WO2010137570A1 - Anti-viral agent or an anti-cancer agent - Google Patents

Abstract

Disclosed are: an anti-viral agent or an anti-cancer agent; and a process for producing the anti-viral agent or the anti-cancer agent. The anti-viral agent or the anti-cancer agent comprises: a fusion product of HBs antigen particles and a liposome; and a small nucleic acid molecule which can mediate RNA interference (RNAi) that enables the cleavage of mRNA for a viral nucleic acid or a target cancer gene and which is encapsulated in the fusion product.

Description

Antiviral or anticancer agent

The present invention relates to an antiviral agent or an anticancer agent, or a production method thereof.

In RNAi (RNA interference), when a double-stranded RNA (dsRNA) that is homologous to the target gene is introduced into the cell, the homologous portion of the mRNA that is the transcription product of the target gene is specifically degraded. This is a phenomenon in which expression is suppressed. Since the discovery of RNAi in nematodes in 1998 (Fire et al., Nature, 391, 806-811, 1998), RNAi phenomena have been observed in various species, including mammals including humans in 2001. However, it was reported that siRNA (small interference RNA), which is a dsRNA of about 21 to 23 bases, can cause an RNAi phenomenon (Elbashirst al., Nature, 411, 494-498, 2001).

Currently, great expectations are placed on the application of RNAi technology to pharmaceuticals. Most diseases are thought to be caused by some kind of protein dysfunction, and siRNA capable of suppressing the expression of the abnormal protein may be applied to a large number of diseases. Moreover, since the gene expression suppression effect by siRNA is high in sequence specificity, it is assumed that the effect appears only in the target causal factor, and almost no side effects occur. Currently, drugs using siRNA (siRNA drugs) are based on a mechanism of action different from conventional drugs, and safety in humans has not been fully established. Development has been ahead as a therapeutic agent for diseases such as cancer and infectious diseases.

Another advantage of siRNA medicines is that siRNA as an active ingredient can be designed if the relationship between the disease and the gene becomes clear, and the active ingredient can be easily synthesized. While conventional small molecule drugs have spent a great deal of time and effort searching for their active ingredients, siRNA drugs can minimize such efforts.

On the other hand, siRNA has a very bad in vivo stability, and it has a big problem that it has poor biological membrane permeability. Yet another major problem is the long-lasting delivery of the required amount of siRNA only to the target cells.

Much research has been conducted on DDS technology for siRNA. In order to introduce siRNA into a cell and to exert its function, it is necessary to clear several steps as follows.
1. Arrival of siRNA complex to cells Since siRNA is easily degraded by lytic enzymes in blood such as exonuclease, it is degraded before reaching the target site. For this reason, it is necessary to protect against degrading enzymes.

2. SiRNA uptake into cells
Since siRNA itself cannot penetrate the cell membrane, it is not taken into the cytoplasm. For this reason, it is carried out through a cellular uptake mechanism such as endocytosis. Membrane-permeable peptides and cationic lipids were thought to act directly on the cell membrane and introduce siRNA into the cell, but at least the membrane-permeable peptide passed siRNA into the cell via a pathway via endocytosis. It is known that cationic lipids are involved in endocytosis in cases other than when used at high concentrations.

3. Escape from the uptake mechanism into the cytoplasm The siRNA taken in through the uptake mechanism enters intracellular vesicles mainly composed of endosomes. Since siRNA does not act as it is, it is necessary to escape from it. For escape from endosomes, those using pH-sensitive lipids, some membrane-permeable peptides, cationic lipids, and the like are used.

When all the above steps are cleared, the knockdown effect by siRNA can be seen.

On the other hand, Patent Documents 1 and 2 describe the delivery of siRNA using HBs antigen particles (HBs antigen particles) containing HBs (hepatitis B virus surface) antigen protein as a constituent element. It is not described that it can be used.

Since siRNA is rapidly degraded even after being introduced into the cell or after being introduced into the cell, it is necessary to continuously introduce the siRNA into the cell.

JP2008-162981 JP2008-029249

The present invention can introduce a small nucleic acid molecule capable of mediating RNA interference (RNAi) into target cells, tissues or organs continuously for a long time, has a low toxicity, and can suppress the expression of the target gene sufficiently. An object is to provide a deliverable antiviral agent or anticancer agent.

The present inventor provides the following antiviral agents or anticancer agents and methods for producing them.
Item 1. An antiviral agent or anticancer agent comprising a small nucleic acid molecule capable of mediating RNA interference (RNAi) capable of cleaving viral nucleic acid or cancer target gene mRNA in a fusion of HBs antigen particles and liposomes.
Item 2. Item 2. The antiviral agent or anticancer agent according to Item 1, wherein the HBs antigen comprises a targeting portion of a cell, organ or tissue.
Item 3. Item 3. The antiviral agent or anticancer agent according to Item 1 or 2, wherein the HBs antigen is an HBs antigen (Q129R, G145R).
Item 4. Item 5. The antiviral agent according to any one of Items 1 to 3, wherein the small nucleic acid molecule capable of mediating RNA interference (RNAi) capable of cleaving viral nucleic acid or hepatocellular carcinoma target gene mRNA is siRNA, miRNA or shRNA. Anticancer drugs.
Item 5. Item 5. The antiviral agent or anticancer agent according to any one of Items 1 to 4, wherein the target gene is a target gene expressed in hepatocytes.
Item 6. Production of antiviral agent or anticancer agent characterized in that liposome and HBs antigen particles are fused in the presence or absence of a surfactant, and a small nucleic acid molecule capable of mediating RNA interference (RNAi) is added to the fusion. Method.
Item 7. Surfactant is MEGA-8, Triton X-100, Tween 20, Tween 80, N-lauroyl sarcosine-sodium salt, lithium dodecyl sulfate, sodium cholate, sodium deoxycholate, SDS, cetylpyridinium chloride, CTAB, CHAPS, Item 7. The method according to Item 6, selected from the group consisting of CHAPSO, sulfobetaine SB10, and sulfobetaine SB16.
Item 8. As an antiviral agent or anticancer agent of a particle comprising a small nucleic acid molecule capable of mediating RNA interference (RNAi) capable of cleaving viral nucleic acid or cancer target gene mRNA in a fusion of HBs antigen particle and liposome Use of.
Item 9. Administering to a mammal an effective amount of a particle comprising a small nucleic acid molecule capable of mediating RNA interference (RNAi) capable of cleaving viral nucleic acid or cancer target gene mRNA in a fusion of HBs antigen particle and liposome. A method for treating viral infection or cancer.

The antiviral agent or anticancer agent of the present invention comprises an antiviral agent in which a small nucleic acid molecule capable of mediating RNA interference (RNAi) capable of cleaving mRNA of viral nucleic acid is encapsulated in a fusion of HBs antigen particles and liposome, It includes both anticancer agents formed by encapsulating small nucleic acid molecules capable of mediating RNA interference (RNAi) capable of cleaving mRNA of a target gene in a fusion of HBs antigen particles and liposomes.

The antiviral agent or anticancer agent of the present invention has been demonstrated that small nucleic acid molecules capable of mediating RNA interference (RNAi) such as siRNA, shRNA, miRNA can actually act in cells to treat viral infections and cancer. It is an ideal therapeutic agent that can be expected to have a high effect on viral infections and cancer, and has substantially no side effects.

The measurement result of mRNA about HepG2 and TE10 is shown. The measurement result of the cell growth ability about HepG2 is shown. 48 hours later luciferase assay with L particles. The results of a luciferase assay after 48 hours using DharmaFECT® transfection reagent are shown. The structure of the gene construct of Example 2 is shown.

The HBs antigen particles of the present invention are nanoparticles having hepatitis B virus HBs antigen protein or a variant thereof as a constituent element.

The fusion of the present invention is obtained by fusing HBs antigen particles and liposomes.
Small nucleic acid molecules that can mediate RNA interference (RNAi) include siRNA (short interfering RNA), shRNA (short hairpin RNA), miRNA (microRNA), siNA (short interfering nucleic acid), dsRNA (double stranded RNA), Antisense RNA is mentioned, Preferably siRNA and shRNA are mentioned.

The active ingredient of the antiviral agent or anticancer agent of the present invention is one in which a small nucleic acid molecule capable of mediating RNA interference (RNAi) is encapsulated in a fusion of HBs antigen particles and liposomes. Here, “encapsulation” means that a small nucleic acid molecule is complexed with a fusion in a state where it is not substantially degraded in a living body such as blood before being introduced into a target cell or the like. Including both when encapsulated inside or in a fusion body and when outside the HBs antigen particle but interacts with a part of the liposome and is not degraded by an enzyme such as RNase . Small nucleic acid molecules that can mediate RNA interference (RNAi) are small nucleic acid molecules that can fuse HBs antigen particles and liposomes in the presence or absence of a surfactant first, and then mediate RNA interference (RNAi) Or when a small nucleic acid molecule capable of mediating RNA interference (RNAi) is mixed and reacted at the same time, the small nucleic acid molecule is present outside the HBs antigen particle. It is thought that it interacts with a part of the liposome and is complexed without being degraded by an enzyme such as RNase. On the other hand, when a liposome encapsulating a small nucleic acid molecule capable of mediating RNA interference (RNAi) and HBs antigen particles are mixed and reacted in the presence of a surfactant as necessary, the small nucleic acid molecule becomes HBs antigen. It can be both encapsulated in the particle and outside the HBs antigen particle, but it can interact with a part of the liposome and fuse or complex without being degraded by an enzyme such as RNase, These can be present alone or as a mixture.

The HBs antigen particle of the present invention comprises an HBs antigen protein having particle-forming ability or a modified form thereof and a lipid membrane as structural elements, for example, the HBs antigen protein having the particle-forming ability or a modified form thereof is spherical, elliptical, or It has a structure complexed with a lipid membrane having a similar shape.

In the present specification, examples of the HBs antigen particles having HBs antigen protein or a modified form thereof as constituent elements include particles having HBs antigen protein (HBsAg) or a modified form thereof and a lipid membrane as constituent elements.

In the present specification, HBs antigen particles include HBs antigen protein or a variant thereof as a main component, and the protein is retained in a lipid membrane. In addition, sugar chains are usually bound to the protein. This sugar chain is bound in a eukaryotic cell when the HBs antigen protein or a modified form thereof is expressed in a eukaryotic cell. However, after the HBs antigen particle is obtained, the sugar chain is chemically bonded by a covalent bond or the like. A sugar chain may be bound to.

Examples of HBs antigen particles include those obtained by expressing an HBs antigen protein or a modified form thereof in eukaryotic cells including mammalian cells such as yeast, insect cells or CHO cells. A method for producing HBs antigen particles is described in JP-A-2001-316298, and a method for preparing HBs antigen is Vaccine. 2001 2001 Apr 30; 19 (23-24): 3154-63. Physicochemical and immunological characterization of hepatitis B virus and envelope particles exclusively consisting of the entire L (pre-S1 + pre-S2 + S) protein. Yamada T, Iwabuki H, Kanno T, Tanaka H, Kawai T, Fukuda H, Kondo A, Seno M, Tanizawa. It is described in.

When an HBs antigen protein is expressed in a eukaryotic cell, the protein is expressed and accumulated as a membrane protein on the endoplasmic reticulum membrane, and is released as a nanoparticle, resulting in a structure having a lipid membrane. Since HBs antigen particles expressed in eukaryotic cells do not contain any HBV genome, they are extremely safe for the human body.

In one preferred embodiment, the HBs antigen particles are composed of 65 to 80 parts by weight of HBs antigen protein or a variant thereof, 8 to 20 parts by weight of lipid, and 5 to 20 parts by weight of a sugar chain. The HBs antigen particles (L particles) used in the examples of the present application comprise about 70 parts by weight of HBs antigen protein or a variant thereof, about 16 parts by weight of sugar chain, and about 13 parts by weight of lipid.

The small nucleic acid molecule of the present invention, for example, siRNA, shRNA (short hairpin RNA), miRNA is not particularly limited as long as it can degrade viral nucleic acid or RNA of cancer cell target gene (especially mRNA). If it is decided, it can be designed according to the ordinary method. The length of siRNA is preferably 21 to 23 bases, but may have a longer or shorter sequence of, for example, 15 to 30 bases as long as the target RNA can be cleaved. shRNA is an appropriate length of two siRNAs (lower limit is 0, 1, 2, 3, 4 or 5 bases, upper limit is 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, or 10 bases) RNA are exemplified.

In a preferred embodiment of the present invention, the liposome HBs antigen particle fusion excluding small nucleic acid molecules after fusion with the liposome is 10 to 60 parts by weight of HBs antigen protein or a variant thereof, 40 to 95 parts by weight of lipid. It may be composed of 1 to 10 parts by weight of sugar chains (including phospholipids).

Examples of lipids constituting HBs antigen particles include membrane components derived from eukaryotic cells such as yeast or animal cells (eg, phospholipids, cholesterol, etc.) and phospholipids constituting liposomes. The sugar chain is introduced into the protein when the HBs antigen protein is expressed in a eukaryotic cell, but the sugar chain may be bound as a site that specifically recognizes a target cell / tissue / organ. Is possible. An example of a sugar chain that can be used as a target recognition site is sialyl Lewis X. Since sialyl Lewis X interacts with a lectin protein present on the surface of an inflamed cell, it can be used for targeting an inflamed site in vivo. A sugar chain as a target recognition molecule such as sialyl Lewis X may be introduced into any particle before and after the introduction of the complex. Alternatively, target a virus-infected cell or cancer cell by introducing an aldehyde group by oxidizing the sugar chain with an oxidizing agent such as NaIO ₄ and binding a ligand that recognizes a specific cell, organ, tissue, etc. Is possible.

The HBs antigen protein contains S protein as an essential component and may further contain two regions, PreS1 and PreS2. S protein (226 amino acids) has the ability to form particles. Pre-S2 consisting of 55 amino acids added to S particles is M protein (M particle constituent protein), and M protein added with Pre-S1 consisting of 108 or 119 amino acids is L protein (L particles) Protein). L protein and M protein have the ability to form particles, as does S protein. Therefore, the two regions of PreS1 and PreS2 may be arbitrarily substituted, added, deleted, or inserted. For example, by using a modified protein in which the hepatocyte recognition site contained in amino acid residues 3 to 77 of the Pre-S1 region is deleted, hollow particles having lost hepatocyte recognition ability can be obtained. In addition, since the PreS2 region includes a site that recognizes hepatocytes via albumin, this albumin recognition site can also be deleted. On the other hand, since the S region (226 amino acids) is responsible for particle forming ability, modification of the S region must be performed so as not to impair the particle forming ability. For example, in the S region, a protein in which Gln at position 129 is substituted with Arg and / or Gly at position 145 is substituted with Arg decreases the antigenicity of HBs antigen particles, and the ability to form particles and siRNA into cells. Since the introduction ability is not impaired, it is preferable. Particularly, in the S region of the L protein, the HBs antigen (Q129R, G145R), which is a protein in which Gln at position 129 is substituted with Arg and Gly at position 145 is substituted with Arg, It is preferable because it has particle forming ability and siRNA introduction ability into cells, and has low immunogenicity.

The present inventors have clarified that the PreS1 region, particularly the amino acids at positions 1 to 50 in the PreS1 region, contribute to fusion with lipids such as liposomes. Accordingly, the HBs antigen protein of the present invention or a variant thereof preferably has a PreS1 region, particularly the 1st to 50th positions of the PreS1 region, and even if this region has alterations such as substitution, addition, deletion, insertion, etc. It is preferable that the mutation does not impair the lipid fusion ability in this region.

Various variants of HBs antigen protein are widely included as long as they have the ability to form particles. Taking HBs antigen protein as an example, any number of substitutions, deletions and additions for PreS1 and PreS2 regions With respect to the S region, one or several or a plurality of, for example, 1 to 120, preferably 1 to 50, more preferably 1 to 20, even more preferably 1 to 10, particularly 1 Up to 5 amino acids may be substituted, added, deleted or inserted. As a method for introducing mutations such as substitution, addition, deletion, insertion and the like, DNA encoding the protein can be obtained by using, for example, cytospecific mutagenesis (Methods in Enzymology, 154, 350, 367-382 (1987); 100 , 468 (1983); Nucleic Acids Res., 12, 9441 (1984)), chemical synthesis means such as phosphate triester method and phosphate amidite method (for example, using a DNA synthesizer) ( J. Am. Chem. Soc., 89, 4801 (1967); 91, 3350 (1969); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981)). Codon selection can be determined by taking into account the codon usage of the host.

In the case of HBs antigen particles composed of proteins capable of recognizing hepatocytes such as L protein as an HBs antigen protein or a variant thereof, it is not necessary to introduce a cell recognition site. On the other hand, HBs antigen particles composed of a modified protein in which the hepatocyte recognition site contained in amino acids 3 to 77 of the Pre-S1 region has been deleted, or a protein in which both the PreS1 and PreS2 regions have been deleted. In this case, since cell recognition cannot be performed as it is, a cell recognition site can be introduced to recognize any cell other than hepatocytes, and the polymer / nucleic acid complex can be introduced into various target cells. Such cell recognition sites for recognizing specific cells include, for example, cell function regulatory molecules consisting of polypeptides such as growth factors and cytokines, cell surface antigens, tissue-specific antigens, receptors and other cells and tissues. These polypeptide molecules, polypeptide molecules derived from viruses and microorganisms, antibodies, sugar chains and the like are preferably used. Specifically, antibodies against EGF receptor and IL-2 receptor overexpressed in cancer cells, EGF, and receptors presented by HBV are also included. Alternatively, a protein capable of binding an antibody Fc domain (for example, a ZZ tag), a strept tag showing biotin-like activity to present a biorecognition molecule labeled with biotin via streptavidin, and the like can also be used.

When the cell recognition site is a polypeptide, the DNA encoding the HBs antigen protein or a variant thereof and the DNA encoding the cell recognition site are linked in-frame via a DNA encoding the spacer peptide as necessary. By incorporating this into a vector and expressing it in eukaryotic cells, HBs antigen particles that recognize any target cell can be obtained.

When the cell recognition site is an antibody, the DNA encoding the HBs antigen protein or a variant thereof and the DNA encoding the ZZ tag are linked in-frame via a DNA encoding a spacer peptide, if necessary, and this is connected to a vector. The HBs antigen particles to which the target antibody is bound can be obtained by mixing the obtained HBs antigen particles and the antibodies capable of recognizing the target cells.

When the cell recognition site is a sugar chain, it can be obtained by linking a sugar chain capable of recognizing cells such as sialyl Lewis X to HBs antigen particles having no cell recognition ability using glycosyltransferase.

Fusion of HBs antigen particles and liposomes can be easily performed by mixing the liposomes and HBs antigen particles in water or an aqueous medium in the presence of a surfactant, if necessary, and stirring or shaking as necessary. (Direct fusion method).

Alternatively, the fusion of HBs antigen particles and liposomes (not including small nucleic acid molecules) is performed by mixing and dialyzing liposomes and HBs antigen particles in the presence of a surfactant in water or an aqueous medium to form a complex. Small nucleic acid molecules may be included.

The size of the fusion of HBs antigen particles containing small nucleic acid molecules and liposomes (HBs antigen fusion particles) as an active ingredient is 50 to 1000 nm, preferably about 60 to 700 nm, more preferably about 70 to 500 nm, particularly preferably. Is about 70 to 300 nm, particularly about 70 to 200 nm. The particle size of the HBs antigen-fused particles affects the half-life and clearance in vivo (particularly in blood).

In the present specification, the size of HBs antigen particles can be measured by a measuring device using a dynamic scattering light method, for example, FPAR1000 (Otsuka Electronics), and the surface charge is measured by Zetasizer Nano-ZS (Malvern Instruments). can do.

When the size of the HBs antigen-fused particles of the present invention is within the above range, it is particularly preferable because the half-life in vivo (especially in blood) is long and small nucleic acid molecules can be introduced into target cells for a long time.

In order to adjust the particle size of the HBs antigen-fused particles encapsulating small nucleic acid molecules, in the direct fusion method, a treatment for minimizing the particle size of the liposome is performed, and then the particles are fused with the HBs antigen particles whose particle size is minimized. . Also in the surfactant method, for example, liposomes adjusted to a small size of, for example, 50 nm to 100 nm and HBs antigen particles with a minimized particle size are prepared, and these are fused in the presence of a surfactant. Further, it is possible by performing a minimization process.

In order to minimize the particle size of the liposome, for example, the particle size can be adjusted by passing it through a filter having a small pore size (for example, 100 nm, 80 nm, 50 nm, etc.) using an extruder. As another method, it is possible to reduce the particle size by processing for several hours with a rod-shaped ultrasonic generator. Of course, there is no problem even if a method for minimizing the liposome particle diameter by another method is used. When minimizing the particle size of HBs antigen particles, for example, the particle size can be reduced by processing for several hours with a rod-shaped ultrasonic generator. There is no problem even if you use. When minimizing the complex by the surfactant method, for example, the particle diameter can be adjusted by passing it through a filter having a small pore diameter (for example, 200 nm, 150 nm, 100 nm, etc.) using an extruder. .

The liposome may be either a multilamellar liposome or a single membrane liposome. The size of the liposome is about 40 to 300 nm, preferably about 50 to 200 nm, particularly about 60 to 150 nm. When the liposome is directly fused with the HBs antigen particle, the size of the liposome is preferably about 0.5 to 2 times that of the HBs antigen particle. When a liposome and HBs antigen particles are complexed using a surfactant, the size of the liposome is arbitrary, but a liposome having the above size is preferably exemplified.

Liposomes can be produced by sonication, reverse phase evaporation, freeze-thaw, lipid dissolution, spray drying, and the like.

Liposomes are prepared and used in an appropriate amount and composition with respect to HBs antigen particles. Specifically, 20 to 1000 parts by weight of liposomes are used per 100 parts by weight of HBs antigen particles.

Examples of liposome components include phospholipids, cholesterols, fatty acids, and the like. Specifically, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid, cardiolipin, sphingomyelin, egg yolk lecithin, soybean In addition to natural phospholipids such as lecithin and lysolecithin, or those hydrogenated by conventional methods, distearoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, dipalmitoyl phosphatidylserine, eleostearoyl phosphatidylcholine, eleostearoyl phosphatidylethanol Amine, eleostearoyl phosphatidyl seri Synthetic phospholipids and the like. It is desirable to use phospholipids in combination with lipids having various degrees of saturation. In addition, cholesterols include cholesterol, phytosterol, and the like, and examples of fatty acids include oleic acid, palmitooleic acid, linoleic acid, and fatty acid mixtures containing these unsaturated fatty acids. Liposomes containing unsaturated fatty acids with small side chains are effective in producing small liposomes because of curvature.

The liposome preferably contains at least one kind of pH-sensitive lipid (for example, CHEMS), a membrane-permeable peptide, and a cationic lipid in order to escape small nucleic acid molecules from the endosome. Cationic lipids include DC-6-14 (O, O'-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride), DODAC (dioctadecyldimethylammonium chloride), DOTMA (N- (2,3-dioleyloxy) propyl-N, N, N-trimethylammonium), DDAB (didodecylammonium bromide), DOTAP (1,2-dioleoyloxy-3-trimethylammonio propane), DC-Chol (3β-N- (N ', N',-dimethyl-aminoethane) -carbamol cholesterol ), DMRIE (1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium), DOSPA (2,3-dioleyloxy-N- [2 (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminum trifluoroacetate), and the like.

In one preferred embodiment, the liposome of the present invention comprises a cationic lipid, a phospholipid, and cholesterol. These ratios are as follows: Cationic lipid: 10 to 60 parts by weight, preferably 20 to 50 parts by weight, more preferably 30 to 40 parts by weight
Phospholipid: 10-50 parts by weight, preferably 15-45 parts by weight, more preferably 20-40 parts by weight,
Cholesterol: 10 to 50 parts by weight, preferably 15 to 45 parts by weight, more preferably 20 to 40 parts by weight.

As the phospholipid, a phospholipid modified with PEG such as DSPE-PG10G and DSPE-PEG350 can also be used.

An example of a method for producing a fusion product of liposomes and HBs antigen particles will be described in detail. For example, the above-described phospholipid, cholesterol and the like are dissolved in an appropriate organic solvent, and the solution is placed in an appropriate container and the solvent is removed under reduced pressure. By distilling off, a phospholipid membrane is formed on the inner surface of the container, and an aqueous solution, preferably a buffer solution, is added thereto and stirred to obtain liposomes. The liposome can be directly or once freeze-dried and then mixed with HBs antigen particles to obtain a fusion of the liposome and HBs antigen particles (direct fusion method). As another method, for example, the above-described phospholipid, cholesterol, and the like are dissolved in a suitable organic solvent, put in a suitable container, and the solvent is distilled off under reduced pressure to form a phospholipid film on the inner surface of the container. Or hydrate in an aqueous medium to produce liposomes. Freeze-thaw the prepared liposomes, adjust the size by ultrasonic treatment, and add a surfactant. A fusion of liposomes and HBs antigen particles can also be obtained by mixing a HBs antigen particle solution containing a surfactant and dialysis treatment (surfactant method). Surfactants include MEGA-8, MEGA-9, MEGA-10, n-octyl-α-D-glucopyranoside, n-octyl-α-D-thioglucoside, n-heptyl-β-D. -Thioglucoside, Triton X-100, Tween 20, Tween 80, N-lauroyl sarcosine-sodium salt, lithium dodecyl sulfate, sodium cholate, sodium deoxycholate, SDS, cetylpyridinium chloride, CTAB, CHAPS, CHAPSO, sulfobetaine And SB10 and sulfobetaine SB16.

In the case of fusion of liposomes and HBs antigen particles by the surfactant method, the concentration of the surfactant is about 0.1 to 5%, preferably about 0.5 to 2%. In this case, HBs antigen particles are used in an amount of about 0.01 to 0.5 parts by weight, preferably about 0.02 to 0.1 parts by weight, and liposomes are about 0.03 to 10 parts by weight, preferably about 0.06 to 5 parts by weight per 100 parts by weight of a solvent such as water. used. The surfactant method is carried out at a temperature of about 5 to 75 ° C., preferably about 10 to 60 ° C. for 10 minutes to 6 hours, preferably about 20 minutes to 3 hours.

In the case of the direct fusion method, HBs antigen particles are used in an amount of about 0.005 to 0.5 parts by weight, preferably about 0.02 to 0.2 parts by weight, and liposomes are about 0.01 to 10 parts by weight, preferably 0.03 to 5 parts per 100 parts by weight of a solvent such as water. About parts by weight are used. The temperature at the time of carrying out the direct fusion method is preferably about 0 to 50 ° C., preferably about 0 to 40 ° C. for 1 minute to 120 minutes, preferably about 2 minutes to 60 minutes.

In the present invention, examples of the virus to be used as an antiviral agent include RNA viruses and DNA viruses such as double-stranded RNA viruses and single-stranded RNA viruses. Infectious diseases of RNA viruses can be exemplified as preferred therapeutic subjects. . The “viral nucleic acid” of the present invention includes nucleic acids derived from these RNA viruses and DNA viruses.

RNA viruses include Arenaviridae (Lassa virus, etc.), Orthomyxoviridae (influenza virus A, B, etc.), Caliciviridae (Norovirus, Sapovirus, etc.), Coronaviridae (SARS virus, avian influenza virus) ), Togaviridae (such as rubella virus), Nodaviridae (such as viral necrosis virus), Paramyxoviridae (such as mumps virus, measles virus, RS virus), Picornaviridae (poliovirus, Coxsackie virus) , Echovirus, etc.), Filoviridae (Marburg virus, Ebola virus, etc.), Bunyaviridae (Crimea-Congo hemorrhagic fever virus, etc.), Flaviviridae (yellow fever virus, Dengue virus, Hepatitis C virus) , Hepatitis G virus), rhabdoviridae (rabies virus), reoviridae, retroviridae (human immunodeficiency virus, human T lymphophilic virus, simian immunodeficiency virus, STLV, etc.).

Cancers to be treated include colon cancer, breast cancer, lung cancer, prostate cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, spleen cancer, kidney cancer, bladder cancer, uterine cancer, testicular cancer, thyroid cancer, pancreatic cancer. Brain tumor, ovarian cancer or blood tumor.

The antiviral agent or anticancer agent of the present invention can introduce a small nucleic acid molecule by targeting a specific organ, tissue, cell or the like.

Cancer target genes include the following:
EGFR, ErbB2, ErbB3, IGF-1R, RET, K-ras, R-ras, CSF-1R, PDGFR-β, FLT-3, Met, EphA2, BRAF, ABL, c-Kit, c-Src, Met, β-catenin, c-Myc, N-Myc, Cycline-D1, PI3K, AKT, IKK-β, NF-κβ, EWS, HIF-1α, XBP-1, HIPV E7, E2F4, HIPV E6, Hdmx, Notch- 1, Delta-like-1, Jagged-1, Cycline B1, Chk1, FLIP, BCL-2, BCL-XL, Survivin, XIAP, Telomerase, Id1, Cks-1, Skp-2, βTRCP1, cathepsin L, VEGF ( AD), PIGF, Angiogenin, Angiotropin, EGF, HGF, SF, FGF, PDGF, TNF-α, IGF-1, Cathepsin, MMP 2, 9, Stromelysin, uPA, c-myc, ras, c-src, v- raf, c-jun, VEGFR1, 2, 3, Thymidine, phospholyrase, RAS-farnesyl, transferase, Geranyl, IL-1, 6, 8, 10, αvβ3 integrin, Ang-1, Angiostatin II, Endothelin, Erythropoietin, iNOS, PAF, Cox-2, Thrombopoietin, CEACAM1, RhoA, CXCR4, IGFBP2, EphA2, EpCAM, TGF-β, IDO, MIC-A, MIC-B, ABCB1, 4, 5, ERCC1.

Mammals to which particles comprising small nucleic acid molecules encapsulated in a fusion of HBs antigen particles and liposomes are administered include humans, cows, pigs, rabbits, rats, mice, dogs, cats, etc. preferable.

The dose of the small nucleic acid molecule to be administered is not particularly limited, and is, for example, about 0.1 ng to about 100 mg / kg / day, preferably about 1 ng to about 10 mg / kg / day per day for a human adult.

The effective amount of the antiviral agent or anticancer agent of the present invention obtained by encapsulating a small nucleic acid molecule in a fusion of HBs antigen particles and liposomes is an amount that can realize the dose of the small nucleic acid molecule.

Hereinafter, although the present invention will be described in more detail based on examples, it is needless to say that the present invention is not limited to these examples.

Example 1
DC-6-14 (O, O'-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride)
DOPE (dioleoylphosphatidylethanolamine)
1. Preparation Method of siRNA Encapsulated BNC-Liposome Chloroform, DC-6-14, DOPE, Cholesterol were dissolved in a 200 ml eggplant type flask to prepare a lipid solution. (Molar ratio of DC-6-14: DOPE: Cholesterol: DSPE-PG10G = 4: 3: 3: 1) Set the prepared eggplant-shaped flask on a rotary evaporator, purge with nitrogen, depressurize and distill off chloroform. A lipid film was prepared. Otsuka distilled water was added to the prepared lipid film for hydration, and the hydrated solution was repeatedly frozen and thawed with liquid nitrogen to prepare liposomes. PCMB 25mm, 2μm (Whatman 110602), PCMB 25mm, 1μm (Whatman 110603), PCMB 25mm, 0.08μm (Whatman 110604), PCMB 25mm, 0.05μm (Whatman 110605) using NLI LIPEX Extruder , PCMB 25 mm, 0.03 μm (Whatman 110606), each three times in order, the particle size was prepared and filter sterilized. 10 μl of the liposome solution was diluted with 240 μl of Otsuka distilled water, and the particle size was measured using FPAR-1000AS (Otsuka Electronics). The liposome particle size was 68-75 nm. Cholesterol concentration was measured by the cholesterol E-test, and the liposome total lipid concentration was calculated from the measured value. The total liposome lipid concentration was 7.55 mg / ml. About 10% of PG-10G was added to the liposome. SiRNA and BNC-ST on the liposome (HBs antigen (Q129R, G145R), a protein in which Gln at position 129 is replaced with Arg and Gly at position 145 is replaced with Arg in the S region of the L protein in L particles) And stealth L particles obtained by expressing the yeast in yeast, and the mixture was quickly mixed and filter sterilized. A mixing ratio of 0.5 to 3 was used for BNC with respect to liposome 1 in terms of weight ratio.

2. Quantification of mRNA by quantitative RT-PCR
2 × 10 ⁵ cells (HepG2, TE10) were seeded in a 6-well dish (Corning 3516).
After 24 hours, siRNA-encapsulated BNC-Liposome was added to a final concentration of 250 nM as siRNA. Another 24 hours later, a medium change was performed. Medium is MEM (SIGMA M4655) with 10% FBS (JRH 12303 500M) added to HepG2 and DMEM (SIGMA D5796) medium for TE10. On the third day after siRNA introduction, RNA was extracted with the QIAGEN RNeasy Mini Kit (74104). Refer to the manufacturer's instruction manual for the protocol. Using ABI's High Capacity RNA-to-cDNA Kit (4387406), cDNA was prepared from approximately 2 μg of RNA, and PCR was performed using the ABI 7300 Real Time PCR System (using TaqMan Gene Expression Assay ABI 436916). The ΔΔcT value was obtained and compared with the cells of.

The results are shown in Figure 1. In FIG.
Ep EpCAM siRNA
Sc scramble siRNA
No Mock siRNA (BNC-Liposome only)
NC negative control (untreated).

The sequence of siRNA is shown below.
EpCAM siRNA sequence (purchased from Ambion ID: 11452)
Sense 5'-GGCAGAAAUGAAUGGCUCA (TT) (SEQ ID NO: 1)
Antisense 5'-UGAGCCAUUCAUUUCUGCC (TT) (SEQ ID NO: 2)
Scramble siRNA sequence (see Osta et.al 2004 Cancer Research)
Sense 5'-UUCUCCGAACGUGUCACGU (TT) (SEQ ID NO: 3)
Antisense 5'-ACGUGACACGUUCGGAGAA (TT) (SEQ ID NO: 4)
As shown in FIG. 1, in HepG2 cells derived from hepatocytes, EpCAM expression was suppressed to 40% by the siRNA introduction method of the present invention. There was no change in EpCAM expression in scrambled siRNA or mock. On the other hand, in the esophageal cancer cell TE10, no apparent decrease in mRNA was observed even when siRNA against EpCAM was used.

3. Cell growth rate measurement
1 × 10 ⁴ cells (HepG2) were seeded in a 96-well dish (Corning 3599). After 24 hours, siRNA-encapsulated BNC-Liposome was added to a final concentration of 250 nM as siRNA. A medium change was performed 24 hours later. Medium is a MEM medium supplemented with 10% FBS as described above. On the third day after the introduction of siRNA, cell proliferation rates were compared using Cell Proliferation ELISA, BrdU Kit (Roche 11 647 229 001) with reference to untreated cells (negative control: NC). The protocol is attached.
The result is shown in figure 2.
As shown in FIG. 2, it was confirmed that the HepG2 cells into which the siRNA of the present invention was introduced had cell growth suppressed and had an antitumor effect.

Example 2
Inhibition of viral growth by an antiviral agent, wherein liposomes and HBs antigen particles are fused in the presence or absence of a surfactant and a small nucleic acid molecule capable of mediating RNA interference (RNAi) is added to the fusion. (1) Cell lines and their subcultured HCV
Replicon cell line OR6 (HuH-7 cell-derived polyclonal OR / C-5B / KE full-length HCV RNA replicating cell) 10% fetal bovine serum (FBS: manufactured by Biowest), penicillin streptomycin (invitrogen) ) And DMEM medium (invitrogen) containing geneticin (300 ug / ml invitrogen) so that it does not become confluent in a 10 cm ² collagen-coated tissue culture dish (BD Biocoat: 354450) It was cultured under conditions of 5% CO ₂ and 37 ° C. with caution, and transferred to a new tissue culture flask using a trypsin-EDTA solution (manufactured by Sigma) during the logarithmic growth phase and subcultured.

(2) Growth inhibition test of OR6HCV replicon cells with siRNA using L particles and commercially available transfection reagents
(I) Transfection protocol and luciferase assay protocol
For 100 nM siRNA, the transfection reagent (DharmaFECT1) (registered trademark) was used.
1. Collagen-coated tissue culture 24-well plate (Collagen coated I 24-Well Plate: BD Biocoat 354408) was seeded with 2 × 10 ⁴ 500 μl cells and the cells were completely antibiotic-free overnight Culture was performed in a DMEM (manufactured by Invitrogen) medium containing 10% FBS (manufactured by Biowest).
Tube 1 preparation: 87.5 μl of 2 μM siRNA solution was added to 87.5 μl of serum free medium. The total volume was 175 μl.
Tube 2 preparation: 3.5 μl of DharmaFECT1® was added to 171.5 μl of serum free medium. The total volume was 175 μl.
2. The contents of tubes 1 and 2 were carefully mixed using a pipette and incubated at room temperature for 20 minutes.
3. 1400 μl of antibiotic-free complete medium was added.
4. The culture medium was removed from each well of the 24-well plate and 500 μl of transfection medium was added.
5. Cells were cultured for 48-72hr (for protein analysis)
6. Cells were cultured for 48-72 hours for Renilla luciferase (RL) assay. Treated cells were harvested with Renilla Lysis Reagent (Promega) and subjected to RL assay according to manufacturer's protocol. Cells were seeded at least in triplicate for each assay.

(II) L particle and luciferase assay protocol for siRNA transfection
1. Collagen-coated tissue culture 24-well plate (Collagen coated I 24-Well Plate: BD Biocoat 354408) was seeded with 2 × 10 ⁴ 500 μl cells and the cells were completely antibiotic-free overnight The medium was cultured in a DMEM (manufactured by Invitrogen) medium containing 10% FBS (manufactured by Biowest).
2. L particle solution containing siRNA was added to the cells.
3. After 1 hour, the cell culture medium was replaced with antibiotic-free complete medium.
4. Cells were cultured for 48 hours.
5. Cells were harvested with Renilla Lysis Reagent (Promega) and subjected to RL assay according to manufacturer's protocol.

SiRNA (ON-TARGETplus Non-targeting siRNA # 1) commercially available from Dharmacon was used as a negative control that does not suppress cell growth, and siRNA against the renilla luciferase gene contained in the replicon was used as a positive control that suppresses HCV. siRNA used as negative control: ON-TARGETplus Non-targeting siRNA # 1
Cat # D-001810-01-05
The nucleotide sequence of siRNA for luciferase is the following sequence:
It consists of 5'- GGCCUUUCACUACUCCUACUU -3 '(SEQ ID NO: 5).
The nucleotide sequence of siRNA for HCV is:
It consists of 5′-GUCUCGUAGACCGUGCAUCAUU-3 ′ (SEQ ID NO: 6).
The luciferase value expressed in the replicon cells and the rate of change of the luciferase expression value when various transfection reagents and SiRNA were introduced were determined.

The results of the luciferase assay after 48 hours using L particles (FIG. 3), 48 hours (FIG. 4) and 72 hours (FIG. 5) using DharmaFECT® transfection reagent are shown. 3-5,
Mock: Add only transfectin agent. Only DharmaFECT1 (registered trademark) or L particles do not contain SiRNA. Nontarget: Dharmacon contains commercially available nontarget siRNA
posi (hluc): Contains siRNA targeting the sequence of the Renilla reporter gene. Used as a positive control because the replicon cell contains the Renilla reporter gene.
siHCV: An siRNA created using the HCV RNA sequence as a target.

When L particles for siRNA transfection are used When the luciferase activity value after 48 hours of the replicon cell line OR6 to which nothing is added is defined as 100%, the transfection reagent (SiRNA is not included) (L particle only) Luciferase activity value when only introduced, Non-targeting siRNA # 1 200 nM as a negative control, siRNA 200 nM for the Renilla reporter gene sequence used as a positive control, siRNA 200 nM for the HCV RNA sequence The luciferase activity values introduced into the cell line OR6 were MOCK (80.1% ± 27%), negative control (42.5% ± 7.2%), positive control (12.1% ± 2.0%), and siRNA against the HCV RNA sequence, respectively. (12.9% ± 2.3%) Virus replication rate decreased to (mean ± standard deviation (SD)).

When the transfection reagent (DharmaFECT1) (registered trademark) is used When the luciferase activity value after 48 hours of the replicon cell line OR6 to which nothing is added is defined as 100%, the transfection reagent (DharmaFECT) ( The ratio of the luciferase activity value when only registered (registered trademark) is introduced, and when non-targeting siRNA # 1 100 nM is introduced as a negative control, siRNA 100 nM against the Renilla reporter gene sequence used as a positive control, siRNA 100 nM against the HCV RNA sequence The luciferase activity values introduced into the introduced replicon cell line OR6 are MOCK (1.51% ± 0.84%), negative control (1.47% ± 1.25%), positive control (0.59% ± 0.11%), siRNA against the sequence of HCVRNA (0.89), respectively. % ± 0.27%) The virus replication rate decreased to (mean ± standard deviation (SD)).

The commercially available transfection reagent itself was highly toxic and siRNA could not be evaluated. On the other hand, when L particles were used, the toxicity was limited to about 20%, suggesting that the toxicity is suppressed to a small extent compared with a commercially available transfection reagent.

The OR6 replicon cell is a cell model in which a chimeric RNA containing a fragment of the HCV genome necessary for the replication of the HCV genome is continuously maintained in the human hepatoma cell line HuH7.

Replicon RNA is originally an ribosome insertion site (IRES) derived from the encephalomyocarditis virus (EMCV) gene downstream of the 5 'UTR downstream of the renyluciferase reporter gene, the cytotoxic neomycin resistance gene in the HCV genome. ) Is inserted. This sequence initiates a translation reaction from the inside of the RNA, and the structural (s) protein and nonstructural (NS) protein are efficiently translated downstream. After introducing RNA synthesized by in vitro transcription reaction into HuH7 cells derived from liver cancer, selection is performed with neomycin. When the introduced RNA is efficiently self-replicated in the cell and a clone is selected with neomycin, the cell becomes viable. In this way, HCV genome sequences that can self-replicate in cells and cells that allow self-replication are selected and replicon-replicating cells are obtained. Specifically, OR6 cells are genotype 1 and strain O-derived replicon cells (FIG. 5).

Claims (9)

An antiviral agent or anticancer agent comprising a small nucleic acid molecule capable of mediating RNA interference (RNAi) capable of cleaving viral nucleic acid or cancer target gene mRNA in a fusion of HBs antigen particles and liposomes. The antiviral agent or anticancer agent according to claim 1, wherein the HBs antigen comprises a targeting portion of a cell, organ or tissue. The antiviral agent or anticancer agent according to claim 1 or 2, wherein the HBs antigen is an HBs antigen (Q129R, G145R). The antiviral agent according to any one of claims 1 to 3, wherein the small nucleic acid molecule capable of mediating RNA interference (RNAi) capable of cleaving viral nucleic acid or mRNA of a hepatoma target gene is siRNA, miRNA or shRNA. Or anticancer drugs. The antiviral agent or anticancer agent according to any one of claims 1 to 4, wherein the target gene is a target gene expressed in hepatocytes. Production of antiviral agent or anticancer agent characterized in that liposome and HBs antigen particles are fused in the presence or absence of a surfactant, and a small nucleic acid molecule capable of mediating RNA interference (RNAi) is added to the fusion. Method. Surfactant is MEGA-8, Triton X-100, Tween 20, Tween 80, N-lauroyl sarcosine-sodium salt, lithium dodecyl sulfate, sodium cholate, sodium deoxycholate, SDS, cetylpyridinium chloride, CTAB, CHAPS, 7. The method of claim 6, selected from the group consisting of CHAPSO, sulfobetaine SB10 and sulfobetaine SB16. As an antiviral agent or anticancer agent of a particle comprising a small nucleic acid molecule capable of mediating RNA interference (RNAi) capable of cleaving viral nucleic acid or cancer target gene mRNA in a fusion of HBs antigen particle and liposome Use of. Administering to a mammal an effective amount of a particle comprising a small nucleic acid molecule capable of mediating RNA interference (RNAi) capable of cleaving viral nucleic acid or cancer target gene mRNA in a fusion of HBs antigen particle and liposome. A method for treating viral infection or cancer.

Images