JP6566368B2 - Hepatitis B virus secretion inhibitor - Google Patents

Description

  The present invention relates to identification of a glycan gene involved in HBV secretion in hepatitis B virus (HBV) -infected hepatocytes, a method for inhibiting HBV secretion targeting the glycan synthesis-related protein or its gene, and the glycan The present invention relates to an HBV therapeutic agent and an HBV therapeutic method comprising an activity inhibitor of synthesis-related protein or a gene expression inhibitor thereof as an active ingredient.

  Hepatitis B virus (HBV) belongs to hepadnavirus and has incomplete double-stranded DNA that specifically infects human liver. The infection is caused by acute hepatitis B, chronic hepatitis, cirrhosis, hepatocytes This is one of the major health problems. Currently there are approximately 1.1 to 1.4 million hepatitis B virus (HBV) carriers in Japan, but there are 350 million infected people worldwide (WHO report) ), HBV therapeutics are expected to be developed worldwide. Despite universal vaccination for HBV in most countries and regions, it is considered difficult to prevent the onset of newly infected patients. In addition to conventional maternal-infant infection, horizontal infection is also spreading.

  Currently, hepatitis B is mainly treated with interferon (IFN) (injection) and nucleic acid analog preparations (lamivudine [LMV], adefovir [ADV], entecavir [ETV], etc.) (internal use). . However, IFN's treatment results for hepatitis B are often poor, and strong side effects may appear. Nucleic acid analog preparations inhibit reverse transcription from pregenomic RNA to genomic DNA in the same way as HIV treatment drugs, and HBV growth is suppressed and treatment results are high, but when the drug is stopped, hepatitis relapses in most cases. Sometimes, acute hate leads to liver failure. Furthermore, in the case of nucleic acid analog preparations, the emergence of drug-resistant viruses due to continuous administration of drugs is also a big problem. HBV genomic DNA is easily mutated. Over 1000 types of base sequences of HBV DNA have been reported so far, and over 8 types of HBV genotypes have been reported. The fact that most of these mutation sites are regions encoding reverse transcriptase is also the main cause of the emergence of resistant strains of nucleic acid analog preparations that inhibit reverse transcription reaction. Therefore, it is essential to search for drug discovery targets that can replace reverse transcriptase.

The development of therapeutic agents for HBV infections in which resistant viruses do not appear is active, and as a low molecular weight compound that can prevent HBV growth or infection, HBV infection inhibitory activity can be prevented by inhibiting capsid formation of HBV pregenomic RNA. Low molecular weight compounds (Patent Document 1), peptides that inhibit binding of HBsAg surface antigen and HBcAg core antigen by peptides that bind to HBV core antigen, etc. (Patent Documents 2 and 3) have been proposed. In addition, as an HBV-inhibiting active substance derived from an extract from a natural product, a polysaccharide component of a medicinal fungus (Patent Document 4), a cyclohexane ketone compound derived from an extract of Benix nokitake (Patent Document 5), a pro extracted from a plant Anthocyanidin oligomers (Patent Document 6) have also been proposed.
In addition, it has been found that one kind of sphingolipid conventionally used as a therapeutic agent for antifungal infection and immune disease is useful as a therapeutic agent for HBV infection (Patent Document 7), and HBsAg surface antigen and HBcAg core antigen are found. A non-natural sialic acid-containing sugar chain compound (Patent Document 8) having an activity of binding to HBsAg and an anti-HBsAg antibody (Patent Document 9) that binds to an HBsAg surface antigen have been proposed.

Recent elucidation of viral infection mechanisms is revealing that sugar chains and glycoproteins are the receptors for various viruses, and sugar chain-related molecules may be greatly involved in HBV adhesion / invasion. Sex is conceivable.
The present inventors showed incomplete formation of infectious HBV having HBV DNA after mutagenesis of the sugar chain binding site on the surface antigen of HBs, and glycosylation on HBs antigen is involved in particle formation / secretion of infectious HBV. It was clarified that it is important (Non-Patent Document 1).
As an attempt to inhibit sugar chain modification of HBs antigen, a method of inhibiting replication and secretion of HBV virion using N-alkyl derivative of 1,5-deoxyimino D-glucitol, which is an α-glucosidase I, II inhibitor (Patent Document 10) has been proposed. Furthermore, led by the present inventors, a sugar chain-related molecule essential for glycosylation of HBs antigens is identified, and the sugar chain-related molecule is used as an inhibition target, leading to the development of inhibitors such as RNA interference and antisense technology. Attempts to do so have also become active (Non-Patent Document 2, etc.).

In recent years, treatment using RNA interference has been studied as a treatment for various diseases (Non-Patent Documents 3 and 4). RNA interference introduces a complementary sequence of mRNA after transcription into the cell, forms a complex with mRNA, and is destroyed by the activity of ribonucleases such as Dicer. As a result, the mRNA expression level and protein level decrease. cause.
Also in HBV therapy, recently, research and development for silencing RNA expression such as double-stranded ribonucleic acid (dsRNA) such as HBV replication and pathogenicity (Patent Documents 11 to 13, Non-Patent Documents 4 and 5). Etc.) are active. Similarly, antisense oligonucleotides and ribozymes for inhibiting HBV replication targeting HBV nucleic acids have also been proposed (Patent Documents 14, 15, etc.). However, since these RNAi and antisense technologies target the genomic sequence of HBV, the problem of emergence of drug-resistant strains due to HBV DNA mutation remains.

WO2013 / 096744 WO98 / 18818 JP2008-120814A JP 2004-91780 A JP2009-13152 JP 2010-155840 A WO2009 / 154248 WO2011 / 046057 WO97 / 47654 WO95 / 19172 WO2013 / 003520 WO2006 / 069064 US2003 / 0206887 WO97 / 03211 WO98 / 57175 U.S. Pat. No. 8,618,277 (US8618277)

Ito K et al. J Virol. 2010 Dec; 84 (24): 12850-61. 2014.12.9-12The 6th ACGG in Hyderabad Abstract Burnett JC et al, Biotechnol J. 2011 Sep; 6 (9): 1130-46. Draz MS et al, Theranostics 2014; 4 (9): 872-92. Chen A et al, World J Gastroenterol. 2014 Jul; 20 (25): 7993-8004. Ishida N et al. J Biochem. 1996 Dec; 120 (6): 1074-8. Dejima K et al., FASEB J. 2009 Jul; 23 (7): 2215-25.

  The present invention provides a method for inhibiting the formation and / or secretion of HBV in infected hepatocytes, a method for preventing and treating HBV infection and thereby a pharmaceutical composition therefor.

  In order to solve the above problems, the present inventors focus on the hepatocyte side infected with HBV, rather than targeting the HBV genome sequence or HBV-derived reverse transcriptase, and in HBV-infected hepatocytes. We tried to search for glycosynthetic proteins related to glycosylation that are essential for HBV particle formation and secretion. HBV particle formation and / or HBV particle secretion in HBV-infected hepatocytes by inhibiting sugar chain modification activity or inhibiting the expression of sugar chain synthesis-related protein targeting such a sugar chain synthesis-related protein or gene thereof He thought that he could prevent or treat hepatitis B by preventing infection of surrounding hepatocytes as a result. In addition, since glycoprotein synthesis-related protein genes (hereinafter also simply referred to as “glycan genes”) are localized in the endoplasmic reticulum or Golgi apparatus in hepatocytes, they are efficient when used as target molecules for inhibiting HBV infection. It is considered advantageous for effective inhibition.

The present inventors recently analyzed the effect of suppressing HBV-DNA in a culture supernatant using an in vitro HBV replication model, and siRNAs corresponding to multiple types of sugar chain genes have a high HBV-DNA inhibitory effect. it was found out (non-Patent Document 2, the other to ^{2014.4.19-20 2 nd TASL-Japan HBV in} Taipei, 2014.5.29-30 50th liver General meeting of the Society workshop 3,2014.9.3-6 Molecular Biology of HBV announced at Los Angeles).
Based on these results, first, total RNA was prepared from each of normal hepatocytes and two types of liver cancer-derived cell lines, a cDNA library was prepared, and comprehensive sequencing was performed. From the mapped sequences, only those mapped to “glycan genes” in the glycan-related gene database (GGDB, Japan) are extracted, and approximately 185 types of glycan genes expressed in hepatocytes are identified. did. Expression profiles were obtained by comparative analysis of the expression levels of these 185 sugar chain genes.
Based on the expression profiles of the 185 types of sugar chain genes, 86 types that are highly expressed or moderately expressed in hepatocytes are extracted, and consist of 3 types of siRNAs corresponding to each gene 3 A seed mixed siRNA was prepared.

Next, after introducing three mixed siRNAs corresponding to these 86 sugar chain genes into hepatocytes, HBV S-HBs antigen cDNA was transfected into each cell, and S-HBs secreted into the culture supernatant The expression level of the antigen and the addition of sugar chains were confirmed.
As a result, with regard to 10 or more of the 86 sugar chain genes, the expression of S-HBs antigen was reduced or the S-HBs antigen sugar was reduced when expression was suppressed by siRNA introduction compared to control siRNA introduction. A decrease in chain addition was shown.

  In order to analyze the effects on HBV replication and secretion, three types of siRNA corresponding to 86 sugar chain genes were introduced into HBV-producing hepatoma cells and the amount of HBV DNA and HBs antigen in the culture supernatant Was measured. As a result, it was found that the sugar chain genes of the SLC35B1 gene, the CHST9 gene, and the ST8SIA3 gene are sugar chain genes that serve as inhibition targets that significantly reduce both the HBV DNA amount and the HBs antigen amount.

Regarding the SLC35B1 gene, first of all, the three siRNAs used for SLC35B1 gene knockdown were confirmed to have the ability to reduce both the amount of HBV DNA and the amount of HBs antigen as well as the ability to inhibit SLC35B1 gene expression. The position on the base sequence was identified. In addition, siRNA (siSLC35B1 # 1), which has the most ability to inhibit SLC35B1 gene expression among the three species, reduces the amount of HBV DNA and HBs antigen, that is, HBV particle formation and HBV secretion in HBV-infected cells. It was also confirmed that the function was inhibited. Next, siRNA (dsRNA) corresponding to multiple other target candidate regions is prepared, and the base sequence of the highly effective target region in the SLC35B1 gene sequence is determined by comparing each SLC35B1 gene expression inhibition ability I was able to. This means that, together with the siRNA corresponding to the target region, other RNAi reagents such as shRNA that generate RNAi in the same target region, antisense, and ribozyme can be used as inhibitors of HBV infection. For example, RNAi reagents such as siRNA and shRNA corresponding to the target region can suppress the growth and secretion of HBV in the liver by using a system that transports siRNA specifically in the liver (Patent Document 16, etc.) It can be used as a therapeutic agent for HBV infection.
The above results also indicate that the SLC35B1 gene and the expressed SLC35B1 protein, as well as the CHST9 and ST8SIA3 genes and the expressed CHST9 and ST8SIA3 proteins themselves are inhibition targets in HBV-infected hepatocytes. That is, SLC35B1, CHST9 and ST8SIA3 binding peptides, anti-SLC35B1 neutralizing antibodies, anti-CHST9 neutralizing antibodies, anti-ST8SIA3 neutralizing antibodies and other SLC35B1, CHST9 and ST8SIA3 activity inhibitors are also HBs surface antigens in HBV-infected hepatocytes. Since it acts as an inhibitor of HBV proliferation and secretion by inhibiting the sugar chain transfer activity, it becomes an active ingredient of a therapeutic agent for HBV infection. Furthermore, all of the hepatocyte glycosylation-related proteins such as SLC35B1 that act on the above-mentioned siRNA are necessary for HBV particle formation and secretion, and inhibition of siRNA expression inhibits HBV particle formation and HBV antigen expression. It was also confirmed that it has little effect on the original proliferation of hepatocytes.
Obtaining these findings completed the present invention.

That is, the present invention includes the following inventions.
[1] An inhibitor of HBV particle formation or HBV secretion in HBV-infected hepatocytes, comprising as an active ingredient an inhibitor of at least one sugar chain gene expression selected from the SLC35B1 gene, CHST9 gene, and ST8SIA3 gene.
[2] The inhibitor according to [1], wherein the sugar chain gene expression inhibitor is an oligonucleic acid preparation selected from RNAi preparations, antisenses, nucleic acid aptamers, and ribozymes.
[3] A nucleic acid comprising the oligonucleic acid preparation comprising a base sequence containing at least 15 consecutive bases in a base sequence of at least one sugar chain gene selected from SLC35B1 gene, CHST9 gene and ST8SIA3 gene The inhibitor according to [2] above, which is contained as an active ingredient.
[4] The oligonucleic acid preparation is an RNAi preparation, and a base sequence in which at least 15 consecutive bases coincide with the complementary strand of mRNA of at least one sugar chain gene selected from the SLC35B1 gene, CHST9 gene and ST8SIA3 gene The inhibitor according to [3] above, comprising a double-stranded RNA containing as an active ingredient.
[5] Regions and sequences of the oligonucleic acid agent at positions 102 to 121, 161 to 179, 311 to 330, 477 to 495, 576 to 594, and 627 to 644 of the base sequence shown in SEQ ID NO: 3 A nucleotide sequence comprising at least 15 consecutive bases of any one or more regions selected from the region of positions 806 to 824 of No. 8 and the region of positions 178 to 196 of SEQ ID NO: 10 or a complementary sequence thereof The inhibitor according to any one of [2] to [4] above,
[6] The sugar chain gene expression inhibitor specifically binds to a region consisting of a base sequence containing at least 15 consecutive bases of SEQ ID NO: 12 in the base sequence of SLC35B1 mRNA. [2] to [4].
[7] An inhibitor of HBV particle formation or HBV secretion in HBV-infected hepatocytes, comprising an inhibitor of sugar chain modification activity of at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9, and ST8SIA3.
[8] For prevention, suppression or treatment of HBV infection, comprising as an active ingredient at least one inhibitor of HBV particle formation or HBV secretion according to any one of [1] to [7] Pharmaceutical composition.
[9] The pharmaceutical composition for prevention, suppression or treatment of HBV infection according to [8] above, which is used in combination or in combination with another anti-HBV agent.
[10] A method for screening an inhibitor of HBV particle formation or HBV secretion, which comprises the following steps (1) to (4);
(1) A step of measuring the expression level of at least one sugar chain gene selected from SLC35B1 gene, CHST9 gene and ST8SIA3 gene in cultured hepatocytes,
(2) introducing a test compound into cultured hepatocytes or administering the test compound into a medium;
(3) measuring the expression level of the sugar chain gene selected in step (1),
(4) When the expression level measured in step (3) decreases compared to the expression level measured in step (1), the test compound is used as a candidate for an inhibitor of HBV particle formation or HBV secretion. Process to select.
Here, the test compound includes a test oligonucleic acid, and in the case of a test oligonucleic acid, it is preferably introduced into cultured hepatocytes using a vector or a liposome.
[11] A method for screening an inhibitor of HBV particle formation or HBV secretion, comprising the following steps (1) to (4);
(1) a step of measuring the amount or activity of at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3 in cultured hepatocytes,
(2) introducing a test compound into cultured hepatocytes or administering the test compound into a medium;
(3) a step of measuring the amount or activity of the sugar chain synthesis-related protein selected in step (1),
(4) Inhibitor of HBV particle formation or HBV secretion when the amount or activity value measured in step (3) decreases compared to the amount or activity value measured in step (1) Selecting as a candidate.
Here, the test compound includes a test oligonucleic acid, and in the case of a test oligonucleic acid, it is preferably introduced into cultured hepatocytes using a vector or a liposome.
In addition, in order to measure the activity of a sugar chain synthesis-related protein, in the case of SLC35B1, for example, the concentration of a sugar chain such as UDP-Gal or the concentration of UDP released from UDP-Gal may be measured. In the case of ST8SIA3, the concentration of inorganic phosphate based on CMP released from CMP-NeuAc containing sialic acid. (For example, manufactured by R & D Systems).
[12] A method for screening an inhibitor of HBV particle formation or HBV secretion, comprising the following steps (1) to (4);
(1) A step of preparing a transformed cell transformed with at least one sugar chain gene selected from the SLC35B1 gene, CHST9 gene and ST8SIA3 gene, and measuring the expression level of the selected sugar chain gene in the cell ,
(2) introducing a test compound into the transformed cell or administering the test compound in a medium;
(3) a step of measuring the expression level of the selected sugar chain gene in the transformed cell of step (2),
(4) When the expression level of the selected glycan gene measured in step (3) decreases compared to the expression level measured in step (1), the test compound is converted into HBV particles or Selecting as a candidate for an inhibitor of HBV secretion.
Here, the test compound includes a test oligonucleic acid, and in the case of a test oligonucleic acid, it is preferably introduced into a transformed cell using a vector or a liposome.
Further, instead of the step of measuring the expression level of the sugar chain gene in steps (1) and (3), the activity of the sugar chain synthesis-related protein expressed in the transformed cell may be measured and compared.
[13] A method for screening an inhibitor of HBV particle formation or HBV secretion, comprising the following steps (1) to (4);
(1) Prepare transformed cells that express at least one labeled sugar chain synthesis-related protein selected from labeled SLC35B1, CHST9, and ST8SIA3 on the inner membrane surface, and measure the amount of labeling on the inner membrane surface The process of
(2) introducing the test compound into the transformed cell or administering the test compound into the medium;
(3) a step of measuring the labeling amount on the surface of the intracellular membrane,
(4) When the amount of label measured in step (3) decreases compared to the amount of label measured in step (1), the test compound is selected as a candidate for an inhibitor of HBV particle formation or HBV secretion Process.
Here, the test compound includes a test oligonucleic acid, and in the case of a test oligonucleic acid, it is preferably introduced into a transformed cell using a vector or a liposome.
[14] The transformed cell is a cell that expresses a tagged sugar chain synthesis-related protein, and is a transformed cell in which the inner membrane surface is fluorescently labeled with a fluorescently labeled anti-tag antibody, and the cell to be measured The method according to [13] above, wherein the labeling amount on the inner membrane surface is a fluorescence amount.
[15] A kit for screening an inhibitor of HBV particle formation or HBV secretion,
A kit comprising a transformed cell transformed with at least one sugar chain gene selected from SLC35B1 gene, CHST9 gene and ST8SIA3 gene.
Furthermore, a kit combining a sugar chain-nucleotide such as UDP-Gal, PAPS, or CMP-NeuAc for measuring the activity of a sugar chain synthesis-related protein may be used.
[16] The sugar chain gene is fused with a gene encoding a marker protein, and the transformed cell has at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9, and ST8SIA3 on the inner membrane surface thereof. The kit according to [15] above, which is a transformed cell expressed in a directly or indirectly labeled state.
Here, as a gene encoding the labeled protein, for example, a fluorescent protein gene such as a GFP gene is preferably used.
[17] A kit for screening an inhibitor of HBV particle formation or HBV secretion, comprising the following (1) and (2);
(1) A transformed cell that expresses at least one sugar chain synthesis-related protein selected from tagged SLC35B1, CHST9, and ST8SIA3,
(2) A labeled anti-tag antibody.
[18] The kit according to [17], wherein the tagged sugar chain synthesis-related protein is a Flag-tagged sugar chain synthesis-related protein, and the anti-tag antibody is an anti-Flag antibody.
[19] A kit for screening for inhibitors of HBV particle formation or HBV secretion, comprising the following (1) and (2);
(1) a substrate on which at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3 is immobilized;
(2) A library comprising nucleic acids containing random base sequences and corresponding peptides.
Here, the length of the random base sequence is usually 21 to 36 bases (7 to 12 amino acids). Typically, it is a peptide library by phage display, and may be a peptide library obtained by translating a nucleic acid library with a cell-free translation system.

The present invention also includes the following aspects.
[20] In an HBV-infected hepatocyte, the method comprises the step of administering an expression inhibitor of at least one sugar chain gene selected from SLC35B1 gene, CHST9 gene and ST8SIA3 gene to HBV-infected hepatocytes. A method of inhibiting HBV granulation and secretion.
For example, the step of administering a sugar chain gene expression inhibitor is a step of introducing an oligonucleic acid preparation selected from RNAi preparations, antisenses, nucleic acid aptamers and ribozymes into cells, and the oligonucleic acid preparation at that time is, for example, A double-stranded RNA comprising at least one base sequence selected from SLC35B1 gene, CHST9 gene and ST8SIA3 gene or a complementary sequence thereof and a base sequence comprising at least 15 consecutive bases as an active ingredient. In particular, regions 102 to 121, 161 to 179, 311 to 330, 311 to 330, 477 to 495, 576 to 594, and 627 to 644 of the base sequence shown in SEQ ID NO: 3, 806 to 824 of SEQ ID NO: 8 And a base sequence containing at least 15 consecutive bases of any one or more regions selected from the region of positions 178 to 196 of SEQ ID NO: 10 or a complementary sequence thereof.
In addition, instead of the SLC35B1 gene, CHST9 gene and ST8SIA3 gene expression inhibitor to be administered, at least one sugar chain synthesis-related protein sugar chain selected from SLC35B1, CHST9 and ST8SIA3 A modified activity inhibitor may be used.
That is, the present invention can be expressed as follows.
[20 ′] A method for inhibiting HBV particle formation and secretion in HBV-infected cells,
Administering an inhibitor of HBV particle formation and secretion to HBV-infected cells,
Here, the inhibitor of HBV particle formation and secretion is an expression inhibitor of at least one sugar chain gene selected from SLC35B1 gene, CHST9 gene and ST8SIA3 gene,
Or a sugar chain modification activity inhibitor of at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3,
A method comprising: as an active ingredient.
And since this invention can prevent, suppress or treat HBV infection by inhibiting HBV particle formation and secretion in HBV-infected cells, the following aspects are also included.
[21] A method for preventing, suppressing or treating HBV infection,
Administering a pharmaceutical composition to a patient in need of the method,
Here, the pharmaceutical composition comprises an inhibitor of expression of at least one sugar chain gene selected from SLC35B1 gene, CHST9 gene and ST8SIA3 gene,
Or a sugar chain modification activity inhibitor of at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3,
A method comprising at least one of the above as an active ingredient.
The present invention also includes the following aspects.
[23] A gene expression inhibitor for use in a method for preventing, suppressing or treating HBV infection, wherein the gene expression inhibitor is at least one selected from the group consisting of SLC35B1 gene, CHST9 gene and ST8SIA3 gene A gene expression inhibitor characterized by suppressing the expression of two target sugar chain genes.
[23 ′] A nucleic acid for use in a method for preventing, suppressing or treating HBV infection, wherein the nucleic acid is at least one sugar chain gene selected from the group consisting of SLC35B1 gene, CHST9 gene and ST8SIA3 gene A nucleic acid characterized by comprising a base sequence comprising at least 15 consecutive bases in the base sequence or its complementary sequence.
[23 ″] A double-stranded RNA for use in a method for preventing, suppressing or treating HBV infection, wherein the double-stranded RNA is a sugar chain gene selected from the SLC35B1 gene, the CHST9 gene and the ST8SIA3 gene A double-stranded RNA characterized in that it comprises a base sequence that matches at least 15 consecutive bases with the complementary strand of at least one of the mRNAs.
[23 ′ ″] Double-stranded RNA for use in a method for preventing, suppressing or treating HBV infection, wherein the double-stranded RNA is located at positions 102 to 121 of the nucleotide sequence represented by SEQ ID NO: 3, From regions 161 to 179, 311 to 330, 477 to 495, 576 to 594 and 627 to 644, region 806 to 824 of SEQ ID NO: 8, and region 178 to 196 of SEQ ID NO: 10 A double-stranded RNA comprising a base sequence comprising at least 15 consecutive bases in any selected region or a complementary sequence thereof.
[24] A sugar chain modification activity inhibitor for use in a method for preventing, suppressing or treating HBV infection, wherein the sugar chain modification activity inhibitor is associated with at least one sugar chain synthesis of SLC35B1, CHST9 and ST8SIA3 A sugar chain-modifying activity inhibitor characterized by specifically binding to a protein.
[24 ′] A peptide or antibody for use in a method for the prevention, suppression or treatment of HBV infection, wherein the peptide or antibody specifically binds to at least one glycosylation-related protein of SLC35B1, CHST9 and ST8SIA3 A peptide or antibody characterized by binding.
Furthermore, the present invention includes the following aspects.
[25] Use of an expression inhibitor of at least one target sugar chain gene selected from the group consisting of SLC35B1 gene, CHST9 gene and ST8SIA3 gene in the manufacture of a pharmaceutical composition for prevention, suppression or treatment of HBV infection.
[25 ′] A nucleic acid comprising a base sequence comprising at least 15 consecutive bases in the base sequence of at least one target sugar chain gene selected from the group consisting of SLC35B1 gene, CHST9 gene and ST8SIA3 gene or its complementary sequence Use in the manufacture of a pharmaceutical composition for the prevention, suppression or treatment of HBV infection.
[25 ″] a double strand comprising a base sequence in which at least 15 consecutive bases coincide with the complementary strand of the mRNA of at least one target sugar chain gene selected from the group consisting of SLC35B1 gene, CHST9 gene and ST8SIA3 gene Use of RNA in the manufacture of a pharmaceutical composition for prevention, suppression or treatment of HBV infection.
[25 ′ ″] regions of positions 102 to 121, 161 to 179, 311 to 330, 477 to 495, 576 to 594, and 627 to 644 of the base sequence shown in SEQ ID NO: 3, SEQ ID NO: 8 A region comprising at least 15 consecutive bases of any one or more regions selected from the region from positions 806 to 824 of SEQ ID NO: 10 and the region from positions 178 to 196 of SEQ ID NO: 10, or a duplex comprising the complementary sequence thereof Use of RNA in the manufacture of a pharmaceutical composition for prevention, suppression or treatment of HBV infection.
[26] Use of a sugar chain modification activity inhibitor of at least one sugar chain synthesis-related protein selected from the group consisting of SLC35B1, CHST9 and ST8SIA3 in the manufacture of a pharmaceutical group for prevention, suppression or treatment of HBV infection.
[26 ′] In the manufacture of a pharmaceutical group for the prevention, suppression or treatment of HBV infection of a peptide or antibody that specifically binds to at least one glycosylation-related protein selected from the group consisting of SLC35B1, CHST9 and ST8SIA3 use.

Unlike the conventional nucleic acid analog preparation and siRNA preparation depending on the HBV genome sequence, the present invention targets the SLC35B1 gene, CHST9 gene or ST8SIA3 gene on the human hepatocyte side, or an expressed protein thereof. Unaffected by mutations, there is almost no possibility of emergence of resistant virus strains. It is also advantageous as a target molecule for inhibiting HBV infection that the sugar chain gene involved in sugar chain modification is localized in the endoplasmic reticulum or Golgi apparatus in hepatocytes. In addition, the SLC35B1, CHST9, and ST8SIA3 sugar chain synthesis-related proteins and genes thereof of the hepatocytes specified in the present invention are all necessary for HBV particle formation and secretion, but they are inherent to hepatocytes. Since these are proteins and genes that have little effect on synthesis, it is expected that inhibition of their expression will not impair hepatocyte function and have extremely low side effects.
By using as a pharmaceutical composition a compound that inhibits the expression of SLC35B1, CHST9 or ST8SIA3 gene expressed in human hepatocytes or a compound that suppresses the activity of SLC35B1, CHST9 or ST8SIA3 related to sugar chain synthesis, in the liver Can suppress the growth and secretion of HBV. Typical examples of the former include siRNA preparations or other RNAi preparations or antisense preparations that can hybridize to a specific region of the SLC35B1 gene, and the latter typical compounds include low-level peptides such as peptides that bind to SLC35B1. There are molecular compounds and anti-SLC35B1 neutralizing antibodies. For example, in the case of the SLC35B1 siRNA preparation, a pharmaceutical composition for HBV infection using a known liver-specific siRNA transport system (eg, Patent Document 16) can suppress the growth and secretion of HBV in the liver, alone or in other cases. It can be used in combination with other therapeutic agents for HBV infection as a pharmaceutical composition for the suppression and treatment of hepatitis B.

A system for measuring the amount of S-HBs antigen secreted into the culture supernatant by the tagged S-HBs antigen and the sugar chain content of the secreted S-HBs antigen Effects of Glycosynthesis inhibitors on S-HBs antigen secretion Expression profile of glycan genes in hepatocytes and hepatoma cells (NGS) Expression profile of glycan genes in hepatocytes and hepatoma cells (qRT-PCR) Screening method of target glycan gene by analysis of effects on S-HBs antigen secretion using siRNA plate Example of analysis of effects of siRNA on S-HBs antigen secretion Screening method for target glycan genes by analysis of effects on HBV DNA secretion and S-HBs antigen levels using siRNA plates Effect of siRNA on HBV DNA secretion (RT-PCR) and S-HBs antigen level (ELISA) Analysis results: In addition, the effect on cell proliferation was also observed by MTT assay. In the figure, No. 15 is CHST9 siRNA, No. 19 is ST8SIA3 siRNA, No. 79 is SLC35B1 siRNA 2nd screening result using siRNA plate (RT-PCR, ELISA) SiRNA target site on SLC35B1: In the figure, the underlined part is the target position of siRNA, which are called a, b, c,..., J in order from the first. The white square (c) shows the target site of siSLC35B1 # 1 and the gray squares (e and g) used in the following figure show the target sites of siSLC35B1 # 2 and siSLC35B1 # 3, respectively. Comparison of HBV inhibitory effect between SLC35B1 siRNA (siSLC35B1 # 1) and nucleic acid analog: In the figure, E represents entecavir and L represents lamivudine. SLC35B1 expression inhibition effect (qRT-PCR value) by siRNA a to j targeting positions a to j (Fig. 7) of SLC35B1 SLC35B1 siRNA (siSLC35B1 # 1) confirmed SLC35B1 expression suppression effect (qRT-PCR) Comparison of the effects of SLC35B1 siRNA (siSLC35B1 # 1) and sugar chain synthesis inhibitor (Tunicamycin) on AFP secretion Effect of SLC35B1siRNA (siSLC35B1 # 1) on glycan synthesis for host cells: In the figure, # 1 is siSLC35B1 # 1, # 2 is siSLC35B1 # 2, and siC is a control siRNA (manufactured by SIGMA) Comparison of lectin array analysis between untreated group and siRNA (siSLC35B1) treated group. (A) Comparison with HepG2 cells (B) Comparison with Huh7 cells. Effect on cells by siRNA (siSLC35B1) treatment. Comparison with TM and BreA treatments is shown. Cytotoxicity assay results of SLC35B1 siRNA (siSLC35B1 # 1) Effect of SLC35B1 siRNA (siSLC35B1 # 1) on the expression level of glycan genes in hepatocytes (NGS) Effect of SLC35B1 siRNA (siSLC35B1 # 1) on the expression level of glycan genes in hepatocytes (volcano plot analysis) Effect of siRNA (siSLC35B1) treatment in Huh7 and HepG2 cells (NGS analysis for off-target search) Effects of siRNA (siSLC35B1) treatment on normal human hepatocytes (NGS analysis for off-target search) Effect of siRNA (siSLC35B1) treatment on HBV expression: (A) SLC35B1 expression level (B) HBV DNA expression level Effect of SLC35B1 siRNA (siSLC35B1 # 1) on concentration-dependent expression of SLC35B1 gene Effect of SLC35B1 siRNA (siSLC35B1 # 1) on concentration-dependent secretion of HBV DNA Reduction of HBs antigen level after siRNA (siSLC35B1) treatment Decrease in the number of HepG2.2.15.7 cells after siRNA (siSLC35B1) treatment Effect of combined use of entecavir on inhibition of HBV secretion by siRNA-SLC35B1 # 1 (A) HBV DNA secretion inhibition effect (B) Change in HBs antigen level Assay method using entecavir resistant strain Secretion inhibitory effect of siRNA-SLC35B1 in entecavir resistant strains (A) Changes in HBs antigen level (B) HBV DNA secretion inhibitory effect Flag-SLC35B1 localization to intracellular endoplasmic reticulum: The Potential TM sequence (presumed transmembrane region) in the SLC35B1 cDNA sequence is shown in bold. The underline indicates the Di-lysine motif. Detection of SLC35B1 using Flag-SLC35B1 and verification of expression inhibition

1. HBV and HBV infection The term “HBV” in the present specification means a virus capable of causing hepatitis B. HBV currently has a genotype from A to H, but HBV to be treated and suppressed in the pharmaceutical composition for HBV infection of the present invention includes all genotypes.
In the present invention, “HBV infection” is typically hepatitis B, and is classified into chronic hepatitis, acute hepatitis, and fulminant hepatitis. In addition to hepatitis B, liver cancers such as cirrhosis, liver fibrosis, and hepatocellular carcinoma are also included as long as they are caused by infection of living organisms including humans with HBV. Whether or not it is an HBV infection, for example, detection of HBs antigen in blood, detection of HBe antigen in blood, measurement of the amount of HBV-DNA in blood and measurement of the amount of HBV DNA polymerase in blood, and This can be determined by a combination of these.
In the present specification, the term “treatment of HBV infection” refers to elimination of HBV, reduction of symptoms due to infection with HBV, calming of hepatitis, and prevention and reduction of progression from hepatitis to cirrhosis, liver fibrosis and liver cancer. Means. In addition, it may be expressed as "treatment or suppression of HBV infection" in order to clarify that the symptoms and progression of HBV infection are included even before the remission of HBV infection is not achieved. . The term “prevention of HBV infection” means preventing the onset of HBV infection such as hepatitis B before or after infection with HBV.

2. HBs antigen
The HBs antigen is an HBV envelope glycoprotein, which starts with a different methionine (Met) from one HBs gene, and there are three types of L-HBs, M-HBs, and S-HBs antigens depending on their size.
HBs antigen undergoes sugar chain modification in the endoplasmic reticulum and Golgi apparatus after translation by intracellular ribosomes. The core (HBc) encapsulating HBV DNA (RNA) is taken up by the HBs antigen in the endoplasmic reticulum and is modified with a sugar chain in the same manner as the HBs antigen to form infectious HBV particles (Schaedler S et al, Viruses. 2009 Sep; 1 (2): 185-209., Grimm D et al, Hepatol Int. 2011 Jun; 5 (2): 644-53.
And this sugar chain modification to HBs antigen and HBc antigen is an essential process essential for particle formation and secretion of infectious HBV (Non-patent Document 1).
In the examples of the present invention, the amount of S-HBs antigen in the culture supernatant is used as an index for screening for sugar chain synthesis inhibitors, and a S-HBs antigen with a Flag tag for purification added to the N-terminal is used. Full-length DNA (SEQ ID NO: 1) was used.

3. Sugar chain synthesis-related proteins and their genes (3-1) What are sugar chain synthesis-related proteins and their genes? Conventionally, various sugar chains and sulfuric acid are added to proteins translated from mRNA in the Golgi apparatus or endoplasmic reticulum in cells. Genes such as enzymes that add, degrade and transfer, transporters, and sugar-nucleotide synthases that synthesize sugar-nucleotides were collectively referred to as Glycogene (glycan genes or sugar chain-related genes) (Taniguchi N, et al. , Handbook of Glycosyltransferases and Related Genes 2014 Springer-Verlag). In the present invention, the gene is referred to as “sugar chain gene” or “sugar chain synthesis-related protein gene”, and the protein encoded by the gene is referred to as “sugar chain synthesis-related protein”. According to the above Taniguchi et al. Document, there are more than 200 types of sugar chain genes that work constitutively in normal human cells, of which known gene names are published in various databases (glycan related gene databases ( (GGDB, Japan) http://jcggdb.jp/rcmg/ggdb/ or Carbohydrate-active enzymes (CAZy) database (http://www.cazy.org), Hansen L et al, Glycobiology, 2015 25 (2): 211-24. See Supplementary Table 1.)

  In the present invention, a group of physiologically active proteins such as enzymes and transporters belonging to these glycosyltransferase-related enzyme groups, and 185 types of sugar chain gene groups in which registration numbers of registered base sequences or amino acid sequences are described. Used as a population. Specifically, 185 types of enzymes and transporter genes belonging to the sugar chain synthesis-related proteins that are the population of the present invention are listed below (Tables 1 to 8) (Ito H et al, J Proteome Res. 2009 Mar; 8 (3): 1358-67 Supplemental Table 1) is a glycan gene described above (excluding duplicate genes).

(3-2) Sugar chain synthesis-related protein essential for HBV particle formation or secretion in human-infected hepatocytes and a method for identifying the gene In the present invention, it is essential for HBV particle formation or secretion in infected hepatocytes. The sugar chain gene was identified by the following procedure.
1) A cDNA library is prepared from total RNA of human hepatocytes (normal hepatocytes and liver cancer-derived cell lines), comprehensively sequenced, and mapped to human cDNA.
2) Extraction of those mapped to “glycan genes” in the glycan-related gene database (GGDB, Japan), and comparative analysis of the expression levels of the 185 types of glycan genes listed above (Tables 1-8) To obtain an expression profile.
3) Based on the expression profiles of the 185 types of sugar chain genes, sugar chain genes that are highly expressed or moderately expressed in hepatocytes are extracted, and corresponding to different regions of each gene sequence 3 Prepare a mixed siRNA composed of three types of siRNA.
4) After introducing 3 types of mixed siRNA into cultured hepatic cell line, HBV S-HBs antigen cDNA is transfected, and the amount of S-HBs antigen expressed and the amount of sugar chain added to the culture supernatant Are selected, and those showing a decrease in the expression of S-HBs antigen or a decrease in the addition of sugar chains of S-HBs antigen are selected as compared with the case of introduction of control siRNA.
5) Simultaneously, each of the three mixed siRNAs is introduced into HBV-producing hepatoma cells, and the amount of HBV DNA and HBs antigen in the culture supernatant is measured. Compared with the control siRNA, the amount of HBV DNA and HBs antigen Select a reduced one.
Here, a known immunological or biochemical analysis method can be applied to the measurement of the amount of HBV DNA, the amount of HBs antigen expressed, the amount of added sugar chain, etc. in the culture supernatant. In the examples of the present invention, the amount of HBV DNA was detected by RT-PCR, the amount of HBs antigen expressed was detected by ELISA, and the amount of added sugar chain was detected by a lectin array, but is not limited thereto.
6) In any of the steps 4) and 5), the sugar chain gene corresponding to the selected siRNA can be identified as a gene essential for HBV particle formation and / or secretion. The gene and the sugar chain synthesis-related protein encoding the gene are the inhibition target molecules of the present invention.
In the present invention, SLC35B1, CHST9 and ST8SIA3 genes, and SLC35B1, CHST9 and ST8SIA3 proteins could be provided as inhibition target molecules.

(3-3) SLC35B1 and its gene One of the target proteins of the present invention is SLC35B1.
SLC35B1 is a protein belonging to solute carrier (SLC) family 35 and cloned as a gene having homology to nucleic acid-sugar transporters (UDP-Gal transporter related protein, UGTrel1, 322aa) (Non-patent Document 6). The human gene is located on chromosome 17 and there are at least 9 exons. mRNA is ubiquitously expressed, and two proteins, 255aa (NP_001265713) and 359aa (NP_005818), are registered. In the present invention, 322aa (P78383: SEQ ID NOs: 3 and 4) whose activity was measured in (Non-patent Documents 6 and 7) using a partial sequence thereof instead of these full-length sequences is used. So far, SLC35B1 homologs have been reported in yeast and nematodes, but their activity as a nucleic acid-sugar transporter has not been confirmed, and there has been no report on their role in HBV secretion or their intracellular functions.
According to the present invention, for the first time, the SLC35B1 protein is constitutively working in hepatocytes, and when it is infected with HBV, a sugar chain that is essential for modifying HBV into particles and secreting it outside the cell It was elucidated to be a synthesis-related protein. In addition, it was confirmed that inhibition of the expression of SLC35B1 and its gene in hepatocytes had almost no effect on the sugar chain synthesis function of hepatocytes themselves and there was no cytotoxicity.

(3-4) Other sugar chain genes In the present invention, CHST9 gene and ST8SIA3 gene have been identified as sugar chain genes that have undergone sugar chain modification essential for making HBV particles and secreting them outside the cell. .
CHST9 protein is a sulfotransferase belonging to the Carbohydrate sulfotransferase family, and ST8SIA3 protein is one of α2,8-sialyltransferase (see Taniguchi N et al., Handbook of Glycosyltransferases and Related Genes 2014). .
In the present specification, hereinafter, expression inhibition mainly targeting the SLC35B1 gene as a typical sugar chain gene is described, but the same applies to the CHST9 gene and the ST8SIA3 gene.

4). Method of controlling expression level of target sugar chain gene or protein activity related to sugar chain synthesis (4-1) Method of determining target region by RNAi in base sequence of target gene HBV particle formation and / or in the above (3-2) A highly effective inhibitory target region in a gene sequence identified as a sugar chain gene essential for secretion can be determined as follows.
The siRNA target sequence can be determined and searched and designed using commercially available software or a website (such as GENETYX or siDirect http://sidirect2.rnai.jp manufactured by Genetics). In this study, we used 3 types of mixed siRNAs combining siRNAs corresponding to 3 different positions on the base sequence of each glycan gene to determine the inhibition target gene.
Each of these 3 types of siRNA was introduced into HBV-infected cells, and the amount of HBV DNA and HBs antigen in the culture supernatant were compared. Identify the region on the gene sequence. This region becomes a target region for inhibiting the expression of the sugar chain gene. Furthermore, by preparing siRNA having a base sequence in the vicinity region and comparing the inhibition ability of each HBV secretion function, a base sequence of a more effective target region can be determined.

(4-2) Method for inhibiting expression of target sugar chain gene If the target region in the base sequence of the target sugar chain gene SLC35B1, CHST9 or ST8SIA3 gene can be determined, siRNA containing a sequence corresponding to the region or By using oligonucleic acid preparations such as RNAi preparations that induce RNAi such as shRNA, antisense RNA or DNA that hybridizes to the region, or antisense preparations or ribozyme preparations that prepare ribozymes, HBV in hepatocytes infected with HBV Particleization and secretion can be inhibited. Since these oligonucleic acid preparations can directly act on HBV-infected cells in the liver using a liver-specific transport system (Patent Document 16, etc.) described later, treatment of HBV infection and It is effective as a pharmaceutical composition for prevention.

That is, a typical RNAi preparation as an oligonucleic acid useful as a pharmaceutical composition for the treatment and prevention of HBV infection of the present invention is the SLC35B1 gene (SEQ ID NO: 3), CHST9 gene (SEQ ID NO: 8) or ST8SIA3 gene (SEQ ID NO: 10) siRNA or shRNA comprising a double-stranded RNA in which at least 15, preferably 17, and more preferably 18 or more consecutive base sequences correspond to a complementary strand corresponding to a specific region in the base sequence of 10) RNAi preparation. That is, it can be said that it is a double-stranded RNA comprising at least 15, preferably 17, and more preferably 18 consecutive base sequences corresponding to a specific region of the SLC35B1 gene, CHST9 gene or ST8SIA3 gene. These double-stranded RNAs, when used as RNAi preparations, act as SLC35B1 gene, CHST9 gene or ST8SIA3 gene expression inhibitors in HBV-infected cells, so they have excellent pharmaceutical composition for treatment, suppression or prevention of HBV infection It becomes an active ingredient of things.
For example, positions 102 to 121 (a), 161 to 179 (b), 311 to 330 (c), 477 to 495 (e), and 576 to 594 of the nucleotide sequence of the SLC35B1 gene (SEQ ID NO: 3) ( g) and at least 15, preferably 17, and more preferably 18 or more of the region corresponding to positions 627 to 644 (i), a double-stranded RNA targeting a continuous nucleotide sequence, particularly the base of the SLC35B1 gene Double-stranded RNA targeting a base sequence comprising at least 15, preferably 17, and more preferably 18 contiguous bases in the region corresponding to positions 311 to 330 of the sequence (SEQ ID NO: 3) (eg siRNA- When used as an RNAi preparation, SLC35B1 # 1) acts as an SLC35B1 gene expression inhibitor in HBV-infected cells and becomes an active ingredient of an excellent pharmaceutical composition for treatment, suppression, or prevention of HBV infection.

On the other hand, for the CHST9 gene, the amount of HBV DNA in CHST9 # 1 (positions 1188 to 1206), CHST9 # 2 (positions 806 to 824) and CHST9 # 3 (positions 588 to 606) used in this example was determined. For the region of positions 806 to 824 corresponding to the position of CHST9 # 2 of the CHST9 gene (SEQ ID NO: 8) significantly decreased, and the ST8SIA3 gene, ST8SIA3 # 1 (positions 178 to 196) used in this Example ST8SIA3 # 2 (positions 354 to 372) and ST8SIA3 # 3 (positions 208 to 226) of ST8SIA3 # 1 (SEQ ID NO: 10) of ST8SIA3 # 1 in which both the amount of HBs antigen and the amount of HBV DNA were significantly reduced Double-stranded RNA targeting a base sequence containing at least 15, preferably 17, and more preferably 18 consecutive bases in the region from position 178 to 196 corresponding to the position is the same as in the case of the SLC35B1 gene .
In addition, not only RNAi preparations, but also antisense preparations containing the above-mentioned specific base sequences, oligonucleic acid preparations such as nucleic acid aptamers or ribozymes also act as inhibitors of the expression of each sugar chain gene. It becomes an active ingredient of a pharmaceutical composition for prevention.
Furthermore, peptides or low molecular weight compounds that specifically bind with the specific region of each sugar chain gene as a target are also active ingredients of a pharmaceutical composition for the treatment and prevention of HBV infection, like double-stranded RNA.

Moreover, as a method for inhibiting gene expression, the gene is disrupted by genome editing technology (Shibata T, Aburatani H. Nat Rev Gastroenterol Hepatol. 2014 Jun; 11 (6): 340-9.). It is also possible to suppress the function.
Furthermore, using the sequence of the promoter region of the target sugar chain gene, the binding peptide to the sequence can be searched by the ChIP method or the like, and the transcription and expression of the gene can be performed using the binding peptide or a transcription factor inhibitor. Can be inhibited.
That is, peptides or low molecular weight compounds that specifically bind to the gene expression control region such as the promoter sequence of the SLC35B1 gene, CHST9 gene or ST8SIA3 gene identified in this way also express the target sugar chain gene in HBV-infected cells. Since it inhibits, it becomes an active ingredient of a pharmaceutical composition for the treatment and prevention of HBV infection.

(4-3) Method for inhibiting target sugar chain synthesis-related protein activity
HBV particle formation and secretion can also be inhibited by inhibiting functions such as the sugar chain transfer activity of SLC35B1, CHST9 or ST8SIA3 protein, which is a target sugar chain synthesis-related protein in HBV-infected hepatocytes.
Specifically, antibodies, peptides, or other low molecular weight compounds that specifically bind to SLC35B1, CHST9, or ST8SIA3 protein are used to bind to the enzyme activity region of these proteins, or to a region close to that region. Then, by changing the structure of the enzyme active region, the function such as sugar chain synthesis activity or sugar chain transfer activity is inhibited.
That is, neutralizing antibodies, peptides, or other low molecular weight compounds that specifically bind to SLC35B1, CHST9 or ST8SIA3 protein act as activity inhibitors of SLC35B1, CHST9 or ST8SIA3 protein in HBV-infected cells. It becomes an active ingredient of the pharmaceutical composition for treatment and prevention.

In order to obtain a peptide that specifically binds to SLC35B1, CHST9, or ST8SIA3 protein, the peptide sequence may be determined by an evolutionary molecular engineering display technique or the like. Typically, random phage display (Smith, GP, et al, Chem. Rev. 1997 Apr 1; 97 (2): 391-410.) Can be applied, but ribosome display, mRNA display, cDNA display, etc. Cell-free translation system display technology (Lohse PA et al., Curr Opin Drug Discov Devel., 2001 Mar; 4 (2): 198-204, Bradbury AR et al., Nat Biotechnol. 2011 Mar; 29 (3) : 245-54 etc.) can also be applied.
In addition, SLC35B1, CHST9 or ST8SIA3 protein-specific antibodies, anti-SLC35B1 antibody, anti-CHST9 antibody or anti-ST8SIA3 antibody can be obtained from commercially available products such as Abcam, and can also be obtained by known immunoengineering techniques. Can do.
Here, the antibody may be a polyclonal antibody, not a monoclonal antibody, and may be a full-length monoclonal antibody, in addition to an antibody fragment such as a Fab fragment in which the antigen recognition site is conserved, a humanized antibody, Single chain antibodies and the like can also be used. Such antibody production methods are also known to those skilled in the art.
Furthermore, a screening system (alpha screen method: Demeulemeester J et al., J Biomol Screen. 2012 Jun; 17 (5): 618-28 that searches for inhibitors that bind to SLC35B1 using regions other than the ER transmembrane domain of SLC35B1. .) And a screening system based on activity (HTS method: Trubetskoy O et al., J Pharm Pharmacol. 2008 Aug; 60 (8): 1061-7.) In addition, low molecular weight compounds that can inhibit the activity can be screened.

5. Composition for preventing, suppressing or treating HBV infection of the present invention (5-1) Method for delivering drug to hepatocytes or their endoplasmic reticulum Sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3 of the present invention In order to act in vivo on a sugar chain gene expression inhibitor such as an oligonucleic acid inhibitor or an oligonucleic acid (hereinafter also referred to as a sugar chain modification inhibitor), the sugar chain modification inhibitor is made into HBV particles and secreted. It is efficient to directly deliver to and inhibit the endoplasmic reticulum (ER) or Golgi apparatus where proteins essential for sugar chain synthesis are localized. For this purpose, an intracellular delivery system (WO2008 / 088581) for an antiviral agent administered by encapsulating in a pH-sensitive liposome containing phosphatidylinositol (PI) lipid can be applied.

  In addition, in the case of sugar chain gene expression inhibitors using oligonucleic acids such as siRNA and shRNA, a liver-specific transport system (Patent Document 16, etc.), or a cationic composed of a glycerol derivative and a phospholipid. Techniques (such as WO99 / 48531) that are complexed with liposomes and accumulated in the liver region by setting the average particle size to 100 nm or more can also be used. Furthermore, as a technique for specifically delivering a compound to the liver, a method of complexing with a hydrophobically modified HBV preS-derived peptide (WO2009 / 092612) can also be applied. In addition, gene therapy approaches including the use of conventionally used non-pathogenic viruses, modified viral vectors such as AAV, and delivery by nanoparticles or liposomes can also be used. (Akhtar, Journal of Clinical Investigation (2007) 117: 3623-3632, Nguyen et al., Current Opinion in Molecular Therapeutics (2008) 10: 158-167, Zamboni, Clin Cancer Res (2005) 11: 8230-8234, or (See Ikeda et al., Pharmaceutical Researeh (2006) 23: 1631-1640, etc.)

  In particular, the delivery system of oligonucleic acid preparations such as siRNA has been actively researched and developed for each target organ to be delivered (Kanasty R et al. Nat Mater. 2013 Nov; 12 (11): 967 -77., Haynes M, et al. Drug Deliv Transl Res. 2014 Feb; 4 (1): 61-73.), And clinical trials in which siRNA preparations were actually administered to the liver have also been reported ( Coelho T et al. N Engl J Med. 2013 Aug 29; 369 (9): 819-29.), These methods can also be applied as appropriate. According to the actual clinical test example, the dosage of oligonucleic acid by intravenous injection is 0.01 to 1.0 mg / kg.

(5-2) Pharmaceutical Composition One of the main embodiments of the pharmaceutical composition of the present invention comprises an oligonucleic acid such as siRNA or shRNA as an active ingredient, and optionally a pharmaceutically acceptable carrier and / or excipient. A pharmaceutical composition for the treatment and prevention of HBV infection. In this case, the oligonucleic acid as the active ingredient may be incorporated into a known liver-specific deliverable vector. In addition, a liposome delivery method such as a pH-sensitive liposome (WO2008 / 088581) that can be delivered specifically in the endoplasmic reticulum is also preferably used.
In the following, formulation of oligonucleic acid such as siRNA (dsRNA) and specific administration methods, dosages, etc. will be mainly described. However, the present invention is not limited to oligonucleic acid, and as a target sugar chain synthesis-related protein activity inhibitor. It can also be applied to low molecular compounds such as working peptides, antibodies, and the like.

  The route of administration may be either oral or parenteral, and in the case of parenteral administration, it is preferably subcutaneous or intravenous injection, by nasal administration such as nasal spray, transdermal administration, inhalation, suppository, etc. There may be. The siRNA can be administered to a patient by intravenous injection, subcutaneous injection, oral delivery, liposome delivery or intranasal delivery and then accumulate in the patient's whole body, liver, or endoplasmic reticulum in hepatocytes.

  “Pharmaceutically acceptable carriers and / or excipients” are known to those skilled in the art, and include any type of encapsulating material such as non-toxic solid, semi-solid or liquid fillers, diluents, liposomes or Contains formulation aids.

Pharmaceutical compositions for parenteral injection include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile injectable solutions just before use. Or it may contain a sterile powder for reconstitution into a dispersion. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles are water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) A), as well as injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained by blending a coating material such as lecithin and a surfactant. In addition, adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents, antibacterial and antifungal agents, isotonic agents such as sugars and sodium chloride may be included.
Alternatively, a depot injectable formulation is prepared by forming a microcapsule matrix with a biodegradable polymer such as polylactide-polyglycolide or encapsulating the pharmaceutical composition in liposomes or microemulsions that are compatible with body tissue be able to. Injectable formulations are made sterile by, for example, filtration through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use. Can be done.

  Solid dosage forms for oral administration include, but are not limited to, capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound comprises at least one component pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or (a) starch, lactose, sucrose Fillers or extenders such as glucose, mannitol and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginate, polyvinylpyrrolidone, sucrose and gum arabic, (c) wetting agents such as glycerol, (d ) Disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, (e) solution retarders such as paraffin, and (f) absorption enhancers such as quaternary ammonium compounds. (G) wetting agents such as acetyl alcohol and glycerol monostearate, (h) Mixed with absorbents such as phosphorus and bentonite clays and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents.

  The solid dosage forms of tablets, dragees, capsules, pills, and granules are prepared with coatings such as enteric coatings and other coatings well known in the pharmaceutical formulating art. Lactose and high molecular weight polyethylene glycol may be used to form soft and hard filled gelatin capsules.

  Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compound, liquid dosage forms can solubilize the agent, eg, inert diluents commonly used in the art such as water or other solvents, as well as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate , Benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oil (especially cottonseed, peanut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol And emulsifiers such as fatty acid esters of sorbitan, and mixtures thereof.

In addition to inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents, the suspension containing the active compound. In addition, suspensions may be included such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sulfitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof. .
Compositions for oral administration include compressed gases such as nitrogen or liquefied gas propellants containing liquid or solid nonionic surfactants to the extent that the active ingredient does not dissolve, or solid anionic surfactants. But you can.

In the case of oligonucleic acids such as siRNA, liposome delivery methods such as pH-sensitive liposomes (WO2008 / 088581) containing phosphatidylinositol (PI) lipids and cationic liposomes (WO99 / 48531 etc.) that can be delivered specifically to the liver are applied. Thus, it is preferable to quickly reach HBV-infected cells in the liver.
Also when using other delivery vehicles, it is preferably administered in the form of liposomes. As is known in the art, liposomes are generally drawn from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present composition in liposome form may contain stabilizers, preservatives, excipients and the like in addition to the compound of the present invention. Preferred lipids are both natural and synthetic phospholipids and phosphatidylcholines (lecithins). Methods for forming liposomes are known in the art (see, eg, Prescott, Meth. Cell Biol. 14: 33-34 (1976)).

  The “therapeutically effective” amount of the pharmaceutical composition for preventing, suppressing or treating HBV infection of the present invention is used to prevent and / or treat liver diseases or liver disorders caused by various HBV infections such as hepatitis B. A sufficient amount and can be determined by the prevention or amelioration of the liver disease being treated, or the detrimental state or symptoms of liver damage. The agent can be administered to a patient in need of treatment for HBV infection as a pharmaceutical composition in combination with one or more pharmaceutically acceptable excipients. The therapeutically effective dose level varies depending on the age, weight, gender, etc. of the patient as well as the HBV subtype, the degree of infection, the degree of symptoms, the dosage form, etc. In the case of oligonucleic acid, it is preferably in the range of 0.01 to 1.0 mg. In the case of other compounds such as antibodies, the dose is preferably 2.5 to 10 mg / kg. In some cases, a dose lower than the above range may be sufficient, and conversely, a dose exceeding the above range may be required. When administering a large amount, it is desirable to divide the dose into several times a day.

The target sugar chain synthesis-related protein activity inhibitor and target sugar chain gene expression inhibitor of the present invention may be used in combination with or in combination with other anti-HBV agents.
That is, the present invention includes an activity inhibitor of a target sugar chain synthesis-related protein selected from SLC35B1, CHST9, and ST8SIA3 or an expression inhibitor of a target sugar chain gene selected from SLC35B1, CHST9 gene, and ST8SIA3 gene and another 1 Also provided is a pharmaceutical composition for treating or preventing HBV infection, comprising a combination of two or more anti-HBV agents. When other anti-HBV agents are combined, they can be administered at the same time, separately or sequentially, so they may be included in the same pharmaceutical composition, and other anti-HBV agents may be used separately. It may be contained in a pharmaceutical composition or a kit. In this case, the dosage forms of two or more separate preparations may be the same or different. For example, any one or more may be a parenteral preparation, an injection, an infusion, or an intravenous infusion.
Here, “other anti-HBV agents” include known interferon (IFN), a therapeutic agent for HBV infection, and nucleic acid analogs (acting that has a nucleic acid partial structure in the molecule and inhibits HBV DNA synthesis) The generic name of compounds having Examples of interferon (IFN) include IFN-α or IFN-β preparations, and examples of nucleic acid analogs include lamivudine [LMV], adefovir [ADV], entecavir [ETV], and the like. The interferon preparation is preferably a PEGylated preparation.

6). HBV secretion inhibitor screening method and kit for the same The present invention also provides a screening method for searching for HBV particle formation and / or HBV secretion inhibitor as a therapeutic agent for HBV infection. Here, since inhibition of HBV particleization results in inhibition of HBV secretion from HBV-infected hepatocytes, the search target of the present invention is sometimes simply referred to as “HBV secretion inhibitor”.
One of the methods for screening for the HBV secretion inhibitor of the present invention is a method for searching for a substance that inhibits the transcription / expression of SLC35B1 gene, CHST9 gene or ST8SIA3 gene expressed in cells. A test substance may be administered intracellularly or in its medium, and the presence or absence of a decrease in the expression level of the SLC35B1, CHST9, or ST8SIA3 gene in the hepatocytes may be observed. At that time, changes in the amount of mRNA can also be observed by RT-PCR, etc., but generally the amount of expression product SLC35B1, CHST9 or ST8SIA3 is measured by an immunological technique such as ELISA or immunoblot, and the expression level is determined. Select the test substance to be reduced.

In order to accurately and easily measure changes in the expression level of glycan genes, instead of cultured hepatocytes, at least one gene selected from SLC35B1, CHST9 and ST8SIA3 genes was introduced and overexpressed It is preferable to use transformed cells.
In particular, more efficient measurement is possible by tagging the SLC35B1 to be expressed and the like to obtain a tagged target sugar chain synthesis-related protein (eg, Flag-SLC35B1). For example, the inhibitory effect of the constructed siRNA can be measured, and a more effective target region can be determined.
It can also be used to screen for inhibitors of glycosylation activity of various target sugar chain synthesis-related proteins (eg, SLC35B1), including low molecular weight compounds such as peptides that bind to target sugar chain synthesis-related proteins (eg, SLC35B1). . As the tag, in addition to the above-mentioned Flag, generally used tag sequences such as a His tag, Myc tag, GFP tag, GST tag, Maltose tag, and S tag can be used. Antibodies that recognize each of these tag sequences (hereinafter also referred to as anti-tag antibodies) are known and are sold as commercial kits, so the amount of expressed target sugar chain synthesis-related protein can be measured as the amount of anti-tag antibodies. is there.
This may involve radioactive, enzymatic or fluorescent labeling followed by detection by a suitable method. Many fluorescent materials are known and can be used as labels. Examples of these fluorescent materials include fluorescein, rhodamine, auramine, and Texas red. An enzyme label includes a conjugate of an enzyme and a molecule of interest, such as a polypeptide, and can be detected by either colorimetric, spectrophotometric or fluorescent spectrophotometric methods.

  In addition, when an enzyme or a fluorescent protein is used as a label for detection, the expression level of the target sugar chain synthesis-related protein can also be changed by using a transformed cell with a fusion of the gene encoding the labeled protein and the target sugar chain gene. It can be measured efficiently.

That is, the method for screening for an HBV secretion inhibitor in the present invention can be described as follows, for example.
Using a transformed cell that expresses at least one labeled sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3 directly or indirectly labeled on the surface of the endoplasmic reticulum or Golgi membrane,
Measuring the amount of labeling on the surface of the intracellular membrane, and administering a test compound in the cell or in the medium,
Selecting a compound that reduces the amount of labeling,
A method of screening for an HBV secretion inhibitor.
For example, the transformed cell is a cell expressing a sugar chain synthesis-related protein tagged with a Flag-tag or the like, and the intracellular membrane surface is fluorescently labeled with an anti-tag antibody such as a fluorescently labeled anti-Flag antibody. It is preferable that the labeling amount of the intracellular membrane surface to be measured is a fluorescence amount because it can be visualized as a decrease in the fluorescence amount.
The test substance at that time may be a nucleic acid such as an oligonucleic acid or a low molecular weight compound such as a peptide. Therefore, the administration method may be introduced into a transformed cell or may be administered in a medium.
Moreover, the kit for screening the HBV secretion inhibitor in that case can be expressed as a kit containing the following (1) and (2).
(1) A transformed cell that expresses at least one sugar chain synthesis-related protein selected from tagged SLC35B1, CHST9, and ST8SIA3,
(2) A fluorescently labeled anti-tag antibody.

The second method of screening for the HBV secretion inhibitor of the present invention is a screening method focusing on the inhibition of the activity of at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3.
Specifically, the method comprises the step of measuring the activity of at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3 in cultured hepatocytes, and introducing a test oligonucleic acid or test compound into the cell or You may select the test substance which reduces the said active amount by administering in a culture medium.
The activities of SLC35B1, CHST9, and ST8SIA3 that can be used as indices in this case are the activity of transporting a sugar chain-nucleotide such as UDP-Gal, the activity of transferring a sulfate group to the 4-position of GalNAc, and the activity of sialic acid 8 The activity of transferring sialic acid to the position.
For example, when measuring the change in SLC35B1 activity, use a reaction solution containing UDP-Gal, which is a sugar chain-nucleotide having UDP or labeled UDP, or measuring the concentration of UDP-Gal in the free or endoplasmic reticulum. The released UDP concentration may be measured with a UDP detection reagent or by the methods described in Non-Patent Documents 6 and 7.
In addition, the HTS method (Trubetskoy O et al., J Pharm Pharmacol. 2008 Aug; 60 (8): 1061-7.), Which is a known screening system using activity as an index, can also be applied.

A peptide sequence that binds to a target glycosylation-related protein such as SLC35B1 is determined by an evolutionary molecular engineering display technique. Typically, random phage display (Smith, GP, et al, Chem. Rev. 1997 Apr 1; 97 (2): 391-410.) Can be applied, but ribosome display, mRNA display, cDNA display, etc. Cell-free translation system display technology (Lohse PA et al., Curr Opin Drug Discov Devel., 2001 Mar; 4 (2): 198-204, Bradbury AR et al., Nat Biotechnol. 2011 Mar; 29 (3) : 245-54 etc.) can also be applied. In this case, for example, in the case of SLC35B1, since the three-dimensional structure is fixed by the ER transmembrane region shown in bold in FIG. 17, the loop formed on the ER surface such as the extra-membrane domain is particularly an epitope candidate.
Kits for this purpose include: (1) Beads on which either SLC35B1, CHST9, or ST8SIA3 sugar chain synthesis-related proteins are immobilized, substrates such as plates, and random phage display for application to random phage displays. Peptide libraries containing nucleic acids containing sequences or corresponding proteins can be used. As a parent base sequence as a template in that case, it is preferable to use a synthetic library of M13 or T7 phage, and the length of the random base sequence is usually 21 to 32 bases (7 to 12 amino acids).

  In addition, anti-SLC35B1 neutralizing antibodies can be obtained by known immunoengineering techniques. Further, according to the alpha screen method (Demeulemeester J et al., J Biomol Screen. 2012 Jun; 17 (5): 618-28.), An inhibitor that binds using a region other than the ER transmembrane domain of SLC35B1 (FIG. 27). Can be screened for low molecular weight compounds that can bind to and inhibit the activity of glycosylation-related proteins. Similarly, an inhibitor that inhibits binding to a probe such as an antibody or an inhibitor that inhibits binding to a substrate can be obtained.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature, the patent gazette, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.

(Example 1) Establishment of HBs antigen-secreting hepatic cell line (1-1) Preparation of plasmid for secreting HBs antigen (membrane protein on HBV)
Three types of HBV envelope-derived HBV antigens, L-HBs antigen (389aa), M-HBs antigen (281 aa), and S-HBs antigen (226 aa) DNA (Accession # is AB246345) are amplified by PCR. Incorporated into pcDNA3.1 (Life Technologies). Then, the DNA encoding the full length of the S-HBs antigen is used for the following experiments, and for easy detection, a secretory expression vector in which a signal peptide and a purification Flag tag are added to the N-terminus. Recombined into pFLAG-CMV3 (manufactured by SIGMA). The DNA and amino acid sequences of the tagged S-HBs antigen are shown as SEQ ID NOs: 1 and 2.

(1-2) Expression of HBs antigen in hepatoma cells Using the secretory expression vector prepared in (1-1) above, S-HBs were obtained from hepatoma cell-derived HuH7 cells (obtained from RIKEN BioResource Center). The antigen was expressed and purified.
Specifically, the expression vector (2 μg) containing the S-HBs antigen DNA was transfected into HuH7 cells (about 2 × 10 ⁶ cells) using Lipofectamine LTX (manufactured by Life Technologies), and 48 hours later. The culture supernatant was collected, and the recombinant HBs antigen was adsorbed using anti-FLAG antibody beads (manufactured by SIGMA). Elution from the anti-FLAG antibody was performed in two steps. First, competitive elution was performed with the FLAG peptide, and then SDS-PAGE sample buffer was added, followed by elution and recovery by heating at 100 ° C. The recovered recombinant HBs antigen was developed by SDS-PAGE and Western blotted with an anti-FLAG antibody.
As a result, by adding a secretory signal sequence to the N-terminal side of the S-HBs antigen, approximately 10 times the amount of purified S-HBs compared to when expressing only the S-HBs antigen without a secretory signal. It became clear that the antigen could be recovered.
In addition, two bands of gp28 with N-type sugar chain and p25 without N-type sugar chain were detected in S-HBs antigen, the amount secreted into the culture supernatant and the sugar chain of secreted S-HBs antigen The system which can measure the content of was established (Fig. 1).

(Example 2) Influence on secretion of HBs antigen by inhibition of sugar chain synthesis system
The expression vector containing the S-HBs antigen DNA prepared in (1-1) above was introduced into HuH7 cells seeded in a 24-well plate, and replaced with RPMI1640 medium containing a sugar chain synthesis inhibitor after 24 hours. After a time, it was recovered from the culture supernatant with anti-FLAG antibody beads. The expression of S-HBs antigen was detected using anti-FLAG antibody. 9 sugar chain synthesis inhibitors (Tunicamycin (TM: Wako Pure Chemical), Benzyl-O-GalNAC (BZ-G: Merck), Brefeldin A (Bre-A: Wako Pure Chemical), Castanospermine (CS: Merck) ), Folimycin (FM: Merck), Deoxyfuconojirimycin (DFJ: SIGMA), Deoxynojirimycin (DNJ: Merck), Kifunensine (Kif: Merck), Mannostatin A (Man: Merck)). As a result, it was confirmed that secretion of S-HBs antigen was significantly reduced when TM, Bz-G, Bre-A, and FM among the sugar chain synthesis inhibitors were used (FIG. 2).

(Example 3) Extraction of sugar chain genes expressed in hepatocytes and hepatoma cells Normal cells derived from human liver (primary culture, Clonetics ™ Human Hepatocyte, hNHEPS ™ Cells: LONZA Inc.) and hepatoma cells Total RNA was prepared from HuH7 cells and HepG2 cells (obtained from RIKEN BioResource Center), and the expression level of glycan gene in each cell was analyzed by qRT-PCR (glycan gene qPCR array system) and transcript Measured by tome analysis (next-generation sequencer).
That is, normal hepatocytes and hepatoma cells (HuH7 cells, HepG2 cells) were cultured in 10 cm ² dishes, and 20 μg or more of total RNA was purified with the RNeasy Plus Mini Kit (QIAGEN), and mRNA DIRECT Micro Kit (Dynabeads) was used to prepare 0.2 μg of mRNA. RNA libraries and cDNA libraries were prepared using Ion Total RNA Seq Kit v2 (Life Technologies) and sequenced using IonPGM (Life Technologies). The sequence of millions of reads obtained by one-time sequencing was mapped to human cDNA, and only those mapped to sugar chain genes were extracted and the expression levels were compared and analyzed (FIG. 3A).

In addition, in order to quantify with a more accurate copy number, total RNA was purified from primary cultured normal hepatocytes, HepG2 cells, and HuH7 cells using RNeasy Plus Mini Kit (QIAGEN). Using 4 μg of total RNA, cDNA was prepared with QuantiTect Reverse Transcription Kit (manufactured by QIAGEN), and qRT-PCR array system (Ito H et al) containing 185 types of sugar chain genes shown in the above (Tables 1 to 8). , J Proteome Res. 2009 Mar; 8 (3): 1358-67.), The expression level of the sugar chain gene expressed in hepatocytes was measured.
As a result of sugar chain gene expression analysis, expression profiles of about 185 kinds of sugar chain genes in two types of liver cancer cell lines (HuH7 cells and HepG2 cells) were obtained (FIG. 3B).

(Example 4) Screening of glycan genes affecting the glycan modification of S-HBs antigen (4-1) Preparation of siRNA plates Based on the expression profiles of the 185 types of glycan genes obtained in Example 3, The glycan genes in hepatocytes were divided into two groups, a “high expression to medium expression” group and a “low expression to no expression” group.
Subsequently, 3 types of siRNA (manufactured by AB / Life Technologies) were obtained for 86 sugar chain genes included in the “high expression to medium expression” group, and 3 types of mixed siRNAs were prepared for each gene. On the other hand, as a 12-well plate for primary screening, 10-well is coated with 100 pmol / well of each siRNA, and the other wells are made with 100 pmol of control siRNA (manufactured by SIGMA) and blank, and then air-dried. Stored at -80 ° C until use.

(4-2) siRNA screening using S-HBs antigen Among 86 sugar chain genes highly expressed or intermediately expressed in hepatocytes, screening was performed for genes that affect the sugar chain modification of S-HBs antigen.
OptiMEMI (manufactured by Life Technologies) and DharmaFECT 4 transfection reagent (manufactured by Thermo) were added to the siRNA plate prepared in (4-1), and HuH7 cells (about 2 × 10 ⁵ cells) were seeded 30 minutes later. After 24 hours, the above-mentioned S-HBs antigen cDNA was transfected with Lipofectamine LTX, and 48 hours later, S-HBs antigen was recovered from the supernatant with anti-Flag antibody beads (manufactured by SIGMA) and S-HBs was obtained by Western blotting. The amount of antigen expression and the addition of sugar chains were confirmed (FIG. 4A).
Of the 86 sugar chain genes, 10 or more genes were found to have reduced S-HBs antigen expression or reduced sugar chain addition compared to control siRNA. The typical case is illustrated in FIG. 4B.

(Example 5) siRNA screening in HepG2.2.15.7 cells (5-1) 1st screening Among sugar chain genes expressed in hepatocytes, screening was performed for genes that affect the secretion of HBV particles.
After adding a transfection reagent to the siRNA plate in the same manner as in (4-2) above, HepG2.2.15.7 cells (distributed by the National Institute of Infectious Diseases), a hepatoma cell that produces HBV (about 2 × 10 ⁵ cells). The culture medium was changed the next day. Three days later, the amount of HBV DNA secreted into the supernatant was measured by RT-PCR (Roche LightCycler 96 real-time PCR system) (FIG. 5A).
RT-PCR conditions used for measurement of the amount of HBV DNA are as follows.
Primer 1: 5'-CTTCATCCTGCTGCTATGCCT-3 '(SEQ ID NO: 5)
Primer 2: 5'-AAAGCCCAGGATGATGGGAT-3 '(SEQ ID NO: 6)
Probe: FAM-ATGTTGCCCGTTTGTCCTCTAATTCCAG-TAMRA (SEQ ID NO: 7)
Reagent: Eagle Taq Master Mix (Roche)
The amount of HBs antigen was quantified by ELISA using a specific antibody (manufactured by Special Immunology Institute) (FIG. 5A). At the time of screening, an MTT assay (Roche) was performed to confirm the influence on cell proliferation. As a result, for the SLC35B1 gene, CHST9 gene and ST8SIA3 gene, it was confirmed that the expression level of HBV DNA was significantly reduced by the action of the mixed siRNA, and it was confirmed that the effect on cell proliferation was extremely low and there was no problem with cytotoxicity. (FIG. 5B).
SLC35B1 (SEQ ID NOs: 3 and 4) is one of nucleic acid-sugar transporter-related genes, and homologs have been reported in yeast and nematodes, but the nucleic acid-sugar transporter activity has not been confirmed. Also, the role in HBV secretion and the function in the cell are unknown.
CHST9 (SEQ ID NOs: 8 and 9) is a sulfotransferase belonging to the Carbohydrate sulfotransferase family, and adds a sulfate group at the 4-position of GalNAc.
ST8SIA3 (SEQ ID NOs: 10 and 11) is one of α2,8-sialyltransferase (sialyltransferase), and further adds sialic acid to the position of the 8-position of sugar chain sialic acid (NeuAc) on glycoprotein. .
Subsequently, 2nd screening was performed for these sugar chain genes.

(5-2) Second siRNA screening siRNAs corresponding to SLC35B1, CHST9, and ST8SIA3 sugar chain genes selected in the first screening in (5-1) above are a mixture of three siRNAs. Therefore, 2nd screening was performed for each siRNA.
After adding a transfection reagent to the siRNA plate as in (4-2) above, HepG2.2.15.7 cells were seeded, and the amount of HBV DNA secreted 3 days later was determined by RT-PCR and the amount of HBs antigen was determined by ELISA. did.
The results are shown in FIG. In the case of SLC35B1 sugar chain gene, siRNA of SRNA35S1 # 1 that targets the mRNA region shown in SEQ ID NO: 12 significantly reduces both HBs antigen amount and HBV DNA amount, and has the highest anti-HBV effect. As shown, siRNA-SLC35B1 # 2 also confirmed the effect of decreasing the amount of HBV DNA, and siRNA-SLC35B1 # 3 confirmed the effect of decreasing both the amount of HBs antigen and the amount of HBV DNA.
Here, the positions on the SLC35B1 gene targeted by siRNA-SLC35B1 # 1 to # 3 are shown in FIG. SLC35B1 # 1 corresponds to the position of c (positions 311 to 330 of SEQ ID NO. 3), SLC35B1 # 2 corresponds to e (positions 477 to 495), and SLC35B1 # 3 corresponds to position g (positions 576 to 594).
In this experiment, it became clear that all of SLC35B1 # 1 to # 3 were effective, and the region of c corresponding to SLC35B1 # 1 in this was excellent as the target region. The regions b, d, f, and h to j are also candidate target regions. As will be described later, it can be said that the regions a to c, e, g, and i are particularly effective.

On the other hand, as siRNA corresponding to the CHST9 sugar chain gene, CHST9 # 1 (positions 1188 to 1206 of SEQ ID NO: 8), CHST9 # 2 (positions 806 to 824) and CHST9 # 3 (positions 588 to 606) are used. It was. Of these, CHST9 # 2 significantly reduced the amount of HBV DNA (FIG. 6), so that the target region of the CHST9 gene (SEQ ID NO: 8) corresponds to the position of 806 to 824 corresponding to the position of CHST9 # 2. Can be said to be effective.
Moreover, as siRNA corresponding to ST8SIA3 sugar chain gene, ST8SIA3 # 1 (positions 178 to 196 of SEQ ID NO: 10), ST8SIA3 # 2 (positions 354 to 372) and ST8SIA3 # 3 (positions 208 to 226) are used. It was. Of these, ST8SIA3 # 1 significantly decreased both the HBs antigen level and the HBV DNA level (FIG. 6), so the target region of the ST8SIA3 gene (SEQ ID NO: 10) corresponds to the position of ST8SIA3 # 1. It can be said that the 178th to 196th region is effective.
In the following example, an experiment was conducted using siRNA-SLC35B1 # 1 (targeting the c region) as a typical SLC35B1 siRNA. However, as shown in Example (6-2) below, other regions were used. Similar results can be expected with siRNAs targeting.

(Example 6) HBV secretion inhibitory effect of siRNA-SLC35B1 (6-1) Comparison between siRNA-SLC35B1 # 1 and entecavir The HBV secretion inhibitory effect of siRNA-SLC35B1 # 1 selected in (5-2) above, It was measured using HepG2.2.15.7 cells and compared with nucleic acid analogs (reverse transcriptase inhibitors) entecavir (Abcam) and lamivudine (SIGMA), which are known to be effective as HBV therapeutics.
Entecavir and lamivudine were added at 10 μM and 100 μM, and SLC35B1 siRNA-SLC35B1 # 1 to # 3 were added at 100 nM to HepG2.2.15.7 cells, respectively, and the amount of HBV DNA and HBs antigen was measured 3 days later. Were compared (FIG. 8).
Although both entecavir and lamivudine have the effect of reducing the amount of HBV DNA, they did not significantly affect the amount of HBs antigen secreted into the culture supernatant. On the other hand, siRNA-SLC35B1 # 1 showed the effect of reducing both the amount of HBV DNA and the amount of HBs antigen to 50% or less.

(6-2) Confirmation of siRNA-SLC35B1 effect
To confirm the SLC35B1 inhibitory effect of siRNA, 100 nM siRNA-SLC35B1 # 1 was transfected into HuH7 cells (approximately 4 × 10 ⁵ cells) in 6-well using DharmaFECT 4 (manufactured by Thermo) for 48 hours. Later, RNA was prepared with RNeasy Plus Mini Kit (manufactured by QIAGEN), cDNA was synthesized from 1 μg of total RNA with QuantiTect Reverse Transcription Kit, and qRT-PCR was performed with the following SLC35B1-specific primers and measured.
Primer 3: 5'-GGGAGCTCTGGGAGTTCTTGA-3 '(SEQ ID NO: 13)
Primer 4: 5'-GCCCAAAGAGCAGGATGTTATAGA-3 '(SEQ ID NO: 14)
Reagent: Taq Man Mix (Roche)

Next, siRNAs (siRNA aj) corresponding to knockout target candidate regions aj (Fig. 7) of SLC35B1 gene calculated using various software were transfected into HuH7 cells, and each SLC35B1 expression level was measured. did. The efficiency of siRNA at that time was calculated with the measured value when transfection of siRNA-negative control (siC, SIGMA) was taken as 100%. The amount of β-actin in each cDNA was used as an endogenous control (FIG. 9).
As a result, as the target region of the base sequence of the SLC35B1 gene (SEQ ID NO: 3), positions 311 to 330 (c), 477 to 495 (e) of SEQ ID NO: 3 corresponding to siRNA-SLC35B1 # 1 to # 3 and In addition to the regions corresponding to 576 to 594 (g), regions corresponding to 102 to 121 (a), 161 to 179 (b), and 627 to 644 (i) are also excellent target regions. It was confirmed that
Similarly, HepG2.2.15.7 cells were also transfected with 100 nM siRNA-SLC35B1 # 1, total RNA was prepared, and qRT-PCR was performed with the SLC35B1 specific primer and measured (FIG. 10).
In HepG2.2.15.7 cells, the amount of SLC35B1 mRNA was reduced to about 10% by siRNA-SLC35B1 # 1 transfection compared to cells transfected with siRNA-negative control. Similarly, it decreased to about 27% in HuH7 cells.

(Example 7) Toxicity test of siRNA-SLC35B1 (7-1) Effect of siRNA-SLC35B1 on cells According to the MTT assay in the screening process of (Example 5), even if siRNA-SLC35B1 is used in a mixture of 3 types, Although it has already been confirmed that the cytotoxicity to cells is low, in this example, individual siRNA preparations were further used, and the effects on the overall sugar chain synthesis function in hepatocytes were mainly observed.
HuH7 cells (approximately 2 × 10 ⁵ cells) were transfected with 100 nM siRNA-SLC35B1 # 1 at 12-well, and 48 hours later, the cells were treated with RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5% Sodium). The protein was recovered by dissolving with deoxycholate, 0.1% SDS, 1% NP40). A soluble fraction was prepared after centrifugation, and the protein was developed by SDS-PAGE and blotted on a PVDF membrane. At the same time, HuH7 cells were treated with tunicamycin (manufactured by Wako Pure Chemical Industries, Ltd.) for 48 hours to detect α-fetoprotein (AFP), a glycoprotein serving as a liver cancer marker. The decrease in AFP expression as observed with tunicamycin was not observed when siRNA-SLC35B1 # 1 was used (FIG. 11).

Furthermore, in order to investigate the effect of siRNA-SLC35B1 # 1 on sugar chain synthesis, HuH7 cells were transfected, and the soluble fraction was developed on SDS-PAGE, and E-PHA (a live lectin that recognizes sugar chains) Lectin blotting was performed using Chemical Industries) and RCA (Vector) (FIG. 12). As a result, no influence on the overall sugar chain synthesis was observed.
Next, siRNA-SLC35B1 # 1 was transfected into HepG2 cells and HuH7 cells to prepare membrane fractions, and lectin array analysis was performed. As a result of statistical analysis based on the three measurement results, it was confirmed that no significant change was observed in the sugar chain profile (FIGS. 13A and 13B).
In order to examine the effect of siRNA-SLC35B1 # 1 treatment on ER stress and cytotoxicity, we compared HuH7 cells with siRNA transfected with those treated with Tunicamycin (TM) and Brefeldin A (BreA) by Western analysis. In the case of siRNA-SLC35B1 # 1 treatment, increased expression of BiP, which is an ER stress marker, was not observed compared with TM and BreA, which are sugar chain synthesis inhibitors. (Fig. 14)
In addition, siRNA-SLC35B1 # 1 was transfected into HepG2.2.15.7 cells and cytotoxicity assay was performed using LDH cytotoxicity detection kit (manufactured by Takara Bio Inc.) to confirm that siRNA-SLC35B1 # 1 did not adversely affect the cells. (FIG. 15).

(7-2) Effect of siRNA-SLC35B1 on gene expression
The effect of siRNA-SLC35B1 # 1 on intracellular gene expression was analyzed. HuH7 cells (about 2 × 10 ⁶ cells) seeded in a 10 cm dish were transfected with 100 nM siRNA-SLC35B1 # 1 with DharmaFECT 4, and 48 hours later, total RNA was prepared with RNeasy Plus Mini Kit. 0.2 μg of mRNA was prepared as described above, a cDNA library was prepared from the fragmented RNA library, and sequenced with IonPGM. The expression level of the sugar chain gene is shown in comparison with the results of transcriptome analysis of HuH7 cells (FIG. 16). Moreover, the result (volcano plot) which analyzed the change of the expression level of all the genes by analysis software Genomics Workbench (made by CLCbio) is shown (FIG. 17). The above analysis results confirmed that the expression level of SLC35B1 was reduced to about 25%, and that the glycan genes whose expression levels were increased or decreased as a result of the decrease in SLC35B1 mRNA were confirmed. It was thought that there was little influence on chain modification. These results indicate that SLC35B1 # 1 has little effect on gene expression in cells (within the range of -2 to 2 on the X axis). At the same time, genes that fluctuated 3 times or more (-3 or less on the X axis and 3 or more ranges) were identified as off-target candidates.

To verify the effect of siRNA-SLC35B1 # 1, off-target searches were analyzed from gene expression from three different experiments. HuH7 cells were transfected with siRNA-SLC35B1 # 1 at 100 nM (Experiment 1), 10 nM (Experiment 2), and HepG2.2.15.7 cells were transfected with 10 nM (Experiment 3), and total RNA was prepared using the RNeasy Plus Mini Kit. Transcriptome analysis was performed. The genes whose expression levels were changed 3 times or more in all experiments were listed as off-targets in comparison with the control (-siRNA) (FIG. 18A). As a result, in the above three experiments, changes were observed in 11 genes other than SLC35B1, but genes that were directly involved in HBV replication / secretion were not identified.
In addition, mouse chimeric human hepatocytes (PXB, manufactured by Phoenix Bio) were transfected with siRNA-SLC35B1 # 1 at 10 nM and compared with the results of transcriptome analysis of HepG2.2.15.7 cells and HuH7 cells. . Genes whose expression levels were changed by 2 times or more in all experiments were listed as off-targets, but only a small number of off-targets were also found (FIG. 18B).
PLC / PRL / 5 cells secreting HBs antigen were transfected with siRNA-SLC35B1 # 1 at 1, 10, 50 nM, and the expression levels of SLC35B1 gene and HBV (HBs antigen gene) were analyzed by qRT-PCR (Fig. 19). It was confirmed that the number of siRNA-SLC35B1 # 1 off-target genes was relatively small and the expression level of the HBs antigen gene was also decreased. From the above results, it was confirmed from the viewpoint of gene expression that there were few side effects when targeting SLC35B1.

(Example 8) Confirmation of effective siRNA-SLC35B1 concentration
After transfection of HuH7 cells with siRNA-SLC35B1 # 1 at concentrations of 100, 10, 1, 0.1 nM, 48 hours later, total RNA was prepared, and qRT-PCR was performed in the same manner as in (6-2) above. SLC35B1 mRNA The amount was measured, and the siRNA concentration having the effect of knockdown was analyzed. The knockdown efficiency of siRNA was compared with the measured value when siRNA-negative control was transfected as 100%. The amount of β-actin in each cDNA was corrected as an endogenous control (FIG. 20). It was confirmed that the amount of SLC35B1 mRNA in HuH7 cells could be suppressed to 1/3 or less at a concentration of 1-100 nM. Similarly, HepG2.2.15.7 cells were transfected with siRNA-SLC35B1 # 1 at concentrations of 100, 50, 10, and 5 nM, and the amount of HBs antigen was measured by ELISA. (FIG. 21).
Further, whether or not HBs antigen accumulates in the cells after siRNA-SLC35B1 # 1 treatment was measured by ELISA using an HBsAgEIA kit (Special Immunology Institute) (FIG. 22). ). In fact, when HepG2.2.15.7 cells were immunostained with HBs antigen using anti-HBs antigen antibody (Abcam) and Alexa Fluor ^(R) 488-anti-horse IgG goat antibody (Jackson ImmunoResearch Laboratories), siRNA-SLC35B1 It was confirmed that the amount of HBs antigen was decreased by # 1 treatment (FIG. 23).
As a result, it was found that the HBs antigen amount was suppressed to 1/8 or less at an siRNA concentration of 10 nM and decreased to 1/4 or less at 5 nM. The above results show that 1-10 nM siRNA-SLC35B1 # 1 is sufficient to reduce the amount of SLC35B1 mRNA, and inhibits HBV secretion in a concentration-dependent manner at 5 nM or more. Yes.

(Example 9) HBV secretion inhibitory effect of siRNA-SLC35B1 (PHH cells)
(9-1) Comparison between siRNA-SLC35B1 # 1 and entecavir
The inhibitory effect of siRNA-SLC35B1 # 1 on HBV secretion was measured using human chimeric liver cells (PHH, manufactured by PhoenixBio) and compared with the nucleic acid analog agent entecavir (1 μM) (FIG. 24). siRNA-SLC35B1 # 1 reduced the amount of HBV DNA as did entecavir. When added at the same time as entecavir, siRNA-SLC35B1 # 1 is considered to be able to be used in combination with a nucleic acid analog agent because it is reduced more than entecavir alone (FIG. 24A).
It was confirmed that entecavir does not affect the amount of secreted HBs antigen, and that siRNA-SLC35B1 # 1 suppresses the secretion of HBs antigen even in human chimeric liver cells (FIG. 24B).

(Example 10) Effect of siRNA-SLC35B1 on entecavir resistant strain (10-1) Assay with entecavir resistant strain
It is known that resistant strains are generated in HBV by using nucleic acid analog agents. It was measured whether siRNA-SLC35B1 had an inhibitory effect on secretion of entecavir-resistant HBV. The S202G mutant strain was used as an entecavir resistant strain. HuH7 cells were transfected with HBV DNA (genotype C or S202G, obtained from Nagoya City University), and treated with siRNA-SLC35B1 and entecavir two days later. Seven days after transfection of HBV DNA, the amount of HBs antigen secreted into the supernatant was measured by ELISA and the amount of HBV DNA was measured by RT-PCR (FIG. 25).
The amount of HBs antigen was not affected by genotype and decreased in the presence of siRNA-SLC35B1 (FIG. 26A). The amount of HBV DNA was also suppressed when siRNA-SLC35B1 was present (FIG. 26B). That is, the effect of siRNA-SLC35B1 to inhibit secretion of entecavir-resistant HBV was confirmed.
Since there is a high possibility of a pathway different from that of the conventional HBV inhibitor (ETV), a combined effect with a conventional drug is expected, and side effects such as cytotoxicity are not observed (Example 7). Therefore, the SLC35B1 gene is The effectiveness of drug expression inhibitors or SLC35B1 protein activity inhibitors such as RNAi preparations including siRNA as a target was confirmed.

(Example 11) Confirmation of intracellular localization of SLC35B1 A cDNA expression vector in which cDNA of human SLC35B1 (38-359 aa) (SEQ ID NO: 2, Non-Patent Documents 5 and 6) was connected to a Flag tag (DYKDDDDK sequence) was used. It was constructed and transfected using Lipofectamine LTX (Life Technologies) and expressed in HuH7 cells. After 48 hours, the cells were fixed with a 4% paraformaldehyde solution, and after washing, the cells were subjected to fluorescent immunostaining with an anti-Flag antibody (manufactured by MBL) to confirm that SLC35B1-Flag was localized in the ER (FIG. 27). . Here, the transmembrane domain is shown in bold, and the signal for localization in the ER is underlined.
By using the SLC35B1-Flag, the inhibitory effect of the constructed siRNA can be visualized as a decrease in the amount of fluorescence corresponding to the expression level of SLC35B1-Flag, and quantitative measurement can be performed, so that a more effective target region can be determined. . Moreover, it can be used for screening of low molecular weight compounds such as peptides that bind to SLC35B1 and other inhibitors that inhibit SLC35B1 activity (FIG. 28).

Claims (17)

An inhibitor of HBV particle formation or HBV secretion in HBV-infected hepatocytes, comprising as an active ingredient an inhibitor of at least one sugar chain gene expression selected from SLC35B1 gene, CHST9 gene and ST8SIA3 gene,
An inhibitor, wherein the sugar chain gene expression inhibitor is an oligonucleic acid preparation selected from RNAi preparations, antisenses, nucleic acid aptamers and ribozymes.   As an active ingredient, the oligonucleic acid preparation comprises a nucleotide sequence containing at least 15 consecutive bases in the nucleotide sequence of at least one sugar chain gene selected from the SLC35B1 gene, CHST9 gene and ST8SIA3 gene or its complementary sequence Inhibitor according to claim 1, characterized in that it contains.   The oligonucleic acid preparation is an RNAi preparation, and comprises two nucleotide sequences that match at least 15 consecutive bases with the complementary strand of mRNA of at least one sugar chain gene selected from the SLC35B1 gene, CHST9 gene, and ST8SIA3 gene The inhibitor according to claim 2, comprising a double-stranded RNA as an active ingredient. The 102-121 position of the nucleotide sequence oligo nucleic manufactured agent is shown in SEQ ID NO: 3, 161-179 positions, 311-330 positions, 477-495 positions, 576-594 position and 627-644 positions of regions, SEQ ID NO: 8 A base sequence comprising at least 15 consecutive bases of any one or more regions selected from the region of positions 806 to 824 and the region of positions 178 to 196 of SEQ ID NO: 10 and / or its complementary sequence The inhibitor according to any one of claims 1 to 3, wherein   The sugar chain gene expression inhibitor specifically binds to a region consisting of a base sequence comprising at least 15 consecutive bases of SEQ ID NO: 12 in the base sequence of SLC35B1 mRNA. The inhibitor as described in any one of these. A pharmaceutical composition for preventing, suppressing or treating HBV infection, comprising as an active ingredient at least one of the inhibitors of HBV particle formation or HBV secretion according to any one of claims 1 to 5 . Furthermore, the pharmaceutical composition for prevention, suppression or treatment of HBV infection according to claim 6 , which is used in combination or in combination with another anti-HBV agent. A method for screening an inhibitor of HBV particle formation or HBV secretion, comprising the following steps (1) to (4);
(1) A step of measuring the expression level of at least one sugar chain gene selected from SLC35B1 gene, CHST9 gene and ST8SIA3 gene in cultured hepatocytes,
(2) introducing a test compound into cultured hepatocytes or administering the test compound into a medium;
(3) measuring the expression level of the sugar chain gene selected in step (1),
(4) When the expression level measured in step (3) decreases compared to the expression level measured in step (1), the test compound is used as a candidate for an inhibitor of HBV particle formation or HBV secretion. Process to select. A method for screening an inhibitor of HBV particle formation or HBV secretion, comprising the following steps (1) to (4);
(1) a step of measuring the amount or activity of at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3 in cultured hepatocytes,
(2) introducing a test compound into cultured hepatocytes or administering the test compound into a medium;
(3) a step of measuring the amount or activity of the sugar chain synthesis-related protein selected in step (1),
(4) Inhibitor of HBV particle formation or HBV secretion when the amount or activity value measured in step (3) decreases compared to the amount or activity value measured in step (1) Selecting as a candidate. A method for screening an inhibitor of HBV particle formation or HBV secretion, comprising the following steps (1) to (4);
(1) A step of preparing a transformed cell transformed with at least one sugar chain gene selected from the SLC35B1 gene, CHST9 gene and ST8SIA3 gene, and measuring the expression level of the selected sugar chain gene in the cell ,
(2) introducing a test compound into the transformed cell or administering the test compound in a medium;
(3) a step of measuring the expression level of the selected sugar chain gene in the transformed cell of step (2),
(4) When the expression level of the selected glycan gene measured in step (3) decreases compared to the expression level measured in step (1), the test compound is converted into HBV particles or Selecting as a candidate for an inhibitor of HBV secretion. A method for screening an inhibitor of HBV particle formation or HBV secretion, comprising the following steps (1) to (4);
(1) Prepare transformed cells that express at least one labeled sugar chain synthesis-related protein selected from labeled SLC35B1, CHST9, and ST8SIA3 on the inner membrane surface, and measure the amount of labeling on the inner membrane surface The process of
(2) introducing the test compound into the transformed cell or administering the test compound into the medium;
(3) a step of measuring the labeling amount on the surface of the intracellular membrane,
(4) When the amount of label measured in step (3) decreases compared to the amount of label measured in step (1), the test compound is selected as a candidate for an inhibitor of HBV particle formation or HBV secretion Process. The transformed cell is a cell that expresses a tagged sugar chain synthesis-related protein, and is a transformed cell in which the inner membrane surface is fluorescently labeled with a fluorescently labeled anti-tag antibody, and the measured inner membrane surface The method according to claim 11 , wherein the amount of labeling is a fluorescence amount. A kit for screening for inhibitors of HBV particle formation or HBV secretion,
A kit comprising a transformed cell transformed with at least one sugar chain gene selected from SLC35B1 gene, CHST9 gene and ST8SIA3 gene. The sugar chain gene is fused with a gene encoding a labeling protein, and the transformed cell has at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3 on the surface of its inner cell membrane, directly or The kit according to claim 13 , which is a transformed cell that is expressed in an indirectly labeled state. A kit for screening for an inhibitor of HBV particle formation or HBV secretion, comprising the following (1) and (2);
(1) A transformed cell that expresses at least one sugar chain synthesis-related protein selected from tagged SLC35B1, CHST9, and ST8SIA3,
(2) A labeled anti-tag antibody. The kit according to claim 15 , wherein the tagged sugar chain synthesis-related protein is a Flag-tagged sugar chain synthesis-related protein, and the anti-tag antibody is an anti-Flag antibody. A kit for screening an inhibitor of HBV particle formation or HBV secretion, comprising the following (1) and (2);
(1) a substrate on which at least one sugar chain synthesis-related protein selected from SLC35B1, CHST9 and ST8SIA3 is immobilized;
(2) A library comprising nucleic acids containing random base sequences and corresponding peptides.

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