WO2011135734A1 - Complex of virus, therapeutic agent comprising same, and therapeutic method - Google Patents

Abstract

Disclosed is a therapeutic agent for diseases including cancer, which can be administered by various routes, such as a systemic route and an intraperitoneal route, exhibits a high therapeutic effect, and has few adverse side effects, and is also effective on the inhibition of cancer metastasis when used for cancer. Also disclosed are: a complex comprising a virus, at least one layer containing at least one cationic polymer, a cationic lipid or an aggregate thereof and a layer containing at least one anionic polymer; a method for producing the complex, which involves a step of mixing a virus, a cationic polymer, a cationic lipid or an aggregate thereof, and an anionic polymer sequentially in this order to form the complex; and a therapeutic method using the complex.

Description

Viral complex, therapeutic agent containing the same, and therapeutic method

The present invention relates to a virus complex, a therapeutic agent such as cancer containing the virus, and a treatment method.

As gene therapy for diseases such as cancer, gene therapy using viruses that incorporate various therapeutic genes and viruses that selectively grow in tumor cells has been tried, but its effects are limited. Have been reported (Patent Documents 1 to 3).
However, even if the virus is administered in vivo as it is, the expression of the introduced therapeutic gene and the antitumor activity of the virus are greatly suppressed due to neutralization by the antibody. In particular, for adenoviruses often used for gene therapy, patients often have antibodies from the beginning, and the effect is greatly suppressed. Even in patients who do not have antibodies, antibodies are produced during multiple administrations, and sufficient effects cannot be obtained.
In order to avoid neutralization by antibodies, gene therapy using carrier cells into which viruses have been introduced has been widely studied.
However, in these methods using carrier cells, the effective route of administration is due to the poor diffusibility of the carrier cells in vivo and tissues and the high toxicity of the naked virus itself from the carrier cells. Was limited to intratumoral administration.
On the other hand, for small tumors that cannot be detected, tumors that cannot be injected directly, or tumor tissue that has metastasized, treatment targeting a wide range of sites by intraperitoneal, arterial, or intravenous administration is required. is there. However, when the virus is administered intraperitoneally or intravenously, side effects may occur due to its toxicity. Furthermore, administration into the bloodstream does not provide sufficient delivery to target tissues / cells due to the hepatic accumulation of the virus.
For these reasons, even in the system using carrier cells, the effective administration site is limited to the local area of the tumor, and there is no report showing a high healing effect by intraperitoneal or intravenous administration.
As described above, the virus used for gene therapy is not effective as it is due to neutralization by the antibody as it is, and has a strong side effect. Therefore, the administration route is limited, and it is extremely small by systemic administration or intraperitoneal administration. Small tumors, tumors that cannot be locally administered, or metastatic lesions cannot be widely targeted.
In order to solve such a problem, a method of binding a polymer (Non-patent Document 1) or mixing a cationic lipid with a virus to prevent blocking of an antibody (Non-patent Document 2) has been proposed.

International Publication No. 2005/037321 International Publication No. 99/45783 International Publication No. 01/23004

“Modification of Adenovirus Gene Transfer Vectors With Synthetic Polymers: A Scientific Review and Technical Guide” www. moleculartherapy. org vol. 16 no. 1 Jan. 2008 “Adenovirus complexed with polyethylene glycol and cationic lipid is shielded from neutralizing antibiotics in vitro” Gene Therapies (1998) 5,995-1002.

As a result of diligent research to solve the above-mentioned drawbacks, the present inventors have found that when a virus is complexed with a cationic polymer or a cationic lipid or an aggregate and an anionic polymer, systemic administration or A therapeutic agent for diseases such as cancer is obtained that can be administered in a wide range such as intraperitoneal administration, exhibits high therapeutic effects, has few side effects, and is effective in suppressing cancer metastasis when used in cancer. As a result, the present invention has been completed.

That is, the present invention relates to a complex comprising a virus; a cationic polymer or a cationic lipid or an assembly containing the same; and an anionic polymer. The present invention also provides a method for producing the above-described complex, which comprises the step of forming a complex by sequentially mixing a therapeutic virus; a cationic polymer or a cationic lipid or an assembly containing the same; and an anionic polymer. Relates to a method comprising:

The complex of the present invention is not easily neutralized by antiviral antibodies and has a high therapeutic effect in vivo. In addition, since the toxicity is low, various administrations such as local administration, intraperitoneal administration, intraarterial administration, intravenous administration and intracranial administration are possible. In addition, preparation and handling are simple and storage is excellent. Furthermore, in one embodiment of the present invention, the efficiency and selectivity of uptake into a target cell can be improved by introducing a ligand for a target cell into the complex.

The zeta potential of the complex obtained when PEI (polyethyleneimine) is added to adenovirus is shown. The time-dependent change of zeta potential of the complex obtained when 0.2 microgram of PEI is added to adenovirus is shown. The zeta potential of the complex obtained when HA (hyaluronic acid) is added to the adenovirus / PEI complex is shown. The zeta potential of the complex obtained when PEI is added to the adenovirus / PEI / HA complex is shown. The zeta potential of the complex obtained when HA is added to the adenovirus / PEI / HA / PEI complex is shown. The zeta potential of the complex obtained when PEI is added to the adenovirus / PEI / HA / PEI / HA complex is shown. The zeta potential of the complex obtained when HA is added to the adenovirus / PEI / HA / PEI / HA / PEI complex is shown. The zeta potential of the complex obtained by alternately adding PEI and CS to adenovirus is shown. The infectivity to the cell in the presence of the antibody of the polymer-coated virus complex is shown. The cytotoxicity of the polymer-coated virus complex in the presence of antibody is shown. The zeta potential of the complex obtained when PRT (protamine) is added to adenovirus is shown. The zeta potential of the complex obtained when HA is added to the adenovirus / PRT complex is shown. The zeta potential of the complex obtained when superfect is added to adenovirus is shown. The zeta potential of the complex obtained when HA is added to the adenovirus / superfect complex is shown. The infectivity to a cell in the presence of an antibody of a virus complex coated with a polyanion having a ligand is shown.

The complex of the present invention includes a virus; a layer containing one or more cationic polymers or cationic lipids or an assembly containing the same (cationic layer); and a layer containing one or more anionic polymers (anionic layer) ) Containing two or more coating layers.
In one embodiment of the complex of the present invention, the virus is coated with a cation layer and further coated with an anion layer (two-layer complex). In another embodiment of the complex, the virus is coated with a cation layer, further coated with an anion layer, and further coated with a cation layer (three-layer complex). Thus, a complex including two or more coating layers in which a virus is coated with a cation layer and an anion layer and a cation layer are alternately coated can be preferably exemplified. As such a complex, for example, a two-layer complex in which a virus is coated in the order of a cation layer and an anion layer, a three-layer complex in which this two-layer complex is further coated with a cation layer, and this three-layer complex A four-layer composite in which the composite is further coated with an anionic layer, a five-layer composite in which the four-layer composite is further coated with a cationic layer, and a five-layer composite that is further coated with an anionic layer 6 Examples include a layer composite and a seven-layer composite in which the six-layer composite is further coated with a cationic layer. A complex including two or more coating layers is preferable from the viewpoint of increasing the infection rate to cultured cells, and a complex including three or more coating layers is preferable from the viewpoint of increasing the infection rate to cells in vivo. The upper limit of the number of layers is not particularly limited, but is preferably 19 layers or less, more preferably 14 layers or less, and particularly preferably 11 layers or less from the viewpoint of increasing the production efficiency of the composite.
Here, in the above cationic layer or anionic layer, one or two or more cationic polymers or cationic lipids or aggregates or anionic polymers containing them are contained. May be coated simultaneously or sequentially. For example, the virus can be coated with RGD-polyethyleneimine into which the RGD peptide has been introduced, then coated with polyethyleneimine, and then with hyaluronic acid.
In addition, when the above cationic layer or anionic layer is present, the types of the cationic polymer or cationic lipid contained in each layer or the aggregate containing the cationic polymer and the anionic polymer are not necessarily the same, and different types. These polymers, cationic lipids, aggregates containing them, or combinations thereof can be modified for each layer.
Furthermore, the above-mentioned layer may cover all or part of the surface of the virus or multilayer complex, but it is preferable to cover the entire surface from the viewpoint of increasing the infection rate to cells. However, even if an anionic polymer or a cationic polymer having a specific adhesion ability to a target cell, for example, RGD or the like, is partially covered, a high infection rate to the target cell can be obtained.
In this complex, the virus forms a complex by ionic bonding with a cationic polymer or a cationic lipid or an aggregate containing the cationic polymer, or a cationic lipid or an aggregate containing the cationic lipid or an anionic polymer. Are also ionically bonded. Depending on the number of times of mixing at the time of preparation, the mixing ratio, the mixing order, etc., which will be described later in the description of the preparation method, the surface is a cationic complex, the surface is an anionic complex, and the surface charge is almost neutral. Any of these complexes can be used.
(Virus)
In the complex of the present invention, the viruses are adenovirus, herpes simplex virus, adeno-associated virus, vaccinia virus, measles virus, Sendai virus, reovirus, foamy virus, Newcastle disease virus, lentivirus, Rabies Virus, Pox virus, Examples include DNA viruses such as myxoma virus or RNA viruses introduced with a therapeutic gene, or their restricted-growth oncolytic viruses, etc., which can be appropriately selected according to the animal species and diseases to be treated it can. For example, for the purpose of human gene therapy, a virus that can be safely used in humans, such as adenovirus or its restricted-growth oncolytic virus, can be appropriately used. Mutant viruses and recombinant viruses introduced with therapeutic genes can also be used.
(Gene to be introduced into the virus)
The therapeutic gene that can be introduced into the virus can be appropriately selected according to the disease that is the target of gene therapy. For example, when cancer diseases are targeted for gene therapy, genes such as p53, thymidine kinase, and GM-CSF can be introduced into the virus. When a muscular dystrophy disease is targeted for gene therapy, a gene such as dystrophin can be introduced into the virus.
The type of virus, the type of gene to be introduced, and the gene introduction method can be appropriately selected according to the type of virus and the gene to be introduced. For example, adenovirus introduced with p53 gene as described in WO / 1995/012660, for example, oncolytic herpes simplex virus as described in Nature Medicine, 1,938-943 (1995) Can do.
(Cationic polymer)
Among the cationic polymers or cationic lipids that can be used in the complex of the present invention or the cationic polymer, the cationic polymer is a naturally-derived or synthetic polymer having a positively charged molecular weight of about 1,000 to 3,000,000. In this case, a polymer having a plurality of functional groups, preferably 5 or more, in a molecule that can form a complex with a virus in a solution can be used. Examples of such a functional group include an optionally substituted amino group or ammonium group or a salt thereof (these groups may be mono- or poly-substituted with, for example, an alkyl group having 1 to 6 carbon atoms or a phenyl group). And an organic amino group such as an imino group, an imidazolyl group, and a guanidino group. Examples of such cationic polymers include positively charged proteins and polypeptides; positively charged dendrimers; positively charged synthetic polymers; and positively charged polysaccharide derivatives, and salts thereof. As well as combinations thereof.
The molecular weight of the positively charged protein or positively charged polypeptide that can be used as the cationic polymer in the complex of the present invention is preferably about 1,000 to 500,000. Specific examples of such proteins and polypeptides include proteins and polypeptides such as protamine, histone, HelΔ1, and gelatin. Polyamino acids containing positively charged amino acid residues are also exemplified. It can be illustrated. Specific examples of such a polyamino acid containing a positively charged amino acid residue include poly-L-lysine, polyarginine, polyornithine and the like. Examples of salts of these proteins and polypeptides include hydrochlorides, sulfates, phosphates, borates and the like.
The positively charged dendrimer having a functional group as described above that can be used as a cationic polymer is an amino group or ammonium group which may be substituted at the end or inside of a branched molecular chain or a salt thereof ( These groups are, for example, dendrimers having an organic amino group such as an alkyl group having 1 to 6 carbon atoms, a phenyl group or the like, which may be mono- or polysubstituted), an imino group, an imidazolyl group, a guanidino group, The molecular weight is preferably about 1,000 to 500,000. Specific examples of dendrimers include polyamidoamine dendrimers and polylysine dendrimers. Examples of the dendrimer salt include hydrochloride, sulfate, phosphate, borate and the like.
A positively charged synthetic polymer that can be used as a cationic polymer is a synthetic polymer having a plurality of functional groups, preferably 5 or more, in one molecule, which can form a complex with a virus in solution as described above. A synthetic polymer having a molecular weight of preferably 1,000 to 3,000,000. Specific examples of synthetic polymers include polyethyleneimine (including linear polyethyleneimine or polybranched polyethyleneimine), 2-dimethylaminoethyl methacrylate polymer or copolymer, and 2-trimethylaminoethyl methacrylate polymer. Examples thereof include a polymer or a copolymer and a salt thereof. The molecular weight of polyethyleneimine, which is an example of a synthetic polymer, is preferably about 1,000 to 500,000, more preferably about 5,000 to 200,000, and most preferably about 10,000 to 100,000. Examples of the polyethyleneimine salt include hydrochloride, sulfate, phosphate, borate and the like.
The positively charged polysaccharide derivative that can be used as a cationic polymer has a plurality of functional groups, preferably 5 or more, in a molecule that can form a complex with a virus in solution, and has a molecular weight of preferably 1000. It is a polysaccharide derivative having a viscosity of ˜3 million, more preferably 5,000 to 500,000. Specific examples of such polysaccharides include chitosan, dextran derivatives introduced with the above functional groups, and salts thereof. Of these, the molecular weight of chitosan is preferably about 1,000 to 500,000, more preferably about 5,000 to 200,000, and most preferably about 10,000 to 100,000. Examples of the salt of chitosan include hydrochloride and acetate. The molecular weight of the dextran derivative is preferably 3,000 to 1,000,000. Specific examples of such dextran derivatives include diethylaminoethyl-dextran.
The cationic polymer may be a polymer that is positively charged by introducing a functional group such as an amino group into a conventionally non-positively charged polymer. Moreover, even if it is not normally positively charged, it can be used as long as it is positively charged at the time of complex formation, and if necessary, it can be further modified with a sugar chain, oligopeptide, antibody or the like. It may be.
Cationic lipids (including cationic cholesterol derivatives) that can be used in the complex of the present invention include DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol), DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DMRI (N- (1,2-dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- ( 2- (sperminecarboxamido) ethyl) -N, N-dimethylammonium trifluoro Cetate), DOTAP (N- (1- (2,3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride), or DOTMA (N- (1- (2,3-dioleoyloxy) ) Propyl) -N, N, N-trimethylammonium chloride), and combinations thereof.
Moreover, as an aggregate | assembly containing a cationic lipid, what mixed the said cationic lipid (for example, DOSPA), and neutral substances, such as DOPE (dioleoylphosphatidylethanolamine), cholesterol, etc. can be used. Examples of aggregates containing cationic lipids include lipofectamine (3: 1 w / w mixture liposome of DOSPA and DOPE), lipofectin (1: 1 w / w mixture liposome of DOTMA and DOPE), or a mixture thereof. Can be preferably mentioned.
In the complex of the present invention, as the cationic polymer, polyethyleneimine; protamine; HelΔ1; dendrimer such as polyamidoamine dendrimer and polylysine dendrimer; chitosan; 2-dimethylaminoethyl methacrylate polymer or copolymer; 2-trimethyl A polymer or copolymer of aminoethyl methacrylate can be preferably used, and polyethyleneimine, polyamidoamine dendrimer, polylysine dendrimer, chitosan, and protamine, particularly polyethyleneimine, polyamidoamine dendrimer, and protamine are particularly preferably used. it can. Moreover, as a cationic lipid or an aggregate containing the same, lipofectamine (the above-mentioned 3: 1 w / w mixture liposome of DOSPA and DOPE) can be preferably used.
(Anionic polymer)
The anionic polymer used in the complex of the present invention is a negatively charged, naturally-occurring or synthetic polymer having a molecular weight of about 5 to 4 million containing an anionic group in the molecule, A polymer having a plurality of functional groups, preferably 5 or more, in one molecule can form a complex with a cation can be used. Examples of such a functional group include a carboxyl group and -OSO. ₃ H group, -SO ₃ Mention may be made of H groups, phosphate groups, and salts thereof. Such anionic polymers include amphoteric polymers.
In the composite of the present invention, the anionic polymer is more specifically a carboxyl group, -OSO. ₃ H group, -SO ₃ Polysaccharides or derivatives thereof having functional groups selected from H groups, phosphate groups, and salts thereof; polyamino acids containing amino acid residues having negatively charged side chains; PEG derivatives having carboxyl side chains; carboxyl Group, -OSO ₃ H group, -SO ₃ Synthetic polymer having a functional group selected from H group, phosphoric acid group, and salts thereof; carboxyl group, -OSO ₃ H group, -SO ₃ A functional group selected from an H group, a phosphate group, and a salt thereof, and an optionally substituted amino group or ammonium group or a salt thereof (for example, these groups include an alkyl group having 1 to 6 carbon atoms, phenyl group, Macromolecules which may be mono- or poly-substituted with groups etc .; as well as combinations thereof.
As the polysaccharide having a functional group as described above or a derivative thereof which can be used as an anionic polymer in the complex of the present invention, glucosaminoglycan can be preferably used. The molecular weight of such a glucosaminoglycan is preferably 1,000 to 4,000,000, more preferably 4,000 to 3,000,000. Specific examples of such glucosaminoglycans include hyaluronic acid, chondroitin, chondroitin sulfate, keratan sulfate, heparin, dermatan sulfate, and salts thereof. Of these, hyaluronic acid can be preferably used. Hyaluronic acid can also be used as its salt or a negatively charged derivative. The molecular weight may be 5,000 or more, preferably 10,000 or more, and more preferably 100,000 to 3,000,000. Examples of the salt of hyaluronic acid include sodium salt, potassium salt, ammonium salt and the like. Examples of the derivatives of hyaluronic acid include those obtained by introducing polyethylene glycol, peptides, sugars, proteins, iodic acid, antibodies or a part thereof into hyaluronic acid, and spermine, spermidine, etc. are introduced. And zwitterionic derivatives having a positively charged moiety.
The polyamino acid containing an amino acid residue having a negatively charged side chain that can be used as an anionic polymer in the complex of the present invention is a carboxyl group, -O-SO. ₃ H group, -SO ₃ A polyamino acid having a molecular weight of 500 to 1,000,000, preferably containing an amino acid residue having a side chain of a group such as an H group, a phosphate group, and a salt thereof. Specific examples of such polyamino acids include polyglutamic acid and polyaspartic acid. In addition, carboxyl group, -O-SO ₃ H group, -SO ₃ Examples of the salt of H group and phosphate group include sodium salt, potassium salt and ammonium salt.
The PEG derivative having a carboxyl side chain that can be used as an anionic polymer in the complex of the present invention has a plurality of carboxyl side chains per PEG molecule, preferably 5 or more, 500 or more, preferably 2,000 or more, A PEG derivative having a molecular weight of 4,000 to 40,000 or a salt thereof is more preferable. A PEG derivative having a carboxyl side chain can also be used as a salt thereof or a negatively charged derivative. Examples of these salts include sodium salts, potassium salts, ammonium salts and the like. Specific examples of such PEG derivatives include those described in J. Org. Biometer. Sci. Polymer Edn. Vol. 14, pp 515-531 (2003).
A carboxyl group that can be used as an anionic polymer in the complex of the present invention, -OSO ₃ H group, -SO ₃ The synthetic polymer having a functional group selected from H group, phosphate group, and salts thereof includes a plurality of, preferably 5 or more, carboxyl groups, —O—SO per molecule. ₃ H group, -SO ₃ A polymer or copolymer having a functional group selected from an H group, a phosphate group, and a salt thereof, and preferably a polymer or copolymer having a molecular weight of 5 to 4 million. Specific examples of such a polymer or copolymer include a polymer or copolymer of acrylic acid or methacrylic acid having a molecular weight of 1,000 to 3,000,000, a sulfate ester of polyvinyl alcohol, or succinimidylated poly-L-lysine. Etc. can be illustrated. In addition, carboxyl group, -O-SO ₃ H group, -SO ₃ Examples of the salt of H group and phosphate group include sodium salt, potassium salt and ammonium salt.
A carboxyl group that can be used as an anionic polymer in the complex of the present invention, -OSO ₃ H group, -SO ₃ A functional group selected from an H group, a phosphate group, and a salt thereof, and an optionally substituted amino group or ammonium group or a salt thereof (for example, these groups include an alkyl group having 1 to 6 carbon atoms, phenyl group, And a polymer having a mono- or poly-substituted group such as a carboxyl group, -OSO per molecule. ₃ H group, -SO ₃ A plurality of, preferably 5 or more, functional groups selected from H groups, phosphate groups, and salts thereof, and optionally substituted amino groups or ammonium groups or salts thereof (these groups have, for example, carbon number PEG derivative having a molecular weight of 500 or more, preferably 2,000 or more, more preferably 4,000 to 40,000, which may be mono- or polysubstituted by 1 to 6 alkyl groups, phenyl groups, etc. And other polymers. Carboxyl group, -OSO ₃ H group, -SO ₃ Examples of the salt of H group and phosphate group include sodium salt, potassium salt and ammonium salt. Examples of the amino group or ammonium group salt include hydrochloride, sulfate and acetate.
Such a polymer is preferably a carboxyl side chain and an amino group or ammonium group having an equivalent amount or less or a salt thereof (these groups are, for example, alkyl groups having 1 to 6 carbon atoms, phenyl groups, etc.). PEG derivatives having (which may be substituted) can be mentioned, specifically, Macromol. Biosci. Vol. 2, pp 251-256 (2002), PEG derivatives that can be prepared, hyaluronic acid in which acetamide groups are partially hydrolyzed, hyaluronic acid in which hydrazine is introduced into some carboxyl groups, etc. Can be illustrated.
The anionic polymer that can be used in the composite of the present invention may be one that has been negatively charged by introducing a functional group such as a carboxyl group into a conventionally non-negatively charged one. Even if it is not negatively charged normally, it can be used as long as it is negatively charged at the time of complex formation, and if necessary, it may be further modified with sugar chain, oligopeptide, antibody, etc. Good.
In the complex of the present invention, as the anionic polymer, hyaluronic acid, a PEG derivative having a carboxyl side chain, an anionic polymer such as polyacrylic acid, or a salt thereof can be preferably used. A PEG derivative having a salt thereof or a salt thereof, particularly hyaluronic acid can be particularly preferably used.
In addition, by using cationic polymers or cationic lipids or aggregates containing them, or anionic polymers that have specific adhesion to target cells, the introduction of therapeutic genes specifically to target cells Can be done. For example, when hyaluronic acid is used as the anionic polymer, cells having cell surface molecules such as CD44 that specifically bind to hyaluronic acid can be targeted. In addition, by using an anionic polymer or cationic polymer into which RGD peptide is introduced, preferably PEG derivative or polyethyleneimine into which RGD peptide is introduced, many types of tumor cells can be targeted, and galactose side chain Hepatocytes or liver-derived cells can be targeted by using a cationic polymer or an anionic polymer into which is introduced.
(Preferable combination of cationic polymer and anionic polymer)
In the complex of the present invention, the combination of the cationic polymer or the cationic lipid or the assembly containing the cationic polymer and the anionic polymer includes polyethyleneimine and hyaluronic acid; protamine and hyaluronic acid; cationic dendrimer and hyaluronic acid; A PEG derivative having a carboxyl side chain; an assembly containing DOSPA (eg, lipofectamine (3: 1 w / w mixture liposome of DOSPA and DOPE)) and hyaluronic acid; an assembly containing DOSPA (eg, lipofectamine) and a carboxyl side chain Preferred examples include PEG derivatives possessed.
(Ratio of virus to cationic polymer)
The mixing ratio between the virus used in the complex of the present invention and the cationic polymer or cationic lipid or an assembly containing the virus is the type of the virus, the type of the cationic polymer or the cationic lipid or the assembly containing the same, or the production process. Can be selected as appropriate depending on which step in the virus, but virus 1 × 10 ⁸ Preferably, 0.001 to 500 μg, more preferably 0.01 to 50 μg, particularly preferably 0.1 to 5 μg of a cationic polymer or cationic lipid or an aggregate containing the same can be mixed with pfu. . For example, when adenovirus is used as the virus and polyethyleneimine is used as the cationic polymer, adenovirus 1 × 10 ⁸ The amount of polyethyleneimine added in each step with respect to pfu is preferably 0.001 to 500 μg, more preferably 0.01 to 50 μg, and particularly preferably 0.1 to 5 μg.
(Ratio of virus to anionic polymer)
The mixing ratio between the virus used in the complex of the present invention and the anionic polymer can be appropriately selected according to the type of virus, the type of anionic polymer, and the step in the production process. 10 ⁸ Preferably, 0.005 to 2000 μg, more preferably 0.05 to 200 μg, and particularly preferably 0.5 to 20 μg of an anionic polymer can be mixed with respect to pfu. For example, when adenovirus is used as the virus and hyaluronic acid is used as the anionic polymer, adenovirus 1 × 10 ⁸ The amount of hyaluronic acid added in each step with respect to pfu is preferably 0.005 to 2000 μg, more preferably 0.05 to 200 μg, and particularly preferably 0.5 to 20 μg.
The preferred mixing ratio of the virus contained in the complex of the present invention; the cationic polymer or the cationic lipid or the aggregate containing the same; and the anionic polymer is as described above. Since the optimum conditions vary depending on the disease and disease, the compounding ratio can be determined appropriately by those skilled in the art.
(Method for producing the composite of the present invention)
The complex of the present invention may be prepared by the step of forming a complex by sequentially mixing the virus described above; a cationic polymer or a cationic lipid or an assembly containing the same; and an anionic polymer in this order. it can. The step of mixing the cationic polymer or the cationic lipid or the aggregate containing the same; and the step of mixing the anionic polymer can be alternately performed as necessary. For example, the virus can be mixed with a cationic polymer or cationic lipid or an assembly comprising it to obtain a one-layer complex, and then mixed with an anionic polymer to form a complex of the present invention that is a two-layer. obtain. Further, this two-layer composite is mixed with a cationic polymer or a cationic lipid or an assembly containing the same to obtain a three-layer composite, and further mixed with an anionic polymer to form a four-layer composite. Can be obtained. Further, this four-layer composite is mixed with a cationic polymer or a cationic lipid or an assembly containing the same to obtain a five-layer composite, and further mixed with an anionic polymer to form a six-layer composite. You can also get
Here, in these mixing steps, one or two or more cationic polymers or cationic lipids or aggregates or anionic polymers containing them are mixed. When two or more are mixed, they are mixed at the same time. Or may be mixed sequentially. For example, the virus can be mixed with RGD-polyethyleneimine into which the RGD peptide has been introduced, then mixed with polyethyleneimine, and then mixed with hyaluronic acid.
In addition, when such a mixing step is repeated, the type of the cationic polymer or cationic lipid added or the aggregate containing the cationic polymer and the anionic polymer need not be the same, and different types of polymers and cationic lipids may be used. Or it is also possible to change and add the aggregate | assembly containing it or its combination for every step.
Further, the amount of the cationic polymer or cationic lipid added or the anionic polymer to be added is preferably such that the addition of the virus or the previously prepared virus complex reverses the charge. However, this does not apply to the last item added. Specifically, a cationic polymer or a cationic lipid or an aggregate containing it; and an anionic polymer can be prepared by adding the above amounts at each step.
In the complex prepared by such a method, the virus forms a complex by ionic bonding with the cationic polymer or cationic lipid or an aggregate containing the cationic polymer, or the cationic polymer or cationic lipid or the same. The assemblage and the anionic polymer are also ionically bonded. Depending on the number of times of mixing, mixing ratio, mixing order, etc., the surface can be a complex with a cationic surface, an anionic surface with a complex, or a complex with an almost neutral surface charge.
(Use of the complex of the present invention)
The complex of the present invention prepared as described above can be used for various gene therapy, immunotherapy for humans and animals, or creation of experimental animals and cells into which each gene has been introduced.
The application amount of the complex of the present invention varies depending on the administration route, the administration site, the type of disease, and the type of virus to be introduced. For example, when adenovirus is used, the amount of virus contained in intraperitoneal administration is, for example, 10 ⁸ ~ 10 ¹² pfu / individual. When herpes simplex virus is used, for example, 1 × 10 ⁴ ~ 5 × 10 ⁵ The pfu / individual can be administered into a subcutaneous tumor or a breast cancer tumor mass. Examples of administration routes include intratumoral, intraperitoneal, hepatic artery, intravenous, intracranial and the like. For example, when an adenovirus or oncolytic herpes simplex virus introduced with the p53 gene is used, it can be administered intratumorally, intraperitoneally, intrahepatic artery, intravenously, or intracranially.
In addition, when the complex of the present invention is used for preparation of a gene-transferred cell, a complex of 1 to 8000 pfu in terms of viral load can be applied per cell.
The virus used in the present invention can be freely selected from adenovirus, herpes simplex virus, adeno-associated virus, and the like depending on the animal species and diseases to be used.
Furthermore, in the above use, additives usually used for injections may be appropriately added and used.
The present invention will be described more specifically with reference to the following examples. In addition, these examples are for demonstrating this invention, Comprising: This invention is not limited at all.

(Reagents and materials)
Polyethyleneimine (hereinafter referred to as PEI) was Polyethyleneimine “Max” (hydrochloride; Mw = 40000) manufactured by Polyscience. As the RGD-added PEI, polyPE-translation jetPEI ^™ -RGD was used. As hyaluronic acid (hereinafter referred to as HA), “microorganism-derived” hyaluronic acid (molecular weight: 1,000,000) from Nacalai Tesque, Inc. was used. As chondroitin sulfate (hereinafter referred to as CS), shark-derived chondroitin sulfate (molecular weight of about 10,000) was used. Protamine (hereinafter referred to as PRT) was Wako Pure Chemical Protamine Sulfate (made by salmon). As Superfect, which is a cationic dendrimer (polyamideamine dendrimer), a product manufactured by QIAGEN was used. The wild type human adenovirus (type V) used was obtained from Microbix. Based on this, human adenovirus (AD-GFP) and oncolytic virus (ADE3-IAI.3B) incorporating the GFP gene were prepared by the same method as Patent Document 1. A human adenovirus (AD-GM-CSF) into which a GM-CSF gene was incorporated was prepared by inserting a mouse GM-CSF gene in place of the GFP gene in the same manner as AD-GFP.
In addition, zeta potential (data is shown in mV unless otherwise stated) is obtained by using MALVERN Zetasizer Nano ZS. Measurements were made by
These are the same in the following examples unless otherwise specified.
(Example 1)
The zeta potential of the resulting complex when PEI was added to adenovirus was measured.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. 800 microliters of this liquid was taken, a predetermined amount of PEI was added and stirred well, and the zeta potential immediately after that was measured.
[result]
The results are shown in FIG.
The zeta potential of the virus gradually increased with the addition of PEI, resulting in a complex with a positive surface charge.
(Example 2)
The time change of the zeta potential of the resulting complex when PEI was added to adenovirus was measured.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. 800 microliters of this liquid was taken, 0.2 micrograms of PEI was added and stirred well, and the zeta potential was measured over time immediately after that.
[result]
The results are shown in FIG.
The zeta potential of the virus showed a positive value immediately after the addition of PEI, but then rose more slowly and became almost constant at about 20 minutes.
(Example 3)
The zeta potential of the resulting complex when HA was added to the adenovirus / PEI complex was measured.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. 800 microliters of this solution was added, 0.2 micrograms of PEI was added and stirred well, allowed to stand for 30 minutes, a predetermined amount of HA was added, and the zeta potential immediately after that was measured.
[result]
The results are shown in FIG.
The zeta potential of the virus / PEI complex decreased again by adding HA, resulting in a complex with a negative surface charge.
(Example 4)
The zeta potential of the resulting complex when PEI was added to the adenovirus / PEI / HA complex was measured.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. Take 800 microliters of this solution, add 0.2 micrograms of PEI, stir well, let stand for 30 minutes, add 2 micrograms of HA, leave it for 30 minutes, add a predetermined amount of PEI, and The zeta potential was measured.
[result]
The results are shown in FIG.
The zeta potential of the adenovirus / PEI / HA complex was increased again by adding PEI, resulting in a complex with a positive surface charge.
(Example 5)
The zeta potential of the resulting complex was measured when HA was added to the adenovirus / PEI / HA / PEI complex.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. Take 800 microliters of this solution, add 0.2 micrograms of PEI, stir well, let stand for 30 minutes, add 2 micrograms of HA, leave it for 30 minutes, add 2 micrograms of PEI, 30 minutes After standing, a predetermined amount of HA was added, and the zeta potential immediately after that was measured.
[result]
The results are shown in FIG.
The zeta potential of the adenovirus / PEI / HA / PEI complex decreased again by adding HA, resulting in a complex with a negative surface charge.
(Example 6)
The zeta potential of the resulting complex was measured when PEI was added to the adenovirus / PEI / HA / PEI / HA complex.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. Take 800 microliters of this solution, add 0.2 micrograms of PEI, stir well, let stand for 30 minutes, add 2 micrograms of HA, leave it for 30 minutes, add 2 micrograms of PEI, 30 minutes After standing, 10 micrograms of HA was added, and after standing for 30 minutes, a predetermined amount of PEI was added, and the zeta potential immediately after that was measured.
[result]
The results are shown in FIG.
The zeta potential of the adenovirus / PEI / HA / PEI / HA complex increased again by adding PEI, resulting in a complex with a positive surface charge.
(Example 7)
The zeta potential of the resulting complex was measured when HA was added to the adenovirus / PEI / HA / PEI / HA / PEI complex.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. Take 800 microliters of this solution, add 0.2 micrograms of PEI, stir well, let stand for 30 minutes, add 2 micrograms of HA, leave it for 30 minutes, add 2 micrograms of PEI, 30 minutes After standing, 2 micrograms of HA was added, and after standing for 30 minutes, 2 micrograms of PEI was added. After standing for 30 minutes, a predetermined amount of HA was added, and the zeta potential immediately after that was measured.
[result]
The results are shown in FIG.
The zeta potential of the adenovirus / PEI / HA / PEI / HA / PEI complex decreased again by adding HA, resulting in a complex with a negative surface charge.
(Example 8)
Similarly, the zeta potential of the complex obtained by alternately adding PEI and CS to adenovirus was measured using CS instead of HA.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in 8 mM phosphate buffer (pH 7.4) at a concentration of 1 × 10 ¹¹ pfu / ml. 200 microliters of this liquid was taken and 100 micrograms of PEI and 200 micrograms of CS were added alternately and stirred well. A small amount was sampled at each step, diluted 40-fold with water, and the zeta potential of the complex was measured.
[result]
The results are shown in FIG.
The zeta potential of adenovirus changes alternately with positive and negative values by alternately adding PEI and CS, and when PEI is added last, a complex with a positive surface charge is formed. In addition, when CS was added last, a composite having a negative surface charge was obtained.
(Example 9)
The ability of the polymer-coated virus complex to infect cells in the presence of antibody was examined.
[Operating procedure]
[1] One day before adding the virus and its complex, A549 cells (obtained from JCRB Cell Bank) were seeded in a 24-well multiplate (50,000 cells / well) and incubated overnight (temperature: 37 ° C .; humidity 99%) Medium: RPMI medium containing 10% FCS).
[2] A complex with a polymer was prepared in the same manner as in Examples 3 to 7, using human adenovirus (AD-GFP) incorporating a GFP gene.
[3] 200 microliters of human immunoglobulin drip solution (obtained from Nippon Pharmaceutical Co., Ltd .; titer: × 6000) was added to half of the wells in advance.
[4] The complex prepared in [2] was added to the well (addition amount: 2 × 10 ⁸ pfu / well).
[5] The cells were incubated for 8 days at 37 ° C. and 5% CO ₂ -95% air.
[6] Cells were observed with a fluorescence microscope on a daily basis, and the number of infected cells was counted.
[result]
The results are shown in FIG.
The complex consisting of adenovirus, PEI and HA showed much higher infectivity in the presence of antibody compared to the original virus alone.
(Example 10)
The cytotoxicity of the polymer-coated virus complex in the presence of antibody was examined.
[Operating procedure]
[1] One day before adding the virus and its complex, A549 cells (obtained from JCRB Cell Bank) were seeded in a 96-well multiplate (2000 cells / well) and incubated overnight (temperature: 37 ° C .; humidity 99% Medium: RPMI medium containing 10% FCS).
[2] Using the oncolytic virus (ADE3-IAI.3B), a complex with the polymer was prepared in the same manner as in Example 9, and a 4-fold dilution series was prepared.
[3] To each well, 20 μl of human immunoglobulin drip solution (obtained from Nippon Pharmaceutical Co., Ltd .; titer: × 6000) was added in advance.
[4] The complex prepared in [2] was added to the well (addition amount: maximum 2 × 10 ⁸ pfu / well).
[5] The cells were incubated for 8 days at 37 ° C. and 5% CO ₂ -95% air.
[6] Live cells were stained and quantified.
[result]
The results are shown in FIG.
The complex consisting of oncolytic virus, PEI and CS showed a much higher cell killing ability in the presence of the antibody than the original virus alone.
(Example 11)
The curative effect on OVHM cell transplanted mice by intraperitoneal injection of a complex consisting of oncolytic virus, PEI and HA was examined.
[Operating procedure]
[1] Oncolytic virus (ADE3-IAI.3B) was suspended in 5% aqueous glucose solution at a concentration of 5 × 10 ⁹ pfu / ml. Take 5 ml of this solution, add 83.3 micrograms of PEI, leave it for 30 minutes, add 833 micrograms of HA, leave it for 30 minutes, add 417 micrograms of PEI, leave it for 30 minutes, and then 1390 micrograms HA was added, and after leaving for 30 minutes, 278 micrograms of PEI was added to prepare a virus / PEI / HA / PEI / HA / PEI complex. Furthermore, after leaving the last PEI addition to the same preparation for 30 minutes, 1390 micrograms of HA was added to prepare a virus / PEI / HA / PEI / HA / PEI / HA complex.
[2] One mouse with OVHM cells ("OV2944-HM-1" described in Jpn. J. Cancer Res. 80, 459-463 (1989)) prepared by the same method as in Example 9, [1] 10 ⁶ cells per week were administered intraperitoneally to female (C57BL / 6 × C3 / He) F1 mice (source: Claire Japan) previously immunized with 10 ¹⁰ pfu AD-GSF 3 weeks ago (Number of examples: 13).
[3] On the 5th day and 9th day after cell administration, the oncolytic virus (ADE3-IAI.3B) complex prepared in [1] is intraperitoneally injected into each mouse and converted into the amount of virus. 2.5 × 10 ¹⁰ pfu were administered per mouse, and the survival days were examined. In addition, the control group was not administered the complex, and the comparative group was a naked virus that did not form a complex, ADE3-IAI. 3B was administered at 2.5 × 10 ¹⁰ pfu per animal.
[result]
In the control group to which nothing was administered, all died within 21 days after tumor transplantation (number of cases: 3). In addition, one mouse out of the four mice in the comparison group that received the same amount of naked virus without addition of polymer intraperitoneally developed shock symptoms 5 seconds after administration, and died within a few minutes, and the remaining 3 mice were 31. Died within days. On the other hand, mice administered intraperitoneally with the oncolytic virus complex survived after 70 days or more without accumulation of ascites (number of cases: 3 each).
(Example 12)
The curative effect on mice transplanted with OVHM cells by intraperitoneal injection of a complex consisting of a virus incorporating the GM-CSF gene, PEI and HA was examined.
[Operating procedure]
[1] Human adenovirus (AD-GM-CSF) incorporating the GM-CSF gene was suspended in 5% aqueous glucose solution at a concentration of 5 × 10 ⁹ pfu / ml. Take 5 ml of this solution, add 41.7 micrograms of PEI, leave for 30 minutes, add 417 micrograms of HA, leave for 30 minutes, add 417 micrograms of PEI, and add virus / PEI / HA / PEI. A complex was prepared. Furthermore, after the final addition of PEI was allowed to stand for 30 minutes, 2085 micrograms of HA was added to the same preparation, and a virus / PEI / HA / PEI / HA complex was prepared.
[2] OVHM cells ("OV2944-HM-1" described in Jpn. J. Cancer Res. 80, 459-463 (1989)) prepared by the same method as in [1] of Example 9 10 ⁶ percentage per animal, female (C57BL / 6 × C3 / He ) F1 mice: were administered intraperitoneally (obtained from CLEA Japan) (example: 9).
[3] On the 5th day and 6th day after cell administration, the virus complex having the GM-CSF gene prepared in [1] is intraperitoneally injected into the mouse, and the amount of virus is converted to 2 per mouse. 5 × 10 ¹⁰ pfu were administered and the number of days of survival was examined. In the control group, the complex was not administered (number of cases: 3).
[result]
The control group to which nothing was administered died within 29 days after tumor transplantation (number of cases: 3). On the other hand, mice administered with the virus complex having the GM-CSF gene intraperitoneally survived after 70 days or more without accumulation of ascites (number of cases: 3 each).
(Example 13)
The zeta potential of the resulting complex when PRT was added to adenovirus was measured.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the same GFP gene as used in Example 1 was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. 800 microliters of this liquid was taken, a predetermined amount of PRT was added and stirred well, and the zeta potential immediately after that was measured.
[result]
The results are shown in FIG.
The zeta potential of the virus gradually increased with the addition of PRT, resulting in a complex with a positive surface charge.
(Example 14)
The zeta potential of the resulting complex when HA was added to the adenovirus / PRT complex was measured.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the same GFP gene as used in Example 1 was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. 800 microliters of this liquid was added, 1.5 micrograms of PRT was added and stirred well, allowed to stand for 30 minutes, a predetermined amount of HA was added, and the zeta potential immediately after that was measured.
[result]
The results are shown in FIG.
The zeta potential of the virus / PRT complex decreased again by adding HA, resulting in a complex with a negative surface charge.
(Example 15)
The zeta potential of the resulting complex was measured when the superfect of cationic dendrimer was added to adenovirus.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the GFP gene was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. 800 microliters of this liquid was taken, a predetermined amount of Superfect was added, and the mixture was well stirred, and the zeta potential immediately after that was measured.
[result]
The results are shown in FIG.
The zeta potential of the virus gradually increased with the addition of Superfect, resulting in a complex with a positive surface charge.
(Example 16)
The zeta potential of the resulting complex was measured when HA was added to the adenovirus / superfect complex.
[Operating procedure]
Human adenovirus (AD-GFP) incorporating the same GFP gene as used in Example 1 was suspended in a 5% aqueous glucose solution at a concentration of 1.5 × 10 ⁸ pfu / ml. 800 microliters of this solution was added, 0.5 microgram of Superfect was added and stirred well, allowed to stand for 30 minutes, a predetermined amount of HA was added, and the zeta potential immediately after that was measured.
[result]
The results are shown in FIG.
The zeta potential of the virus / superfect complex decreased again by adding HA, resulting in a complex with a negative surface charge.
(Example 17)
The infectivity of cells in the presence of antibodies of the virus complex coated with the ligand-conjugated polymer was examined.
[Operating procedure]
[1] One day before adding the virus and its complex, A549 cells (obtained from JCRB Cell Bank) were seeded in a 24-well multiplate (50,000 cells / well) and incubated overnight (temperature: 37 ° C .; humidity 99%) Medium: RPMI medium containing 10% FCS).
[2] A complex with PEI was prepared in the same manner as in Example 1 using human adenovirus (AD-GFP) incorporating the GFP gene. Further, a complex with PRT was prepared in the same manner as in Example 11.
[3] RGD-PEG-Suc (synthesized by the method described in Biomedicine & Pharmacotherapy 62 (2008) 448), which is an anionic PEG derivative having an RGD side chain, is added to the obtained complex (addition amount: 10). Microgram), a virus complex carrying RGD was prepared.
[4] 200 microliters of human immunoglobulin drip solution (obtained from Nippon Pharmaceutical Co., Ltd .; titer: x6000) was added to half of the wells in advance.
[5] The complex prepared in [3] was added to the well (addition amount: 1 × 10 ⁸ pfu / well).
[6] The cells were incubated at 37 ° C. and 5% CO ₂ -95% air for 2 days.
[7] Cells were observed with a fluorescence microscope on a daily basis (measurement conditions: blue filter excitation), and the number of infected cells was counted.
[result]
The results regarding the number of infected cells in the presence of human antibodies are shown in FIG.
The complex consisting of adenovirus, PEI and PEG derivative with RGD side chain showed much higher infectivity even in the presence of antibody compared to the original virus alone. In addition, the complex composed of adenovirus, PRT, and PEG derivative having RGD side chain also showed significantly higher infectivity in the presence of the antibody compared to the original virus alone.
(Example 18)
The infectivity of cells in the presence of antibodies of the virus complex polymer-coated using PEI with RGD side chain was examined.
[Operating procedure]
[1] One day before adding the virus and its complex, A549 cells (obtained from JCRB Cell Bank) were seeded in a 24-well multiplate (50,000 cells / well) and incubated overnight (temperature: 37 ° C .; humidity 99%) Medium: RPMI medium containing 10% FCS).
[2] A complex with a polymer was prepared in the same manner as in Examples 6 and 7, using human adenovirus (AD-GFP) incorporating a GFP gene. However, instead of the last PEI added, 3.6 microliters of jetPEI ^™ -RGD was added, and a virus complex having an RGD ligand, ie, virus / PEI / HA / PEI / HA / RGD-PEI complex (5-layer complex) Body) and virus / PEI / HA / PEI / HA / RGD-PEI / HA complex (6-layer complex).
[3] 200 microliters of human immunoglobulin drip solution (obtained from Nippon Pharmaceutical Co., Ltd .; titer: × 6000) was added to half of the wells in advance.
[4] The complex prepared in [2] was added to the well (addition amount: 2 × 10 ⁸ pfu / well).
[5] The cells were incubated for 8 days at 37 ° C. and 5% CO ₂ -95% air.
[6] Cells were observed with a fluorescence microscope on a daily basis, and the number of infected cells was counted.
[result]
The complex consisting of adenovirus, PEI, HA and RGD-PEI shows much higher infectivity than the original virus alone in the presence of antibody, with 40% for the 5 layer complex and 6 layer complex. In the body, 30% of the cells were infected.

Claims (46)

Two or more layers including a virus; a layer containing one or more cationic polymers or a cationic lipid or an assembly containing the same (cation layer); and a layer containing one or more anionic polymers (anion layer) A composite comprising a coating layer. The complex according to claim 1, wherein the virus is coated with a cation layer, and the cation layer and the anion layer are alternately coated. The cationic layer is formed by coating two or more kinds of cationic polymers or cationic lipids or aggregates containing the same simultaneously or sequentially; and / or the anionic layer is coated with two or more kinds of anionic polymers simultaneously or The composite according to claim 1 or 2, which is formed by sequentially coating. The cationic polymer is selected from a positively charged protein or polypeptide; a positively charged dendrimer; a positively charged synthetic polymer; a positively charged polysaccharide derivative; and their salts and combinations thereof The complex according to any one of claims 1 to 3. A protein or polypeptide in which the cationic polymer or the cationic lipid or aggregates thereof is selected from protamine, histone, HelΔ1, gelatin; dendrimers selected from polyamidoamine dendrimers, polylysine dendrimers; linear or poly-branched Synthetic polymer selected from polyethyleneimine, 2-dimethylaminoethyl methacrylate polymer or copolymer, 2-trimethylaminoethyl methacrylate polymer or copolymer; polysaccharide derivative selected from chitosan, diethylaminoethyl-dextran DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol), DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DM RI (N- (1,2-Dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- (2- (sperminecarboxamido) ethyl) -N, N-dimethylammonium trifluoro Acetate), DOTAP (N- (1- (2,3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride), or DOTMA (N- (1- (2,3-dioleyl) A cationic lipid selected from oxy) propyl) -N, N, N-trimethylammonium chloride); Ekutamin, aggregate comprising a cationic lipid selected from lipofectin; and their salts as well as selected combinations thereof, conjugate of any one of claims 1-3. A cationic polymer or a cationic lipid or an assembly comprising it,
6. A composite according to claim 5 selected from polyethyleneimine, polyamidoamine dendrimer, and protamine. An anionic polymer is a polysaccharide or derivative thereof having a functional group selected from a carboxyl group, —OSO ₃ H group, —SO ₃ H group, phosphate group, and a salt thereof; and having a negatively charged side chain Polyamino acid containing amino acid residue; PEG derivative having carboxyl side chain; synthetic polymer having functional group selected from carboxyl group, -OSO ₃ H group, -SO ₃ H group, phosphate group, and salts thereof A functional group selected from a carboxyl group, —OSO ₃ H group, —SO ₃ H group, phosphoric acid group, and salts thereof, and an optionally substituted amino group or ammonium group or salt thereof (these groups Is a polymer having an alkyl group having 1 to 6 carbon atoms or a phenyl group which may be mono- or polysubstituted), and combinations thereof The complex according to any one of claims 1 to 6. A polysaccharide or a derivative thereof, wherein the anionic polymer is selected from hyaluronic acid, chondroitin, chondroitin sulfate, keratan sulfate, heparin, dermatan sulfate; polyamino acid selected from polyglutamic acid, polyaspartic acid; carboxyl side chain per PEG molecule 5 or more PEG derivatives having a molecular weight of 4,000 to 40,000; polymers or copolymers of acrylic acid or methacrylic acid having a molecular weight of 1,000 to 3,000,000, sulfate ester of polyvinyl alcohol, or succinimidylated poly- Synthetic polymer selected from L-lysine; 5 or more functional groups selected from carboxyl groups, —OSO ₃ H groups, —SO ₃ H groups, phosphate groups, and salts thereof, and substitution per molecule Amino group or ammoni which may be A PEG derivative having a molecular weight of 4,000 to 40,000, having an alkyl group or a salt thereof (these groups may be mono- or polysubstituted by an alkyl group having 1 to 6 carbon atoms or a phenyl group); The complex according to any one of claims 1 to 6, which is selected from a combination thereof. The composite according to claim 8, wherein the anionic polymer is hyaluronic acid. 10. The complex according to any one of claims 1 to 9, wherein the virus is selected from a DNA virus, an RNA virus, or a restricted-proliferating oncolytic virus thereof. The virus is selected from adenovirus, herpes simplex virus, adeno-associated virus, vaccinia virus, measles virus, Sendai virus, reovirus, foamy virus, Newcastle disease virus, lentivirus, Rabies virus, Pox virus, or myxoma virus selected. 10. The complex according to any one of claims 1 to 9, which is a virus or an RNA virus, or a restricted propagation oncolytic virus thereof. 10. The complex according to any one of claims 1 to 9, wherein the virus is a mutant virus or a recombinant virus. The method for producing a complex according to claim 1, wherein the complex is formed by sequentially mixing the virus; the cationic polymer or the cationic lipid or the aggregate containing the virus; and the anionic polymer in this order. Including methods. The method according to claim 13, comprising alternately mixing the step of mixing the cationic polymer or the cationic lipid or the aggregate containing the same; and the step of mixing the anionic polymer. The method according to claim 13 or 14, wherein two or more cationic polymers or cationic lipids or aggregates containing the same are mixed simultaneously or sequentially; and / or two or more anionic polymers are mixed simultaneously or sequentially. A complex comprising a virus; a cationic polymer or a cationic lipid or an aggregate containing the virus; and an anionic polymer produced by the method according to any one of claims 13 to 15. Two or more layers including a virus; a layer containing one or more cationic polymers or a cationic lipid or an assembly containing the same (cation layer); and a layer containing one or more anionic polymers (anion layer) A therapeutic method comprising administering an effective amount of a complex comprising a coating layer. The treatment method according to claim 17, wherein the virus is coated with a cation layer, and the cation layer and the anion layer are alternately coated. The cationic layer is formed by coating two or more kinds of cationic polymers or cationic lipids or aggregates containing the same simultaneously or sequentially; and / or the anionic layer is coated with two or more kinds of anionic polymers simultaneously or The treatment method according to claim 17 or 18, wherein the treatment method is formed by sequentially coating. The cationic polymer is selected from a positively charged protein or polypeptide; a positively charged dendrimer; a positively charged synthetic polymer; a positively charged polysaccharide derivative; and their salts and combinations thereof The therapeutic method according to any one of claims 17 to 19. A protein or polypeptide in which the cationic polymer or the cationic lipid or aggregates thereof is selected from protamine, histone, HelΔ1, gelatin; dendrimers selected from polyamidoamine dendrimers, polylysine dendrimers; linear or poly-branched Synthetic polymer selected from polyethyleneimine, 2-dimethylaminoethyl methacrylate polymer or copolymer, 2-trimethylaminoethyl methacrylate polymer or copolymer; polysaccharide derivative selected from chitosan, diethylaminoethyl-dextran DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol), DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DM RI (N- (1,2-Dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- (2- (sperminecarboxamido) ethyl) -N, N-dimethylammonium trifluoro Acetate), DOTAP (N- (1- (2,3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride), or DOTMA (N- (1- (2,3-dioleyl) A cationic lipid selected from oxy) propyl) -N, N, N-trimethylammonium chloride); Ekutamin, aggregate comprising a cationic lipid selected from lipofectin; and their salts as well as selected combinations thereof, a method of treating any one of claims 17-20. A cationic polymer or a cationic lipid or an assembly comprising it,
The method of treatment according to claim 21, wherein the method is selected from polyethyleneimine, polyamidoamine dendrimer, and protamine. An anionic polymer is a polysaccharide or derivative thereof having a functional group selected from a carboxyl group, —OSO ₃ H group, —SO ₃ H group, phosphate group, and a salt thereof; and having a negatively charged side chain Polyamino acid containing amino acid residue; PEG derivative having carboxyl side chain; synthetic polymer having functional group selected from carboxyl group, -OSO ₃ H group, -SO ₃ H group, phosphate group, and salts thereof A functional group selected from a carboxyl group, —OSO ₃ H group, —SO ₃ H group, phosphoric acid group, and salts thereof, and an optionally substituted amino group or ammonium group or salt thereof (these groups Is a polymer having an alkyl group having 1 to 6 carbon atoms or a phenyl group which may be mono- or polysubstituted), and combinations thereof The treatment method according to any one of claims 17 to 22. A polysaccharide or a derivative thereof, wherein the anionic polymer is selected from hyaluronic acid, chondroitin, chondroitin sulfate, keratan sulfate, heparin, dermatan sulfate; polyamino acid selected from polyglutamic acid, polyaspartic acid; carboxyl side chain per PEG molecule 5 or more PEG derivatives having a molecular weight of 4,000 to 40,000; polymers or copolymers of acrylic acid or methacrylic acid having a molecular weight of 1,000 to 3,000,000, sulfate ester of polyvinyl alcohol, or succinimidylated poly- Synthetic polymer selected from L-lysine; 5 or more functional groups selected from carboxyl groups, —OSO ₃ H groups, —SO ₃ H groups, phosphate groups, and salts thereof, and substitution per molecule Amino group or ammoni which may be A PEG derivative having a molecular weight of 4,000 to 40,000, having an alkyl group or a salt thereof (these groups may be mono- or polysubstituted by an alkyl group having 1 to 6 carbon atoms or a phenyl group); The treatment method according to any one of claims 17 to 22, which is selected from a combination thereof. The treatment method according to claim 24, wherein the anionic polymer is hyaluronic acid. The treatment method according to any one of claims 17 to 25, wherein the virus is selected from a DNA virus, an RNA virus, or a restricted-proliferating oncolytic virus thereof. The virus is selected from adenovirus, herpes simplex virus, adeno-associated virus, vaccinia virus, measles virus, Sendai virus, reovirus, foamy virus, Newcastle disease virus, lentivirus, Rabies virus, Pox virus, or myxoma virus selected. The treatment method according to any one of claims 17 to 26, which is a virus or RNA virus, or a restricted-proliferating oncolytic virus thereof. The treatment method according to any one of claims 17 to 27, wherein the virus is a mutant virus or a recombinant virus. The complex of claim 1 is produced by a method comprising the step of forming a complex by sequentially mixing a virus; a cationic polymer or a cationic lipid or an assembly comprising the same; and an anionic polymer in this order. The treatment method according to claim 17, wherein 30. The treatment method according to claim 29, comprising alternately mixing the step of mixing the cationic polymer or the cationic lipid or the assembly containing the same; and the step of mixing the anionic polymer. The therapeutic method according to claim 29 or 30, wherein two or more cationic polymers or cationic lipids or aggregates containing the same are mixed simultaneously or sequentially; and / or two or more anionic polymers are mixed simultaneously or sequentially. Two or more layers including a virus; a layer containing one or more cationic polymers or a cationic lipid or an assembly containing the same (cation layer); and a layer containing one or more anionic polymers (anion layer) A therapeutic agent comprising a complex comprising a coating layer. The therapeutic agent according to claim 32, wherein the virus is coated with a cation layer, and the cation layer and the anion layer are alternately coated. The cationic layer is formed by coating two or more kinds of cationic polymers or cationic lipids or aggregates containing the same simultaneously or sequentially; and / or the anionic layer is coated with two or more kinds of anionic polymers simultaneously or 34. The therapeutic agent according to claim 32 or 33, which is formed by sequential coating. The cationic polymer is selected from a positively charged protein or polypeptide; a positively charged dendrimer; a positively charged synthetic polymer; a positively charged polysaccharide derivative; and their salts and combinations thereof The therapeutic agent according to any one of claims 32 to 34. A protein or polypeptide in which the cationic polymer or the cationic lipid or aggregates thereof is selected from protamine, histone, HelΔ1, gelatin; dendrimers selected from polyamidoamine dendrimers, polylysine dendrimers; linear or poly-branched Synthetic polymer selected from polyethyleneimine, 2-dimethylaminoethyl methacrylate polymer or copolymer, 2-trimethylaminoethyl methacrylate polymer or copolymer; polysaccharide derivative selected from chitosan, diethylaminoethyl-dextran DC-Chol (3β- (N- (N ′, N′-dimethylaminoethane) carbamoyl) cholesterol), DDAB (N, N-distearyl-N, N-dimethylammonium bromide), DM RI (N- (1,2-Dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide), DODAC (N, N-dioleyl-N, N-dimethylammonium chloride), DOGS (diheptadecylamidoglycylspermidine), DOSPA (N- (1- (2,3-dioleyloxy) propyl) -N- (2- (sperminecarboxamido) ethyl) -N, N-dimethylammonium trifluoro Acetate), DOTAP (N- (1- (2,3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride), or DOTMA (N- (1- (2,3-dioleyl) A cationic lipid selected from oxy) propyl) -N, N, N-trimethylammonium chloride); Ekutamin, aggregate comprising a cationic lipid selected from lipofectin; and their salts as well as selected combinations thereof, the therapeutic agent of any one of claims 32-35. A cationic polymer or a cationic lipid or an assembly comprising it,
38. The therapeutic agent of claim 36, selected from polyethyleneimine, polyamidoamine dendrimer, and protamine. An anionic polymer is a polysaccharide or derivative thereof having a functional group selected from a carboxyl group, —OSO ₃ H group, —SO ₃ H group, phosphate group, and a salt thereof; and having a negatively charged side chain Polyamino acid containing amino acid residue; PEG derivative having carboxyl side chain; synthetic polymer having functional group selected from carboxyl group, -OSO ₃ H group, -SO ₃ H group, phosphate group, and salts thereof A functional group selected from a carboxyl group, —OSO ₃ H group, —SO ₃ H group, phosphoric acid group, and salts thereof, and an optionally substituted amino group or ammonium group or salt thereof (these groups Is a polymer having an alkyl group having 1 to 6 carbon atoms or a phenyl group which may be mono- or polysubstituted), and combinations thereof The therapeutic agent according to any one of claims 32 to 37. A polysaccharide or a derivative thereof, wherein the anionic polymer is selected from hyaluronic acid, chondroitin, chondroitin sulfate, keratan sulfate, heparin, dermatan sulfate; polyamino acid selected from polyglutamic acid, polyaspartic acid; carboxyl side chain per PEG molecule 5 or more PEG derivatives having a molecular weight of 4,000 to 40,000; polymers or copolymers of acrylic acid or methacrylic acid having a molecular weight of 1,000 to 3,000,000, sulfate ester of polyvinyl alcohol, or succinimidylated poly- Synthetic polymer selected from L-lysine; 5 or more functional groups selected from carboxyl groups, —OSO ₃ H groups, —SO ₃ H groups, phosphate groups, and salts thereof, and substitution per molecule Amino group or ammoni which may be A PEG derivative having a molecular weight of 4,000 to 40,000, having an alkyl group or a salt thereof (these groups may be mono- or polysubstituted by an alkyl group having 1 to 6 carbon atoms or a phenyl group); The therapeutic agent according to any one of claims 32 to 38, which is selected from a combination thereof. The therapeutic agent according to claim 39, wherein the anionic polymer is hyaluronic acid. The therapeutic agent according to any one of claims 32 to 40, wherein the virus is selected from a DNA virus, an RNA virus, or a restricted-proliferating oncolytic virus thereof. The virus is selected from adenovirus, herpes simplex virus, adeno-associated virus, vaccinia virus, measles virus, Sendai virus, reovirus, foamy virus, Newcastle disease virus, lentivirus, Rabies virus, Pox virus, or myxoma virus selected. The therapeutic agent according to any one of claims 32 to 41, which is a virus or RNA virus, or a restricted-proliferating oncolytic virus thereof. 43. The therapeutic agent according to any one of claims 32 to 42, wherein the virus is a mutant virus or a recombinant virus. The complex of claim 1 is produced by a method comprising the step of forming a complex by sequentially mixing a virus; a cationic polymer or a cationic lipid or an assembly comprising the same; and an anionic polymer in this order. The therapeutic agent according to claim 32, wherein 45. The therapeutic agent according to claim 44, comprising alternately mixing a step of mixing a cationic polymer or a cationic lipid or an aggregate containing the same; and a step of mixing an anionic polymer. The therapeutic agent according to claim 44 or 45, wherein two or more cationic polymers or cationic lipids or aggregates containing the same are mixed simultaneously or sequentially; and / or two or more anionic polymers are mixed simultaneously or sequentially.

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