Composition for treating hepatitis B and method for evaluating the replication activity of hepatitis B virus

Abstract

To provide a replication activity evaluation system of HBV which can visualize and digitize HBV DNA replication inexpensively, safely and quickly, and in a short time, and an evaluation method using the system, and also a novel HBV replication inhibitory composition having a different mechanism of action from conventional anti-HBV medicine.SOLUTION: A therapeutic agent of hepatitis B having an HBV-Pol activity inhibitor, which consists of a phosphorization inhibitor inhibiting the phosphorization of a TxY motif present in a terminal protein region of HBV-Pol, as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for treating hepatitis B, which contains a MAP kinase inhibitor as an active ingredient, a system for evaluating the replication activity of hepatitis B virus, and a method for evaluating the replication activity using the system. [Background technology]

[0002] Hepatitis B is a viral hepatitis caused by infection with the hepatitis B virus (often abbreviated as "HBV" in this specification). Hepatitis B is transmitted through the blood and bodily fluids of HBV-infected individuals, and the main routes of infection are known to be vertical infection (mother-to-child infection) in which a child is infected through the blood of an HBV-infected mother at birth, and horizontal infection through sexual contact, tattooing, sharing of syringes in blood transfusions and mass vaccinations, needlestick injuries, etc. (Non-Patent Document 1).

[0003] HBV infection is roughly divided into transient infection and persistent infection. When infected at age 5 or older, HBV infection usually does not progress to persistent infection, but takes the form of a transient infection, since the immune function is fully developed, and then lifelong immunity is acquired. However, it is estimated that about 20 to 30% develop acute hepatitis B, of which 1% develop fulminant symptoms. On the other hand, persistent infections are mostly mother-to-child infections, but they can also occur when the patient is infected at age 3 or younger, when the immune system is immature, due to medical procedures, family infections, etc. Patients with persistent HBV infection are called HBV carriers, and 10 to 15% of them develop chronic hepatitis, which may progress to liver cirrhosis or liver cancer. The number of HBV carriers is estimated to be 1.5 million in Japan and 300 to 400 million worldwide (Non-Patent Document 2).

[0004] Currently, nucleic acid analogue preparations such as lamivudine and entecavir are widely used as therapeutic agents for chronic hepatitis B. These therapeutic agents have achieved a certain degree of effectiveness in reducing the amount of virus in the blood and delaying the occurrence and progression of liver cirrhosis and hepatocellular carcinoma. However, since they cannot eliminate HBV DNA in liver cells, when the administration of the agent is discontinued, the HBV DNA in the blood rises again and hepatitis relapses. This requires long-term administration of the therapeutic agent. In addition, relapse during long-term treatment using the nucleic acid analogue preparation is accompanied by the emergence of drug-resistant viruses. This makes chronic hepatitis B treatment even more difficult (Non-Patent Documents 3 and 4).

[0005] For the above reasons, the development of a new HBV treatment drug with a mechanism of action different from that of conventional nucleic acid analogue preparations is desired. For the development of such a treatment drug, an experimental system capable of accurately quantifying and evaluating HBV infection and HBV replication is essential. There are several evaluation systems for analyzing HBV infection and HBV replication, but all of them have problems such as safety issues and difficulty in handling large amounts of samples because they use infectious viruses. Furthermore, at the current stage, cells that can be efficiently infected with HBV are limited to primary human hepatocyte cultures or expensive HepaRG (registered trademark) cells, and there is a significant restriction on the cells that can be used. In addition, although infection can be established in cells that overexpress NTCP, which is considered to be the HBV receptor, the infection efficiency is very low, and even if a cell line (HepG2.2.15, HepAD38, etc.) into which the HBV genome has been inserted is used, it takes a long period of culture of 7 to 12 days before replication can be detected. Therefore, there is a major problem of lack of throughput. Such problems in HBV infection experiments make it difficult to develop new drugs for HBV. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Aspinall EJ et al., 2011, Occup Med (Lond), 61: 531-540. [Non-Patent Document 2] Fattovich G., et al., 2008, J Hepatol, 48: 335-352. [Non-Patent Document 3] Lau DT et al., 2000, Hepatology, 32: 828-834. [Non-Patent Document 4] Koumbi L., 2015, World J Hepatol, 7: 1030-1040. Summary of the Invention [Problem to be solved by the invention]

[0007] The object of the present invention is to develop and provide an evaluation system for HBV replication activity that can inexpensively, safely, and quickly visualize and quantify HBV DNA replication in a short period of time using conventional cells without using infectious viruses, and an evaluation method using the same.

[0008] Another object of the present invention is to apply the HBV replication activity evaluation system to a screening method to isolate novel anti-HBV compounds having a mechanism of action different from that of conventional anti-HBV drugs, and to provide them as hepatitis B therapeutic agents that are also effective against recurrent hepatitis B. [Means for solving the problem]

[0009] In order to solve the above problems, the inventors have constructed an HBV replication activity evaluation system that can visualize HBV genome replication in a short period of time and quantify its activity using conventional cells without using an infectious virus.

[0010] In addition, using this HBV replication activity evaluation system, anti-HBV natural compounds were searched for in a fungal extract library, and hypothemycin was obtained as a compound that inhibited HBV replication in a concentration-dependent manner. Hyposemycin is an inhibitor of MAPK kinase (often abbreviated as "MAPKK" in this specification) that contributes to the MAPK (mitogen-activated protein kinase) pathway, but it was previously unknown that MAPK kinase inhibitors have HBV replication inhibitory activity. Therefore, four known MAPK kinase inhibitors were examined for their HBV replication inhibitory activity, and it was revealed that all of them had the activity. In addition, when constitutively active MEK-1 was overexpressed in HBV-infected host cells, HBV replication was significantly promoted. This result indicated that MAPK kinase activity in host cells plays an important role in HBV replication. Furthermore, analysis of the amino acid sequence of HBV-derived proteins revealed that all HBV genotypes have a MAPK kinase phosphorylation motif, TxY (T represents a threonine residue, x represents any amino acid residue, and Y represents a tyrosine residue), in the terminal protein region of HBV-Pol. It is therefore believed that MAPK kinase phosphorylates this phosphorylation motif to activate HBV-Pol, and it was speculated that MAPK kinase inhibitors inhibit the phosphorylation of HBV-Pol by MAPK kinase, thereby inactivating HBV-Pol and inhibiting HBV replication.

[0011] The present invention is based on the above-mentioned new findings and provides the following.

[0012] (1) A method for inhibiting HBV replication, comprising the step of inhibiting phosphorylation at the threonine residue (T) and tyrosine residue (Y) of the TxY motif (x is any amino acid residue) present in the terminal protein region of HBV polymerase (HBV-Pol).

[0013] (2) A composition for inhibiting HBV replication, comprising a MAPK (mitogen-activated protein kinase) kinase inhibitor.

[0014] (3) The composition for inhibiting HBV replication according to (2), wherein the MAPK kinase inhibitor is one or more selected from the group consisting of hypothemycin represented by formula 1, trametinib represented by formula 2, PD98059 represented by formula 3, and PD184352 represented by formula 4.

[0015] [ka]

[0016] (4) A composition for treating hepatitis B, comprising a MAPK kinase inhibitor.

[0017] (5) A composition for treating hepatitis B according to (4), which is used in combination with a nucleic acid analog for treating hepatitis B.

[0018] (6) A nucleic acid comprising an epsilon sequence derived from HBV and comprising at least one reporter sequence containing an intron on the 3'-terminal side of the epsilon sequence.

[0019] (7) The nucleic acid according to (6), further comprising a direct repeat 1 sequence derived from Hepatitis B virus on the 3'-end side of the epsilon, and the reporter sequence is contained between the epsilon sequence and the direct repeat 1 sequence.

[0020] (8) The nucleic acid according to (6), comprising a first epsilon sequence, a direct repeat 2 sequence, a direct repeat 1 sequence, and a second epsilon sequence derived from a Hepatitis B virus in the above-mentioned order from the 5'-end side, and the reporter sequence is contained at any position between the respective sequences.

[0021] (9) A vector comprising a promoter capable of inducing gene expression in a host cell, and the nucleic acid according to any one of (6) to (8) positioned downstream of the promoter in an expressible state.

[0022] (10) A host cell transformed with the vector according to (9).

[0023] (11) An evaluation system for evaluating HBV replication activity for evaluating HBV replication activity, comprising the vector described in (9), an HBV-Pol expression vector in which the HBV P gene is positioned in an expressible state downstream of a promoter capable of inducing gene expression in a host cell, and an HBc expression vector in which the HBV C gene is positioned in an expressible state downstream of a promoter capable of inducing gene expression in a host cell.

[0024] (12) The evaluation system according to (11), further comprising an HBx expression vector in which the HBV X gene is positioned in an expressible state downstream of a promoter capable of inducing gene expression in a host cell.

[0025] (13) The evaluation system according to (11) or (12), further comprising a primer pair designed to enable detection of the intron-removed reporter sequence.

[0026] (14) A method for evaluating HBV replication activity, comprising an introduction step of introducing into a host cell the vector described in (9), an HBV-Pol expression vector in which the HBV P gene is positioned in an expressible state downstream of a promoter capable of inducing gene expression in a host cell, and an HBc expression vector in which the HBV C gene is positioned in an expressible state downstream of a promoter capable of inducing gene expression in a host cell, a culture step of culturing the host cell after the introduction step, an extraction step of extracting DNA from the host cell after the culture step, and a detection step of detecting a gene product of the reporter sequence of the vector, which may be contained in the DNA obtained in the extraction step, and which has an intron-removed reporter sequence.

[0027] (15) The method according to (14), wherein in the transfection step, an HBx expression vector in which the HBV X gene is positioned in an expressible state downstream of a promoter capable of inducing gene expression in the host cell is further transfected into the host cell.

[0028] This specification includes the disclosure of Japanese Patent Application No. 2016-158252, which is the priority basis of this application. Effect of the Invention

[0029] According to the method for evaluating HBV replication activity of the present invention, HBV genome replication can be visualized in a short time, safely, and quickly, and its activity can be quantified, without using an infectious virus, using general cells. Furthermore, by using the method for evaluating HBV replication activity of the present invention as a screening tool, it becomes possible to search for anti-HBV compounds having HBV replication inhibitory activity.

[0030] According to the composition for inhibiting HBV replication or the composition for treating hepatitis B containing the MAPK kinase inhibitor of the present invention, it is possible to provide a composition for inhibiting HBV replication or a composition for treating hepatitis B, which has a mechanism of action different from that of conventional nucleic acid analogs for treating hepatitis B, as well as a method for inhibiting HBV replication.

[0031] In addition, by using the composition for treating hepatitis B of the present invention in combination with a conventional nucleic acid analog for treating hepatitis B, a synergistic effect of anti-HBV activity can be expected.

[0032] Furthermore, a composition for inhibiting HBV replication or a composition for treating hepatitis B comprising the MAPK kinase inhibitor of the present invention exhibits anti-HBV activity even against drug-resistant HBV, and therefore can be an effective therapeutic agent for hepatitis B. [Brief description of the drawings]

[0033] [Figure 1]A: A conceptual diagram of the HBV genomic DNA structure. The part indicated by the thick black line in the center is the HBV incomplete circular double-stranded genomic DNA (rcDNA) consisting of about 3.2 Kb. The thick grey lines indicate the ORFs (open reading frames) of four genes encoded in the HBV genomic DNA, where C indicates the position of the C gene, P indicates the position of the P gene, S indicates the position of the S gene, and X indicates the position of the X gene. The thin black lines on the outer edge indicate the pregenomic RNA (often referred to as "pgRNA" in this specification), which is the longest 3.5 kb mRNA among the mRNAs synthesized using the (-) strand of the HBV genomic DNA as a template. B: A conceptual diagram of the pgRNA structure. In the figure, DR1 and DR2 indicate the direct repeat 1 and 2 sequences, respectively, and ε indicates the ε sequence. The nucleic acid for evaluating the HBV replication activity of the present invention (HBV replication activity evaluation nucleic acid) was prepared based on the base sequence of this pgRNA. [Diagram 2] A: A conceptual diagram showing an example of a nucleic acid for evaluating HBV replication activity of the present invention. B: A conceptual diagram of reporter pgRNA when an HBV replication activity evaluation vector in which the nucleic acid for evaluating HBV replication activity shown in A is incorporated into an expression vector is introduced into a cell and expressed. In the reporter pgRNA, an intron in the reporter sequence has been removed by pre-mRNA splicing. C: A conceptual diagram of reporter negative strand DNA (reporter (-)DNA) synthesized by the reverse transcription activity of HBV-Pol using the reporter pgRNA shown in B as a template. The nucleic acid for evaluating HBV replication activity and the reporter negative strand DNA can be distinguished by the presence or absence of an intron. [Diagram 3] FIG. 1 is a conceptual diagram showing an example of various expression vectors constituting the HBV replication activity evaluation system of the present invention. A: A conceptual diagram of pBB-intron, an example of a vector for evaluating HBV replication activity of the present invention. B: A conceptual diagram of pCI-HBV-Pol, an expression vector for the P gene encoding HBV-Pol. C: A conceptual diagram of pCI-HBc, an expression vector for the C gene encoding HBc. D: A conceptual diagram of pCI-HBx, an expression vector for the X gene encoding HBx. [Figure 4]A: The results of agarose gel electrophoresis. After the HBV replication activity evaluation vector was transfected into HeLa cells together with the HBV protein expression vectors in the combinations shown in the figure (C: HBc, P: HBV-Pol, X: HBx), DNA was extracted and PCR was performed using a primer pair that specifically detects reporter minus strand DNA. The PCR product (131 bp) is shown. B: A standard curve for reporter minus strand DNA. DNA extracted from cells was serially diluted and prepared using real-time PCR. C: The results of real-time PCR performed on the DNA sample used in the experiment in A, and quantitative analysis using the standard curve in B. D: A quantitative analysis of the correlation between the expression levels of HBV proteins (HBc, HBV-Pol, and HBx) and DNA replication using real-time PCR. [Diagram 5] FIG. 2 shows the HBV DNA replication inhibitory effect of entecavir, using the HBV replication activity evaluation method of the present invention. [Figure 6] FIG. 1 shows the effects of natural compounds isolated from a filamentous fungal culture extract library on HBV DNA replication, genomic DNA (G3PDH exon 8) content, and mitochondrial DNA (ND5) content. [Figure 7] 1 shows the effect of MAPK kinase inhibitors on HBV DNA replication. The effects of four different MAPK kinase inhibitors (PD98059, PD184352, trametinib, hypothemycin) and the positive control anti-HBV drug entecavir on HBV DNA replication are shown (n=3, *P<0.01, **P<0.001, ***P<0.00001). [Figure 8] This figure shows the effects of various MEK1 expressions on HBV DNA replication, genomic DNA (G3PDH exon 8) amounts, mitochondrial DNA (ND5) amounts, pgRNA and host mRNA transcription when wild-type (Wt), constitutively active (2E and 2D), and dominant-negative mutant (2A) human MEK1 expression vectors were each introduced into HeLa cells using the method for evaluating HBV replication activity of the present invention. [Figure 9]A: A schematic diagram showing each region of HBV-Pol in genotype C. B and C: Comparison of the base sequences of the TxY motif in HBV-Pol predicted in genotypes A to H. The TxY motif (TKY) in the terminal protein region is conserved in all genotypes A to H, but the TxY motif (284TAY286) in the spacer region was only found in genotype C. [Figure 10] A: A schematic diagram of non-phosphorylated mutant HBV / C-Pol based on HBV / C-Pol of genotype C. B: A diagram showing the promoting activity of HBV replication by forced expression of constitutively active MEK by non-phosphorylated mutant HBV / C-Pol. [Figure 11] FIG. 1 shows the synergistic effect of the combination of hypothemycin and entecavir on the inhibitory activity of HBV DNA replication. [Figure 12-1] A schematic diagram of drug-resistant HBV-Pol. L stands for leucine, T for threonine, S for serine, M for methionine, I for isoleucine, and G for glycine. [Figure 12-2] FIG. 1 shows the results of the inhibitory effect of a MAPK kinase inhibitor on the HBV replication activity of drug-resistant HBV-Pol. [Figure 13] This figure shows the effect of various deletion-type HBV replication activity evaluation vectors on reverse transcription of reporter pgRNA when they were transfected into HeLa cells together with HBV protein expression vectors CP (C: HBc, P: HBV-Pol) or CPX (C: HBc, P: HBV-Pol, X: HBx). In the figure, Δε1 indicates that pBB-intron (Δε1), ΔDR2 indicates that pBB-intron (ΔDR2), ΔDR1(2) indicates that pBB-intron (ΔDR1(2)), and Δε2 indicates that pBB-intron (Δε2) were transfected into HeLa cells as the deletion-type HBV replication activity evaluation vector ΔpBB-intron. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] 1. Hepatitis B virus polymerase activity inhibitor (HBV-Pol activity inhibitor) 1-1. Overview The first aspect of the present invention is an activity inhibitor of hepatitis B virus polymerase (often referred to as "HBV-Pol" in the present specification). The HBV-Pol inhibitor of the present invention comprises a phosphorylation inhibitor that inhibits phosphorylation of the activation site in HBV-Pol. The HBV-Pol inhibitor of the present invention can inhibit HBV replication by inhibiting HBV-Pol activity. 1-2.Definition "Hepatitis B virus (HBV)" is a DNA virus belonging to the Orthohepadovirus genus of the Hepadnaviridae family, and is the causative virus of hepatitis B. There are eight known genotypes of HBV (genotypes A, B, C, D, E, F, G, and H) due to differences in gene sequences. These genotypes have differences in regional distribution and pathology. For example, in Japan, genotype C (often referred to as "HBV / C" in this specification, and the same applies to other genotypes) traditionally accounted for the majority of infections, followed by HBV / B infections, but in recent years, the number of HBV / A infections has been increasing. On the other hand, in Europe and the United States, there are many HBV / A and HBV / D infections. It is known that about 20 to 30% of HBV / A patients develop chronic hepatitis after contracting acute hepatitis, but the chronicity rate after contracting acute hepatitis is low for HBV / B and HBV / C.

[0035] "Hepatitis B virus polymerase" (HBV polymerase: HBV-Pol, or HBV-DNA Pol) is a DNA polymerase with reverse transcriptase activity encoded by the P gene of the HBV genome, and is involved in the replication of HBV (Summers J., & Mason WS, 1982, Cell, 29:403-415). The amino acid sequence of HBV-Pol also differs slightly depending on the genotype. For example, HBV-Pol of HBV / A (often referred to as "HBV / A-Pol" in this specification, the same applies to other genotypes) consists of the amino acid sequence shown in SEQ ID NO: 1, HBV / B-Pol consists of the amino acid sequence shown in SEQ ID NO: 2, HBV / C-Pol consists of the amino acid sequence shown in SEQ ID NO: 3, HBV / D-Pol consists of the amino acid sequence shown in SEQ ID NO: 4, HBV / E-Pol consists of the amino acid sequence shown in SEQ ID NO: 5, HBV / F-Pol consists of the amino acid sequence shown in SEQ ID NO: 6, HBV / G-Pol consists of the amino acid sequence shown in SEQ ID NO: 7, and HBV / H-Pol consists of the amino acid sequence shown in SEQ ID NO: 8.

[0036] HBV-Pol contains three functionally essential regions in its amino acid sequence: the terminal protein region, the reverse transcriptase region, and the RNase H region (FIG. 9A).

[0037] The "terminal protein region" is present on the N-terminal side of HBV-Pol, and for example, in genotype C, it consists of 183 amino acids. This region is thought to bind to the vicinity of direct repeat 1 (DR1), which is involved in viral replication, and function as a primer during reverse transcription (Bartenschlager R. & Schaller H., 1988, EMBO J., 7:4185-4192). The terminal protein region contains the TxY motif, which is the activation site of HBV-Pol, as described below.

[0038] The "reverse transcriptase region" is present on the C-terminal side of the terminal protein region, and in genotype C, for example, it consists of 344 amino acids. This region shows high homology to the amino acid sequence of the reverse transcriptase in retroviruses, and during HBV replication, the negative strand of HBV DNA is synthesized by the reverse transcription activity of this region using the pregenomic RNA transcribed from the HBV genome as a template. Furthermore, the positive strand of HBV DNA is synthesized by DNA polymerase activity using this negative strand as a template.

[0039] The "RNase H region" is present on the C-terminal side of HBV-Pol, and is composed of 153 amino acids in genotype C. This region has the activity of degrading the pregenomic RNA used as a template for reverse transcription during HBV DNA replication.

[0040] In addition to the above three regions, there is a spacer region between the terminal protein domain and the reverse transcriptase domain, which is not essential for the function of HBV-Pol. 1-3.Configuration The HBV-Pol activity inhibitor of the present invention comprises a phosphorylation inhibitor that inhibits phosphorylation of the activation site in HBV-Pol.

[0041] The activation site in HBV-Pol is a TxY motif present in the terminal protein region of HBV-Pol. The "TxY motif" is a conserved sequence consisting of three amino acid residues: T (threonine), x (any amino acid residue, such as lysine [K], glutamic acid [E], proline [P], and glycine [G]), and Y (tyrosine). In HBV-Pol, for example, in genotype C, it is present at positions 120 to 122 when the initiation methionine is set to position 1 (FIGS. 9A and B). In genotype C, in addition to the terminal protein region, there is one TxY motif-like sequence at positions 284 to 286 in the spacer region (FIGS. 9A and C). However, from the results of the Examples described later, this sequence is not phosphorylated and is not directly involved in the activity of HBV-Pol. Therefore, it is considered not to be an activation site.

[0042] It is believed that HBV-Pol is activated by phosphorylation of the T and Y residues in the TxY motif in the terminal protein region, and functions as a DNA polymerase during HBV replication. Therefore, the HBV-Pol activity inhibitor of the present invention may be a substance (phosphorylation inhibitor) that has the effect of inhibiting the phosphorylation of the T and Y residues in the TxY motif. Examples of such phosphorylation inhibitors include substances that inhibit the catalytic activity of a kinase that phosphorylates the TxY motif, and substances that bind to the TxY motif and inhibit the binding between the kinase and the TxY motif.

[0043] Kinds of kinases targeted by HBV-Pol activity inhibitors are not limited as long as they can phosphorylate the T and / or Y residues in the TxY motif. In general, the TxY motif is known as an activation site of the MAPK (Mitogen-Activated Protein Kinase) family, such as ERK (Extracellular signal-Regulated Kinase), p38, and JNK (c-Jun N-terminal Kinase). Factors that phosphorylate the TxY motif in MAPK to activate MAPK are MAPK kinases (MAPKK). MAPK kinases include MEK1 (MAPKK1), MEK2 (MAPKK2), MKK3 (MAPKK3), MKK4 (MAPKK4), MKK5 (MAPKK5), MKK6 (MAPKK6), and MKK7 (MAPKK7). As shown in the examples below, it is believed that HBV-Pol is also activated by phosphorylating the TxY motif by MAPK kinases. Therefore, MAPK kinase can be a target kinase for the HBV-Pol activity inhibitor of the present invention.

[0044] When the HBV-Pol activity inhibitor of the present invention is composed of a kinase inhibitor that inhibits the catalytic activity of a target kinase, the kinase inhibitor may be a MAPK kinase inhibitor, such as hypothemycin shown in the following formula 1, trametinib shown in formula 2, PD98059 shown in formula 3, PD184352 shown in formula 4, and U-126 shown in formula 5.

[0045] [ka]

[0046] 2. Method for inhibiting the replication of hepatitis B virus (method for inhibiting the replication of HBV) A second aspect of the present invention is a method for inhibiting Hepatitis B virus replication (method for inhibiting HBV replication). The method of the present invention includes a step of inhibiting phosphorylation of threonine and tyrosine residues of the TxY motif present in the terminal protein region of HBV-Pol (inhibition step).

[0047] The inhibition step is a step of administering a substance capable of inhibiting phosphorylation of threonine residues and tyrosine residues in the TxY motif to cells infected with HBV. Specific examples of such substances include the HBV-Pol activity inhibitors described in the first aspect. MAPK kinase inhibitors are particularly suitable. Examples include known MAPK kinase inhibitors represented by the above formulas 1 to 5.

[0048] As described above, HBV-Pol is believed to be activated by phosphorylation of the threonine and tyrosine residues of the TxY motif present in the terminal protein region. Since HBV-Pol is involved in HBV replication, inhibition of phosphorylation of both amino acid residues in the TxY motif inhibits HBV replication. The present invention is a method for inhibiting HBV replication based on this mechanism. 3. Composition for inhibiting replication of Hepatitis B virus (composition for inhibiting replication of HBV) 3-1. Overview The third aspect of the present invention is a composition for inhibiting Hepatitis B virus replication (composition for inhibiting HBV replication). The composition for inhibiting HBV replication of the present invention contains the HBV-Pol activity inhibitor described in the first aspect, in particular a MAP kinase inhibitor, as an essential active ingredient, and can inhibit the replication of HBV genomic DNA to suppress the proliferation of HBV. Therefore, it can be used as an anti-HBV composition. 3-2.Configuration 3-2-1. Components The components of the composition for inhibiting HBV replication of the present invention will be described below. (1) Active ingredient The composition for inhibiting HBV replication of the present invention comprises, as an essential active ingredient, an HBV-Pol activity inhibitor, particularly a MAPK kinase inhibitor, described in the first aspect.

[0049] The active ingredient may be only the HBV-Pol activity inhibitor described in the first embodiment, or may be a combination composition in which two or more other known anti-HBV agents are combined. Also, the HBV-Pol activity inhibitor described in the first embodiment may contain not only one type, but also multiple types, for example, two types, hypothemycin and trametinib.

[0050] The known anti-HBV agent is not particularly limited, but a nucleic acid analog for treating hepatitis B is suitable. Examples of nucleic acid analogs for treating hepatitis B include entecavir (ETV), lamivudine (LAM), adefovir, tenofovir, telbivudine, and clevudine. All of them are compositions for inhibiting HBV replication that inhibit the reverse transcriptase activity of HBV. As shown in Example 8 described later, the HBV-Pol activity inhibitor described in the first embodiment has a different mechanism of action from nucleic acid analogs for treating hepatitis B such as entecavir and lamivudine, and therefore a synergistic effect on HBV replication inhibition can be obtained by combining the two.

[0051] The content of the active ingredient contained in the HBV replication inhibiting composition is not particularly limited. In general, the content varies depending on the type of active ingredient, the dosage form, and the type of solvent and carrier, which are other components described later. Therefore, it may be appropriately determined taking into account each condition. It is sufficient that an effective amount of the active ingredient is contained in a single application amount of the HBV replication inhibiting composition. However, when it is necessary to administer a large amount of the HBV replication inhibiting composition to a subject to obtain the pharmacological effect of the active ingredient, it can be administered in several divided doses to reduce the burden on the subject. In this case, the amount of the active ingredient may be an effective amount in total. An "effective amount" refers to an amount necessary to exert the function of the active ingredient and to give little or no harmful side effects to the subject to which it is applied. This effective amount may vary depending on various conditions such as information on the subject, the application route, and the number of applications. Therefore, when the HBV replication inhibiting composition is used as a medicine, the content of the active ingredient is ultimately determined by the judgment of a doctor or pharmacist.

[0052] As used herein, the term "subject" refers to an object to which the HBV-Pol inhibitor according to the first aspect, the composition for inhibiting HBV replication according to this aspect, or the composition for treating hepatitis B according to the fourth aspect is applied. For example, it is a cell (including cultured cell), a tissue, an organ, or an individual. In the case of an individual, a human individual is preferred, and a person infected with hepatitis B is particularly preferred.

[0053] In this specification, "subject information" refers to various information related to the characteristics and condition of a subject. For example, when the subject is a human individual, information includes age, weight, sex, general health condition, presence or absence of disease, progression or severity of disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment. (2) Solvent The composition for inhibiting HBV replication of the present invention can be dissolved in a pharma- ceutically acceptable solvent as necessary. The term "pharma-ceutically acceptable solvent" refers to a solvent commonly used in the field of formulation technology. Examples of the solvent include water or an aqueous solution, or an organic solvent. Examples of the aqueous solution include physiological saline, an isotonic solution containing glucose or other auxiliary agents, a phosphate buffer, and a sodium acetate buffer. Examples of the auxiliary agents include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. Examples of the organic solvent include ethanol. (3) Carrier The composition for inhibiting HBV replication of the present invention may contain a pharma- ceutically acceptable carrier as necessary. The term "pharma-ceutically acceptable carrier" refers to additives commonly used in the field of formulation technology. Examples include excipients, binders, disintegrants, fillers, emulsifiers, flow additive regulators, lubricants, human serum albumin, etc.

[0054] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metallic salts, citric acid, tartaric acid, glycine, polyethylene glycol, pluronics, kaolin, silicic acid, or combinations thereof.

[0055] Binders include, for example, starch paste using vegetable starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof.

[0056] Disintegrants include, for example, the above-mentioned starches, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearate monoglyceride, or salts thereof.

[0057] Examples of fillers include petrolatum, the aforementioned sugars and / or calcium phosphate.

[0058] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0059] Examples of flow regulators and lubricants include silicates, talc, stearates or polyethylene glycol.

[0060] In addition to the above, if necessary, the composition may appropriately contain solubilizers, suspending agents, diluents, dispersing agents, surfactants, soothing agents, stabilizers, absorption promoters, bulking agents, moisturizing agents, humectants, adsorbents, flavorings, deodorants, disintegration inhibitors, coating agents, colorants, preservatives, antiseptics, antioxidants, fragrances, flavorings, sweeteners, buffers, isotonicity agents, and the like that are commonly used in pharmaceutical compositions.

[0061] Carriers are used to avoid or suppress decomposition of the active ingredient by enzymes and the like in the subject, as well as to facilitate formulation and administration methods and maintain the dosage form and pharmacological efficacy, and may be used appropriately as needed. 3-2-2. Dosage form The dosage form of the composition for inhibiting HBV replication of the present invention is not particularly limited, as long as it is in a form that allows the active ingredient to be delivered to the target site in the body of a subject without being inactivated.

[0062] The specific dosage form varies depending on the application method described below. The application method can be roughly divided into parenteral administration and oral administration, so a dosage form suitable for each administration method may be used.

[0063] For example, if the administration method is parenteral administration, the preferred dosage form is a liquid that can be administered directly to the target site or systemically via the circulatory system. An example of a liquid is an injection. The injection can be formulated by appropriately combining the above-mentioned excipients, emulsifiers, suspending agents, surfactants, stabilizers, pH regulators, etc., and mixing them in a unit dose form required for generally accepted pharmaceutical practice.

[0064] If the method of administration is oral administration, preferred dosage forms include solid agents (including tablets, capsules, drops, and lozenges), granules, powders, powders, and liquid agents (including oral waters, emulsions, and syrups). If the agent is a solid agent, it can be made into a dosage form with a coating known in the art, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets, as necessary.

[0065] The specific shape and size of each of the above dosage forms are not particularly limited as long as they are within the range of dosage forms known in the art. The method for producing the composition for inhibiting HBV replication of the present invention may be carried out by formulating the composition according to a conventional method in the art. 3-2-3. Application method The composition for inhibiting HBV replication of the present invention may be administered orally or parenterally. Oral administration is generally systemic administration, while parenteral administration can be further divided into local administration and systemic administration. Local administration includes, for example, intramuscular administration, subcutaneous administration, tissue administration, and organ administration, while systemic administration includes administration into the circulatory system, for example, intravenous administration (intravenous injection), intraarterial administration, and intralymphatic administration. When the composition for inhibiting HBV replication of the present invention is administered locally, it may be administered directly to the liver by injection or the like. When the composition is administered systemically, it may be administered into the circulatory system, for example, by intravenous injection. The dosage may be an amount effective for the active ingredient to be effective. The effective amount is appropriately selected according to the subject information as described above.

[0066] In addition, the composition for inhibiting HBV replication of the present invention can also be used in combination with two or more other known anti-HBV agents, particularly nucleic acid analogs for treating hepatitis B. 4. Composition for hepatitis B treatment 4-1. Overview A fourth aspect of the present invention is a composition for treating hepatitis B. The composition for treating hepatitis B of the present invention contains the HBV-Pol activity inhibitor described in the first aspect, in particular a MAPK kinase inhibitor, as an essential active ingredient, and inhibits HBV replication after HBV infection, thereby suppressing HBV proliferation and treating hepatitis B.

[0067] As used herein, the term "treatment" refers to alleviating or eliminating symptoms associated with the disease, and / or preventing or inhibiting the progression of the disease, as well as curing the disease. As used herein, the term "disease" refers to hepatitis B, unless otherwise specified. 4-2.Configuration The third and fourth aspects differ only in their applications. That is, while the composition for inhibiting HBV replication described in the third aspect is intended to inhibit HBV replication in host cells, the composition for treating hepatitis B in this aspect is intended to treat hepatitis B-infected individuals through the inhibition of HBV replication. Therefore, the basic configuration in this aspect is substantially the same as the content described in "3-2. Configuration" of the composition for inhibiting HBV replication in the third aspect. Therefore, a detailed explanation thereof will be omitted here. 4-3. Hepatitis B treatment method Hepatitis B can be treated by administering the composition for treating hepatitis B of the present invention to a subject. Such a method for treating hepatitis B using the composition for treating hepatitis B utilizes the activity of the HBV-Pol activity inhibitor, particularly the MAP kinase inhibitor, described in the first aspect, which is an active ingredient of the composition for treating hepatitis B, and therefore includes a step of inhibiting phosphorylation of the threonine and tyrosine residues of the TxY motif present in the terminal protein region of HBV-Pol (inhibition step) in the same manner as the method for inhibiting HBV replication in the second aspect. 5. Hepatitis B virus polymerase activity promoter (HBV-Pol activity promoter) 5-1. Overview A fifth aspect of the present invention is a hepatitis B virus polymerase activity promoter (HBV-Pol activity promoter). The HBV-Pol activity promoter of this aspect exhibits an opposite effect to that of the HBV-Pol activity inhibitor of the first aspect, and is composed of a compound that phosphorylates an active site present in the terminal protein region of HBV-Pol.

[0068] The HBV-Pol activity promoter of the present invention, when administered to cells infected with HBV, can activate HBV-Pol and increase the amount of HBV replication in the host cells. 5-2.Configuration The HBV-Pol activity promoter of the present invention comprises a compound that phosphorylates the activation site in HBV-Pol, that is, the T residue and / or the Y residue in the TxY motif.

[0069] HBV-Pol is activated by phosphorylation of the TxY motif in the terminal protein region. As described above, compounds that phosphorylate the TxY motif include, for example, MAPK kinase. Therefore, the HBV-Pol activity promoter may be composed of a MAPK kinase. Specific examples of MAPK kinases include the MAPKK family, such as MEK1 (MAPKK1), MEK2 (MAPKK2), MKK3 (MAPKK3), MKK4 (MAPKK4), MKK5 (MAPKK5), MKK6 (MAPKK6), and MKK7 (MAPKK7), as described above. The MAPK kinase may be a constitutively active MAPK kinase into which a mutation has been introduced. For example, in the case of human MEK1, constitutively active MEK1 in which serine (S) at positions 218 and 222 has been replaced with glutamic acid (E) and constitutively active MEK1 in which serine (S) at positions 218 and 222 has been replaced with aspartic acid (D) are included. 6. Hepatitis B virus replication activator 6-1. Overview The sixth aspect of the present invention is a hepatitis B virus replication activator (HBV replication activator). The HBV replication activator of this aspect contains the HBV-Pol activity promoter described in the fifth aspect as an essential active ingredient, and can promote the replication of HBV genomic DNA in host cells after HBV infection, thereby enhancing its proliferation. Therefore, the HBV replication activator of the present invention has an action effect opposite to that of the HBV replication inhibitor composition of the third aspect.

[0070] In general, HBV has an extremely low infection efficiency, and the cells that can be efficiently infected are limited to primary cultures of human hepatocytes or HepaRG cells, and there are no established cell lines with improved HBV infection efficiency. The limited availability of HBV-infected cells has been a cause of delays in research on HBV infection and development of compositions for treating hepatitis B. By using the HBV replication activator of the present invention, it is possible to improve the proliferation activity and infection efficiency of HBV, which can contribute to research on HBV infection and development of compositions for treating hepatitis B. 6-2.Configuration The HBV replication activator of the present invention includes the HBV-Pol activity promoter described in the fifth aspect as an active ingredient.

[0071] The active ingredient may be only the HBV-Pol activity promoter described in the fifth aspect, or may be a combination composition in which two or more other known HBV proliferation promoters are combined. In addition, the HBV-Pol activity promoter described in the fifth aspect may contain not only one type, but also multiple types, for example, two types, MEK1 and MKK3.

[0072] The content of the active ingredient contained in the HBV replication activator of the present invention corresponds to the content of the active ingredient in the HBV replication inhibiting composition of the third embodiment. 7. Hepatitis B virus replication activity evaluation nucleic acid (HBV replication activity evaluation nucleic acid) 7-1. Overview A seventh aspect of the present invention is a nucleic acid molecule capable of evaluating the replication activity of hepatitis B virus (nucleic acid for evaluating HBV replication activity). The nucleic acid for evaluating HBV replication activity of the present invention is a reporter gene mimicking the structure of a gene encoding HBV pregenomic RNA (pgRNA), and is constructed so as to be capable of quantifying the amount of minus-strand DNA synthesized by the reverse transcription activity of HBV-Pol using pgRNA as a template. 7-2.HBV replication mechanism As shown in Figure 1A, the HBV genome has a partially single-stranded relaxed circular DNA (rcDNA) of about 3.2 Kb, in which the plus strand ((+) strand) is shorter than the minus strand ((-) strand). HBV invades host hepatocytes via an unknown HBV-specific receptor, and after infection is established, the single-stranded portion is repaired by endogenous DNA polymerase in the nucleus of the host cell to form a completely double-stranded DNA (covalently closed circular DNA (cccDNA)). The (-) strand of this cccDNA serves as a template, and four types of mRNA of different lengths (3.5 kb, 2.4 kb, 2.1 kb, and 0.7 kb) are synthesized by RNA polymerase II (RNA pol II) derived from the host cell. The longest of these, 3.5 kb mRNA, is called pregenomic RNA (pgRNA), and serves as a template for HBV genome DNA.

[0073] As shown in FIG. 1B, pgRNA has an HBV encapsidation signal epsilon (RNA encapsidation signal epsilon: often abbreviated as "ε" in this specification) sequence at both the 5' and 3' ends. HBV-Pol interacts with this ε sequence and is incorporated into the nucleocapsid formed by HBc. In the nucleocapsid, HBV (-) strand DNA is synthesized using pgRNA as a template by the reverse transcription activity of the reverse transcription region of HBV-Pol and the primer function of the terminal protein region. After reverse transcription, pgRNA is rapidly degraded by the RNase H activity of the RNase H region of HBV-Pol, leaving 17 bases at the 5' end.

[0074] The 5' end of the pregenomic RNA remaining after negative strand DNA synthesis pairs with Direct Repeat 2 (DR2) of the negative strand DNA. This serves as a primer to initiate synthesis of positive strand DNA using the negative strand DNA as a template. This synthesis is terminated midway, and the nucleocapsid is enveloped and released outside the cell. 7-3.Configuration An example of the structure of the nucleic acid for evaluating HBV replication activity of the present invention is shown in Figure 2A. The nucleic acid for evaluating HBV replication activity of the present invention is a gene encoding HBV reporter pgRNA (Figure 2B), and its basic structure is based on HBV genomic DNA. The nucleic acid for evaluating HBV replication activity of the present invention contains, as essential components, an epsilon sequence (ε sequence) derived from HBV pgRNA, and an optional reporter sequence located on the 3'-terminal side thereof and containing an intron. In addition, as an optional component, it contains a direct repeat 1 sequence derived from HBV pgRNA. Furthermore, it may contain a direct repeat 2 sequence and / or another epsilon sequence as necessary.

[0075] "Direct Repeat 1 (herein often referred to as "DR1") sequence" is, for example, a sequence consisting of 11 bases as shown in SEQ ID NO: 9 in genotype C, and is present at two locations on the 5'-end and 3'-end of pgRNA as shown in Figure 1B. Herein, the 5'-end DR1 sequence and the 3'-end DR1 sequence are referred to as the first DR1 sequence and the second DR1 sequence, respectively.

[0076] The "Direct Repeat 2 (herein often referred to as "DR2") sequence" is a sequence consisting of 11 bases shown in SEQ ID NO: 10, which is the same as the above-mentioned DR1 sequence, and is present on the pgRNA between the first ε sequence described below and the second DR1 sequence, as shown in Figure 1B.

[0077] The "epsilon (ε) sequence" is present immediately downstream of the DR1 sequence on the pgRNA as shown in FIG. 1B. ε consists of a base sequence that differs depending on the genotype. Specifically, genotype A is a base sequence consisting of 61 bases shown in SEQ ID NO: 11, genotypes B to F are base sequences consisting of 61 bases shown in SEQ ID NO: 12, genotype G is a base sequence consisting of 61 bases shown in SEQ ID NO: 13, and genotype H is a base sequence consisting of 61 bases shown in SEQ ID NO: 14.

[0078] In pgRNA, the ε sequence self-folds to form a secondary structure consisting of a stem structure, a bulge structure, and a loop structure as shown in FIG. 1B. As described above, the ε sequence functions as an HBV encapsidation signal, and HBV-Pol interacts with this ε sequence and is incorporated into the nucleocapsid. In this specification, the ε sequence located downstream of the first DR1 sequence in pgRNA is referred to as the first ε sequence, and the ε sequence located downstream of the second DR1 sequence is referred to as the second ε sequence. Of these, it has been revealed by the examples of the present application that the first ε sequence is essential for the activity of HBV replication.

[0079] The DR1 sequence, DR2 sequence, and ε sequence in the nucleic acid for evaluating HBV replication activity may be sequences derived from any genotype.

[0080] The "reporter sequence" is a sequence specific to the nucleic acid for evaluating HBV replication activity of the present invention, and has a structure containing at least one intron that can be removed by pre-mRNA splicing within an arbitrary base sequence. The base sequence of the reporter sequence is not particularly limited. For example, it may be a base sequence derived from between the first ε sequence and the DR2 sequence of pgRNA, or a base sequence derived from between the DR2 sequence and the first DR1 sequence of pgRNA, or it may be an arbitrary gene sequence. The base length including the intron is also not particularly limited. For example, it may be 150 bases or more, 200 bases or more, 250 bases or more, 300 bases or more, 350 bases or more, or 400 bases or more. There is no upper limit, but 1000 bases or less, 900 bases or less, 800 bases or less, 700 bases or less, 600 bases or less, or 500 bases or less will suffice. The base length of the intron within the reporter sequence is not particularly limited, but may be within the range of 30 bases or more, 40 bases or more, 50 bases or more, 60 bases or more, 70 bases or more, 80 bases or more, 90 bases or more, 100 bases or more, 110 bases or more, 120 bases or more, 130 bases or more, 140 bases or more, 150 bases or more, 160 bases or more, 170 bases or more, 180 bases or more, 190 bases or more, or 200 bases or more, 300 bases or less, 290 bases or less, 280 bases or less, 270 bases or less, 260 bases or less, or 250 bases or less.

[0081] The reporter pgRNA transcribed from the HBV replication activity evaluation nucleic acid has a structure that mimics pgRNA as a whole, as shown in FIG. 2B. For example, when the reporter pgRNA contains an ε sequence and a DR1 sequence, the ε sequence (corresponding to the first ε sequence) and the DR1 sequence (corresponding to the second DR1 sequence) can be arranged in this order from the 5'-end side. When the reporter pgRNA contains a DR2 sequence and another ε sequence in addition to the ε sequence and the DR1 sequence, each sequence can be arranged in this order from the 5'-end side: the ε sequence (corresponding to the first ε sequence), the DR2 sequence, the DR1 sequence (corresponding to the second DR1 sequence), and the other ε sequence (corresponding to the second ε sequence). The position of the reporter sequence containing an intron is not particularly limited, but can be located, for example, between the ε sequence and the DR1 sequence (corresponding to the "first ε / second DR1 sequence"), between the ε sequence and the DR2 sequence (corresponding to the "first ε / DR2 sequence"), and / or between the DR2 sequence and the DR1 sequence (corresponding to the "DR2 / second DR1 sequence"). The first ε / DR2 sequence or the DR2 / second DR1 sequence other than the reporter sequence may contain a spacer sequence. The base sequence of the spacer sequence is not particularly limited. In addition, the spacer sequence between each sequence does not need to have the same base sequence or base length. A suitable spacer sequence is a base sequence that is present at the same position in pgRNA. For example, the second DR1 / second ε sequence may have the same base sequence as the second DR1 / second ε sequence in pgRNA. In addition, the first ε / DR2 sequence or the DR2 / second DR1 sequence other than the reporter sequence can also have the same base sequence as that in pgRNA. However, in the nucleic acids for evaluating replication activity, the base sequences must be converted to DNA by substituting U (uracil) with T (thymine) in the base sequence.

[0082] The HBV replication activity evaluation nucleic acid can exert its function by being incorporated into an appropriate gene expression system such as the HBV replication activity evaluation vector described in the eighth embodiment described below. As described above, HBV synthesizes pgRNA by RNA pol II of the host cell using the negative strand of cccDNA as a template. Then, HBV resynthesizes the negative strand DNA of HBV using the synthesized pgRNA as a template by the reverse transcription activity of HBV-pol. The HBV replication activity evaluation nucleic acid of the present invention also serves as a template when expressed in the host cell, and a reporter pgRNA (FIG. 2B) is synthesized by RNA pol II, during which the intron in the reporter sequence is removed (spliced ​​out) by pre-mRNA splicing (FIG. 2B). Then, the DNA shown in FIG. 2C is synthesized by the reverse transcription activity of HBV-pol using the reporter pgRNA as a template. In this specification, the DNA synthesized by the reverse transcription reaction from this reporter pgRNA is referred to as "reporter minus strand DNA" or "reporter negative strand DNA". This reporter (-) strand DNA corresponds to the (-) strand DNA of HBV. In other words, the reporter (-) strand DNA is generated only when the HBV replication reaction proceeds, so the amount of reporter (-) strand DNA synthesized reflects the amount of (-) strand DNA synthesized using pgRNA as a template due to the reverse transcription activity of HBV-Pol. Here, the HBV replication activity evaluation nucleic acid has an intron in the reporter sequence, whereas the reporter (-) strand DNA has no intron in the reporter sequence. Therefore, the template HBV replication activity evaluation nucleic acid and the newly replicated reporter (-) strand DNA can be distinguished by the presence or absence of an intron. Therefore, it is possible to quantify the replication activity of HBV by detecting the reporter sequence from which the intron has been removed in the host cell. Specifically, it is possible to detect it using a primer pair for detecting the reporter minus strand DNA described later. 8. Hepatitis B virus replication activity evaluation vector (HBV replication activity evaluation vector) 8-1. Overview The eighth aspect of the present invention is a vector capable of evaluating the replication activity of hepatitis B virus (HBV replication activity evaluation vector). The HBV replication activity evaluation vector of the present invention is composed of an expression vector containing the HBV replication activity evaluation nucleic acid described in the seventh aspect in an expressible state. This HBV replication activity evaluation vector is a central expression vector in the HBV replication activity evaluation system of the ninth aspect described below, and by detecting the reporter minus strand DNA generated by reverse transcription of the reporter pgRNA expressed from this expression vector, it becomes possible to quantify the replication activity of HBV. 8-2.Configuration The vector for evaluating HBV replication activity in this embodiment is an expression vector, and contains, as essential components, a promoter and the nucleic acid for evaluating HBV replication activity described in the seventh embodiment.

[0083] The term "expression vector" refers to a vector that contains a gene or a gene fragment in an expressible state and includes an expression unit that can control the expression of the gene or the like. In this specification, "expressible state" refers to a state in which a gene to be expressed is placed in the downstream region of a promoter under the control of the promoter. In this specification, the term refers to a vector that contains the nucleic acid for evaluating HBV replication activity described in the seventh aspect in an expressible state. As vectors, plasmid vectors, virus vectors, etc. are known, and any vector can be used. Usually, a plasmid vector that is easy to manipulate for gene recombination is sufficient. The expression vector may be a commercially available expression vector for mammalian cells. For example, pCI vector or pSI vector from Promega Corporation can be used. The expression vector may also be a shuttle vector that can be replicated between mammalian cells and bacteria such as E. coli.

[0084] The promoter in the HBV replication activity evaluation vector of this embodiment is a promoter capable of inducing gene expression in a host cell. Since the host cell into which the HBV replication activity evaluation vector is introduced is, in principle, a mammalian cell, particularly a cell derived from a human or chimpanzee, any promoter capable of expressing a downstream gene in the cell may be used. Examples of such promoters include a CMV promoter (CMV-IE promoter), an SV40 early promoter, an RSV promoter, an EF1α promoter, and an Ub promoter.

[0085] In the HBV replication activity evaluation vector, the nucleic acid for evaluating HBV replication activity according to the seventh embodiment is placed downstream of a promoter in an expressible state. Expression of the nucleic acid for evaluating replication activity is induced by activation of the promoter.

[0086] The HBV replication activity evaluation vector may contain, in addition to the above-mentioned essential components, an enhancer, a terminator, a multicloning site, a selection marker, a replication origin, and the like.

[0087] In principle, the HBV replication activity evaluation vector requires only transient expression in mammalian cells, and therefore does not require a replication origin for mammalian cells. However, when the HBV replication activity evaluation vector is expressed in bacteria such as E. coli as a shuttle vector, the replication origin is essential. A known sequence can be used as the replication origin. For example, the f1 origin can be used as a replication origin for E. coli. 9. Hepatitis B Virus Replication Assessment System (HBV Replication Assessment System) 9-1. Overview The ninth aspect of the present invention is a system capable of evaluating the replication activity of hepatitis B virus (HBV replication activity evaluation system). The HBV replication activity evaluation system of this aspect is an assay system that mimics the replication mechanism of HBV, and is composed of an expression vector containing several auxiliary genes in addition to the HBV replication activity evaluation vector described in the eighth aspect. The expression vector is introduced into a host cell, and after culturing, a reporter minus strand DNA generated by reverse transcription of the reporter pgRNA derived from the HBV replication activity evaluation vector described in the eighth aspect in the cell is detected and quantified, whereby the replication activity of HBV can be evaluated in a numerical value. 9-2.Configuration The HBV replication activity evaluation system has as essential components the HBV replication activity evaluation vector described in the eighth embodiment and expression vectors for the HBV C gene and P gene, and has as optional components an expression vector for the HBV X gene and a primer pair for detecting reporter minus strand DNA. Each of the components of the HBV replication activity evaluation vector, HBc expression vector, HBV-P expression vector, HBx expression vector, and primer pair for detecting reporter minus strand DNA will be described below.

[0088] (1) HBV replication activity evaluation vector The configuration of the HBV replication activity evaluation vector is as described in the eighth embodiment, and therefore, a detailed description thereof will be omitted here.

[0089] (2) HBc expression vector An "HBc expression vector" is an expression vector that contains the C gene of HBV in an expressible state. The "C gene" is a gene essential for the replication of HBV that encodes the core protein HBc constituting the nucleocapsid of HBV. The C gene also consists of a base sequence that differs depending on the genotype. For example, HBc of HBV / A (often referred to as "HBc / A" in this specification, and the same applies to other genotypes) consists of the amino acid sequence shown in SEQ ID NO: 16, HBc / B consists of the amino acid sequence shown in SEQ ID NO: 17, HBc / C consists of the amino acid sequence shown in SEQ ID NO: 18, HBc / D consists of the amino acid sequence shown in SEQ ID NO: 19, HBc / E consists of the amino acid sequence shown in SEQ ID NO: 20, HBc / F consists of the amino acid sequence shown in SEQ ID NO: 21, HBc / G consists of the amino acid sequence shown in SEQ ID NO: 22, and HBc / H consists of the amino acid sequence shown in SEQ ID NO: 23. The C gene is composed of a base sequence that codes for each of the above HBcs. One example is the C gene of genotype C consisting of the base sequence shown in sequence number 24 which encodes HBc / C shown in sequence number 18, or the human codon-optimized sequence shown in sequence number 25 which was artificially designed based on it.

[0090] The HBc expression vector may be any expression vector capable of expressing the contained C gene in a host cell. The basic structure may be similar to that of the above-mentioned HBV replication activity evaluation vector. The promoter used for the expression of the C gene is, in principle, a promoter capable of inducing the expression of a downstream gene in a mammalian cell, similar to the HBV replication activity evaluation vector. In addition, a commercially available expression vector for mammalian cells may be used. Examples include pCI vector and pSI vector from Promega.

[0091] (3) HBV-P expression vector The "HBV-P expression vector" is an expression vector that contains the P gene of HBV in an expressible state. The "P gene" is a gene that encodes the DNA polymerase HBV-Pol of HBV, which is essential for the replication of HBV, as described above. As described above, eight genotypes, A to H, are known for HBV-Pol, and the amino acid sequences of each are different as shown in SEQ ID NOs: 1 to 8. However, the P gene contained in the HBV-P expression vector may be composed of a base sequence that encodes any of the HBV-Pols. An example is the base sequence of the P gene encoding HBV / C-Pol shown in SEQ ID NO: 26, or the human codon-optimized sequence artificially designed based on the base sequence shown in SEQ ID NO: 27.

[0092] The HBV-P expression vector may be any expression vector capable of expressing the contained P gene in a host cell. The basic structure may be similar to that of the above-mentioned HBV replication activity evaluation vector. The promoter used for the expression of the P gene is, in principle, a promoter capable of inducing the expression of a downstream gene in a mammalian cell, similar to the HBV replication activity evaluation vector. In addition, a commercially available expression vector for mammalian cells may be used. Examples include pCI vector and pSI vector from Promega.

[0093] (4) HBx expression vector An "HBx expression vector" is an expression vector that contains the X gene of HBV in an expressible state. The "X gene" is a gene that encodes the protein HBx, which transactivates the transcription of other genes in HBV. The X gene also consists of a base sequence that differs depending on the genotype. For example, HBx of HBV / A (often referred to as "HBx / A" in this specification, and the same applies to other genotypes) consists of the amino acid sequence shown in SEQ ID NO: 28, HBx / B consists of the amino acid sequence shown in SEQ ID NO: 29, HBx / C consists of the amino acid sequence shown in SEQ ID NO: 30, HBx / D consists of the amino acid sequence shown in SEQ ID NO: 31, HBx / E consists of the amino acid sequence shown in SEQ ID NO: 32, HBx / F consists of the amino acid sequence shown in SEQ ID NO: 33, HBx / G consists of the amino acid sequence shown in SEQ ID NO: 34, and HBx / H consists of the amino acid sequence shown in SEQ ID NO: 35. The X gene is composed of a base sequence that encodes each of the HBxes. An example of such a sequence is the base sequence shown in SEQ ID NO: 36 which encodes HBx / C shown in SEQ ID NO: 30, or a human codon-optimized sequence artificially designed based thereon and shown in SEQ ID NO: 37. Although the HBx expression vector is not an essential component of the HBV replication activity evaluation system, the presence of HBx can promote reverse transcription activity by HBc and HBV-Pol, and therefore the HBx expression vector can be added to the HBV replication activity evaluation system as necessary.

[0094] The HBx expression vector may be any expression vector capable of expressing the contained X gene in a host cell. The basic structure may be similar to that of the HBV replication activity evaluation vector. The promoter used for the expression of the X gene is, in principle, a promoter capable of inducing the expression of a downstream gene in a mammalian cell, similar to the HBV replication activity evaluation vector. In addition, a commercially available expression vector for mammalian cells may be used. For example, pCI vector or pSI vector from Promega may be used.

[0095] The genes contained in each expression vector of the HBV replication activity evaluation vector, the HBc expression vector, the HBV-P expression vector, and the HBx expression vector may be contained in different expression vectors, or two or more genes may be contained in one expression vector. For example, the C gene and the P gene are contained in one expression vector, the C gene and the X gene are contained in one expression vector, the P gene and the X gene are contained in one expression vector, or the C gene, the P gene and the X gene are contained in one expression vector. Alternatively, the HBV replication activity evaluation vector may contain one or more of the C gene, the P gene, and the X gene. In addition, when two or more genes are expressed in one expression vector, each gene may be placed under the control of the same promoter, or may be placed under the control of different promoters. For example, when the C gene and the P gene are contained in one expression vector, the C gene and the P gene may be placed under the control of the same CMV promoter, or the C gene may be placed under the control of the CMV promoter and the P gene may be placed under the control of the SV40 promoter.

[0096] (5) Primer pair for detecting reporter negative strand DNA The "primer pair for detecting reporter minus strand DNA" is configured so as to be capable of detecting the presence or absence of an intron in the reporter sequence contained in the vector for evaluating HBV replication activity by a nucleic acid amplification method. The design of the primer pair is not particularly limited. For example, the primer pair may be designed so as to amplify only the reporter sequence in the reporter minus strand DNA.

[0097] In order to design the reporter sequence in the reporter minus strand DNA, i.e., the reporter sequence from which the intron has been removed, to be amplified, either the forward primer or the reverse primer may be designed to span the exon junction site in the reporter sequence. In this case, if the forward primer is designed to span the exon junction site by 2 to 3 bases in the extension direction, this is convenient because it can reduce background due to non-specific amplification. For example, if the forward primer is designed to span the exon junction site, only the 2 to 3 bases on the 3' end of the forward primer may be designed to be the 2 to 3 bases at the 5' end of the downstream exon.

[0098] The genotypes of the genes derived from each HBV constituting the HBV replication activity evaluation vector, HBc expression vector, HBV-P expression vector, and HBx expression vector may be any combination of genotypes, but it is preferable to use genes of the same genotype. For example, when the replication activity evaluation nucleic acid contained in the HBV replication activity evaluation vector is of genotype C, the C gene contained in the HBc expression vector, the P gene contained in the HBV-P expression vector, and the X gene contained in the HBx expression vector may each be derived from a different genotype, but are preferably of the same genotype C as the replication activity evaluation nucleic acid.

[0099] The HBV replication activity evaluation system of the present invention can also be used as a kit, which may contain, in addition to the above-mentioned essential and optional components, a DNA extraction reagent, a nucleic acid amplification reagent, a spin column, and instructions for use. 10. Method for evaluating hepatitis B virus replication activity (method for evaluating HBV replication activity) 10-1. Overview A tenth aspect of the present invention is a method for evaluating replication activity of hepatitis B virus (HBV replication activity evaluation method). According to the HBV replication activity evaluation method of the present invention, HBV replication can be analyzed safely, inexpensively, quickly, and with high throughput by using various cells derived from mammals other than humans and other than hepatocytes, without using infectious HBV virus. 10-2. Method The method for evaluating HBV replication activity in this embodiment includes, as essential steps, an introduction step, a culture step, an extraction step, and a detection step. Each step will be described below. (1) Introduction process The "introduction step" is a step of introducing into a host cell the HBV replication activity evaluation vector and each expression vector in the HBV replication activity evaluation system according to the ninth aspect. In this step, a cell line capable of evaluating HBV replication activity is prepared.

[0100] The essential expression vectors to be introduced are the HBV replication activity evaluation vector, the HBc expression vector, and the HBV-P expression vector, and the HBx expression vector may be introduced as necessary. The ratio of expression vectors to be introduced is not limited, but it is preferable that the C gene is the most abundant among the C, P, and X genes, followed by the P gene and then the X gene. An example of the ratio is C:P:X=9:3:1. The ratio of HBV replication activity evaluation nucleic acid to other genes to be introduced is also not limited, but it is preferable that the HBV replication activity evaluation nucleic acid is the most abundant. An example of the ratio is HBV replication activity evaluation nucleic acid:C+P+X=1:1.

[0101] The host cell may be a mammalian cell. Preferably, the cell is derived from a human or chimpanzee, which is the host of HBV. The cell type is not important. Cells derived from various organs or tissues other than hepatocytes, which are HBV-infected cells, can be used. The host cell may be either a cell line or a primary culture cell line. Specific examples of the host cell include HeLa cells, HEK293 cells, NIH3T3 cells, CHO cells, etc.

[0102] The method of introducing each expression vector into a host cell is not particularly limited. A gene introduction method (transformation method) known in the art described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc. may be used. For example, the lipofectin method (PNAS, 1989, 86: 6077; PNAS, 1987, 84: 7413), electroporation method, calcium phosphate method (Virology, 1973, 52: 456-467), DEAE-Dextran method, etc. may be used for introduction. (2)Culture process The "culture step" is a step of culturing the host cells after introduction. In this step, various proteins of HBc, HBV-Pol, and HBx are expressed from the introduced expression vector, and reporter pgRNA derived from the nucleic acid for evaluating HBV replication activity is reverse transcribed, thereby producing reporter minus-strand DNA in the cells.

[0103] The medium used in this step may be a medium generally used in cell culture and known in the art. Usually, a standard cell culture medium is sufficient. The term "standard cell culture medium" as used herein refers to a versatile basic medium that is mainly used for culturing various types of cells derived from mammals. Specific examples include Eagle Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F10 (Ham's Nutrient Mixture F10) medium, Ham's Nutrient Mixture F12 (Ham's Nutrient Mixture F12) medium, M199 medium, High Performance Medium 199 (High Performance Medium 199), RPMI-1640 (Roswell Park Memorial Institute-1640) medium, and DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F12) medium. In the case of DMEM / F12 medium, the mixing ratio is not particularly limited. Preferably, DMEM and F12 are mixed in a weight concentration ratio of the components in the range of 6:4 to 4:6.

[0104] The specific composition of standard cell culture media is known in the art, and therefore may be prepared based on the composition described in an appropriate document (e.g., Kaech S. and Banker G., 2006, Nat. Protoc., 1(5): 2406-2415). Alternatively, media commercially available from manufacturers such as Thermo Fisher Scientifics and Wako Pure Chemical Industries may be used.

[0105] The method for culturing the cells in this step may be a culture method known in the art that is appropriate for the type and origin of the host cells. Usually, it is sufficient to culture the cells in a cell culture medium at 37° C. under 5% CO2.

[0106] The culture period varies somewhat depending on the type and origin of the host cell, but in order to achieve the above-mentioned object of this step, it is usually 12 to 72 hours, preferably 24 to 48 hours. (3) Extraction process The "extraction step" is a step of extracting DNA from the host cells after the culture step. The purpose of this step is to extract DNA containing a reporter minus strand DNA synthesized by reverse transcription of the reporter pgRNA derived from the nucleic acid for evaluating HBV replication activity from the host cells.

[0107] The DNA may be extracted by any method known in the art for extracting DNA from cultured cells. For example, the method described in Green & Sambrook (mentioned above) may be used. Alternatively, a kit for extracting nucleic acids from mammalian cells available from various manufacturers may be used. (4) Detection process The "detection step" is a step of detecting the intron-removed reporter sequence in the reporter minus strand DNA obtained in the extraction step. In this step, the intron-removed reporter sequence is detected and quantified, allowing quantitative analysis of HBV replication activity.

[0108] The DNA obtained in the extraction step contains a mixture of the genomic DNA of the host cells, various HBV-derived genes and HBV replication activity evaluation nucleic acids introduced in the introduction step, and reporter minus strand DNA synthesized by reverse transcription of reporter pgRNA derived from the HBV replication activity evaluation nucleic acid, etc. In this step, only the reporter sequence from which the intron of the reporter minus strand DNA has been removed is specifically detected.

[0109] The detection method is not particularly limited as long as it can detect the reporter sequence from which the intron of the reporter minus strand DNA has been removed, and examples of such a method include a nucleic acid amplification method in which a primer pair is used to amplify the reporter sequence, and a hybridization method in which a probe is used to detect the exon junction site.

[0110] (4-1) Nucleic acid amplification method The term "nucleic acid amplification method" refers to a method in which a specific region of a target nucleic acid is amplified by a nucleic acid polymerase using a primer pair. Examples of such methods include PCR, NASBA, ICAN, and LAMP (registered trademark) (including RT-LAMP). PCR is preferred. In particular, when measuring the amount of reporter minus strand DNA present in the DNA obtained in the extraction step, it is desirable to use a quantitative nucleic acid amplification method such as real-time PCR.

[0111] The primer pair used may be the above-mentioned primer pair for detecting the reporter minus strand DNA, and is preferably a primer pair that amplifies only the reporter sequence in the reporter minus strand DNA.

[0112] The reaction conditions of real-time PCR may be determined by a method known in the art, and may refer to the method described in Green & Sambrook (mentioned above). Generally, the reaction conditions may be determined appropriately according to the known PCR method, since they vary depending on the base length of the nucleic acid fragment to be amplified, the amount of template nucleic acid, the base length and Tm value of the primer used, the optimal reaction temperature and optimal pH of the nucleic acid polymerase used, etc. As an example, the denaturation reaction is usually performed at 94 to 95°C for 5 seconds to 5 minutes, the annealing reaction is performed at 50 to 70°C for 10 seconds to 1 minute, and the extension reaction is performed at 68 to 72°C for 30 seconds to 3 minutes, and this is regarded as one cycle, which is repeated for about 15 to 40 cycles, and finally, the extension reaction is performed at 68 to 72°C for 30 seconds to 10 minutes. When using a commercially available kit, the protocol attached to the kit may be followed in principle.

[0113] The nucleic acid polymerase used in real-time PCR is a DNA polymerase, particularly a thermostable DNA polymerase. Various types of such nucleic acid polymerases are commercially available, and they can also be used.

[0114] (4-2) Hybridization method The "hybridization method" is a method in which a nucleic acid fragment having a sequence complementary to all or part of the base sequence of the target nucleic acid to be detected is used as a probe, and a target nucleic acid or a fragment thereof is detected and quantified by utilizing base pairing between the nucleic acid and the probe. Several hybridization methods with different detection means are known, but since the reporter minus strand DNA of the target nucleic acid is DNA, for example, the Southern hybridization method, the RNA microarray method, the surface plasmon resonance method, or the quartz crystal microbalance method are suitable.

[0115] "Southern hybridization" is the most common method for analyzing DNA. DNA extracted from a sample is separated by electrophoresis using agarose gel or polyacrylamide gel under denaturing conditions, and transferred (blotted) onto a filter. The target DNA is then detected using a probe containing a sequence complementary to a base sequence specific to the target DNA. By labeling the probe with an appropriate marker such as a fluorescent dye or a radioisotope, it is also possible to quantify the target DNA using a measuring device such as a chemiluminescence (chemiluminescence) imaging analyzer (e.g., Light Capture; ATTO), a scintillation counter, or an imaging analyzer (e.g., FUJIFILM: BAS series). Southern hybridization is a well-known and well-known technique in the field, and can be found in, for example, Green, MR and Sambrook, J. (see above).

[0116] The probe may be a probe that hybridizes only to the reporter sequence in the reporter minus strand DNA, or a combination of a probe that specifically hybridizes to an intron and a probe that hybridizes only to one exon or to the entire reporter sequence, with the former probe being preferred. For example, a probe that hybridizes only to the reporter sequence in the reporter minus strand DNA may have a base sequence complementary to the exon junction after mRNA-splicing in the reporter sequence, and the base sequence may be designed so that it cannot hybridize only to one exon under stringent conditions. EXAMPLES

[0117] <Example 1: Establishment of a method for evaluating HBV replication activity> (the purpose) We will develop a safe, inexpensive, and rapid method for evaluating HBV replication activity that can quickly visualize and quantify HBV genome replication using standard cells without using infectious HBV. (Method) (1) Construction of HBV replication activity evaluation vector (pBB-intron) A nucleic acid for evaluating HBV replication activity shown in SEQ ID NO: 15 according to the seventh aspect of the present invention was prepared by replacing the 99 bases at the 5' end including the first DR1 sequence and the first ε sequence (corresponding to positions 32 to 130 of HBV pgRNA) and the 349 bases at the 3' end including the DR2 sequence, the second DR1 sequence and the second ε sequence (corresponding to positions 3013 to 3215 and positions 1 to 146 of HBV pgRNA), i.e., the first ε / DR2 sequence (corresponding to positions 131 to 3012 of HBV pgRNA), with a 320-base reporter sequence including an intron ( FIG. 2A ). This reporter sequence is derived from the base sequence of the 483 to 453 positions (31 base pairs) of the pCRE-MetLuc2 vector (Clontech), the 857 to 989 positions (133 base pairs) of the pCI vector (Promega), and the 452 to 337 positions (116 base pairs) of the pCRE-MetLuc2 vector.

[0118] A CMV promoter (742 base pairs) and a TATA box (46 base pairs) were inserted upstream of the prepared nucleic acid for evaluating HBV replication activity, and a synthetic polyA signal sequence (49 base pairs) was inserted downstream. All of these sequences were inserted into the backbone of the pCI vector (containing only the phage f1 origin, ampicillin resistance gene, and replication origin) to prepare pBB-intron, an HBV replication activity evaluation vector described in the eighth aspect of the present invention (Figure 3A).

[0119] When this HBV replication activity evaluation vector is introduced into a host cell, mRNA is synthesized by the host cell's RNA-pol II. After this mRNA is synthesized as pre-mRNA, the intron in the reporter sequence is immediately removed by pre-mRNA splicing in the host cell. The mature mRNA with the intron spliced ​​out corresponds to the reporter pgRNA (Figure 2B). The reporter pgRNA is reverse transcribed by the action of HBV-Pol, HBc, and HBx expressed from the HBV-P expression vector, HBc expression vector, and HBx expression vector described below, and a reporter minus-strand DNA containing a reporter sequence different from the reporter sequence of the original HBV replication activity evaluation vector, i.e., a reporter sequence with the intron removed, is synthesized (Figure 2C). (2) Construction of HBV-P expression vector (pCI-HBV-P) The sequence shown in sequence number 27, which was artificially designed based on the HBV P gene shown in sequence number 26, was inserted under the control of the CMV promoter of the pCI vector (Promega) to prepare pCI-HBV-P, which is the HBV-P expression vector described in the eighth aspect of the present invention (Figure 3B). (3) Construction of HBc expression vector (pCI-HBc) The sequence shown in sequence number 25, which was artificially designed based on the HBV C gene shown in sequence number 24, was inserted under the control of the CMV promoter of the pCI vector (Promega) to prepare pCI-HBc, an HBV-C expression vector described in the eighth aspect of the present invention (Figure 3C). (4) Construction of HBx expression vector (pCI-HBx) The sequence shown in SEQ ID NO: 37, which was artificially designed based on the HBV X gene shown in SEQ ID NO: 36, was inserted under the control of the CMV promoter of the pCI vector (Promega) to prepare pCI-HBx, which is the HBx expression vector described in the eighth aspect of the present invention (FIG. 3D). (5) Method for assessing HBV replication activity The HBV replication activity evaluation vector, HBV-P expression vector, HBc expression vector, and HBx expression vector were introduced into HeLa cells in various combinations (transduction step). The combination patterns are as shown in Figure 4A. The electroporation method was used for gene introduction into HeLa cells. Using an electroporator Nepa21 (Nepa Gene Co., Ltd.), 10 μg of DNA was injected at 125 V / 2.5 ms plus length, resulting in approximately 1 × 10 6 The vectors were introduced into HeLa cells. The ratio of HBc expression vector:HBV-P expression vector:HBx expression vector was 9:3:1, and when some expression vectors were not introduced due to the combination, the same ratio of empty vector (pCI) as the expression vectors not introduced was introduced. The total ratio of the HBV replication activity evaluation vector and other expression vectors was 1:1 (5μg:5μg), and for the expression vectors not introduced, pCI was introduced in the same ratio.

[0120] Furthermore, to confirm whether the amount of reverse-transcribed reporter minus-strand DNA reflects the functions of HBc, HBV-Pol, and HBx in cells, the ratio of HBV replication activity evaluation vector:HBc / HBV-P / HBx expression vector was changed stepwise from 1:0 (5 μg:pCI 5 μg), 1:0.004 (5 μg:0.02 μg), 1:0.016 (5 μg:0.08 μg), 1:0.062 (5 μg:0.31 μg), 1:0.25 (5 μg:1.25 μg), and 1:1 (5 μg:5 μg) without changing the ratio of each expression vector for HBc / HBV-P / HBx (HBc:HBV-P:HBx=9:3:1), and the effect on the reverse transcription reaction was analyzed based on the amount of reporter minus-strand DNA.

[0121] After gene transfer, the HeLa cells were cultured in 2 mL of DMEM supplemented with 10% FBS in the presence of 5% CO2 at 37°C for 48 hours (culture step).

[0122] After the culture, DNA was extracted from the HeLa cells using DNeasy Mini (Qiagen). A primer pair consisting of a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 38 and a reverse primer consisting of the nucleotide sequence shown in SEQ ID NO: 39 was used. The two bases at the 3' end of this forward primer match the two bases at the 5' end of the downstream exon, but do not match the two bases at the 5' end of the intron. Therefore, it functions as a primer only when a reporter minus strand DNA having a reporter sequence from which the intron has been removed is present, and a 131-base DNA fragment is amplified using the reporter minus strand DNA as a template.

[0123] In addition, for DNA derived from cells into which each expression vector was introduced in the combination shown in FIG. 4A, the PCR products after PCR were separated by electrophoresis on a 2% agarose gel and stained with ethidium bromide.

[0124] Furthermore, to prepare a standard curve, DNA derived from the transfected HeLa cells was serially diluted so that the ratio of HBc:HBV-P:HBx expression vectors was 9:3:1, and the ratio of HBV replication activity evaluation vector:HBc / HBV-P / HBx expression vectors was 1:1, to prepare template DNA, and real-time PCR was performed. (result) The results of Example 1 are shown in FIG.

[0125] A shows the results of 2% agarose gel electrophoresis of PCR products derived from cells transfected with expression vectors in each combination, followed by staining with ethidium bromide. C indicates HBc expression vector containing the C gene, P indicates HBV-P expression vector containing the P gene, and X indicates HBx expression vector containing the X gene. + indicates transfection into HeLa cells, and - indicates no transfection. A 131 bp PCR product derived from the reporter minus strand DNA was observed only when the HBc expression vector and HBV-P expression vector were transfected. This demonstrated that HBc and HBV-Pol are essential for reverse transcription of pgRNA. It was also confirmed that HBx is not essential for reverse transcription of pgRNA, but significantly promotes reverse transcription by HBc and HBV-Pol.

[0126] B is a standard curve generated from the results of real-time PCR of serially diluted DNA. As shown in this figure, R 2 A precise calibration curve with a qPCR yield of 0.9996 was obtained, demonstrating that the amount of reverse transcribed reporter negative strand DNA can be quantified by real-time PCR.

[0127] C shows the results of quantitative analysis using real-time PCR and the standard curve in B. The DNA sample used in A above was amplified by real-time PCR, and then quantitatively analyzed using the standard curve shown in B above. The results were almost the same as those obtained by agarose gel electrophoresis in A.

[0128] FIG. 1D is a diagram showing the correlation between the expression level of HBV proteins (HBc, HBV-P, HBx) and HBV DNA replication, i.e., the amount of reporter minus strand DNA. Reverse transcription of pgRNA increased exponentially according to the expression level of HBV proteins. From this result, it was revealed that the HBV replication activity evaluation method using the HBV replication activity evaluation system of the present invention can measure and quantify the reverse transcription activity, i.e., replication activity, reflecting the function of HBV proteins in host cells. <Example 2: Verification of the method for evaluating HBV replication activity using entecavir> (the purpose) The HBV replication activity evaluation system of the present invention will be used to verify the efficacy of entecavir, an existing anti-HBV drug. (method) According to the HBV replication activity evaluation method described in Example 1, the HBV replication activity evaluation vector, the HBV-P expression vector, the HBc expression vector, and the HBx expression vector were introduced into HeLa cells. The introduction ratio of each expression vector of HBc:HBV-P:HBx was 9:3:1, and the introduction ratio of the HBV replication activity evaluation vector and the expression vector of HBc+HBV-P+HBx was 1:1 (5μg:5μg). After the introduction step, 2mL of DMEM supplemented with 10% FBS was added to the HeLa cells, and the cells were dispensed into a 96-well plate at 0.1mL / well, and then 0.04μM / well, 0.08μM / well, 0.16μM / well, 0.31μM / well, 0.63μM / well, 1.25μM / well, 2.50μM / well, and 5.00μM / well of entecavir was added to each medium, and the cells were cultured at 37°C for 24 hours in the presence of 5% CO2. Entecavir is a potent nucleoside analogue drug known to act on HBV-Pol and inhibit reverse transcription (Billich A., 2001, Curr Opin Investig Drugs., 2(5):617-21).

[0129] After the incubation, 2% NP-40 / Proteinase K was added to each well to extract DNA from the HeLa cells, and real-time PCR was carried out according to the method of Example 1. (result) After amplification by real-time PCR, the reporter minus strand DNA was quantitatively analyzed using the standard curve (FIG. 4B) prepared in Example 1. The amount of the reporter minus strand DNA was reduced depending on the concentration of entecavir. This result suggests that the replication of HBV DNA was inhibited depending on the concentration of entecavir. Therefore, it was demonstrated that the HBV replication activity evaluation system of the present invention can quantify the HBV replication process in an extremely short time of 24 hours compared to the conventional method, without using infectious HBV particles or producing HBV. <Example 3: Search for natural compounds derived from filamentous fungi that selectively inhibit HBV replication> (the purpose) Using the HBV replication activity evaluation system of the present invention, novel anti-HBV compounds that inhibit HBV replication are screened. (method) (1) Discovery of natural compounds with anti-HBV activity Using the HBV replication evaluation system of the present invention, a search for natural compounds having anti-HBV activity was carried out from a filamentous fungal culture extract library (ExMyco: HyphaGenesis).

[0130] The HBV replication activity evaluation vector and each of the expression vectors for HBV-P, HBc, and HBx were introduced into HeLa cells at the ratios described in the HBV replication activity evaluation method described in Example 2, and then the cells were cultured at 37°C under 5% CO2 in a 96-well plate containing a culture medium supplemented with 0.25% n-butanol extract of filamentous fungus culture. After 24 hours, WST-8 (Nacalai) was added to the culture medium, which was then cultured for an additional 2 hours, and the cell viability was evaluated by measuring the absorbance at 450 nm / 650 nm.

[0131] After removing the culture medium, DNA in the cytoplasm was recovered by treatment with 50 μL of Buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.05% NP40, pH 7.9), and real-time PCR was performed according to the method of Example 1. As a result of primary screening of 800 extracts (200 strains, 4 cultures), 162 extracts that inhibited HBV DNA replication (reverse transcription) by 25% or more and had cytotoxicity of 80% or less were selected as primary hits.

[0132] Next, the 162 extracts that were the primary hits were serially diluted and added to the evaluation system to compare their anti-HBV activity and cytotoxic activity. As a result, one filamentous fungal strain (FA233 strain) was obtained that produced an extract that inhibited HBV DNA replication in a concentration-dependent manner and did not show cytotoxic activity when cultured in four types of media (GP medium, MV8 medium, KGC medium, and SC medium). Furthermore, this FA233 strain was mass-cultured in KGC medium, and compounds with anti-HBV activity were isolated using silica gel chromatography and HPLC, and the compounds were serially diluted and re-examined for their anti-HBV activity and cytotoxic activity. (result) The results are shown in Figure 6. The compound inhibited HBV DNA replication in a concentration-dependent manner (Figure 6A). However, it did not affect the amount of genomic DNA (G3PDH exon8) or mitochondrial DNA (ND5) (Figures 6B and 6C). Therefore, this compound was selected as an inhibitor of HBV DNA replication. Example 4: Identification of HBV DNA replication inhibitors (the purpose) The chemical structure of the HBV DNA replication inhibitor obtained in Example 3 is identified. (method) The NMR sample was prepared at 10 mg / CDCl3 (35 mm height) (approximately 0.4 mL). Then, various NMR (H-1, C-13, DEPT, COSY, HSQC, HMBC, and NOESY) were measured using a JEOL ECA 500 (JEOL RESONANCE). (result) The NMR measurement results and the comparison results with the literature values ​​described in the reference literature are shown in Table 1. In the table, lit means the reference literature (Agatsuma T., et al. 1993, Chem. Pharm. Bull., 41: 373-375).

[0133] [Table 1]

[0134] Signal assignments from various NMR measurement data showed agreement with the assignments of hypothemycin described in the above literature (C-13: 0.1 or less, H-1: 0.04 or less). Therefore, it was concluded that the compound obtained in Example 3 was hypothemycin represented by formula 1.

[0135] [ka]

[0136] <Example 5: Verification of the HBV replication inhibitory effect by MAPK kinase inhibitor> (the purpose) Hypothemycin, identified as an HBV DNA replication inhibitor in Example 4, is known as an inhibitor of MEK, i.e., MAPK / ERK kinase (MAPKK), which plays an important role in the classical MAPK pathway (Fukazawa H, et al., 2010, Biol Pharm Bull., 33(2):168-73). However, it has not been known that MAPKK inhibitors have any pharmacological effect as anti-HBV drugs.

[0137] Therefore, the inhibitory activity of hypothemycin against HBV DNA replication will be re-examined, and the HBV replication activity evaluation system of the present invention will be used to examine whether other known MAPKK inhibitors have the same inhibitory activity against HBV replication as hypothemycin. (method) After introducing the HBV replication activity evaluation vector and each expression vector of HBV-P, HBc and HBx into HeLa cells at the ratio of the HBV replication activity evaluation method described in Example 2, 5 μM of MAPKK inhibitor was added and cultured at 37 ° C under 5% CO2. Four types of MAPKK inhibitors were used, including hypothemycin, PD98059, PD184352 and trametinib. In addition, no MAPKK inhibitor was added as a negative control, and entecavir, which is not a MAPKK inhibitor but is known as an anti-HBV drug, was added at 5 μM as a positive control for HBV replication inhibition activity and cultured under the same conditions. After 24 hours, DNA was extracted from the culture solution according to the method described in Example 1, and real-time PCR was performed. (result) The results are shown in Figure 7. All four MAPKK inhibitors tested showed significant HBV DNA replication inhibitory activity at a concentration of 5 μM. These results confirmed the novel finding that MAPKK inhibitors are effective as anti-HBV drugs. Example 6: Effect of MAPK kinase on HBV DNA replication (the purpose) The results of Example 5 demonstrated that MAPKK inhibitors have HBV DNA replication inhibitory activity. These results strongly suggest that MAPK kinase activity plays an important role in HBV DNA replication. Therefore, the effect of MAPK kinase on HBV DNA replication was examined. (method) Various human MEK1s were forced to be expressed in cells to examine the effect of MAPK kinases on HBV DNA replication. Specifically, HBV replication activity evaluation vectors and HBV-P, HBc, and HBx expression vectors were introduced into HeLa cells at the ratios used in the HBV replication activity evaluation method described in Example 2, and wild type (referred to as "Wt"), constitutively active type (S 218 E / S 222 E: written as "2E", S 218 D / S 222 D: denoted as "2D"), or dominant negative mutant type (K 97 A / S 222A: Various human MEK1 expression vectors (referred to as "2A") were introduced into the cells. After the introduction of the various expression vectors, the cells were cultured at 37°C under 5% CO2. After 24 hours of culture, DNA was extracted from each culture medium according to the method described in Example 1, and real-time PCR was carried out to examine the effect of MEK1 expression on the amount of HBV DNA, genomic DNA, and mitochondrial DNA due to the expression of MAPK kinase (MEK). (result) The results are shown in Figure 8. In cells (2D, 2E) in which constitutively active human MEK1 was forcibly expressed, HBV replication was found to be promoted nearly 10-fold (Figure 8A). In particular, in the constitutively active 2D, it was promoted by more than 45-fold. On the other hand, forced expression of human MEK did not affect the amount of genomic DNA (Figure 8B) or mitochondrial DNA (Figure 8C). Furthermore, it did not affect the transcription of reporter pgRNA or host mRNA (G3PDH).

[0138] These results confirmed that MEK activity in host cells is important for HBV DNA replication. Example 7: Phosphorylation of HBV proteins by MAPK kinase (the purpose) To elucidate the mechanism by which MAPK kinase promotes HBV DNA replication, we will examine where MAPK kinase activity acts in HBV DNA replication. (method) MEK, a MAPK kinase, is a phosphorylating enzyme that plays an important role in the classical MAPK signaling pathway. The MAPK family (ERK, JNK1, and p38), which are substrates of MEK, have a conserved sequence of three amino acids called the "TxY motif," and the MAPK family is activated when the T and Y residues in this motif are phosphorylated by MEK (Morrison DK, 2012, MAP kinase pathways. Cold Spring Harb Perspect Biol., 4(11)). Therefore, it was speculated that in HBV, one of the HBV proteins involved in HBV DNA replication is phosphorylated by MEK.

[0139] Analysis of the amino acid sequences of HBV proteins involved in HBV DNA replication (HBc, HBV-Pol, and HBx) revealed the presence of a TxY motif at positions 120-122 in the terminal protein region of HBV-Pol and at positions 284-286 in the spacer region (Figure 9A). 120 TKY 122 ) was conserved in all genotypes A to F examined, whereas the TxY motif in the spacer region ( 284 TAY 286 ) was only observed in genotype C (HBV / C) (Fig. 9B).

[0140] Therefore, we created mutant P genes encoding three mutant HBV / C-Pols (TAYF-1, TAYF-2, and TAYF-1 / 2) in which T in the TxY motif of HBV-Pol was replaced with A (alanine) and Y with F (phenylalanine) (Figure 10A).

[0141] "TAYF-1" is a terminal protein domain 120 TKY 122 Instead of 120 AKF 122 The mutant HBV / C-Pol contains the spacer region of 284 TAY 286 Instead of 284 AAF 286 and "TAYF-1 / 2" is a mutant HBV / C-Pol containing the terminal protein region 120 TKY 122 and the spacer region 284 TAY 286 Instead, 120 AKF 122 and 284 AAF 286The expression vectors containing genes encoding the mutant HBV / C-Pols TAYF-1, TAYF-2, and TAYF-1 / 2 were designated HBV / CP-TAYF-1, HBV / CP-TAYF-2, and HBV / CP-TAYF-1 / 2, respectively.

[0142] For HBV replication evaluation, similarly to the HBV replication activity evaluation method described in Example 1, an HBV replication activity evaluation vector and expression vectors for wild-type HBV / CP (Wt) or each mutant HBV-P, HBc, and HBx were introduced at the same ratio as in Example 2, and 5 μM of a 2D expression vector for forced expression of constitutively active human MEK1 was introduced, followed by culture at 37° C. under 5% CO2. After 24 hours of culture, DNA was extracted from each culture solution according to the method described in Example 1, and real-time PCR was performed to verify HBV replication activity. (result) The results are shown in FIG. 10B. 120 TKY 122 In TAYF-1 and TAYF-1 / 2, which had mutations in the spacer region, the activity of MEK1 was significantly suppressed compared to the activity of constitutively active MEK1 in the expression of wild-type HBV / CP. 284 AAF 286 These results suggest that HBV-Pol is a ubiquitous HBV-like protein that is located in the terminal protein region. 120 TKY 122 These results suggest that the IL-11 protein is activated by phosphorylation with MAPK kinase in the host cell.

[0143] The results of Examples 6 and 7 suggest that MAPKK inhibitors including hypothemycin inhibit the phosphorylation of HBV-Pol, which is its substrate, through inhibition of MAPKK activity, thereby suppressing the activation of HBV-Pol and inhibiting HBV DNA replication. <Example 8: Synergistic effect of known anti-HBV drugs and MAPKK inhibitors in inhibiting HBV DNA replication> (the purpose) The inhibitory effect of HBV DNA replication when a known anti-HBV drug is used in combination with a MAPKK inhibitor will be verified. (method) The known anti-HBV drug used was entecavir, which is known as a reverse transcription inhibitor in HBV replication, and the MAPKK inhibitor used was hypothemycin.

[0144] The method for evaluating HBV replication activity of the present invention was essentially based on the method described in Example 1. The introduction ratio of each expression vector was based on the ratio described in Example 2. After the expression vector was introduced into the cells, all combinations (4 x 4 = 16 combinations) of entecavir and hypothemycin at 0 μM, 1.25 μM, 2.50 μM, and 5.00 μM were added together with DMEM supplemented with 10% FBS, and the cells were then cultured at 37° C. in the presence of 5% CO2.

[0145] After 24 hours, DNA was extracted from the culture medium according to the method described in Example 1, and real-time PCR was carried out. (result) The results of the synergistic effect are shown in Figure 11. It was found that the HBV DNA replication inhibitory effect was enhanced in a concentration-dependent manner when both entecavir and hypothemycin were added, regardless of the concentration, compared to when entecavir or hypothemycin was added alone. This result demonstrated that hypothemycin and entecavir synergistically enhance the HBV DNA replication inhibitory effect. <Example 9: HBV DNA replication inhibitory effect of MAPKK inhibitor against nucleic acid analogue drug-resistant HBV> (the purpose) We will verify the inhibitory effect of MAPKK inhibitors on HBV DNA replication against mutant HBV-Pol, which is responsible for the emergence of HBV resistant to nucleoside analogues. (method) As described in the Background Art section, conventional anti-HBV drugs consisting of nucleic acid analogs such as entecavir and lamivudine cause the emergence of drug-resistant HBV when administered for a long period of time. Mutations in the reverse transcriptase (RT) region of HBV-Pol have been reported as the cause of the emergence of such drug-resistant HBV. For example, as shown in FIG. 12A, lamivudine-resistant HBV (LAMr) has an M204I mutation in which methionine (M) at position 204 located in the RT region is replaced with isoleucine (I), and entecavir-resistant HBV (ETVr) has an L180M / T184G / S202I / M204I mutation in which leucine (L) at position 180 located in the RT region is replaced with methionine, T at position 184 is replaced with glycine (G), serine (S) at position 202 is replaced with I, and M at position 204 is replaced with I, resulting in a total of four amino acid substitutions.

[0146] Therefore, we prepared mutant HBV-Pol expression vectors carrying the M204I and L180M / T184G / S202I / M204I mutations by standard methods, and named them HBV-P-LAMr and HBV-P-ETVr, respectively. Hyposemycin was used as the MAPKK inhibitor. Entecavir, an existing anti-HBV drug, was used as a negative control for drug-resistant HBV.

[0147] For HBV replication evaluation, similarly to the HBV replication activity evaluation method described in Example 1, the HBV replication activity evaluation vector and the expression vectors of wild-type HBV-P or each mutant HBV-P (HBV-P-LAMr or HBV-P-ETVr), HBc and HBx were introduced at the same ratio as in Example 2, and hypothemycin or entecavir was introduced at 0.000 μM, 0.020 μM, 0.039 μM, 0.078 μM, 0.156 μM, 0.313 μM, 0.625 μM, 1.250 μM, 2.500 μM, and 5.000 μM, and cultured at 37° C. under 5% CO2. After 24 hours of culture, DNA was extracted from each culture solution according to the method described in Example 1, and real-time PCR was performed to verify the inhibitory effect of HBV replication activity. (result) Figure 12B shows the results of the inhibitory effect on HBV replication activity. Entecavir concentration-dependently inhibited reverse transcription by wild-type HBV-Pol (b), but had a weak inhibitory effect on the reverse transcription activity of lamivudine-resistant mutant HBV-Pol (d), and hardly inhibited the reverse transcription activity of entecavir-resistant mutant HBV-Pol (f). On the other hand, hypothemycin concentration-dependently inhibited the reverse transcription activity of both wild-type (a) and two drug-resistant mutant HBV-Pol (HBV-Pol-LAMr (c) and HBV-Pol-ETVr (e)). These results demonstrate that the mechanism of action of MAPKK inhibitors such as hypothemycin in inhibiting HBV replication is different from that of anti-HBV activity by nucleic acid analogs, that is, the HBV replication inhibitory effect based on the inhibition of the reverse transcription activity of HBV-Pol.

[0148] In chronic hepatitis B treatment, a current problem is "reactivation," i.e., viral reactivation. In particular, reactivation during long-term treatment with nucleotide analogues such as entecavir is accompanied by the emergence of drug-resistant HBV, making subsequent treatment extremely difficult. Substances that inhibit the phosphorylation of T and Y residues in the TxY motif present in the terminal protein region of HBV-Pol, such as MAPKK inhibitors, have a different mechanism of action from nucleotide analogues, and are therefore applicable to reactivation accompanied by the emergence of currently known drug-resistant HBV, and their effectiveness is expected.

[0149] Therefore, it is strongly suggested that the therapeutic agent for hepatitis B containing the MAPKK inhibitor of the present invention as an active ingredient is extremely useful as a novel anti-HBV drug. <Example 10: HBV replication evaluation experiment using a deletion-type HBV replication activity evaluation vector> (the purpose) HBV replication evaluation experiments will be conducted using various deletion-type HBV replication activity evaluation vectors (ΔpBB-intron) to identify the HBV genome-derived sequences contained in pBB-intron that play an important role in reverse transcription of pgRNA. (method) The following four types of ΔpBB-intron were prepared.

[0150] (1) pBB-intron(Δε1): pBB-intron lacking the first ε sequence, prepared in Example 1, (2) pBB-intron(ΔDR2): DR2-deleted pBB-intron obtained by deleting the DR2 sequence from pBB-intron prepared in Example 1 (3) pBB-intron (ΔDR1(2)): DR1(2)-deleted pBB-intron obtained by deleting the second DR1 from pBB-intron prepared in Example 1 (4) pBB-intron(Δε2): pBB-intron lacking the second ε sequence, prepared in Example 1, pBB-intron (referred to as wild-type pBB-intron for ΔpBB-intron) and any one of the four types of ΔpBB-intron were transfected into HeLa cells together with HBV protein CP (C: HBc, P: HBV-Pol) or CPX (C: HBc, P: HBV-Pol, X: HBx). After 24 hours of culture, DNA was extracted and the amount of reverse-transcribed reporter HBV DNA was quantified by real-time PCR. The values ​​are expressed as a ratio to the amount of genomic DNA (G3PDH exon 8) of the host (HeLa cells) quantified by real-time PCR. (result) The results are shown in FIG. 13. In pBB-intron (Δε1) in which the first ε sequence located at the 5' end was deleted, the reporter HBV DNA was almost completely lost, indicating that the first ε sequence is an essential element for reverse transcription of pgRNA. In addition, in pBB-intron (ΔDR1(2)) in which the second DR1 sequence located at the 3' end was deleted, the amount of reporter HBV DNA produced was reduced, indicating that the second DR1 sequence is not essential for reverse transcription of pgRNA, but may play an important role. On the other hand, in pBB-intron (ΔDR2) in which the DR2 sequence was deleted and in pBB-intron (Δε2) in which the second ε sequence was deleted, the amount of reporter HBV DNA produced was not significantly different from that of wild-type pBB-intron (wt). This result revealed that the DR2 sequence and the second ε sequence are not particularly important, at least in reverse transcription of pgRNA.

[0151] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for evaluating replication activity of hepatitis B virus, comprising the steps of: A vector comprising a promoter capable of inducing gene expression in a host cell and a nucleic acid comprising an epsilon sequence derived from Hepatitis B virus, the nucleic acid further comprises an arbitrary reporter sequence including an intron on the 3'-terminal side of the epsilon sequence, and the nucleic acid is arranged downstream of the promoter in an expressible state; A hepatitis B virus polymerase expression vector in which the P gene of the hepatitis B virus is arranged in an expressible state downstream of a promoter capable of inducing gene expression in a host cell, and a hepatitis B virus core protein expression vector in which the hepatitis B virus C gene is arranged in an expressible state downstream of a promoter capable of inducing gene expression in a host cell; an introduction step of introducing the vector into a host cell; a culturing step of culturing the host cells after the introduction step; An extraction step of extracting DNA from the host cells after the culturing step; and a detection step of detecting a gene product of the reporter sequence of the vector using a primer pair consisting of a forward primer and a reverse primer designed to be capable of detecting the reporter sequence in the reporter minus strand DNA, the gene product being a reporter sequence obtained by removing an intron from a reporter minus strand derived from pre-mRNA, which may be contained in the DNA obtained in the extraction step; Including, In the method, either one of the forward primer or the reverse primer is designed to span the exon junction site in the reporter sequence by 2 to 3 bases in the extension direction, thereby amplifying only the reporter sequence in the reporter minus strand DNA.

2. 2. The method according to claim 1, wherein in the transfection step, a hepatitis B virus X protein expression vector in which the X gene of the hepatitis B virus is arranged in an expressible state downstream of a promoter capable of inducing gene expression in the host cell is further transfected into the host cell.

3. The method of claim 1, wherein the reporter negative strand corresponds to the negative strand DNA of Hepatitis B virus.

4. The method according to claim 1, wherein a nucleic acid containing an epsilon sequence derived from Hepatitis B virus serves as a template during expression in a host cell, and a reporter pregenomic RNA is synthesized by RNA polymerase II, during which an intron within the reporter sequence is removed by pre-mRNA splicing.

5. The method according to claim 1, wherein reporter negative-strand DNA is produced only when the replication reaction of the Hepatitis B virus progresses, and the amount of reporter negative-strand DNA synthesized reflects the amount of negative-strand DNA synthesized using the pregenomic RNA as a template by the reverse transcription activity of the Hepatitis B virus polymerase.

6. The method according to claim 1, wherein the forward primer is designed to span the exon junction site in the reporter sequence and to align only 2 to 3 bases on the 3'-end of the forward primer to 2 to 3 bases at the 5'-end of the downstream exon.

7. The method of claim 6, wherein the forward primer comprises a base sequence consisting of aggt.

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