D These results indicate that the antiviral effects of F7 and poly-U / UC PAMP against HBV are specifically signaled via RIG-I, and each can be defined as a RIG-I agonist that activates IRF3 to inhibit cccDNA.

[0133] IRF3-activated RIG-I signaling inhibits HBV infection in primary human hepatocytes.

[0134] To validate the antiviral effect of RIG-I signaling activated by IRF3 via polyU / UC PAMP, HBV infection was assessed in non-immortalized and terminally differentiated primary human hepatocytes (PHHs) with hepatocyte marker gene expression maintained at levels comparable to human liver tissue. PHH cultures were treated with polyU / UC PAMP in a dose-responsive manner, and innate immune activation was assessed. Treatment of PHH cultures with 50–200 ng / ml polyU / UC PAMP induced IRF3 activation, marked by the accumulation of phosphoserine 386IRF3 and IFIT1 expression. Treatment with 100 ng / ml X-RNA did not induce innate immune activation in PHHs, but cells responded fully to acute infection using a SenV control. Figure 7AThis indicates that PHH possesses the complete RIG-I pathway. Furthermore, PHH treatment with poly-U / UC PAMP instead of X-RNA strongly induced the expression of innate immune genes. Figure 7B Then, cccDNA levels were assessed in HBV-infected PHH cells treated with poly-U / UC PAMP. PHH cultures were infected with HBV and treated with poly-U / UC PAMP once at 1 dpi. Cells were harvested at 3 dpi, DNA was extracted, and Southern blot analysis was performed. Figure 7C As shown, treatment with poly-U / UC PAMP suppressed cccDNA levels in infected PHH to levels similar to those achieved with CsA treatment. Therefore, poly-U / UC treatment, which triggers IRF3-mediated RIG-I signaling and innate immune activation, guides the cccDNA-suppressing response in HBV-infected PHH. These results demonstrate the efficacy of therapeutics targeting the RIG-I pathway in controlling the virus in HBV-infected primary cells.

[0135] discuss

[0136] The persistence of cccDNA in the nuclei of HBV-infected hepatocytes is key to mediating chronic HBV infection, with recent analyses indicating that a given cccDNA library persists in vivo. 1 / 2The duration is approximately 5–21 weeks (Huang, Q. et al. (2020), ibid.). The problem is that current treatments for chronic HBV infection neither significantly reduce nor eliminate cccDNA (Maynard, M. et al. (2005). Sustained HBs seroconversion during lamivudine and adefovir dipivoxil combination therapy for lamivudine failure. J Hepatol 42, 279–281; Werle-Lapostolle et al. (2004). Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 126, 1750–1758; Zoulim, F., and Durantel, D. (2015). Antiviral therapies and prospects for a cure of chronic hepatitis B. ColdSpring Harb Perspect Med 5). Members of the current nucleoside analogue class of HBV treatment agents are administered long-term, often for life, to maintain viral suppression in patients.Furthermore, these therapies have vulnerabilities in their ability to completely shut down viral replication, thus the nuclear cccDNA library (Huang, Q. et al. (2020), ibid.) persists even after long-term treatment (Gish, R. et al. (2012). Selection of chronic hepatitis B therapy with high barrier to resistance. Lancet Infect Dis 12, 341-353; Werle-Lapostolle et al. (2004), ibid.; Zoulim, F., and Locarnini, S. (2009). Hepatitis B virus resistance to nucleos(t)ide analogues. Gastroenterology 137, 1593-1608, e1591-1592), however, importantly, it has been demonstrated that inhibition of cccDNA during acute HBV infection can occur through a non-cytolytic mechanism driven by cytokines guided by IFN-α, IFN-γ, or tumor necrosis factor-α, associated with the expression of APOBEC3 deaminase (Lucifora et al. (2014), ibid.; Xia, Y. et al. (2016). Interferon-gamma and Tumor Necrosis Factor-alpha Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis. Gastroenterology 150, 194-205). Pharmacological induction of intrahepatic cytokine responses has been considered an ideal treatment for chronic hepatitis B (Chang, J. et al. (2012). The innate immune response to hepatitis B virus infection: implications for pathogenesis and therapy. Antiviral Research 96, 405-413).While this approach utilizes innate immunity and immune-regulating signaling programs driven by specific cytokines to induce their action and suppress cccDNA gene expression, cytokine therapy for chronic HBV faces the hurdle of systemic toxicity from widespread off-target effects (non-hepatic effects of cytokine therapy) (Kwon, H., and Lok, AS (2011). Hepatitis B therapy. Nature reviews Gastroenterology & Hepatology 8, 275-284; Locarnini, S. et al. (2015). Strategies to control hepatitis B: Public policy, epidemiology, vaccine and drugs. Journal of Hepatology 62, 76-86), thus highlighting the need for target-guided therapy strategies that can also be combined with current NA therapies for HBV.

[0137] This study demonstrates that targeting the RIG-I and RLR pathways to activate IRF3 (using the F7 small molecule of RIG-I delivered to hepatocytes and a polyU / UC PAMP agonist as proof of concept) guides a specific RIG-I-dependent innate immune response via IRF3, which effectively inhibits HBV cccDNA in a cell culture model of HBV infection. Furthermore, mechanistic studies show that administration of F7 or polyU / UC PAMP inhibits de novo cccDNA biosynthesis and enhances cccDNA degradation, rather than inhibiting the production of newly synthesized HBV rcDNA as current NA HBV drugs do. Administration of F7 and polyU / UC PAMP induces IRF3 activation and expression of IRF3 target genes. Figure 1A-1DTherefore, the potential mechanism by which F7 and poly-U / UC PAMP inhibit cccDNA may involve the action of IRF3 target genes to block cccDNA synthesis and confer the role of promoting cccDNA destabilization and degradation to deplete it from infected cells. Notably, F7 treatment does not induce the expression of type I or type III IFNs because the RIG-I activating properties of these compounds do not signal to NF-κB but specifically activate downstream IRF3 (Bedard et al. (2012), ibid.; Probst et al. (2017), ibid.). Thus, in the case of F7, the antiviral effect of cccDNA inhibition is independent of IFN action but acts through IRF3-responsive genes. These results suggest that poly-U / UC PAMP also induces strong IRF3 target gene expression and low levels of IFN that can induce ISG. This includes the APOBEC3 gene, a known antiviral effector against HBV replication (Bonvin et al. (2006). Interferon-inducible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication. Hepatology (Baltimore, Md) 43, 1364-1374; Turelli, P. et al. (2004). Inhibition of Hepatitis B Virus Replication by APOBEC3G. Science 303, 1829).IRF3 activation drives the expression and production of various immunomodulatory cytokines and chemokines, including CXCL10, a chemical inducer for T cells (Sankar, S. et al. (2006). IKK-i signals through IRF3 and NFkappaB tomediate the production of inflammatory cytokines. Cell Signal 18, 982-993; Zhai, Y. et al. (2008). CXCL10 regulates liver innate immune response against ischemia and reperfusion injury. Hepatology 47, 207-214), as well as guiding the expression of factors that regulate ubiquitination (Maelfait, J., and Beyaert, R. (2012). Emerging role of ubiquitination in antiviral RIG-I signaling. Microbiol Mol Biol Rev 76, 33-45) and various cell signaling processes (Zhou, Y. et al. (2017). Post-translational regulation of antiviral innate signaling. EurJ). Immunol 47, 1414-1426. Therefore, in addition to the known functions of the APOBEC gene, IRF3 target genes have been proposed to include a variety of anti-cccDNA effectors. These effector genes then confer pleiotropic effects to i) inhibit cccDNA amplification, and ii) destabilize cccDNA and / or enhance cccDNA degradation. Indeed, cccDNA t was observed when cells were treated with F7 or poly-U / UC PAMP. 1 / 2The reduction was significant, and this reduction was further enhanced when cells were treated with any of these drugs plus entecavir. Furthermore, for poly-U / UC PAMP, the antiviral effect against cccDNA may also include IFN-mediated effects induced by low levels of IFN (Isorce, N. et al. (2016). Antiviral activity of various interferons and pro-inflammatory cytokines in non-transformed cultured hepatocytes infected with hepatitis B virus. Antiviral research 130, 36-45; Lucifora, J. et al. (2014), ibid.; Phillips, S. et al. (2017). Peg-Interferon Lambda Treatment Induces Robust Innate and Adaptive Immunity in Chronic Hepatitis B Patients. Frontiers in Immunology 8; Robek, MD et al. (2005). Lambda Interferon Inhibits Hepatitis B and C Virus Replication. Journal of Virology 79, 3851-3854; Xu, F. et al. (2018). Type III interferon-induced CBFβ inhibits HBV replication by hijacking HBx. Cellular & Molecular Immunology. Interestingly, these effects of IFN and specific ISG generated by treatment with poly-U / UC PAMP, compared to treatment with F7, may contribute to the inhibition of cccDNA, resulting in a moderately shortened half-life. Mechanistically, it is proposed that the antiviral effects of F7 and poly-U / UC PAMP may also include altering cellular DNA repair mechanisms that would otherwise contribute to cccDNA biosynthesis.Because HBV utilizes host DNA repair factors to repair discontinuities in RC-DNA and convert them into cccDNA that allows transcription, several cellular DNA repair proteins involved in cccDNA metabolism are known, including TDP2 (Koniger, C. et al. (2014). Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses. Proceedings of the National Academy of Sciences of the United States of America 111, E4244-4253), DNA ligases (Long, Q. et al. (2017). The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation. PLoS pathogens 13, e1006784), and DNA topoisomerases (Sheraz, M. et al. (2019). Cellular DNA Topoisomerases Are Required for the Synthesis of Hepatitis B Virus Covalently Closed Circular DNA. Journal of Virology 93) and DNA polymerases (polα,λ,κ) (Qi, Y. et al. (2016). DNA Polymerase kappa Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus. PLoSpathogens 12, e1005893; Tang, L. et al. (2019). DNA Polymerase alpha is essential for intracellular amplification of hepatitis B virus covalently closed circular DNA. PLoS pathogens 15, e1007742) may be candidates regulated by RIG-I / IRF3 signaling.

[0138] Current research indicates that RIG-I and IRF3 can be specifically targeted to activate the RLR innate immune program to control HBV infection by inhibiting cccDNA, resulting in reduced t-cell immunity compared to weeks or months without treatment. 1 / 2 The duration of infection can be reduced to several days. Therefore, targeting the innate immune system and the RLR pathway provides an effective strategy for novel antiviral therapies against HBV, which can be offered alone or in combination with NA for HBV treatment. Identifying innate immune targets guided by RIG-I and IRF3, which lead to cccDNA depletion, will contribute to a deeper understanding of the mechanisms of action and unique antiviral properties of these novel drug candidates for HBV cure.

[0139] Methods and Materials

[0140] Table 3: PCR resources.

[0141]

[0142]

[0143] *MT-CO3: Mitochondrial cytochrome c oxidase subunit 3

[0144] Experimental Model and Topic Details

[0145] Cell culture: Human NTCP (a subclone of HepG2) stably expressing the human hepatocellular carcinoma cell line C3A was maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 10% heat-inactivated FBS, 1x Glutamax (GIBCO), 100 U / ml penicillin and 100 μg / ml streptomycin, and selected / amplified with medium containing 1 μg / ml puromycin, as previously described (Guo, F. et al. (2017). HBV core protein allosteric modulators differentially altercccDNA biosynthesis from de novo infection and intracellular amplification pathways. PLoS pathogens 13, e1006658; Ko, C. et al. (2014a). DDX3 DEAD-Box RNAHelicase Is a Host Factor That Restricts Hepatitis B Virus Replication at the Transcriptional Level. Journal of Virology 88, 13689-13698). The HepAD38 cell line, which generates HBV via tetracycline (TET) induction, was maintained as described above (Watashi, K. et al. (2013). Interleukin-1 and tumor necrosis factor-alpha trigger restriction of hepatitis B virus infection via a cytidine deaminase activation-induced cytidine deaminase (AID). The Journal of Biological Chemistry 288, 31715-31727).Human hepatoma progenitor cell line HepaRG was cultured in complete William's E medium supplemented with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, hydrocortisone 21-hemisuccinate (Cayman), human insulin (Sigma), and 1 x Glutamax (GIBCO) (Gripon, P. et al. (2002). Infection of a human hepatoma cell line by hepatitis B virus. Proceedings of the National Academy of Sciences of the United States of America 99, 15655-15660). Primary human hepatocytes were freshly isolated from chimeric mice with humanized livers reconstructed with PHH. The recovered PHH was cultured in DMEM supplemented with 10% heat-inactivated FBS, 15 μg / ml L-proline, 25 ng / ml insulin, 50 nM dexamethasone, 5 ng / ml EGF and 0.1 mM L-ascorbic acid 2-phosphate, as previously described (Ishida, Y. et al. (2015). Novel robust in vitro hepatitis B virus infection model using fresh human hepatocytes isolated from humanized mice. The American journal of pathology 185, 1275-1285).

[0146] The HepG2-hNTCP-NT / RIG-I / MDA5 KO cell line was generated using a CRISPR system: To express the human taurocholate sodium cotransport polypeptide (hNTCP), the gene coding sequence was amplified from cDNA clones prepared from dHepaRG cells. A C9 tag was added via PCR amplification. Transduced cells were selected using 20 μg / ml blastcin, and single-cell clones with the best growth potential were screened for their ability to support HBV infection. For CRISPR / Cas-mediated gene knockout, a guide RNA (gRNA) sequence was designed using CRISPR tools from Benchling (Biology Software, 2017, https: / / benchling.com). The gRNA target oligonucleotide was cloned into the Cas9-t2a-pRRL lentiviral vector using the In-Fusion cloning kit (Takara). The gRNA sequences used for gene knockout were gRIG-I:5'-GGGTCTTCCGGATATAATCC-3' (SEQ ID NO:28) and gMDA5:5'-GTGGTTGGACTCGGGAATTCG-3' (SEQ ID NO:29) (Esser-Nobis, K. et al. (2019). Comparative Analysis of African and Asian Lineage-Derived Zika Virus Strains: Reveals Differences in Activation of and Sensitivity to Antiviral Innate Immunity. Journal of Virology 93). After transduction, cells were maintained under continuous selection with 10 μg / ml puromycin, and knockout was confirmed by Western blotting.

[0147] Method details

[0148] HBV infection: HBV (genotype D) was purified from the supernatant of HepAD38 cells using PEG concentrations followed by a sucrose gradient, as previously described (Ko, C. et al. (2014). DDX3 DEAD-Box RNA Helicase Is a Host Factor That Restricts Hepatitis B Virus Replication at the Transcriptional Level. Journal of Virology 88, 13689-13698; Watashi, K. et al. (2013), ibid.). For HBV infection, cells were seeded into collagen-coated plates. One day later, cells were infected with HBV in DMEM containing 4% polyethylene glycol 8000 (PEG-8000). The multiplicity of infection (indicated as viral genome equivalents / cell) is shown in each figure. After 24 hours, the inoculum was removed and the infected culture was kept in complete DMEM containing 2.5% DMSO until harvest, as previously described (Ni, Y. et al. (2014). Hepatitis B and D viruses exploit sodium taurocholate co-transporting polypeptide for species-specific entry into hepatocytes. Gastroenterology 146, 1070-1083).

[0149] Reagent: F7 is a small molecule based on a benzothiazole core structure identified in high-throughput screening of IRF3 agonists (US9884876; Probst et al. (2017), ibid.). F7 (N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthalene-2-carboxamide) (see [link to product]). Figure 1A The structure was obtained from US 9884876 and synthesized de novo by Medchem Source, Inc. for use in this invention. Working stock of Sendai virus (SenV) strain Cantell was generated as previously described (Loo, YM et al. (2008). Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity. Journal of Virology 82, 335-345). Mirus Trans-IT mRNA transfection reagent was used to treat cells with X-RNA and PAMP-RNA. Cyclosporin A (C1832) and entecavir (SML1103) were purchased from Sigma Aldrich.

[0150] In vitro transcription: PolyU / UC PAMP-RNA and X-RNA were each transcribed into polyU / UC PAMP RNA (forward: 5'-TAATACGACTCACTATAGGCCATCCTGTTTTTTTCCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTCCTTTTTTTTTTTTTTTTTTTTCTCCTTTTTTTTTTT-3' (SEQ ID NO: 30), reverse: 5'-AAAGGAAAGAAAAGGAAAAAAAGAGGAAAAAAAAAGGAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAGGGAAAAAAACAGGATGGCCTATAGTGAGTCGTATTA-3' (SEQ ID NO: 30) Synthesis of complementary oligonucleotides linked to the T7 promoter of X-RNA (forward: 5'-TAATACGACTCACTATAGGTGGCTCCATCTTAGCCCTAGTCACGGCTAGCTGTGAAAGGTCCGTGAGCCGCTTGACTGCAGAGAGTGCTGATACTGGCCTCTCTGCAGATCAAGT-3' (SEQ ID NO:32), reverse: 5'-ACTTGATCTGCAGAGAGGCCAGTATCAGCACTCTCTGCAGTCAAGCGGCTCACGGACCTTTCACAGCTAGCCGTGACTAGGGCTAAGATGGAGCCACCTATAGTGAGTCGTATTA-3' (SEQ ID NO:33)), as previously described (Kell, A. et al. (2015). Pathogen-Associated Molecular Pattern Recognition of Hepatitis C Virus Transmitted / Founder Variants by RIG-I IsDependent on U-Core Length. Journal of Virology). 89, 11056-11068; Saito, T. et al. (2008, ibid.). RNA products were generated using T7 RNA polymerase and the T7 MEGAshortscript kit (Ambion) according to the manufacturer's instructions. 10 μg of the oligonucleotide mixture was annealed using a gradient PCR program (95 °C for 2 min, then gradually decreasing the temperature by 1 °C / 30 sec to 50 °C).After annealing, as described by the manufacturer, the reaction mixture was assembled in a microcentrifuge tube containing 7.5 mM of each nucleotide, 10x reaction buffer, 2 μg template DNA, and T7 enzyme without RNase, and incubated at 37°C for 4 hours to allow for in vitro transcription. The DNA template was then removed by Turbo DNAase treatment, and unincorporated nucleotides and proteins were removed by phenol-chloroform extraction. RNA was precipitated using ethanol and ammonium acetate as described by the manufacturer and resuspended in nuclease-free water. RNA concentration was determined by absorbance using a Nanodrop spectrophotometer. RNA quality and purity were assessed on a denatured 2% formaldehyde agarose gel.

[0151] Quantitative reverse transcription real-time qPCR (RT-qPCR) analysis: Total cellular RNA was extracted from cells using TRIZOL reagents and the manufacturer's protocol (Invitrogen). cDNA was synthesized from purified RNA using the iScript Select cDNA Synthesis Kit (Biorad, Inc.) via random and oligonucleotide (dT) initiation. For HBV cccDNA expression analysis, total DNA was extracted using the DNeasy kit (QIAGEN). For selective cccDNA PCR analysis, isolated DNA was treated with 10 units of T5 exonuclease (NEB) in a 10 μl reaction volume at 37°C for 30 min, followed by heat inactivation at 95°C for 5 min and dilution 4-fold with nuclease-free water. For extracellular HBV DNA quantification, PCR was programmed using external HBV plasmid standards (Ko, C., et al. (2018), ibid.). The relative mRNA levels of all target genes were quantified by RT-qPCR using the ΔΔCT method, and expression levels were normalized to housekeeping genes. Real-time PCR assays were performed using the SYBR green method (Applied Biosystems) on an Applied Biosystems 7300 thermal cycler. Table 3 provides primer sequence information for RT-qPCR analysis of human and HBV genes.

[0152] Southern blot analysis of HBV DNA: Southern blot analysis was performed on DNA isolated from the cytoplasmic viral capsid, exactly as described above (Ko, C. et al. (2014). DDX3DEAD-Box RNA Helicase Is a Host Factor That Restricts Hepatitis B Virus Replication at the Transcriptional Level. Journal of Virology 88, 13689-13698; Ko, C. et al. (2014b). Residues Arg703, Asp777, and Arg781 of the RNase H Domain of Hepatitis B Virus Polymerase Are Critical for Viral DNA Synthesis. Journal of Virology 88, 154-163). To detect protein-free forms of HBV-DNA, including cccDNA, a modified Hirt extraction method was used, as previously described (Cai, D. et al. (2013). A southern blot assay for detection of hepatitis B virus covalently closed circular DNA from cell cultures. Methods in Molecular Biology (Clifton, NJ) 1030, 151-161; Guo, H. et al. (2007). Characterization of the Intracellular Deproteinized Relaxed Circular DNA of Hepatitis B Virus: an Intermediate of Covalently Closed Circular DNA Formation. Journal of Virology 81, 12472-12484). The protein-free DNA preparation extracted by Hirt was digested with a plasmid-safe ATP-dependent DNase (Epicentre). The extracted viral DNA was separated on a 1.2% agarose gel and transferred to a positively charged nylon membrane (GE Healthcare, Amersham) via upward capillary transfer, and then hybridized with a digoxigenin-labeled HBV-specific DNA probe. The DNA signal was detected using a DIG luminescence detection kit (Roche).

[0153] Immunoblot analysis: Immunoblot analysis was performed essentially as described (Lee, S. et al. (2016). Hepatitis B virus X protein enhances Myc stability by inhibiting SCF(Skp2)ubiquitin E3 ligase-mediated Myc ubiquitination and contributes to tooncogenesis. Oncogene 35, 1857-1867). Cells were lysed with RIPA buffer containing 0.1% sodium dodecyl sulfate in the presence of a mixture of protease and phosphatase inhibitors (Sigma-Aldrich). Lysates were separated by SDS-PAGE and then electrotransferred to a nitrocellulose membrane. The membrane was probed overnight at 4°C with an appropriate primary antibody, followed by probed with the corresponding HRP-conjugated secondary antibody. The following primary antibodies were used in this study: rabbit anti-IRF3 phosphoserine 386 (CellSignaling), rabbit anti-IRF3 (Cell Signaling), rabbit anti-IFIT1 (antibody 972; generated in rabbit targeting the IFIT1 437-490aa peptide sequence), rabbit anti-RIG-I (antibody 969; generated in rabbit targeting the RIG-I aa 1-227 peptide sequence), rabbit anti-MDA5 (Enzo Life Sciences), rabbit anti-lamin B1 (Abcam), mouse anti-calcinin (Abcam), and mouse anti-α-tubulin (Cell Signaling).

[0154] Immunofluorescence analysis: Immunofluorescence analysis was performed essentially as described (Lee, S. et al. (2016), ibid.). Briefly, cells seeded on collagen-coated 24-mm coverslips were fixed with 3% paraformaldehyde and permeabilized with 0.2% Triton-X 100 in PBS. Cells were then incubated with the IRF3-specific mouse monoclonal antibody AR1 (Rustagi, A. et al. (2013). Two new monoclonal antibodies for biochemical and flow cytometric analyses of human interferon regulatory factor-3 activation, turnover, and depletion. Methods (San Diego, Calif) 59, 225-232) and rabbit anti-human NTCP (Invitrogen), followed by specific secondary antibodies conjugated to Alexa Flour 594- or 488- (Invitrogen) and 4',6-bisamidinyl-2-phenylindole (DAPI), respectively. After immunostaining, coverslips were secured with Prolong Gold anti-fade reagent (Life Technologies) and images were collected using a Nikon Elipse-Ti confocal microscope.

[0155] Cytotoxicity assay: Cytotoxicity of HepG2-C3A-hNTCP and dHepaRG cell cultures was assessed using CellTiter-Glo as described (Edwards, TC et al. (2019). Inhibition of HBV replication by N-hydroxyisoquinolinedione and N-hydroxypyridinedinoneribonuclease H inhibitors. Antiviral Research 164, 70-80). Cells were seeded in 96-well plates in DMEM medium and incubated with or without serially diluted compounds or poly-U / UC PAMP. CellTiter-Glo was used according to the manufacturer's instructions. TM The reagent (Promega) measured cytotoxicity for each cell type using ATP content as a measure of cell viability. A luminescent plate reader (Berthold) was used to read the plates, and the relative luminescent units (RLU) data produced for each well were calculated as a percentage signal compared to the untreated control. The values ​​are expressed as CC. 50Values ​​(50% cytotoxicity concentration; the concentration of the compound or poly-U / UC PAMP that results in a 50% decrease in absorbance compared to untreated cells). Tests were performed in triplicate, with each experiment repeated three times. To calculate the selectivity index (SI), a CC value greater than 40 was considered acceptable. 50 The value is assigned a maximum of 40. The selectivity index (SI) of a compound is calculated as follows: SI = CC 50 / IC 50 .

[0156] HBV entry assay and cell fractionation: Cells were seeded with HBV for 6 h at 4 °C in the presence of 4% PEG. To assess HBV entry, the inoculum was removed by washing with PBS containing proteinase K, and cells were transferred to 37 °C after attachment. After incubation and treatment, cells were lysed in hypotonic buffer (100 mM HEPES, 15 mM MgCl2, 100 mM KCl, and Nonidet P-40) and homogenized using a dounce homogenizer. Cytoplasmic fractions were separated from nuclear pellets by centrifugation (6,000 × g, 4 °C, 5 min). The nuclear pellets were resuspended in extraction buffer (20 mM HEPES, 15 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, and 25% (v / v) glycerol, including a mixture of DTT and protease inhibitors). Each cell fraction was mixed with 1% SDS (v / v) and protein-free DNA was extracted using the Hirt extraction method.

[0157] ELISA: For ELISA, collect the cell supernatant, centrifuge at 10,000 x g for 5 minutes, and recover the liquid fraction for analysis. Perform HBsAg ELISA on the recovered supernatant using the Hepatitis B virus antigen (HBsAg) detection kit (AlphaLISA; PerkinElmer) according to the manufacturer's instructions.

[0158] cccDNA half-life analysis model: To analyze the decay of cccDNA under treatment, the following mathematical model is used: If t≤τ, C(t)=C(0), otherwise C(t)=C(0)e -λ(t-τ), where C(t) is the amount of cccDNA at time t after treatment, C(0) is the cccDNA at the start of treatment, λ is the decay rate of cccDNA, and τ is the delay before treatment causes cccDNA decay. λ and τ were estimated using MATLAB R2017b by simultaneously fitting three replicates of data from each of the four treatments. The half-life of cccDNA was calculated as ln(2) / λ and is reported in Table 2. Using the MATLAB function nlparci, which is based on the asymptotic normal approximation method of least squares estimators (Vandeginste, B. (1989). Nonlinear regression analysis: Its applications, DMBates and DGWatts, Wiley, New York, 1988. ISBN 0471-816434. Price: £34.50. Journal of Chemometrics 3, 544-545), the 95% confidence intervals of parameters λ and τ were estimated and reported in Table 2.

[0159] Statistical analysis: Statistical analysis and multiple comparisons were performed using Graphpad software. Continuous variables were reported as mean ± standard deviation (SD). For all tests, a p-value ≤ 0.05 was considered statistically significant.

[0160] Although illustrative embodiments have been described and illustrated, it should be understood that various changes may be made therein without departing from the spirit and scope of the invention. sequence list <110> University of Washington Gale, Jr., Michael J. Lee, Sooyoung <120> Compositions and methods for treating hepatitis B virus infection <130> UWOTL-1-72750 <150> US 62 / 909,321 <151> 2019-10-02 <160> 125 <170> PatentIn version 3.5 <210> 1 <211> 105 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 1 60 uuuuuucucc uuuuuuuuuc cucuuuuuuu ccuuuucuuu ccuuu 105 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 2 gcctattgat tggaaagtat gt 22 <210> 3 <211> 16 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 3 gctgaggcgg tatcta 16 <210> 4 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 4 ctcctccagc ttatagacc 19 <210> 5 <211> 17 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 5 gtgagtgggc ctacaaa 17 <210> 6 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 6 agaagcaggc aatcacagaa aa 22 <210> 7 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 7 ctgaaaccga ccatagtgga aat 23 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Synthetic <400> 16 aaggactgca agtgccgct 19 <210> 17 <211> 18 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 17 gctggtccaa gacatccc 18 <210> 18 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 18 tcacaggcgc attactgcc 19 <210> 19 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 19 ggatttgaac caatcgctgg a 21 <210> 20 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 20 gagaagggac aagcacatgg 20 <210> twenty one <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> twenty one tggatccatc aagtgtctgg 20 <210> twenty two <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> twenty two ccgaggaccc gaaggttac 19 <210> twenty three <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> twenty three tccaacagtg ctgaaattcg 20 <210> twenty four <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> twenty four acaactttgg tatcgtggaa gg 22 <210> 25 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 25 gccatcacgc cacagtttc 19 <210> 26 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 26 ccccacaaac cccattacta aaccca 26 <210> 27 <211> 26 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 27 tttcatcatg cggagatgtt ggatgg 26 <210> 28 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 28 gggtcttccg gatataatcc 20 <210> 29 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 29 gtggttggac tcgggaattc g 21 <210> 30 <211> 122 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 30 taatacgact cactataggc catcctgttt ttttcccttt ttttttttct tttttttttt 60 tttttttttt tttttttttt tttctccttt ttttttcctc tttttttcct tttctttcct 120 tt 122 <210> 31 <211> 122 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 31 aaaggaaaga aaaggaaaaa aagaggaaaa aaaaaggaga aaaaaaaaaa aaaaaaaaaa 60 aaaaaaaaaa aaagaaaaaa aaaaagggaa aaaaacagga tggcctatag tgagtcgtat 120 ta 122 <210> 32 <211> 115 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 32 taatacgact cactataggt ggctccatct tagccctagt cacggctagc tgtgaaaggt 60 ccgtgagccg cttgactgca gagagtgctg atactggcct ctctgcagat caagt 115 <210> 33 <211> 115 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 33 acttgatctg cagagaggcc agtatcagca ctctctgcag tcaagcggct cacggacctt 60 tcacagctag ccgtgactag ggctaagatg gagccaccta tagtgagtcg tatta 115 <210> 34 <211> 105 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 34 ggccauccug uuuuuuuccc uuuuuuuuuu ucuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60 uuuuuucucc uuuuuuuuuc cucuuuuuuu ccuuuucuuu ccuuu 105 <210> 35 <211> 107 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 35 60 uuuuuucccu cuuucuuccc uucucaucuu auucuacuuu cuuucuu 107 <210> 36 <211> 131 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 36 ggccauccug uuuuuuuccc uuuuuuuuuu ucuuuuuuuu uuuuuuuuuu uuuuuuuuuuu 60 uuuuuucuccc uuuuuuuuuc cucuuuuuuu ccuuuucuuu ccuuuccccc cccccccccc 120 cccccccccc c 131 <210> 37 <211> 105 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 37 ggccauccug uuuuuuuccc uuuuuuuuuu ucuuuuuuuu uuuuuuuuuu uuuuuuuuuuu 60 uuuuuucuccc uuuuuuuuuc cccccccccc cccccccccc ccccc 105 <210> 38 <211> 105 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 38 ggccauccug uuuuuuuccc uuuuuuuuuu ucuuuuuuuu uuuuuuuuuu uuuuuuuuuuu 60 uuuuuuuucc uuuuuuuuuc cucuuuuuuu ccuuuucuuu ccuuu 105 <210> 39 <211> 105 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 39 60 uuuuuuvuuu uuuuuuuuuu uuuuuuuuuuuuuuuuuuuuu uuuuu 105 <210> 40 <211> 105 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 40 ggccauccug uuuuuuuccc uuuuuuuuuu ucuuuuuuuu cuccuuuuuu uuuccucuuu 60 uuuuccuuuu cuuuccuuuc cccccccccc cccccccccc ccccc 105 <210> 41 <211> 105 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 41 60 uuccucuuuu uuuccuuuuc uuuccuuucc cccccccccc ccccc 105 <210> 42 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 42 ggccauccug uuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuucuccuu uuuuuuuccu 60 cu 62 <210> 43 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 43 cccccccccc uuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuucuccuu uuuuuuuccu 60 cu 62 <210> 44 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 44 ggccauccug uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuccccc cccccccccc 60 cc 62 <210> 45 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 45 60 uu 62 <210> 46 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 46 60 uu 62 <210> 47 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 47 60 cu 62 <210> 48 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 48 60 cu 62 <210> 49 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 49 60 cu 62 <210> 50 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 50 60 uu 62 <210> 51 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 51 ggccauccug uuuuuuuuuu cuuuuuuuuu ucuuuuuuuu uucucuccuu uuuuuuuccu 60 cu 62 <210> 52 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 52 ggccauccug uuuuuuuuuu uuuuucuuuu uuuuuuuuuu ucuucuccuu uuuuuuuccu 60 cu 62 <210> 53 <211> 62 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 53 ggccauccug uuuuuuuuuu cccuuuuuuu uuucccuuuu uuuucuccuu uuuuuuuccu 60 cu 62 <210> 54 <211> 62 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 54 ggccauccug uuuuuuuuuu uuuuuuuucc ccccuuuuuu uuuucuccuu uuuuuuuccu 60 cu 62 <210> 55 <211> 105 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 55 ggccauccug uuuuuuuccc uuuuuuuuuu ucuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60 uuuuuucucc uuuuuuuuuc cucuuuuuuu ccuuuucuuu ccuuu 105 <210> 56 <211> 35 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 56 ggccauucuu uuuuuuuuuu uuuucuuucu ucuuu 35 <210> 57 <211> 116 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 57 ggccauuccc uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 116 <210> 58 <211> 101 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 58 ggccguuccu uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuuuuuu uuucuuuucc ccuuuuuuau uuuucuuuuc u 101 <210> 59 <211> 105 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 59 ggccauccug uuuuuuuguu uuuucuuuuu uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuuuuuucu uuuuuucccu uuuuuuuuuu uuuuuuuuuu uuuuu 105 <210> 60 <211> 131 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 60 ggccauccc cuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuucc cuuuuuuucc 120 uuuuuuuuu u 131 <210> 61 <211> 116 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 61 ggccguucug uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuccuuu uuuuuauucc ucuucu 116 <210> 62 <211> 123 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 62 ggccauccc uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 120 very 123 <210> 63 <211> 87 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 63 ggccauuccu uuuuuuuu uuuuuuuu uuuucuuuuu uuuccuuuu uuuuuuuu 60 uuuuuuuuuu ccuuuucuuu cuuuuu 87 <210> 64 <211> 87 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 64 60 uuuuuuuuuu ccuuuucuuu cuuuuu 87 <210> 65 <211> 96 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 65 60 uccuucuuuu uuuccuuucu uuuucccuuc uuuaau 96 <210> 66 <211> 101 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 66 60 uuuuuuuucc uuuccuucuu uuuuuuuuuu ucccuuua u 101 <210> 67 <211> 87 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 67 60 ucccuuuuuc uuucuuccuu cuuuaau 87 <210> 68 <211> 100 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 68 60 100 <210> 69 <211> 109 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 69 60 uuuuuuuuuu uuccuuuuuu ucuuuuuuuu uuuuuuuuuc cuuccuuuu 109 <210> 70 <211> 34 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 70 ggccauccug uuuuuuuuuu uuuuuucuuu cuuu 34 <210> 71 <211> 74 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 71 60 uuuuucuuuu cuuu 74 <210> 72 <211> 81 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 72 60 uuhuruuucu cuucuccuuu u 81 <210> 73 <211> 107 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 73 60 107 <210> 74 <211> 109 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 74 60 ucuuuuuuuu uuccuuuuuu uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 109 <210> 75 <211> 35 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 75 ggccauucuu uuuuuuuuuu uuuucuuucu ucuuu 35 <210> 76 <211> 48 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 76 ggccaucccc uucuuuuuuu uuuuuuuuuu uuuuuccuuu ucuucuuu 48 <210> 77 <211> 100 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 77 ggccauccug uuuuuuuccc uuuuuuuuuu ucuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60 ucuccuuuuu uuuuccucuu uuuuuccuuu ucuuuccuuu 100 <210> 78 <211> 112 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 78 60 112 <210> 79 <211> 145 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 79 60 120 ccuuucuuuu ccuuccuucu uuaau 145 <210> 80 <211> 95 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 80 60 cuuucuuuu uuuuuuuuuu ccuuucuucu uuaau 95 <210> 81 <211> 72 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 81 ggccauuucc uguuuuuuuu uuuuuuuuuu uucuuuccuu cuuuuuuccu uucuuuuccu 60 uccuucuuua au 72 <210> 82 <211> 87 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 82 60 ucccuuuuuc uuucuuccuu cuuuaau 87 <210> 83 <211> 143 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 83 60 120 uuucuuuucc uuccuucuuu aau 143 <210> 84 <211> 108 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 84 60 uuuuuucuuu ccuucuuuuu uuuccuuucu uuuccuuccu ucuuuaau 108 <210> 85 <211> 82 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 85 ggcauccugu uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uu <210> 86 <211> 130 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 86 acacuccauu ucuuuuuuug uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uu uuccuuuuuu 130 <210> 87 <211> 129 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 87 guccuucugu uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 120 ccuuacuuu 129 <210> 88 <211> 57 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 88 ggguccccuu guuuuuuuuu uuucuuuccu ucuuuccuuu ccuaaucuuu cuuucuu 57 <210> 89 <211> 96 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 89 60 uccuucuuuu uuuccuuucu uuuucccuuc uuuaau 96 <210> 90 <211> 115 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 90 60 uuuuuuuuuu uuuuuuuuuu uuuccuuucc uuuuuuuuuu uuuuucccuu uuuau 115 <210> 91 <211> 107 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 91 ggccauccug uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuuuu cuuuuuuuuu 60 cuucuuuuuc uuuccuuuuu uuuuuuuu uuuuuuuuc uucuuuc 107 <210> 92 <211> 96 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 92 ggccauuucc uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuucccucu uuuucuucuc uuuuuccuuc uuuaau 96 <210> 93 <211> 111 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 93 gcuaacuguu ccuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuuuuuuuu cccucuuucu uccccuucuca ucuuauucua cuuucuuuu u 111 <210> 94 <211> 107 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 94 gcuaacuguu ccuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu cuuuuuuuuu 60 uuuuuucccu cuuucuuccc uucucaucuu auucuacuuu cuuucuu 107 <210> 95 <211> 92 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 95 60 uuucuuucuu accuuacuuu acuuucuuuu cu 92 <210> 96 <211> 111 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 96 60 uuuuuuuauu uucuuuuccu uucuuucuca ccuuacauua cuuucuuucu u 111 <210> 97 <211> 77 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 97 60 cacuuuaccu ucuuucu 77 <210> 98 <211> 124 <212> RNA <213> Artificial sequence <220> <223> synthetic <400> 98 60 uuuuuuuuuu uuuuuuuuuu uuuuuccuuu ccuuucuuuc uuaccuuacu uuacauucuu 120 uucu 124 <210> 99 <211> 119 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 99 60 uuuuuuuuuu uuuuuuuuuu uucuuuccuu ccuuucucac cuucuuuuac uucuuuccu 119 <210> 100 <211> 93 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 100 60 uuccuuuccu ucuuacucua cuuuacuuuu ucu 93 <210> 101 <211> 129 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 101 gcuaacuguu cuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuuuuuu uuuuuuuu uuuuuuuuuc uuuuccuucu ucuuucuuac cuuauuuucc 120 uuuuuuuu 129 <210> 102 <211> 86 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 102 gcuaacuguu uuuuuuuu uuuuuuuu uuuuuuuucu uuuuuucu uuucuuuccu 60 ucuuaccuua cuuuacuuuc uuuuucu 86 <210> 103 <211> 133 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 103 gcuaacuguu uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 uuuuuuuuuuuuuuuuuuuuuuuuuuuuuc cuuuuuccuu uuccuucucu uuuuaccuua 120 133 <210> 104 <211> 144 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 104 60 120 uuaccuuauu uuacuuucuu uccu 144 <210> 105 <211> 119 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 105 60 uuuuuuuuuu uguuucuuuu ccuucucauu uccuucuuau cuuaauuacu uccuuuccu 119 <210> 106 <211> 88 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 106 60 cuuuuaccu uacuuuauuu ucuuuccu 88 <210> 107 <211> 101 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 107 gcuaacuguu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60 uucuuuucuu ucuuuucuca ccuuacuuuua cuuccuuuuc u 101 <210> 108 <211> 86 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 108 gcuaguuuuc uuuuuuuu uuuuuuuu uuuuguuuuu uuuuuuuuc cucuuuuucc 60 wow wow wow 86 <210> 109 <211> 105 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 109 ggccauccug uuuuuuuccc uuuuuuuuuu ucuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60 uuuuuucucc uuuuuuuc cucuuuuuuu ccuuuucuuu ccuuu 105 <210> 110 <211> 79 <212> RNA <213> artificial sequence <220> <223> Synthesis <400> 110 ccauuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu 60 cuuuuaauuu uccuucuua 79 <210> 111 <211> 116 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 111 60 uuguuuuuuu uuuuuuuuuu cuuuccuucu uuccugacuu uuaauuuucc uucuua 116 <210> 112 <211> 84 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 112 60 uuuuuuuuuu ccuucuucuu uaau 84 <210> 113 <211> 74 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 113 60 ccuucuucuu uaau 74 <210> 114 <211> 38 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 114 ggccauuuuc uguuuuuuuu uuuuuuuauuu ucuuuaau 38 <210> 115 <211> 53 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 115 ggccauuuuc uuuuuuuuuu cucuuuuuuu uuuuuuuuuu uauuuucuuu aau 53 <210> 116 <211> 82 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 116 60 uuuuuuuuuu auuuucuuua au 82 <210> 117 <211> 82 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 117 60 uuuuuuuuuu auuuucuuua au 82 <210> 118 <211> 85 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 118 60 uuuuuuuuuu uuuauuuucu ucuuu 85 <210> 119 <211> 59 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 119 gguccuaagu uuuuuuuuuu uucuuccuuc cuucuuuccu uuucuaauuu uccuucuuu 59 <210> 120 <211> 62 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 120 gguccuaagu uguuuuuuuu uuuuuuuccu uccuucuuuc ccuuuucuaa uuuuccuucu 60 uu 62 <210> 121 <211> 74 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 121 60 aauuuuccuu cuuu 74 <210> 122 <211> 95 <212> RNA <213> Artificial sequence <220> <223> Synthetic <400> 122 60 cuuuuuccuu uucuuuuuuu uuuucucccu uuaau 95 <210> one two three <211> 70 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> one two three 60 ucccuuuaau 70 <210> 124 <211> 70 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 124 ggccauccug uuuuuuuccc uuuuuuuuuu ucuccuuuuu uuuuccucuu uuuuuccuuu 60 ucuuuccuuu 70 <210> 125 <211> 53 <212> RNA <213> artificial hierarchy <220> <223> synthetic <400> 125 ggccauuuuc uuuuuuuuuu cucuuuuuuu uuuuuuuuuu uauuuucuuu aau 53

Claims

1. Use of the composition in the preparation of a reagent for inhibiting the level of covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) in infected cells, said composition comprising: RIG-I agonists, Mediators for intracellular delivery, and Pharmaceutically acceptable carriers The RIG-I agonist mentioned therein is the nucleic acid molecule of SEQ ID NO:

34.

2. The use according to claim 1, wherein the composition further comprises a nucleoside reverse transcriptase inhibitor (NRTI).

3. The use according to claim 2, wherein the NRTI is selected from lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, tenofovir alafenamide (TAF), clavidine, Besivo, thymosin alpha 1 and remdesivir.

4. Use of the composition in the preparation of a reagent for treating hepatitis B virus (HBV) infection in a subject by inducing retinoic acid-induced gene I (RIG-I)-like receptor (RLR) signaling pathway, the composition comprising: RIG-I agonists, Mediators for intracellular delivery, and Pharmaceutically acceptable carriers The RIG-I agonist mentioned therein is the nucleic acid molecule of SEQ ID NO:

34.

5. The use of claim 4, wherein the composition further comprises a benzothiazole derivative molecule having the chemical formula N-(6-benzoylamino-1,3-benzothiazole-2-yl)naphthalene-2-carboxamide.

6. The use of claim 4, wherein the composition further comprises a nucleoside reverse transcriptase inhibitor (NRTI).

7. The use of claim 5, wherein the composition further comprises a nucleoside reverse transcriptase inhibitor (NRTI).

8. The use of claim 6 or 7, wherein the NRTI is selected from lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir, tenofovir alafenamide (TAF), clavidine, Besivo, dazometidine, and remdesivir.

9. Use according to any one of claims 4-7, wherein the RIG-I agonist is incorporated into the medium.

10. The use of claim 9, wherein the medium is a nanoparticle, exosome, microparticle, or lipid particle.

Images