JP2009513109A - In vitro culture of caveolin-3 and hepatitis C virus - Google Patents

Abstract

Caveolin-3 has been identified as a cellular factor associated with HCV proteins E1 and E2, and is proposed as a cell limiting factor. As an alternative possibility, caveolin-3 may function as a cellular receptor for HCV acting with CD81. In either case, cells with increased expression of caveolin-3 are more suitable for in vitro culture of HCV and have therapeutic potential in blocking the interaction between caveolin-3 protein and HCV protein.

Description

  All references cited herein are incorporated by reference in their entirety.

(Technical field)
The present invention falls within the field of hepatitis C virus (HCV) culture and its in vitro culture.

(Background technology)
One challenge in research on HCV was the lack of an efficient in vitro culture system.

  Subgenomic replicons containing the majority of HCV nonstructural proteins have been shown to replicate in hepatoma cell lines (Non-Patent Document 1), and subgenomic replicons with improved transfection efficiency are also disclosed ( Non-Patent Document 2 and Patent Document 1). These systems have allowed detailed studies on the mechanism of HCV replication, but the lack of structural genes in the replicon means that virion assembly is not possible. Non-Patent Document 3 describes a method for permanent and transient replication of the full-length HCV genome in cell culture, by which the mutant “selectable full-length” is selected. The (sfl) genome described that it was able to stably replicate in 21-5 cells (from the Huh-7 cell line) and express all viral proteins. These 21-5 cells were found not to produce virus particles.

Due to the lack of intracellular particle assembly, it has been proposed that cellular factors exist that limit HCV maturation and assembly. If this limiting factor can be identified, its intracellular level can be artificially increased, thereby allowing virion assembly in vitro. Furthermore, if the factor is important for virion assembly, there is therapeutic potential in preventing or interfering with the factor's interaction with HCV proteins.
International Publication No. 01/89364 Pamphlet Lohmann et al., Science 1999, 285: 110-113. Blight et al., Science 2000, 290: 1972-1974. Pieschmann et al., J Virol 2002, 76: 4008-21

(Disclosure of the Invention)
Caveolin-3 has been identified as a cellular factor associated with HCV proteins E1 and E2, and has been proposed as a cell-limiting factor. As another possibility, caveolin-3 may function as a cellular receptor for HCV acting with CD81. In either case, cells with increased caveolin-3 expression are more suitable for in vitro culture of HCV and have therapeutic potential in blocking the interaction between caveolin-3 and HCV proteins. Furthermore, by providing increased caveolin-3 expression, animal models of HCV infection can be improved.

  HCV has been previously associated with caveolin-1 and caveolin-2. For example, references 5-7 report that caveolin-1 was overexpressed in cirrhotic liver associated with HCV. Furthermore, it has been found previously that HCV RNA is co-fractionated with caveolin-2 [8]. However, there has been no report associating HCV with caveolin-3 so far. Both caveolin-1 and caveolin-2 are ubiquitously expressed [9], but caveolin-3 expression has been reported to be muscle specific (evidence suggesting expression in astrocytes). There are some [10]). This expression pattern is inconsistent with the tissue tropism of HCV, and thus the relationship between HCV and caveolin-3 is surprising.

  Accordingly, the present invention provides (i) a hepatitis C virus genome and / or a nucleic acid encoding a hepatitis C virus genome; and (ii) a cell that expresses caveolin-3.

  The invention also provides a cell comprising (i) a hepatitis C virus genome and / or a nucleic acid encoding hepatitis C virus genome, and (ii) a nucleic acid encoding caveolin-3. The nucleic acid is preferably an mRNA transcript encoding caveolin-3 (ie, the nucleic acid is expressed).

  The present invention also provides an in vitro cell culture comprising the cells of the present invention.

  The present invention relates to a process for propagating hepatitis C virus comprising: (a) a cell that expresses caveolin-3 and contains a hepatitis C virus genome and / or a nucleic acid encoding a hepatitis C virus genome. Providing a process; and (b) culturing the cell. The process for producing HCV may then include the step of (c) purifying the HCV virion.

  The present invention also provides a process for propagating hepatitis C virus comprising: (a) (i) a nucleic acid encoding caveolin-3; and (ii) hepatitis C virus genome and / or hepatitis C virus genome. A process comprising transfecting a cell with a nucleic acid encoding; and (b) culturing the cell. The process for producing HCV may then include the step of (c) purifying the HCV virion.

  The present invention also provides a process for propagating hepatitis C virus comprising the steps of: (a) transfecting a cell with a vector encoding caveolin-3, wherein the cell comprises the hepatitis C virus genome and / or Or a process comprising a nucleic acid encoding the hepatitis C virus genome; and (b) culturing the cell. The process for producing HCV may then include the step of (c) purifying the HCV virion.

  The present invention also provides a process for propagating hepatitis C virus comprising the steps of: (a) transfecting a cell with a nucleic acid encoding caveolin-3; (b) infecting the cell with hepatitis C virus. And (c) providing a process comprising culturing the cell. Steps (a) and (b) can be performed in any order. The process for producing HCV may then include (d) purifying the HCV virion.

  The present invention also provides a process for propagating hepatitis C virus comprising: (i) an endogenous caveolin-3 gene; and (ii) a hepatitis C virus genome and / or a nucleic acid encoding the hepatitis C virus genome. A process comprising culturing a cell comprising a condition under conditions in which the caveolin-3 gene is expressed. The process for producing HCV can then include the step of purifying the HCV virion.

  The present invention also provides a process for modifying a cell that does not express caveolin-3, wherein (i) the cell is transfected with a vector encoding caveolin-3; or (ii) endogenous caveolin-3. Providing a process comprising initiating expression of caveolin-3 in the cell by a method selected from altering a regulatory component of the endogenous gene in the cell to initiate gene expression . This modified cell can then be used for HCV growth.

  The present invention is a process for propagating hepatitis C virus comprising: (a) (i) a caveolin-3 gene; and (ii) a hepatitis C virus genome and / or a nucleic acid encoding a hepatitis C virus genome; Providing a cell comprising: and (b) maintaining the cell under conditions of serum deprivation. The process for producing HCV may then include the step of (c) purifying the HCV virion. This caveolin-3 gene is preferably endogenous.

  The present invention also provides an animal comprising the cell of the present invention. The cells may be transplanted into animals or endogenous.

  The present invention provides a proteinaceous complex comprising (i) CD81; (ii) HCV protein; and (iii) caveolin-3. The complex can also contain cholesterol.

  The present invention is a method for screening a compound that inhibits the interaction between caveolin-3 and HCV protein, and evaluates the inhibition of the interaction between caveolin-3 and HCV protein in the presence of a candidate compound. A method comprising the steps of: Candidate compounds that inhibit this interaction may have antiviral activity. The screening method comprises the steps of: (i) mixing caveolin-3, HCV protein and one or more candidate compounds; (ii) incubating the mixture to caveolin-3, HCV protein and the candidate compound Allowing any interaction; and (iii) assessing whether the interaction between caveolin-3 and the HCV protein is inhibited. The caveolin-3, the HCV protein, and the candidate compound in step (i) can be mixed in any order.

  The present invention also provides a compound that inhibits the interaction between caveolin-3 and HCV protein, obtained or obtainable by the screening method of the present invention.

  The present invention also provides an antibody that can specifically bind to caveolin-3.

  The present invention also provides compounds that can downregulate caveolin-3 expression in target cells. This is typically a nucleic acid (eg, antisense nucleic acid, ribozyme, locked nucleic acid or siRNA) that can act in a sequence specific manner on caveolin-3.

(Hepatitis C virus genome)
The cell of the present invention comprises an HCV genome and / or a nucleic acid encoding the HCV genome. Thus, these cells can contain a positive-strand single-stranded RNA genome or can contain a nucleic acid that can be transcribed directly or indirectly to give a + -strand RNA genome. For example, transcription of a DNA copy of a correctly oriented genome (although the native HCV life cycle does not include a DNA step) can act as an HCV genome (ie, subsequently processed as found in the normal HCV life cycle). RNA that can direct the expression of HCV polyproteins.

The genomic RNA of HCV naturally contains a 5 ′ cap (m ⁷ G5′ppp5′A), no poly-A tail, a 5 ′ non-coding region (NCR) and a 3 ′ non-coding region. Have both. Although these elements are typically present in the HCV genome used in accordance with the present invention, the HCV genome and / or the nucleic acid encoding the genome may contain one or more elements not found in the natural genome. . For example, an HCV genome typically used in an in vitro replication system includes one or more of the following: a selectable marker (eg, a neomycin resistance marker) upstream of the polyprotein (eg, near the 5 ′ end of the genome). Which allows for the translation of the polyprotein even if other upstream coding sequences may be present, eg, an internal ribosome entry site (IRES) upstream of the sequence encoding the polyprotein (eg, EMCV or Poliovirus-derived IRES). A construct with a 5 ′ T7 promoter at the 5 ′ end of the genome has been described [11], but this is not preferred.

  The HCV genome can be of any type (eg, 1, 2, 3, 4, 5, 6) or subtype (eg, 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i, 1j, 1k). 1l, 1m, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2k, 2l, 2m, 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 3k, 4a 4c, 4d, 4e, 4f, 4g, 4h, 4k, 4l, 4m, 4n, 4o, 4p, 4q, 4r, 4s, 4t, 5a, 6a, 6b, 6d, 6f, 6g, 6h, 6i, 6j 6k, 6l, 6m, 6n). This name is the current standard as presented in NIAID Hepatitis C Virus (HCV) Sequence Database [12], which reclassifies the previous genotypes 7-9 as a subtype of type 6 based on reference 13. Therefore, this new classification is the previous classification I, II, III, IV, V, VI, 4α, 4β, 7a, 7b, 7c / NGII / VII, 7d, NGI, 8a, 8b, 9a, 9b, 9c. 10a / TD3 and 11a. One HCV strain suitable for use according to the present invention is the JFH1 strain of genotype 2a [14].

  The HCV genome can comprise a hybrid of one or more mutations (including insertions, deletions and / or substitutions) relative to the wild type genome, or two or more wild type genomes (eg, subtype 2a strains (eg, J6 ) And subtype 1a strains (eg, chimeras of H77). Since viral RNA replicases do not have proofreading activity, mutations in the genome are frequent and, as a result, mutant genomes (ie, “quasi-species”) can be easily obtained and analyzed. Some mutations have been reported to improve the ability of the genome to replicate within certain cells. Reference 4 describes variants for cell-adaptation (including E1202G, T1280I, K1846T and S2197P). Reference 3 describes HCV variants containing mutations (eg, R1164G, S1172C, S1172P, A1174S, S1179I) that increase transfection efficiency and the ability to survive subpassage. Other known mutations include, but are not limited to L1757I, N2109D, P2327S and K2350E. Certain mutations may be suitable for use with certain host cells and vice versa. The HCV polyprotein preferably includes all of C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B, but larger deletions and insertions (eg, mature coding regions (eg, non- It is also possible to delete the mature coding region) of the structural protein, or to insert non-HCV sequences. The present invention also provides subgenomic HCV (ie, HCV shorter than the complete genome (typically including nonstructural proteins but not structural proteins (eg, having no complete C, E1, E2)) The main advantage of the present invention is that it allows full-length genome replication.

  Although these and other mutations may be present in the HCV genome used according to the present invention, in general, these mutants do not remove the ability to induce expression of the HCV polyprotein of this genome, and this HCV After the polyprotein is expressed, it can replicate the HCV genome that expressed the HCV polyprotein.

  HCV RNA or a nucleic acid encoding this HCV RNA can be introduced into a cell as part of the process of the invention, or can already be part of a cell (eg, the 21-5 cell line [4] Already infected with HCV).

  If the cell contains a nucleic acid encoding the HCV genome, the nucleic acid can take the form of DNA or RNA and can be either chromosomal or extrachromosomal (eg, episomal). A DNA plasmid encoding the HCV genome can be conveniently used.

  DNA encoding both the HCV genome and the caveolin-3 protein can be used. This DNA usually has two different transcripts: (i) mRNA encoding HCV genome (or possibly antigenome) and (ii) caveolin-3. However, this DNA can also have a single transcript that contains (i) the HCV genome (or antigenome) and caveolin-3 coding sequences together with an IRES to control the translation of downstream sequences.

  The HCV genome is typically included in the form of a replicon (ie, a nucleic acid that can induce the production of its own copy). Cell-based HCV replication systems that use genomic or subgenomic replicon systems are well known. Although the replicon is based on the sense strand of the viral RNA, the present invention may also utilize complementary sequences that can be converted to the sense strand to provide the replicon. The replicon can also include non-HCV genes (eg, reporter genes).

(Caveolin-3)
Caveolin is a defined protein component of caveolae [9]. Caveolae are classically defined as plasma membrane invaders with a characteristic diameter of about 50 nm to 100 nm, but it is now known that such morphological descriptions are inappropriate. . Caveolae can be flattened into the plasma membrane plane or the isolated vesicle plane. In addition, caveolae can coalesce to form grape-like structures and tubules that are significantly larger in size than 100 nm. Morphologically, they are abundant in endothelium, muscle cell types, adipocytes and lung epithelial cells. Caveolae-like structures are also present in the nervous system.

  Caveolae has a unique liquid composition. Caveola mainly consists of cholesterol and sphingolipids. On the other hand, the non-caveolar region of the plasma membrane is mainly composed of phospholipids. Caveolin is a family of 21 kDa to 25 kDa integral membrane proteins that are implicated in diverse cellular functions. It is known that there are at least three caveolin genes. These caveolin genes are thought to promote the formation of invaginated caveolae by their interaction with cholesterol. Caveolin appears to associate with the membrane through a central 33 amino acid hydrophobic domain, allowing both the N-terminal and C-terminal domains to remain completely cytosolic. Caveolin binds directly to cholesterol.

  For caveolin, three related important functional roles have been described: (i) the major structural protein components of the caveolae membrane; (ii) shuttle proteins in the transport of cholesterol biosynthesis from the ER to the plasma membrane; and (Iii) Scaffolding proteins that integrate and inactivate signaling molecules that concentrate on the cytoplasmic surface of caveolae membranes.

  Caveolin is functionally related to a wide range of signaling processes. In general, caveolin-1 and caveolin-3 inhibit the enzymatic activity of a wide range of signaling molecules, whereas caveolin-2 has little or no effect. One notable exception is the insulin receptor tyrosine kinase; caveolin-1 and caveolin-3 increase the ability of the insulin receptor to phosphorylate IRS-1, one of its major substrates.

  Caveolin-3 localizes to the muscle cell membrane (muscle cell plasma membrane) and is consistent with the distribution of dystrophin, another muscle-specific plasma membrane marker protein. Caveolin-3 is fractionated with membranes of cytoplasmic signaling molecules (G protein and Src-like kinase) and dystrophin complexes (dystrophin, α-sarcoglycan and β-dystroglycan).

  Caveolin-3 has also been shown to form a physical complex with dystrophin, suggesting an important role for caveolin-3 in muscle biology. Caveolin-3 protein expression is dramatically induced during differentiation of C2C12 skeletal myoblasts in culture. In addition, caveolin-3 temporarily associates with T tubules during myogenesis and may be involved in the early development of the T tubule system.

  Caveolin-3 also interacts with phosphofructokinase-M in muscle. This interaction is (i) highly regulated by the extracellular concentration of glucose, and (ii) a number of related intracellular metabolites (eg, known allosteric activators of phosphofructokinase-M). Certain fructose 1,6-bisphosphate and fructose 2,6-bisphosphate). Recruitment of activated phosphofructokinase-M to the glucose-dependent plasma membrane by caveolin-3 may play an important role in regulating energy metabolism in skeletal muscle fibers.

  Genetic evidence highlights the importance of caveolin-3 in muscle function. A form of autosomal dominant limb girdle muscular dystrophy (LGMD) that has been associated with a severe deficiency of caveolin-3 in muscle fibers has been reported. The gene for human caveolin-3 is mapped onto chromosome 3p25 and two mutations within the gene are identified: a missense mutation in the transmembrane region (P → L) and a microdeletion in the scaffold domain (ΔTFT). It was. These mutations can prevent caveolin-3 oligomerization and can also prevent caveolae formation in the muscle cell plasma membrane. However, the molecular mechanism by which these two mutations cause muscular dystrophy is not yet known.

  The phenotypic behavior of these caveolin-3 mutations was investigated by using heterologous expression. Wild-type caveolin-3 or a mutant of caveolin-3 was transiently expressed in NIH 3T3 cells. The LGMD-1C mutation results in the formation of unstable high molecular weight aggregates of caveolin-3, which are retained in the Golgi complex and not led to the plasma membrane. Consistent with the autosomal dominant form of genetic transmission, the LGMD-1C mutant of caveolin-3 behaves in a dominant-inhibitory manner, resulting in the retention of wild-type caveolin-3 at the level of the Golgi complex. These data provide a molecular explanation for why caveolin-3 levels are downregulated in patients with LGMD-1C.

  Caveolin-3 has been reported to be a chaperonin [16].

  It has now been found that caveolin-3 expression is not found only in muscle. Surprisingly, this was detected in the Huh-7 cell line with the HCV replicon [15] and was further found to be localized intracellularly with particulate HCV protein structures. Expression does not appear to be induced by HCV infection but appears to be present at a low endogenous level.

  The Caveolin-3 GeneCard registration can be found using the code CAV3. GenCard reveals that this protein was also called LGMD1C, M-caveolin and VIP-21. Ten SNPs have been described so far. The reference human CAV3 sequence is 151mer and has GI number GI: 4502589 and accession number NP_001225 (SEQ ID NO: 1).

疾病関連単一アミノ酸変異は、残基２７（Ｒ／Ｑ）、２９（Ｄ／Ｅ）、３３（Ｎ／Ｋ）、４４（Ｖ／Ｅ）、４６（Ａ／ＴおよびＡ／Ｖ）、５６（Ｇ／Ｓ）、６４（Ｔ／Ｐ）、７２（Ｃ／Ｗ）、８７（Ｌ／Ｐ）、９３（Ａ／Ｔ）、および１０５（Ｐ／Ｌ）におけるものが開示された。残基６４〜６６の欠失も報告された。切断型のカベオリン−３もまた、報告された（例えば、参考文献１６のｃａｖＤＧＶ切断）。

Disease-related single amino acid mutations are residues 27 (R / Q), 29 (D / E), 33 (N / K), 44 (V / E), 46 (A / T and A / V), 56 (G / S), 64 (T / P), 72 (C / W), 87 (L / P), 93 (A / T), and 105 (P / L) were disclosed. A deletion of residues 64-66 was also reported. A truncated form of caveolin-3 has also been reported (eg, cavDGV cleavage in ref. 16).

  Additional human CAV3 sequences in the database include registrations such as AF043101, AAC14931.1, Y14747, CAA75042.1, AF036367, AAC39758.1, AF036366, AAC39758.1, AF036365, AAC39756.1, BC069368, AAH69368.1. It is done. Human caveolin-3 is 95% identical to mouse and rat caveolin-3 protein sequences. Human caveolin-3 shows 82% similarity to human caveolin-1 and 58% similarity to human caveolin-2.

  In the context of such functional characterization and sequence information, one skilled in the art can readily identify whether a given sequence is that of caveolin-3. A preferred caveolin-3 protein of the invention is at least 50% (eg 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94) relative to SEQ ID NO: 1. %, 95%, 96%, 97%, 98%, 99% or more). Other preferred caveolin-3 proteins comprise a fragment of at least n contiguous amino acids of SEQ ID NO: 1, where n is 7 or more (eg, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more). This fragment may exclude signal peptides and the like. This fragment may be due to caveolin-3 cleavage at the C-terminus and / or N-terminus. Preferred fragments include the central region of amino acids 84-104, which is a transmembrane domain. Another preferred fragment includes the caveolin-scaffold domain of caveolin-3 (ie, at least amino acids 55-73).

  Compared to SEQ ID NO: 1, these polypeptides have one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) conservative amino acid substitutions (ie, related Substitution of one amino acid with another amino acid having a side chain). Genetically encoded amino acids are generally divided into four families: (1) acidic (ie, aspartate, glutamate); (2) basic (ie, lysine, arginine, histidine); (3) nonpolar (ie alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and (4) uncharged polarity (ie glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes classified together as aromatic amino acids. In general, substitution of a single amino acid in these families does not have a significant effect on biological activity. These polypeptides may have one or more single amino acid deletions (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) compared to SEQ ID NO: 1. . These polypeptides also have one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) insertions (eg, 1 amino acid each, 2 amino acids, 3 amino acids, 4 amino acids, or 5 amino acids).

(Cell type)
The cells of the invention have two essential characteristics: (1) they can express the HCV genome to support the HCV life cycle and its replication; (2) they express caveolin-3. It can be expressed. These cells can already have both of these characteristics, or one or both of these characteristics can be introduced. Thus, for example, the cells can be infected with HCV or a nucleic acid encoding HCV and can be transfected with a vector encoding caveolin-3 (either before, after or simultaneously with the introduction of HCV).

  Since all wild-type human cells contain the gene encoding caveolin-3, the cells (eg muscle cells) can already express this protein. If the cell does not naturally express caveolin-3 from an endogenous gene, the ability to express this protein is expressed and / or expressed by activating expression of the endogenous gene (eg, by replacing a promoter). Can be achieved by providing additional copies. This further copy may be episomal or chromosomal.

  Expression of endogenous caveolin-3 can also be achieved by stimulating cells that express the endogenous gene. One known method of stimulating caveolin-3 expression uses serum deprivation. This treatment typically involves replacing a “high mitogen” growth medium (eg, containing 10% fetal calf serum) with a low or no serum “low mitogen” growth medium (eg, Using 2% horse serum). A “low mitogen” growth medium is described in ref. 17 for use in differentiating muscle cells.

  In addition to containing the HCV genome, the cell may contain a nucleic acid encoding one or both of the hepatitis C virus proteins E1 and E2. As disclosed in reference 18, the ability to express E1 and / or E2 proteins separately from the E1 and E2 proteins produced during the HCV life cycle promotes virion assembly. The nucleic acid encoding E1 and / or E2 can be DNA or RNA. A DNA vector is preferred. The nucleic acid can be either chromosomal or extrachromosomal (eg, episomal). A preferred nucleic acid vector for introducing E1 and E2 into a cell is a retroviral vector, which can be an integrating vector. Thus, the E1 and E2 sequences can be introduced as inserts into the retroviral RNA genome. After reverse transcription (and integration, if applicable), the E1 and E2 sequences are in DNA form within the cell. Other suitable vectors include DNA plasmids. In addition to expressing E1 and E2, the nucleic acid can express p7.

  Since HCV naturally infects eukaryotic cells, the present invention generally uses eukaryotic cells. It is preferred to use mammalian cells (eg, primate cells (including human cells)). Other animals whose cells can be used for HCV studies include, but are not limited to: mice; rats; woodchucks; shrews (eg, camellia); chimpanzees; gibbons; tamarins; and marmosets.

  Any suitable caveolin-3 expressing cells (including muscle cells) can be used instead, but because HCV naturally infects liver cells, use cells derived from the liver (eg, hepatocytes). Is convenient. Muscle cells (including myocytes) are a convenient cell type because caveolin-3 expression can be upregulated by a simple process of serum deprivation. However, the present invention may use cells that are not muscle cells, particularly non-muscle cells that express caveolin-3.

  Typically, the cell used in the present invention is a cell line, preferably a packaging cell line. A preferred cell line for use in the present invention is derived from a hepatocellular carcinoma (ie, a human hepatoma cell line [15] known as “Huh7”). A particularly preferred cell line is the Huh7 derived cell line known as “21-5”, which supports the full length HCV replicon. Other preferred cell lines are Huh-7.5 and Huh-7.8, which are sub-strains of Huh-7 [19, 20] that can support complete HCV replication in cell culture, and Huh -7.5 is preferred. Cells obtained by subculture of Huh7 cells (and their derivatives) can also be used as well as cells obtained by treating Huh7 cells with α-interferon and / or γ-interferon. As disclosed in reference 20, a cell line that tolerates HCV can be prepared by a process that includes the following steps: (a) culturing cells infected with HCV; (b) HCV cells. Treating; and (c) identifying a sub-line of the treated cell that is permissive for HCV replication.

  Other established human hepatoma and hepatoblastoma cell lines include HuH-6 cl-5, PLC / PRF / 5, huH-1 and huH-4. Huh7 cells are like complete DMEM (eg Dulbecco's modified minimal basal medium supplemented with 2 mM L-glutamine (non-essential amino acid), 100 U / ml penicillin, 100 μg / ml streptomycin, 10% fetal calf serum). It can be grown as a monolayer on fresh medium.

  Certain cell types may be preferred for use with certain virus strains and vice versa.

  The cells of the present invention express a polyprotein derived from the HCV genome. As a result, these cells contain protein components for expressing HCV particles, including virions and virus-like particles (VLPs). Thus, virions and VLPs can be prepared from the cells of the invention. These may or may not contain the RNA genome. These cells can also express a complex of E1 and E2. These cells can also express a complex of E1, E2, NS3 and NS5a.

As described in more detail above, the cells of the invention comprise an HCV genome and / or a nucleic acid encoding an HCV genome. The HCV genome can be introduced into cells by viral infection or can be generated in situ either after viral infection (real or artificial infection) or after transcription from a DNA copy of the genome. Methods for introducing HCV genome or DNA encoding HCV genome into cells are routine in the art. The in vitro transcribed HCV genome can be introduced into Huh7 cells using electroporation as disclosed in ref. 4 (1 μg of purified in vitro transcript on 4 × 10 ⁶ Huh-7 cells. Introduced by electroporation, or introduced into 2.4 × 10 ⁷ Huh-7 cells by 60 μg HCV RNA by electroporation).

  The cells of the invention can be grown in in vitro culture. Details about culturing these cells in conditions under which HCV replication occurs are known in the art. Methods for purifying HCV virions after viral propagation are also known in the art.

  When the HCV genome is expressed according to the present invention, dot-like particles can be seen when immunofluorescent staining is used. If desired, the release of these particles from the cell can be facilitated by treating the cell with an appropriate release factor (eg, to cause release, such as exosomes). Such factors include alcohol, stress conditions and the like. Non-covalent E1 / E2 heterodimers have been detected in the endoplasmic reticulum and can be recovered from this cell compartment.

(Animal model)
Various animal models for HCV infection have been described (eg refs. 21-29).

  If the efficiency of HCV infection is lower than desired, the efficiency can be increased by upregulating the expression of caveolin-3 in the animal. This can be achieved in various ways. For example, the cells of the present invention can be transplanted or injected into a host animal, where foreign cells can express caveolin-3 and promote HCV growth. Alternatively, genetic changes can be introduced into the animal's endogenous cells (eg, using gene therapy techniques). Yet another method is to introduce such genetic changes into the cells from which the animal is produced (eg, into embryonic cells, stem cells, embryonic stem cells, nuclear transfer donor cells, etc.), thereby making the whole animal allogenic. Can give. As yet another method, the animal can be subjected to a treatment that stimulates expression of its endogenous caveolin-3 gene.

  Thus, the expression of the endogenous caveolin-3 gene can be upregulated and / or the exogenous caveolin-3 gene can be introduced. If an exogenous caveolin-3 gene is introduced, this gene can be derived from the same organism as the animal (eg, introducing a mouse caveolin-3 gene into a mouse) or from a different organism. Obtain (eg, introduce human caveolin-3 gene into mice).

  The animal of the invention preferably has a liver whose cells are thus modified. More preferably, all the liver cells are modified in this way.

  The animal of the present invention may not be infected with HCV, in which case the animal may later be exposed to HCV infection for study; or it may be infected with HCV, in which case This animal is useful for HCV virological studies (eg, cell entry, infection progression, replication, viral assembly, efficacy of antiviral compounds, etc.).

  The animal can be immune deficient (eg, a SCID animal or a irradiated animal). Irradiated SCID mice are particularly useful.

  Any suitable animal may be used, but a typical model is selected from the group consisting of mice; rats; woodchucks; shrews (eg, Tupaia berangeri); chimpanzees; gibbons; tamarins; and marmosets Based on mammals.

  Cells of one organism can be introduced into different organisms for the generation of animal models. For example, it is known to transplant human hepatocytes into the liver of a mouse host [30, 31].

(Screening method)
As described above, the present invention provides a screening method for identifying a compound that can inhibit the interaction between caveolin-3 and HCV protein. The HCV protein is typically an E1, E2, or oligomer comprising E1 and / or E2, or may be a core protein or a nonstructural protein. These screening methods involve assessing the interaction between HCV protein and caveolin-3, and the ability of test compounds to disrupt this interaction indicates potential therapeutic activity that prevents HCV maturation. .

(Direct screening)
Inhibition of the HCV / caveolin-3 interaction in the presence of the candidate compound can be assessed directly. A variety of methods are available for direct detection of protein / protein interactions.

  For example, one or both of HCV and caveolin-3 can be detected such that the interaction between HCV and caveolin-3 can be detected by the intrinsic fluorescence change that occurs when the HCV / caveolin-3 complex is formed or destroyed. It can be labeled with a fluorescent label. For example, when HCV and caveolin-3 interact, the HCV protein is bound to the FRET donor so that a fluorescence resonance energy transfer (FRET) donor stimulus excites the FRET acceptor, thereby allowing photons to be emitted, Caveolin-3 can be bound to a FRET acceptor (or vice versa). Interaction can also be detected by fluorescent labeling of HCV protein and / or caveolin-3 so that fluorescence is quenched when HCV protein and caveolin-3 form a complex.

  Another method for assessing the interaction between HCV and caveolin-3 protein is co-immunoprecipitation. For example, anti-HCV antibodies can be used to immunoprecipitate the relevant HCV protein from the mixture, and the precipitated material can be detected for the presence of caveolin-3 (eg, by Western blot or presence of tag in target protein). Can be tested). The converse can also be used (ie, immunoprecipitated with anti-caveolin-3 antibody and investigated for co-precipitated HCV protein).

  Other methods for assessing the interaction between HCV and caveolin-3 can include determining whether a complex is present using NMR when a candidate compound is present.

  The presence of the HCV / caveolin-3 complex can also be detected as a band at a particular location when performed on a gel. Complex disruption due to the addition of a candidate compound can be detected by the presence of bands at different locations on the gel.

  The interaction of HCV and caveolin-3 proteins also detects the availability of peptide sequences (eg, epitopes) on HCV and / or caveolin-3 proteins that are masked when they form a complex Can be evaluated.

(Indirect screening of inhibitors using a two-hybrid system)
Whether the interaction between HCV protein and caveolin-3 protein is inhibited in the presence of a candidate compound can also be assessed indirectly. One indirect method of screening for inhibition of the interaction between HCV protein and caveolin-3 protein in the presence of a candidate compound involves using a two-hybrid system. HCV protein is fused to the activation domain of the transcription factor and caveolin-3 is fused to the DNA binding domain of the transcription factor (or vice versa), and these interactions promote transcription of the reporter gene in the cell.

  Accordingly, the present invention is a method of screening for a compound that inhibits the interaction between HCV protein and caveolin-3 protein, comprising: (i) a cell comprising a nucleic acid molecule comprising a promoter operably linked to a reporter gene. (A) a first fusion protein comprising one of HCV protein and caveolin-3 fused to the active domain of a transcription factor, (b) the other of HCV protein and caveolin-3 fused to a DNA binding domain of the transcription factor And (iii) contacting with a candidate compound; and (ii) assessing the level of expression of the reporter gene. In this method, the interaction between HCV protein and caveolin-3 protein promotes transcription of the reporter gene by activating the promoter.

  This method can be used to assess the interaction of HCV and caveolin-3 proteins in any eukaryotic cell. Preferably, this method is used to assess the interaction between HCV protein and caveolin-3 protein in yeast cells or mammalian cells. When the candidate compound is an organic compound and a yeast two-hybrid system is used, preferably the permeability of the yeast cell wall is increased (eg, by using a chemical (eg, polymyxin B)).

  The expression level of the reporter gene in the 2-hybrid system indicates the level of interaction between HCV protein and caveolin-3 protein. Candidate compounds that inhibit this interaction reduce the expression level of the reporter gene or stop expression.

  Preferably, the reporter gene is easily assayed. For example, the reporter gene can provide a detectable signal (eg, a visible signal). The reporter gene can encode a protein that gives a visible signal itself, or a protein that catalyzes a reaction that gives a visible change (eg, a fluorescent protein or an enzyme). The reporter gene may encode an enzyme such as β-galactosidase or peroxidase, both of which are generally used with a colored substrate and / or colored product. The reporter gene can encode a fluorescent protein (eg, green fluorescent protein (GFP) or a fluorescent derivative thereof (eg, YFP or CFP) [32] The reporter gene encodes a photoprotein (eg, luciferase). The reporter gene may drive intracellular DNA replication [33] or may encode a drug resistance marker [34].

  The reporter gene may encode a protein that allows positive selection of cells in which the interaction between HCV protein and caveolin-3 protein is inhibited. For example, the reporter gene can encode a toxic or cytostatic protein, so that only cells that do not express this protein can remain or proliferate ("reverse 2-hybrid" or "MAPPIT" assay [35 ~ 37]). As a result, only the remaining cells are cells in which the interaction between the HCV protein and the caveolin-3 protein is inhibited by the candidate compound, and as a result, the reporter gene is not expressed. Examples of this type of reporter gene that can be used in yeast include URA3, LYS2, and CYH2 [38]. The protein encoded by the reporter gene can also prevent cell growth in the absence of or in the presence of certain amino acids or other components in the cell culture medium. For example, the reporter gene can encode a DNA binding protein, Tn10 tetracycline, which represses transcription of the TetRop-HIS3 gene, so that yeast cells in which the reporter gene is expressed are in the absence of histidine. Does not grow in [39]. In contrast, yeast cells in which the interaction between HCV protein and caveolin-3 protein is disrupted do not express TN10 tetracycline and can therefore grow in the absence of histidine.

  These proteins encoded by the reporter gene can be in the form of fusion proteins. Methods for the production of fusion proteins are standard in the art and are known to those skilled in the art.

  The expression level of the reporter gene can also be evaluated by measuring the level of mRNA transcribed from the reporter gene or the level of protein translated after the transcription.

(System for carrying out screening methods)
The screening methods of the invention can be performed in a cell-free system or in cells or tissues. In particular, the indirect screening methods described above can be performed in cell-free systems, in cells or in tissues. This cell-free system must contain all components necessary for transcription of the reporter gene whose expression level is detected by measuring the mRNA level, and the expression level is evaluated by measuring the protein level. Must contain all components necessary for transcription and translation of the reporter gene.

  The screening method of the present invention is preferably performed in a cell-free system because high-throughput screening of candidate compounds is promoted.

  The indirect screening methods of the present invention are preferably performed in eukaryotic cells (eg, mammalian (eg, human) cells or yeast cells). They can also be performed in mammalian (eg, human) tissue. A typical cell is a liver cell.

(Reference criteria)
A reference standard (eg, control) is typically required to detect whether the interaction between HCV protein and caveolin-3 protein is inhibited. In order to detect whether the candidate compound inhibits the interaction between the HCV protein and the caveolin-3 protein, the interaction between the HCV protein and the caveolin-3 protein in the presence of the candidate compound is determined in the absence of the candidate compound. Can be compared with the interaction of HCV protein with caveolin-3 protein.

  The above criteria may be determined before performing the method of the present invention, determined while the method is performed (eg, in parallel), or determined after the method is performed. It can be done. The criteria can be absolute criteria derived from previous studies.

(Candidate compound)
Typical candidate compounds for use in all of the screening methods of the invention include peptides, peptoids, proteins, lipids, metals, small organic molecules, RNA aptamers, antibiotics and other known formulations, polyamines, antibodies or Antibody derivatives such as, but not limited to, antigen binding fragments, single chain antibodies (including scFv), and combinations or derivatives thereof. Small organic molecules have a molecular weight of greater than about 50 daltons and less than about 2,500 daltons, most preferably from about 300 daltons to about 800 daltons. Candidate compounds can be derived from large libraries of synthetic or natural compounds. For example, synthetic compound libraries are available from MayBridge Chemical Co. (Revillet Cornwall, UK) or Aldrich (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be used. In addition, candidate compounds can be synthesized using combinatorial chemistry as individual compounds or as mixtures.

  In some cases, it may be desirable to perform a preliminary screening step that reduces the number of candidate compounds used in the methods of the invention. Compounds that bind to each of the HCV and caveolin-3 proteins appear to inhibit the interaction of the HCV and caveolin-3 proteins essentially more than compounds that do not bind them.

  Therefore, the present invention is a method for screening for a compound that inhibits the interaction between HCV protein and caveolin-3 protein, and further by identifying a compound that binds to either HCV protein or caveolin-3 protein. Methods are provided that include a preliminary step of screening a compound.

(Confirmation of function of identified compound in vivo)
Once a compound has been identified as an inhibitor of the interaction between HCV protein and caveolin-3 protein, it may be desirable to perform further experiments to confirm the in vivo antiviral function of the compound.

  Accordingly, the present invention provides a method for assessing the in vivo antiviral effect of a compound obtained or obtainable by any of the above methods comprising the step of administering this compound to a test animal and the HCV life cycle. There is provided a method comprising the step of assessing the effect on. Non-human studies can be used.

(Compound identified by screening method)
The present invention provides compounds that inhibit the interaction of HCV protein and caveolin-3 protein obtained or obtainable by any of the above methods. Preferably, these compounds of the present invention are organic compounds.

  Also provided is a composition comprising a compound that inhibits the interaction of HCV protein and caveolin-3 protein obtained or obtainable by any of the above methods.

  Compounds that are found to inhibit HCV activity can be useful antiviral drugs by themselves or can lead to compounds for the development of new contraceptives. These compounds are also useful in antiviral research.

(Pharmaceutical use of identified compounds)
Once a compound has been identified using one of the methods of the invention, further research on its pharmaceutical properties may need to be performed. For example, the compound may need to be modified to improve pharmacokinetic properties or bioavailability. The present invention extends to any compounds identified by the methods of the invention and modified to improve pharmacokinetic properties, and compositions comprising those compounds.

  The present invention further provides compounds obtained or obtainable using the methods of the present invention and compositions comprising those compounds for use as antiviral agents. The present invention also provides the use of compounds obtained or obtainable using the methods of the invention, or compositions comprising these compounds, in the manufacture of antiviral agents. An antiviral comprising the step of administering to a mammal (preferably a human) a compound obtained or obtainable by any one of the methods of the invention, or a composition comprising such a compound A method is also provided.

(Therapeutic antibody)
As described above, the present invention provides antibodies that can specifically bind to caveolin-3. Reference 40 describes a mouse monoclonal antibody that recognizes the N-terminal region of caveolin-3, but does not describe other members of the caveolin family. Research Diagnostics Inc. Thus, mouse anti-caveolin-3 and rabbit anti-caveolin-3 antibodies with catalog numbers RDI-CAVEO3abm and RDI-CAVEOL3abrX are available for in vitro research applications. AbCam rabbit polyclonal antibody ab2912 is also available. Previously, for investigation of sodium channels, anti-caveolin-3 antibody was injected into the cytoplasm of cells via a pipette [41].

  The antibodies of the present invention preferably bind to an epitope located in caveolin-3 and this binding prevents caveolin-3 interaction with the HCV protein. Accordingly, these antibodies can be used in therapeutic methods that prevent HCV maturation. It has been reported that some caveolin-3 epitopes can be masked as long as the bound cholesterol is not depleted [42, 43]. Thus, some antibodies recognize caveolin-3 in the plasma membrane, some antibodies recognize caveolin-3 in the Golgi apparatus, and some antibodies recognize both. Appropriately reactive antibodies can be selected by routine testing.

  The antibodies of the invention can be polyclonal or monoclonal and can be produced by any suitable means (eg, recombinant expression). To increase compatibility with the human immune system, these antibodies can be chimeric or humanized (eg, [refs. 44 and 45]) or fully human antibodies can be used. . The antibodies of the present invention are preferably neither natural mouse antibodies nor rabbit antibodies.

  Soluble antibodies that retain binding activity in the intracellular environment are preferred.

  The antibodies of the present invention are preferably provided in a purified or substantially purified form. Typically, the antibody is a composition that is substantially free of other polypeptides (eg, less than 90% by weight of the composition, usually less than 60% by weight, more usually 50%). Less than (% by weight) is composed of other polypeptides).

  The antibodies of the invention can be of any isotype (eg, IgA, IgG, IgM, ie, α heavy chain, γ heavy chain or μ heavy chain), but are generally IgG. Of the IgG isotypes, the antibody can be an IgG1 subclass, IgG2 subclass, IgG3 subclass or IgG4 subclass. An antibody of the invention may have a kappa or lambda light chain.

The antibodies of the present invention can be of various forms (complete antibodies, antibody fragments (eg, F (ab ′) ₂ and F (ab) fragments), Fv fragments (noncovalent heterodimers), single chain antibodies (eg, , Single chain Fv molecules (scFv)), minibodies, oligobodies, etc.). The term “antibody” does not imply any particular source and includes antibodies obtained by non-conventional processes (eg, phage display).

(Therapeutic nucleic acid)
As an alternative to blocking caveolin-3 activity after translation (eg, by using an antibody), protein expression can be blocked at an earlier stage by preventing transcription or preventing translation of transcribed mRNA. . Techniques for achieving this effect include antisense, ribozyme, and gene silencing.

  Accordingly, the present invention provides a nucleic acid capable of inhibiting caveolin-3 expression in a cell, wherein the nucleic acid is selected from an antisense nucleic acid; a ribozyme, a locked nucleic acid; and a gene silencing nucleic acid. These nucleic acids are particularly useful for therapeutic purposes. They can be administered directly or can be generated in situ from expression in this cell. Thus, the present invention also provides a nucleic acid that can be transcribed in a cell to provide an active molecule.

  Antisense technology generally involves the use of a nucleic acid sequence that is complementary to the transcribed (sense) mRNA sequence of a gene. Antisense molecules can be targeted to target regulatory, 5 ', or regulatory regions (signal sequences, promoters, enhancers and introns). Inhibition can also be achieved using a “triple helix” method that can inhibit the ability of the double helix to open sufficiently to the binding of polymerases, transcription factors, or regulatory molecules. Inhibition can also be achieved by preventing the transcript from binding to ribosomes and the like by hybridization of hybridized mRNA and antisense molecules.

  Ribozymes (enzymatic RNA molecules) can also be used to catalyze specific cleavage of RNA. These ribozymes may be natural or synthetic. Synthetic ribozymes can be designed to specifically cleave mRNA at selected positions, thereby preventing translation of the mRNA into a functional polypeptide. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule with a complementary target RNA, followed by endonucleolytic cleavage. Examples of those that can be used include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of target sequences. A specific ribozyme cleavage site within any potential RNA target is first obtained by scanning the target molecule for a ribozyme cleavage site (eg, a ribozyme cleavage site comprising a GUA trinucleotide, a GUU trinucleotide, or a GUC trinucleotide). Identified. Once identified, a short RNA sequence of 15-20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for secondary structural features that can render the oligonucleotide inoperable. The suitability of a candidate target can also be assessed by testing its availability for hybridization with a complementary oligonucleotide using a ribonuclease protection assay.

  Locked nucleic acid (LNA) is described in more detail in reference 46. These locked nucleic acids have a high binding affinity for complementary DNA and RNA. Modified LNA oligonucleotides and chimeric LNA oligonucleotides were applied. LNA oligonucleotides are commercially available, can be transfected using standard techniques, are non-toxic, can be designed to increase target availability, activate RNase H, and steric inhibition approaches It works in (static block approach). LNA antisense (including gapmer LNA containing a central DNA or phosphorothioate-DNA segment adjacent to the LNA gap) is particularly useful.

  Various gene silencing techniques are available. The most commonly used technique is RNA interference (RNAi) through double-stranded RNA (dsRNA) molecules. Appropriately sized dsRNA molecules that are homologous to the target transcript (eg, siRNA having 21-23 nucleotides per strand, ie, small interfering RNA), are rapidly In addition to siRNA, stRNA can be used to split genes by suppressing translation, and μRNA can also cause gene silencing. The preferred target site of the sequence begins with the AA dinucleotide sequence downstream of the start codon and lasts for the next 19 3 'nucleotides Kits and algorithms for siRNA design and delivery are readily available (eg, See references 47 and 48)

  Since RNA molecules can be degraded in vivo, natural RNA nucleotides can be replaced with artificial nucleotides if the technology allows it. For example, RNA molecules may be synthesized by the use of phosphorothioate or 2′O-methyl rather than phosphodiesterase linkages within the backbone of the molecule, or by non-conventional bases (eg, inosine, queeosine and wybutosine), and acetyl Adenine, acetylcytidine, acetylguanine, acetylthymine and acetyluridine, methyladenine, methylcytidine, methylguanine, methylthymine and methyluridine, thioadenine, thiocytidine, thioguanine, thiothymine and thiouridine, similarly modified forms of adenine, cytidine and guanine , Thymine and uridine). Nucleotide analogs such as peptide nucleic acids (PNA) can also be used.

(Pharmaceutical composition)
The antibodies and nucleic acids of the invention can be provided as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. Such compositions can be prepared using a process that includes mixing the active ingredient with a pharmaceutically acceptable carrier.

  Exemplary “pharmaceutically acceptable carriers” include any carrier that does not itself induce the production of antibodies that are detrimental to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules (eg, proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates (eg, oil droplets or liposomes). )). Such carriers are well known to those skilled in the art. Vaccines can also include diluents (eg, water, saline, glycerol, etc.). In addition, adjuvants such as wetting or emulsifying agents, pH buffering substances, sucrose and the like may be present. Sterile pyrogen-free phosphate buffered saline is a typical carrier (eg, based on an injectable solution). A detailed review of pharmaceutically acceptable excipients is available in reference 49.

  The compositions of the present invention are typically in an aqueous form (eg, a solution or suspension) rather than a dry form (eg, lyophilized form). Aqueous compositions are also suitable for reconstitution of other materials from lyophilized form. When the composition of the present invention is used for such an immediate reconstitution, the present invention may also include a kit that may contain two vials, or one ready-filled syringe and one vial. A kit is provided wherein the aqueous contents of the syringe can be used to reactivate the dry contents of the vial prior to injection.

  The composition of the present invention can be provided in a vial or can be provided in a prefilled syringe. The syringe can be provided with or without a needle. The composition may be packaged in unit dose form or multiple dose form. Generally, a syringe will contain a single dose of the composition and a vial may contain a single dose or multiple doses. Thus, vials are preferred over prefilled syringes for multiple dose forms.

  Effective dosage volumes can be routinely set, but a typical human dose of the composition (eg, for intramuscular injection) has a volume of about 0.5 ml.

  The pH of the composition is preferably 6-8, more preferably 6.5-7.5 (eg about 7). The appropriate pH can be maintained by the use of buffers such as Tris buffer, phosphate buffer, or histidine buffer. The composition of the present invention generally comprises a buffer. Where the composition comprises an aluminum hydroxide salt, it is preferred to use a histidine buffer (eg, 1 mM to 10 mM, preferably about 5 mM) [50]. The composition may be sterile and / or pyrogen free. The composition of the present invention may be isotonic to humans.

  The composition may be prepared as an injectable material (either as a solution or a suspension). The composition may be prepared for pulmonary administration (eg, as an inhaler using a fine powder or spray). The composition can be prepared as a suppository or vaginal suppository. The composition can be prepared for nasal, otic or ophthalmic administration (eg, as a spray, drop, gel or powder) [eg refs. 51 and 52]. Injectable materials for intramuscular administration are typical.

  The compositions of the invention may contain antimicrobial substances, particularly when packaged in a multiple dose format. Antibacterial substances (eg, thiomersal and 2-phenoxyethanol) are commonly found in vaccines, but it is desirable to use a mercury-free preservative or no preservative.

  The composition of the present invention may include a surfactant (eg, Tween (polysorbate) (eg, Tween 80)). Surfactants are generally present at low levels (eg, <0.01%).

  The composition of the present invention may include a sodium salt (eg, sodium chloride) to provide tonicity. A concentration of 10 ± 2 mg / ml NaCl is typical. The concentration of sodium chloride is preferably about 9 mg / ml.

(Method of treatment)
The present invention also provides a method for treating a mammal comprising the step of administering to the mammal a pharmaceutical composition of the present invention. The mammal is preferably a human.

  The present invention also provides the antibodies and nucleic acids of the present invention for pharmaceutical use. The invention also provides the use of an antibody or nucleic acid of the invention in the manufacture of a medicament for generating an immune response in a mammal.

  These uses and methods preferably relate to the prevention and / or treatment of diseases caused by HCV (eg, hepatitis). Methods for investigating the efficacy of therapeutic treatment of hepatitis are known in the art.

  The compositions of the invention are generally administered directly to the patient. Direct delivery can be by parenteral injection (eg, subcutaneous injection, intraperitoneal injection, intravenous injection, intramuscular injection, or injection into the interstitial space of the tissue) or rectal, oral, vaginal, topical It can be achieved by administration, transdermal administration, intranasal administration, ocular administration, otic administration, pulmonary administration or other transmucosal administration. Intramuscular administration to the thigh or upper arm is preferred. Injection can be via a needle (eg, a hypodermic needle), but needle-free injection can alternatively be used. A typical intramuscular dose is 0.5 ml. Dosage treatment can be a single dose schedule or a multiple dose schedule.

(General)
The term “comprising” includes not only “including” but also “consisting” (eg, can a composition “comprising” X consist entirely of X)? , Or something additional (eg, X + Y).

  The term “about” for the numerical value x means, for example, x ± 10%.

  The word “substantially” does not exclude “completely” (eg, a composition that is “substantially free” of Y may not be completely free of Y). Where necessary, the word “substantially” may be excluded from the definition of the invention.

  Identity between polypeptide sequences is preferably performed as Smith-Waterman as performed in the MPSRCH program (Oxford Molecular) using an affine gap search with the parameters gap open penalty = 12 and gap extension penalty = 1. Determined by homology search algorithm.

  Detailed information about hepatitis C virus (including life cycle, replication, culture conditions, genome, polyprotein, proteolytic processing, etc.) can be found in chapters 32-34 of reference 53 .

(Mode for carrying out the invention)
Reference 18 describes an improved method for HCV replicon expression in addition to E1 and E2 resulting from translation of the HCV genome, in which the E1 and E2 proteins are expressed separately. . The lentiviral vector provides E1 and E2 together with the p7 protein (E1E2p7 protein) to 21-5 cells [4], which lentiviral vector integrates into the chromosome to give 21-5_R809 cells. Confocal microscopy shows that core protein, E1 protein, E2 protein, NS3 protein, NS5a protein co-localize with the HCV genome in these cells to form a dot-like structure.

  Further studies on co-localized materials are now being carried out and it has surprisingly been found that caveolin-3 is present. There is no suggestion in the prior art that caveolin-3 is expressed in liver cells.

  Confocal microscopy was performed on Huh-7 and 21-5 cells, and the anti-caveolin-3 antibody showed only a hazy label with no defined pattern. However, by Western blot, the protein is found in naive Huh-7 cells, in Huh-7 cells transfected with HCV replicon, and in Huh-7 cells transfected with both replicon and lentiviral vectors. I was able to. These results suggest that caveolin-3 is expressed at low levels in Huh-7 cells and accumulates in dots containing E1 / E2 rather than being induced by HCV infection. is doing.

  FIG. 1 shows immunofluorescence microscopy of 21-5 and 21-5_R809 cells. HCV E1 / E2 protein is not found in naive 21-5 cells (FIG. 1C), but is found after introduction of the R809 lentiviral vector (FIG. 1F). Caveolin-3 is diffusely detected in naive 21-5 cells (FIG. 1B), but bright spots can be found in cells infected with R809 (FIG. 1E). The overlay of FIGS. 1E and 1F (FIG. 1B) shows that E1 / E2 and caveolin-3 co-localize. NS3, NS5a and viral RNA are also found in these respects.

  The usual stimulus for the induction of caveolin-3 in muscle cells is serum deprivation. This technique is therefore used for 21-5 and 21-5_R809 cells, both of which have full length HCV replicons. This technique was also used for these cells along with transfection with lentiviral vectors.

  FIG. 2 shows the immunofluorescence of 21-5 cells infected with R809 and 21-5 cells not infected with R809 72 hours after serum deprivation. Compared to FIG. 1, the expression of E1 / E2 can be found and caveolin-3 shows the expression at the spot. E1 / E2 and caveolin-3 are colocalized.

  FIG. 3 is a Western blot of various Triton X-100 cell extracts stained with anti-caveolin-3 serum. Mouse muscle cells (lanes 1 to 4) show caveolin-3 expression. The resulting cells (lane 1 and lane 2) show expression, with increased expression 48 hours (lane 3) or 96 hours (lane 4) after serum deprivation. Huh-7 cells (lane 5) without horse serum (2%) or Huh-7 cells with horse serum (2%) (lane 6) show no detectable caveolin-3 expression. Huh-7 after infection with R809 shows similar results (lanes 7 and 8). Neither 21-5 cells infected with R809 (lane 9 and lane 10) nor 21-5 cells infected with R809 (lane 11 and lane 12) show detectable caveolin-3.

  As described in reference 18, E1 and E2 expression is not found in natural 21-5 cells. However, by immunofluorescence, expression of E1 and E2 could be found after serum deprivation and also showed a dot-like structure associated with caveolin-3. Western blot analysis showed that the overall expression level of caveolin-3 in 21-5 cells did not increase during serum deprivation (unlike the effect seen in muscle cells) and therefore Instead, it seems that the diffused protein concentrates on a specific structure, so it can be visualized by immunofluorescence.

  Overall, these results suggest that caveolin-3 appears to play a role in HCV maturation.

  It will be understood that the invention has been described by way of example only and modifications may be made within the scope and spirit of the invention.

FIG. 1 shows the results of immunofluorescence using naive 21-5 cells (FIGS. 1A, 1B, 1C) and 21-5 cells infected with R809 (FIGS. 1D, 1E, 1F). This staining antibody is either anti-E1 / E2 (FIG. 1C, FIG. 1F) or anti-caveolin-3 (FIG. 1B, FIG. 1E). FIG. 1A is an overlay of FIGS. 1B and 1C. FIG. 1D is an overlay of FIGS. 1E and 1F. FIG. 2 is similar to FIG. 1 but shows cells after 72 hours of serum deprivation. FIG. 3 is a Western blot of cell extracts. The markers in lane 13 are 193 kDa, 102.9 kDa, 59.9 kDa, 41.2 kDa, 27.6 kDa, 20.7 kDa and 15.4 kDa.

Claims (23)

A cell comprising (i) a hepatitis C virus genome and / or a nucleic acid encoding a hepatitis C virus genome; and (ii) expressing caveolin-3. A cell comprising (i) a hepatitis C virus genome and / or a nucleic acid encoding hepatitis C virus genome, and (ii) a nucleic acid encoding caveolin-3. The cell according to claim 2, wherein the nucleic acid is an mRNA transcript encoding caveolin-3. An in vitro cell culture comprising the cell according to claim 1. A process for propagating hepatitis C virus, comprising: (a) providing a cell that expresses caveolin-3 and comprises a hepatitis C virus genome and / or a nucleic acid encoding the hepatitis C virus genome; And (b) a process comprising culturing the cell. A process for propagating hepatitis C virus comprising: (a) (i) a nucleic acid encoding caveolin-3; and (ii) a nucleic acid encoding hepatitis C virus genome and / or hepatitis C virus genome. Transfecting the cells; and (b) culturing the cells. A process for propagating hepatitis C virus comprising the steps of: (a) transfecting a cell with a vector encoding caveolin-3, wherein the cell is a hepatitis C virus genome and / or hepatitis C virus. A process comprising a nucleic acid encoding the genome; and (b) culturing the cell. 8. The process of claims 5-7, further comprising (c) purifying HCV virions. A process for propagating hepatitis C virus, comprising: (a) transfecting the cell with a nucleic acid encoding caveolin-3; (b) infecting the cell with hepatitis C virus; and (c ) A process comprising culturing the cells. A process for growing hepatitis C virus comprising: (i) an endogenous caveolin-3 gene; and (ii) a hepatitis C virus genome and / or a nucleic acid encoding a hepatitis C virus genome, Culturing under conditions where the caveolin-3 gene is expressed. A process for growing hepatitis C virus comprising: (a) a cell comprising (i) a caveolin-3 gene and (ii) a hepatitis C virus genome and / or a nucleic acid encoding a hepatitis C virus genome. Providing a process; and (b) maintaining the cell under conditions of serum deprivation. 12. Process according to claims 9-11, further comprising the step of purifying HCV virions. A process for modifying a cell that does not express caveolin-3, comprising (i) transfecting the cell with a vector encoding caveolin-3; or (ii) initiating expression of an endogenous caveolin-3 gene Initiating caveolin-3 expression in the cell by a method selected from altering a regulatory component of the endogenous gene in the cell to cause An animal comprising the cell of the present invention. The animal according to claim 15, wherein the animal is a mouse. A proteinaceous complex comprising (i) CD81; (ii) HCV protein; and (iii) caveolin-3. A process for screening for compounds that inhibit the interaction between caveolin-3 and HCV protein, the method comprising inhibiting the interaction between caveolin-3 and HCV protein in the presence of a candidate compound A process comprising the step of evaluating. 18. The process of claim 17, wherein (i) mixing caveolin-3, HCV protein and one or more candidate compounds; (ii) incubating the mixture to caveolin-3, HCV protein and Allowing any interaction of the candidate compound; and (iii) assessing whether the interaction between caveolin-3 and the HCV protein is inhibited. 19. A cell, process, culture, animal or complex according to any of claims 1 to 18, wherein the cell comprises a nucleic acid encoding the HCV genome in the form of DNA or RNA. 20. The cell, process, culture, animal or complex of any of claims 1-19, wherein the caveolin-3 comprises the amino acid sequence GI: 4502589. 14. A cell, process or culture according to any one of claims 1 to 13, wherein the cell is a mammalian cell. 24. The cell, process or culture of claim 21, wherein the cell is a human cell. 23. The cell, process or culture of claim 21 or claim 22, wherein the cell is derived from the liver.

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