WO2005070459A1 - Method of continuing expression of vector repeatedly administered - Google Patents

Method of continuing expression of vector repeatedly administered Download PDF

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Publication number
WO2005070459A1
WO2005070459A1 PCT/JP2005/000777 JP2005000777W WO2005070459A1 WO 2005070459 A1 WO2005070459 A1 WO 2005070459A1 JP 2005000777 W JP2005000777 W JP 2005000777W WO 2005070459 A1 WO2005070459 A1 WO 2005070459A1
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vector
complement
administration
expression
gene
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PCT/JP2005/000777
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French (fr)
Japanese (ja)
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WO2005070459A8 (en
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Hiroto Hara
Yasuji Ueda
Makoto Inoue
Mamoru Hasegawa
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Dnavec Research Inc.
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Publication of WO2005070459A8 publication Critical patent/WO2005070459A8/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin

Definitions

  • the present invention relates to a method and a drug for continuing gene expression from a vector with repeated administration, and particularly to a method and a drug using a complement inhibitor.
  • virus vectors There are two main types of virus vectors. One type is incorporated into the chromosome to execute gene expression such as transcription and replication, and the other type is to perform gene expression such as transcription and replication outside the chromosome.
  • the present invention enables repeated administration of a vector within a short period of time. It is an object of the present invention to provide a method capable of continuing gene expression and a drug capable of performing the method.
  • the present invention is based on these findings, and is specifically as follows.
  • a vector expression continuation agent comprising a complement inhibitor as an active ingredient.
  • a drug for gene therapy comprising a complement inhibitor as an active ingredient.
  • a repeated dose vector system comprising a complement inhibitor and a repeated dose vector.
  • FIG. 1 A graph showing a decrease in gene expression by 2 administrations of SeV shown as Reference Example 1.
  • FIG. 2 A graph showing an enhancement of SeV neutralization activity in the presence of complement shown as Reference Example 2. so is there.
  • A Inhibition activity of expression of genes mounted on SeV when complement is added or not added to mouse anti-SeV serum.
  • B As a control experiment, the results using mouse non-immune serum are shown.
  • FIG. 3 Draft showing the result of restoring the GFP gene expression of vector force by the complement inhibitor nafamostat mesilate (may be indicated as “FUT175”) in the in vitro analysis system.
  • A Suppressive activity of GFP expression in the experimental group supplemented with guinea pig complement (final concentration 1%) and complement inhibitor nafamostat mesilate at different concentrations.
  • B SeV-loaded GFP expression-inhibiting activity in experimental groups supplemented with different concentrations of the complement inhibitor nafamostat mesilate without complement.
  • FIG. 5 is a graph showing the anti-SeV antibody titer of the host just before the second administration of SeV vector.
  • FIG. 6 is a graph showing the results of restoring gene expression with vector inhibitor by complement inhibitor C1INH in an in vitro analysis system.
  • A Inhibition activity of SeV-loaded GFP expression in experimental groups supplemented with guinea pig complement (final concentration 1%) and different concentrations of complement inhibitor C1INH.
  • B SeV-loaded GFP expression-inhibiting activity in experimental groups in which complement inhibitors C1INH with different concentrations were added to non-complement-added cells.
  • the present invention firstly provides a method of continuing gene expression of a repetitive administration type vector force using a complement inhibitor.
  • the repetitive administration type vector means a vector that may be repeatedly administered within a short period of time during which the elimination action by the host immune system works for reasons such as high expression of the on-board gene, and such a host. It is a vector that is expected to increase its usefulness, such as the spread of applicable diseases, if repeated administration is possible within a short period of time during which the immune system eliminates.
  • Examples of such vectors that exist outside the chromosome, such as the cytoplasm after introduction into the host are Sendai virus vectors and -Eucastle disease virus (
  • NDV vectors such as Paramyxoinoles vector, Baculoinores vector, Box virus vectors such as wild virus vectors, alphavirus vectors, herpes virus vectors, influenza virus vectors, etc. There is.
  • paramyxovirus vectors are suitable examples of the repeated administration vector of the present invention.
  • Sendai virus vectors belonging to this species can be repeatedly administered at long intervals, but if they can be repeatedly administered within a short period of time, they are one of the vectors that are expected to expand the applicable diseases of gene therapy.
  • the paramyxovirus vector may be, for example, a complex of paramyxovirus genomic RNA and viral protein, ie, ribonucleoprotein (RNP).
  • RNP can be introduced into cells in combination with, for example, a desired transfection reagent.
  • Such an RNP is specifically a complex containing the paramyxovirus genomic RNA, N protein, P protein, and L protein.
  • the viral protein functions to transcribe the cistron that encodes the viral protein from the genomic RNA, and the genome itself replicates to form a daughter RNP.
  • the paramyxovirus vector may be a gene lacking any of the genes of the wild-type paramyxovirus.
  • a paramyxovirus vector containing a M, F, or HN gene, or a combination thereof can be suitably used as the paramyxovirus vector of the present invention.
  • Such reconstitution of a viral vector can be performed, for example, by supplying a defective gene product exogenously.
  • the virus vector produced in this way adheres to the host cell in the same way as the wild type virus and is then introduced into the cell by fusing the cell membrane and the vector envelope.
  • the vector genome introduced into the cell is the virus.
  • Daughter virus particles with infectivity similar to the wild type are not formed because of the gene defect.
  • genes that also lack genomic power include the F gene and the Z or HN gene.
  • Viral vectors can be reconstituted by transfecting cells (International Publication Nos. WO00 / 70055 and WO00 / 70070; Li, H.-0. Et al., J. Virol. 74 (14)
  • a virus can be produced using a host cell in which the F gene is integrated into the chromosome.
  • the amino acid sequence is not the virus-derived sequence as it is, but if the activity in introducing the nucleic acid is equal to or higher than that of the wild type, a mutation is introduced or other Substituting with the homologous gene of the virus.
  • the vectors are heterogeneous, they may be repeatedly administered between vectors having the same cross-reactivity. As long as both can be recognized as the same antigen by the host immune system, the method of the present invention is effective.
  • the method for administering these vectors to the host is not particularly limited, and is administered by a method suitable for the type of vector.
  • the administration route can be selected as appropriate, but it can be performed, for example, transdermally, intranasally, transbronchially, intramuscularly, intraperitoneally, intravenously, intraarticularly, intrathecally, or subcutaneously, but is not limited thereto. . It can also be administered locally or systemically.
  • the amount of vector administered is generally about 10 5 ClU / ml to about 10 11 CIU / mU, preferably about 10 7 CIU / ml to about 10 9 CIU / ml, more preferably about 1 X 10 8 CIU.
  • an amount in the range of from / ml to about 5 ⁇ 10 8 CIU / ml is administered in a pharmaceutically acceptable carrier.
  • the dose per dose is 2 X 10 5 CIU — 2 X 10 10 CIU is preferred, and can be given once or multiple times within the range of clinically acceptable side effects. The same applies to the number of administrations.
  • a complement inhibitor is used in order to continuously express a gene introduced into a host via a repeated administration type vector as described above.
  • Complement is a group of proteins belonging to a natural immune system that recognizes and eliminates foreign substances such as pathogens that have invaded a living body.
  • the antigen-antibody conjugate formed by capturing the foreign body with the antibody is complemented by the antigen-antibody conjugate formed by a part of the complement system.
  • a series of systems This enhances the elimination of foreign substances by antibodies.
  • complement system components There are about 20 types of complement system components, of which nine are called complement components, C1 to C9.
  • complement inhibitors also inhibit the classical pathway, alternative pathway, or both pathways (Makrides SC, Pharmacol Reviews 1998 50 (l: 59-87, bahu A et al. Immunopharmacology 2000 49: 133-148).
  • the complement inhibitor that can be used in the invention is not particularly limited as long as it can continuously express a gene introduced into a host via a repeated administration vector.
  • Anti-C5 antibody formulation Antibody formulation against necrotic components such as pexelizumab (Alexion Pharmaceuticals Inc)
  • Solubilized protein factors Solubilized Clq receptors such as TP10, TP20 (Clq inhibition, Marsh H and Ryan Ub, 1997, Xenotransplantation: The Transplantation of Organs ana Tissues Between Species (Cooper DKC, Kemp E, Piatt JL and White DJG, eds) 2nd ed, pp 437-455, Springer, Berlin), solubilized CD59 (Suppression of MAC complex formation, Suzuki H, et al. 1996 FEBS Lett 399: 272-276), solubilized DAF (C3 / C5Convertase Inhibition, Moran P et al. 1992 J Immunol 149: 1736-1743) and solubilized MCP (C3 / C5 Convertase inhibition, Christiansen D et al. 1996 Eur J Immunol 26: 578-585)
  • Peptide inhibitors C089 (C5 receptor antagonist, Konteatis ZD et al. 1994 J Immunol 153: 4200-4205), Compstatin (C3 inhibitor, Sahu A, et al. J Immunol 1996 157 (2): 884-91)
  • a complement inhibitor can be used as a complement inhibitor of the present invention as long as it suppresses complement that enhances neutralizing activity against a repetitive administration type vector and can continue expression of vector force.
  • Preferable examples include complement inhibitors that can suppress C or C5 among complements.
  • Complement inhibitors that can suppress C1 include those listed as “C1 suppression” in the complement inhibitors listed above, as well as anti-C1 antibodies and C1 receptor antagonists.
  • Complement inhibitors that can suppress C5 are described as “C5 suppression”, “anti-C5 antibody”, “C5 receptor antagonist”, “C3 / C5 convertase inhibition” among the complement inhibitors listed above. Or those having functions equivalent to these.
  • the timing of the use of a complement suppressor to continue gene expression from a repeated dose vector is at the time of vector administration, preferably at the repeated dose where the vector is administered again after the first administration of the vector. .
  • the beta be administered repeatedly after the second dose.
  • “during administration” in “vector administration” or “repetitive administration” means mixing into a vector solution and administering it to the host at the same time, or separately from the vector solution and before and after vector administration. Or administration by different routes.
  • the method of administration of the complement inhibitor is not necessarily the same as the method of administration of the vector and the administration site, and as an example, the complement inhibitor is administered orally to the intravenous administration of the vector. Another route or form of administration Any administration method capable of continuing gene expression from the vector is included in the present invention.
  • the complement inhibitor is not limited to one type of use, and a plurality of types may be used in combination. Alternatively, the type may be changed for each vector administration. Further, the dosage of the complement inhibitor may be constant or may be changed for each vector administration.
  • continuation of expression means enabling or facilitating gene expression from repeatedly administered vectors.
  • the expression level after repeated administration as a result of this continuation of expression may be the same level or higher than the expression level after the first vector introduction, or less. Even in this case, it is possible to include a deviation in the case where a significantly higher expression can be maintained as compared with the case where a conventional complement inhibitor is not administered.
  • the use of a complement suppressor enables gene expression from a repeatedly administered vector in a repeat-administration vector, thereby complement suppression. Significantly higher gene expression can be maintained than when no agent is used. Therefore, it is possible to continuously impart the gene function loaded on the vector to the host. Therefore, according to the present invention, the therapeutic effect of gene therapy is improved when used in the medical field such as gene therapy, and more stable when used in the research field such as the creation of transgenic animals. To provide a representation.
  • Another aspect of the present invention relates to a vector expression continuation agent comprising a complement inhibitor as an active ingredient.
  • the complement suppressor can be used in the repeated administration of a repeated administration type vector to continue the expression of the gene loaded on the vector. Therefore, this complement inhibitor can be used as a vector expression continuation agent.
  • the vector expression continuation agent means a reagent for suppressing the second and subsequent expression reduction generally observed in vectors repeatedly administered and continuing the expression.
  • This vector expression continuation agent contains a complement inhibitor capable of continuing gene expression from the vector.
  • Whether or not the expression of the gene of complement inhibitory vector power can be continued is described in the Examples. It can be evaluated using the method shown. That is, after introducing the first vector into a laboratory animal such as a mouse, a candidate complement inhibitor is administered when the second vector is introduced. Evaluate the expression of the loaded gene after the second and subsequent vector introductions based on the expression of the reporter gene. Then, the expression level of the loaded gene in the host to which the drug is not administered is compared with the expression level of the loaded gene in the host to which the complement inhibitor has been administered. When the expression level is high, it is evaluated that the complement inhibitor can be used as a vector expression continuation agent.
  • this complement inhibitor when used as a vector expression continuation agent, it may be composed of one type of complement inhibitor force or a mixture of plural types of complement inhibitors. Note that complement inhibitors that can be used as a vector expression continuation agent are as described above, and a description thereof is omitted here. Moreover, other components can be contained in addition to the complement inhibitor.
  • anti-inflammatory drugs cortisol, dexamethasone, prednisolone, triamcinolone, etc.
  • immunosuppressants cyclophosphamide, azathioprine, 6-mercaptopurine, methotrexate, mizoribine, cyclosporine, tacrolimus, OKT-3, Baslliximab, Zenapax, Remicade
  • it may be diluted with a physiological saline or a buffer that may contain a preservative or the like.
  • the vector expression continuation agent of the present invention can be used as a reagent for producing a transgenic animal.
  • a vector expression continuation agent is used for the production of a transgenic animal, the expression of a foreign gene introduced through the vector can be continued, so that a stable phenotype can be imparted.
  • Yet another embodiment of the present invention is a gene therapy drug comprising a complement inhibitor as an active ingredient.
  • the complement suppressor can be used for repeated administration of a repeated-dose vector, so that the expression of the gene loaded on the vector can be continued. It can be used as a drug used in combination with gene therapy. That is, the drug for gene therapy comprising the complement suppressant of the present invention as an active ingredient is a repetitive administration vector carrying a gene having a function desired to be given to a patient that does not itself exert the effect of gene therapy. In combination with the agent of the present invention, the expression of the gene loaded on the vector is continued. It has an effect.
  • a pharmaceutical in order to prepare a complement inhibitor as an auxiliary drug for the above gene therapy, a pharmaceutical can be combined with a desired pharmacologically acceptable carrier or vehicle as necessary.
  • this carrier or medium is a suspending agent, surfactant, stabilizer, killing agent as long as it is within the range of materials that do not significantly inhibit the activity that can continue the expression of the onboard gene on the vector by the complement inhibitor.
  • Biological agents, preservatives, and other additives can be added.
  • the gene therapy drug can be used for the treatment of humans and animals other than humans as an adjunct to gene therapy using a repetitive administration type vector.
  • gene expression from the repeated administration vector can be continued and the therapeutic effect can be improved.
  • This drug can be used at the time of the first administration of the vector. For example, if you are naturally infected with a virus that can induce neutralizing activity that is the same or different from the vector but crossed before gene therapy with a repeated dose vector, Even at the time of vector administration, gene expression from the vector may be suppressed. Therefore, if neutralization activity that crosses the vector used for treatment is detected in the patient's serum, this gene therapy drug can be used at the first vector introduction to allow expression of the on-board gene. It ’s good.
  • the present invention can also be provided as a repeated administration vector system comprising a complement inhibitor and a repeated administration vector.
  • a complement inhibitor for continuing expression from this vector is provided, so that research fields such as the production of transgenic animals, medical fields such as gene therapy, etc.
  • the present invention can be made easier to use.
  • the expression level of the vector-borne gene was examined when the second administration of the SeV vector was performed.
  • the Ffly-deficient SeV vector was loaded with the firefly luciferase gene as a reporter gene.
  • This SeV vector (5xl0 6 CIU / mouse) was intranasally administered to Balb / cA mice as the first, and eight days later, the same vector lxlO 8 CIU was administered into the thigh muscle.
  • muscles were collected and homogenized, and luciferase activity in the homogenate was determined for the Luciferase Assay System (Promega).
  • the luciferase activity at the second administration was almost reduced to the background level as compared with the first time. This indicates that the host has acquired the neutralizing activity of the SeV vector by multiple administrations.
  • SeV neutralization activity was enhanced by complement.
  • anti-SeV serum Seb vector (2xl0 7 CIU / mouse) was intraperitoneally administered to Balb / cA mice (60 mice), and the same amount of vector was additionally administered on days 14 and 21. .
  • whole blood was collected from all the heads and pooled to obtain anti-SeV serum.
  • the non-immune serum used as a control was pooled from the serum of 30 untreated mice.
  • the serum was previously heated at 56 ° C for 30 minutes.
  • Guinea pig complement (CedarLane CL5000-1) was used as the complement, and was used at a final concentration of 1% in the experimental system.
  • Neutralization activity was measured by modifying a known method (neutralization test, pp261-274, revised second edition, Virus Experimental Studies, General Review, National Institute of Preventive Hygiene, Alumni Association, 1973 Maruzen).
  • a dilution series of anti-SeV serum or non-immune serum was prepared. Complement was added to this serum dilution series as necessary, and then mixed with a certain amount of the GFP gene-loaded F gene-deficient SeV betater. This mixed solution was kept at 37 ° C. for 1 hour. After incubation the mixture was added LLC-MK cells (about 2xl0 5 cells / well) ⁇ this in 96 well, cells were infected with SeV.
  • the horizontal axis represents the serum dilution rate
  • the vertical axis represents the neutralizing activity.
  • Neutralizing activity was shown relative to 0% of the fluorescence in wells without vector addition and 100% of the fluorescence in wells without vector addition-antiserum. In the graph, the more the curve is shifted to the right, the stronger the neutralization activity.
  • Neutralization activity was measured in the same manner as described in Reference Example 2 except that a complement inhibitor was added.
  • nafamostat mesilate (Core Inhibitor for Injection 10, Shimizu Pharmaceutical) is a mixture of antiserum and complement (or antiserum) in Reference Example 2 above so that the final concentration is 0.1 mg / m or O.Olmg / ml. Only) and then the vector solution was added to this mixture.
  • Complement suppression 2J administration increases SeV vector expression at the second administration
  • a peptide-type complement inhibitor 2J (Roos A, et al. J Immunol. 2001 167 (12): 7052-9) that suppresses C1 of the classical pathway was used as a complement inhibitor.
  • the 2J peptide used in this example was synthesized based on the amino acid sequence published in the literature of Roos A et al.
  • the GFP gene-loaded F gene-deficient SeV vector was first administered intranasally at 5xl0 6 CIU per Balb / cA mouse.
  • 250 g of each 2J peptide dissolved in 100 1 DPBS (-) was intravenously injected into the tail.
  • Type SeV vector (5xl0 6 CIU / mouse) was mixed with 2J peptide (1.25 / zg) and administered by auricle.
  • the pinna was cut and collected, and the Luciferase activity in the homogenate was measured using the Luciferase Assay System (Promega).
  • the single-dose group was prepared in the same manner as the double-dose group except that the first administration of GFP-loaded SeV / dF vector was not performed.
  • DPBS ( ⁇ ) was administered in place of 2J in each of the twice-administered group and the once-administered group was performed in parallel.
  • Example 2 the anti-SeV antibody titer in the plasma collected just before the second administration on the 14th day was measured by ELISA (Presym Seika HVJ, Denka Seken). In the single dose group, the antibody titer is measured before the first vector introduction.
  • C1INH complement inhibitory action of C1INH was determined in Example 1, except that instead of nafamostat mesilate, the complement inhibitor C1INH “Belinaito! 3 ” (Aventis Pharma) was used. It carried out similarly by activity measurement. “Berinaito! 3 ” was added so that the final concentration was equivalent to 0, 3, 6, 12-fold concentrated plasma.
  • Figure 5 (A) shows the experimental group with guinea pig complement added at a final concentration of 1%, and (b) shows the experimental group with no complement added.

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Abstract

It is found out that, in the elimination a vector from the immune system of a host, a complement potentiates the function. By administering a complement inhibitor in the second administration of the vector and thereafter, the gene expression from the vector can be significantly continued compared with the case of not administering the complement inhibitor. A method, which is based on the above finding, for continuing the gene expression of a vector to be repeatedly administered twice or more characterized in that a complement inhibitor is administered in the second administration of the vector and thereafter. Moreover, the complement inhibitor is provided as a reagent or a drug whereby the expression from a vector to be repeatedly administered can be continued.

Description

明 細 書  Specification
繰返し投与を伴うベクターの発現を継続させる方法  Method of continuing the expression of a vector with repeated administration
技術分野  Technical field
[0001] 本発明は、繰返し投与を伴うベクターからの遺伝子発現を継続させる方法および薬 剤、特に、補体抑制剤を利用した方法および薬剤に関する。  [0001] The present invention relates to a method and a drug for continuing gene expression from a vector with repeated administration, and particularly to a method and a drug using a complement inhibitor.
背景技術  Background art
[0002] 遺伝性疾患の根本治療法としてあるいは癌、虚血性疾患等の重症性疾患の治療 法として遺伝子治療に対する期待は大きい。中でもウィルスに基づくベクターを用い る方法は遺伝子発現効率の高さから繁用されている。ウィルスベクターには、大きく 二つの種類が存在する。一方は染色体内に組み込まれて転写'複製などの遺伝子 発現を実行するタイプと、他方は染色体外で転写'複製などの遺伝子発現を実行す るタイプとがある。  There is a great expectation for gene therapy as a fundamental therapy for genetic diseases or as a therapy for severe diseases such as cancer and ischemic diseases. Among them, the method using a virus-based vector is frequently used because of its high gene expression efficiency. There are two main types of virus vectors. One type is incorporated into the chromosome to execute gene expression such as transcription and replication, and the other type is to perform gene expression such as transcription and replication outside the chromosome.
[0003] このうち、後者のタイプでは、染色体内への組み込みが生じないため染色体異常に よる癌化または不死化などの安全面における問題が生じない一方で、ウィルスベクタ 一に搭載された治療用遺伝子の発現期間には限界がある。そのため、この種のベタ ターを用いて遺伝子発現を継続させる場合、繰返し投与が必要とされることがある。 しかし、多くのウィルスベクターはベクター自体の免疫原性のために繰返し投与は困 難であり、臨床応用上解決すべき問題となっている。また、ベクターの中には長期の 間隔をあければ繰返し投与に適用できるものもある力 短期間内の繰返し投与が可 能となれば、そのベクターを利用した遺伝子治療の適用疾患の拡大が期待される。  [0003] Of these, the latter type does not cause integration into the chromosome, so there are no safety issues such as canceration or immortalization due to chromosomal abnormalities. There is a limit to the duration of gene expression. Therefore, repeated administration may be required when gene expression is continued using this type of beta. However, many viral vectors are difficult to administer repeatedly due to the immunogenicity of the vector itself, and this is a problem to be solved in clinical application. In addition, some vectors can be applied to repeated administration at long intervals. If repeated administration is possible within a short period of time, the use of the vector for gene therapy is expected to expand. The
[0004] そのため、宿主免疫反応の制御により短期間内での繰返し投与を可能とする一般 的方法が確立されればベクターの治療効果が大幅に改善され、適用範囲も大きく広 力 Sると考えられる。し力しながら、そのような技術はいまだ確立されておらずその開発 が待ち望まれている。  [0004] Therefore, if a general method that enables repeated administration within a short period of time by controlling the host immune response is established, the therapeutic effect of the vector will be greatly improved, and the scope of application will be greatly increased. It is done. However, such a technology has not yet been established and its development is awaited.
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0005] そこで、本発明は、短期間内のベクターの繰返し投与を可能とし、ベクターからの 遺伝子発現を継続させ得る方法および当該方法の実行を可能とする薬剤を提供す ることを課題とする。 [0005] Therefore, the present invention enables repeated administration of a vector within a short period of time. It is an object of the present invention to provide a method capable of continuing gene expression and a drug capable of performing the method.
課題を解決するための手段  Means for solving the problem
[0006] 上記課題に鑑み、本発明者らは、鋭意研究を重ねた結果、ベクターが宿主免疫系 により排除される際、補体がその作用を増強していることを見出した。そこで、短期間 内に複数回ベクターを繰返し投与する場合、その 2回目以降のベクター導入時に補 体抑制剤を投与すると、補体抑制剤非投与群に比してベクターからの遺伝子発現を 上昇させ得ることに成功した。  [0006] In view of the above problems, the present inventors have conducted extensive research and found that complement enhances its action when a vector is eliminated by the host immune system. Therefore, when a vector is administered repeatedly several times within a short period of time, if a complement inhibitor is administered at the second and subsequent vector introductions, gene expression from the vector is increased compared to the group not administered with the complement inhibitor. Succeeded in getting.
[0007] すなわち、本発明はこれら知見に基づくものであり、具体的には以下の通りである。 That is, the present invention is based on these findings, and is specifically as follows.
1. 所望の遺伝子を担持したベクターを繰返し投与することにより遺伝子を継続的に 発現させる方法において、ベクター投与時に補体抑制剤が投与される、方法。  1. A method in which a complement inhibitor is administered at the time of vector administration in a method of continuously expressing a gene by repeatedly administering a vector carrying a desired gene.
2. ベクター投与時がベクターが繰返し投与される時である、上記 1記載の方法。 2. The method according to 1 above, wherein the administration of the vector is when the vector is administered repeatedly.
3.ベクター投与時が 2回目以降にベクターが投与される時である、上記 1記載の方 法。 3. The method according to 1 above, wherein the vector is administered at the second and subsequent times.
4.補体抑制剤が補体成分 C1または C5を抑制し得る、上記 1記載の方法。  4. The method according to 1 above, wherein the complement inhibitor can inhibit complement component C1 or C5.
5.補体抑制剤を有効成分とする、ベクター発現継続剤。  5. A vector expression continuation agent comprising a complement inhibitor as an active ingredient.
6.補体抑制剤が補体成分 C1または C5を抑制し得る、上記 5記載のベクター発現継 続剤。  6. The vector expression continuation agent according to 5 above, wherein the complement inhibitor can suppress complement component C1 or C5.
7.補体抑制剤を有効成分とする、遺伝子治療用薬剤。  7. A drug for gene therapy, comprising a complement inhibitor as an active ingredient.
8.補体抑制剤が補体成分 Cほたは C5を抑制し得る、上記 7記載の遺伝子治療用 薬剤。  8. The gene therapy drug according to 7 above, wherein the complement inhibitor can inhibit complement components C and C5.
9.補体抑制剤と繰返し投与型ベクターとを含む、繰返し投与ベクターシステム。 9. A repeated dose vector system comprising a complement inhibitor and a repeated dose vector.
10.補体抑制剤が補体成分 C1または C5を抑制し得る、上記 9記載の繰返し投与べ クタ一システム。 10. The repeated dose vector system according to 9 above, wherein the complement inhibitor can suppress complement component C1 or C5.
図面の簡単な説明  Brief Description of Drawings
[0008] [図 1]参考例 1として示した SeV2回投与による遺伝子発現の低下を示すグラフである [図 2]参考例 2として示した補体存在下における SeV中和活性の増強を示すグラフで ある。 (A)マウス抗 SeV血清に補体添加あるいは非添カ卩した場合の SeVに搭載された 遺伝子発現の阻害活性を示す。（B)対照実験として、マウス非免疫血清を用いた結 果を示す。 [0008] [Fig. 1] A graph showing a decrease in gene expression by 2 administrations of SeV shown as Reference Example 1. [Fig. 2] A graph showing an enhancement of SeV neutralization activity in the presence of complement shown as Reference Example 2. so is there. (A) Inhibition activity of expression of genes mounted on SeV when complement is added or not added to mouse anti-SeV serum. (B) As a control experiment, the results using mouse non-immune serum are shown.
[図 3]In vitro解析系において、補体抑制剤 nafamostat mesilate(「FUT175」として表示 される場合がある)によりベクター力 の GFP遺伝子発現を回復させた結果を示すダラ フである。（A)モルモット補体 (最終濃度 1%)および異なる濃度の補体抑制剤 nafamostat mesilateを添カ卩した実験群における SeV搭載 GFP発現の抑制活性を示す 。 （B)補体非添加にお 、て異なる濃度の補体抑制剤 nafamostat mesilateを添カ卩した 実験群における SeV搭載 GFPの発現抑制活性を示す。  [Fig. 3] Draft showing the result of restoring the GFP gene expression of vector force by the complement inhibitor nafamostat mesilate (may be indicated as “FUT175”) in the in vitro analysis system. (A) Suppressive activity of GFP expression in the experimental group supplemented with guinea pig complement (final concentration 1%) and complement inhibitor nafamostat mesilate at different concentrations. (B) SeV-loaded GFP expression-inhibiting activity in experimental groups supplemented with different concentrations of the complement inhibitor nafamostat mesilate without complement.
[図 4]SeVベクター 2回目投与時に補体抑制剤 2Jを投与した際のベクター搭載遺伝子 [Fig.4] Vector-loaded genes when complement inhibitor 2J is administered at the second administration of SeV vector
(ルシフェラーゼ遺伝子）の発現上昇を示したグラフである。 It is the graph which showed the expression increase of (luciferase gene).
[図 5]SeVベクター 2回目投与直前の宿主の抗 SeV抗体価を示すグラフである。  FIG. 5 is a graph showing the anti-SeV antibody titer of the host just before the second administration of SeV vector.
[図 6]In vitro解析系において、補体抑制剤 C1INHによりベクター力もの遺伝子発現を 回復させた結果を示すグラフである。 (A)モルモット補体 (最終濃度 1 %)および異な る濃度の補体抑制剤 C1INHを添加した実験群における SeV搭載 GFP発現の抑制活 性を示す。 (B)補体非添カ卩にお 、て異なる濃度の補体抑制剤 C1INHを添加した実 験群における SeV搭載 GFPの発現抑制活性を示す。  FIG. 6 is a graph showing the results of restoring gene expression with vector inhibitor by complement inhibitor C1INH in an in vitro analysis system. (A) Inhibition activity of SeV-loaded GFP expression in experimental groups supplemented with guinea pig complement (final concentration 1%) and different concentrations of complement inhibitor C1INH. (B) SeV-loaded GFP expression-inhibiting activity in experimental groups in which complement inhibitors C1INH with different concentrations were added to non-complement-added cells.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0009] 以下、本発明について、実施の形態を用いて、より詳細に説明する。 Hereinafter, the present invention will be described in more detail using embodiments.
[0010] 本発明は第一に、補体抑制剤を用いて繰返し投与型ベクター力もの遺伝子発現を 継続させる方法を提供する。 [0010] The present invention firstly provides a method of continuing gene expression of a repetitive administration type vector force using a complement inhibitor.
[0011] ここで繰返し投与型ベクターとは、搭載遺伝子を高度に発現させるためなどの理由 から宿主免疫系による排除作用が働く短期間内に繰返して投与される可能性がある ベクターや、こうした宿主免疫系による排除作用が働く短期間内に繰返し投与が可 能になれば更に適用疾患が広がるなどの有用性が増すと期待されるベクターである 。こうしたベクターは宿主への導入後、細胞質などの染色体外に存在するベクターが 多ぐ例を挙げれば、センダイウィルスベクターおよび-ユーキャッスル病ウィルス（[0011] Here, the repetitive administration type vector means a vector that may be repeatedly administered within a short period of time during which the elimination action by the host immune system works for reasons such as high expression of the on-board gene, and such a host. It is a vector that is expected to increase its usefulness, such as the spread of applicable diseases, if repeated administration is possible within a short period of time during which the immune system eliminates. Examples of such vectors that exist outside the chromosome, such as the cytoplasm after introduction into the host, are Sendai virus vectors and -Eucastle disease virus (
NDV )ベクターなどのパラミクソゥイノレスベクター、バキュロウイノレスベクター、ヮクシ- ァウィルスベクターなどのボックスウィルスベクター、アルファウィルスベクター、ヘル ぺスウィルスベクター、インフルエンザウイルスベクターなどのオルソミクソウィルスべ クタ一、ラブドウィルスベクター、アデノアソシエイテッドウィルス（AAV )ベクター、ァ デノウィルスべクタ一などがある。 NDV) vectors such as Paramyxoinoles vector, Baculoinores vector, Box virus vectors such as wild virus vectors, alphavirus vectors, herpes virus vectors, influenza virus vectors, etc. There is.
[0012] 上記繰返し投与型ベクターのうちパラミクソウィルスベクターは、本発明の繰返し投 与型ベクターとして好適な例である。この種に属するセンダイウィルスベクターは長期 に間隔をあければ繰返し投与が可能であるが、短期間内の繰返し投与が可能となれ ば遺伝子治療の適用疾患の拡大が期待されるベクターの一つである。このパラミクソ ウィルスベクターは、例えばパラミクソウィルスのゲノム RNAとウィルス蛋白質力 なる 複合体、すなわちリボヌクレオプロテイン (RNP)であってもよい。 RNPは、例えば所望 のトランスフエクシヨン試薬と組み合わせて細胞に導入することができる。このような RNPは、具体的にはパラミクソウィルスのゲノム RNA、 N蛋白質、 P蛋白質、および L蛋 白質を含む複合体である。 RNPは細胞内に導入されると、ウィルス蛋白質の働きによ りゲノム RNAからウィルス蛋白質をコードするシストロンが転写されると共に、ゲノム自 身が複製され娘 RNPが形成される。  [0012] Of the above repeated administration vectors, paramyxovirus vectors are suitable examples of the repeated administration vector of the present invention. Sendai virus vectors belonging to this species can be repeatedly administered at long intervals, but if they can be repeatedly administered within a short period of time, they are one of the vectors that are expected to expand the applicable diseases of gene therapy. . The paramyxovirus vector may be, for example, a complex of paramyxovirus genomic RNA and viral protein, ie, ribonucleoprotein (RNP). RNP can be introduced into cells in combination with, for example, a desired transfection reagent. Such an RNP is specifically a complex containing the paramyxovirus genomic RNA, N protein, P protein, and L protein. When RNP is introduced into a cell, the viral protein functions to transcribe the cistron that encodes the viral protein from the genomic RNA, and the genome itself replicates to form a daughter RNP.
[0013] パラミクソウィルスベクターは野生型パラミクソウィルスが持つ遺伝子の 、ずれかを 欠損したものであってよい。例えば、 M、 F、または HN遺伝子、あるいはそれらの組み 合わせが含まれて!/ヽな ヽパラミクソウィルスベクターも、本発明のパラミクソウィルスべ クタ一として好適に用いることができる。このようなウィルスベクターの再構成は、例え ば、欠損している遺伝子産物を外来的に供給することにより行うことができる。このよう にして製造されたウィルスベクターは、野生型ウィルスと同様に宿主細胞に接着した のち細胞膜とベクターのエンベロープが融合することによって細胞内に導入されるが 、細胞に導入されたベクターゲノムはウィルス遺伝子に欠損を有するため、野生型と 同じような感染力を持つ娘ウィルス粒子は形成されない。このため、一回限りの遺伝 子導入力を持つ安全なウィルスベクターとして有用である。ゲノム力も欠損させる遺 伝子としては、例えば F遺伝子および Zまたは HN遺伝子が挙げられる。例えば、 F遺 伝子が欠損した組み換えパラミクソウィルスベクターゲノムを発現するプラスミドを、 F 蛋白質の発現ベクターならびに NP、 P、および L蛋白質の発現ベクターと共に宿主細 胞にトランスフエクシヨンすることにより、ウィルスベクターの再構成を行うことができる（ 国際公開番号 WO00/70055および WO00/70070; Li, H.- 0. et al.， J. Virol. 74(14)[0013] The paramyxovirus vector may be a gene lacking any of the genes of the wild-type paramyxovirus. For example, a paramyxovirus vector containing a M, F, or HN gene, or a combination thereof can be suitably used as the paramyxovirus vector of the present invention. Such reconstitution of a viral vector can be performed, for example, by supplying a defective gene product exogenously. The virus vector produced in this way adheres to the host cell in the same way as the wild type virus and is then introduced into the cell by fusing the cell membrane and the vector envelope. The vector genome introduced into the cell is the virus. Daughter virus particles with infectivity similar to the wild type are not formed because of the gene defect. Therefore, it is useful as a safe viral vector with a one-time gene transfer ability. Examples of genes that also lack genomic power include the F gene and the Z or HN gene. For example, a plasmid that expresses a recombinant paramyxovirus vector genome deficient in the F gene together with an F protein expression vector and NP, P, and L protein expression vectors. Viral vectors can be reconstituted by transfecting cells (International Publication Nos. WO00 / 70055 and WO00 / 70070; Li, H.-0. Et al., J. Virol. 74 (14)
6564-6569 (2000))。また、例えば、 F遺伝子が染色体に組み込まれた宿主細胞を用 いてウィルスを製造することもできる。これらの蛋白質群を外から供給する場合、その アミノ酸配列はウィルス由来の配列そのままでなくとも、核酸の導入における活性が 野生型のそれと同等かそれ以上ならば、変異を導入したり、あるいは他のウィルスの 相同遺伝子で代用してもょ 、。 6564-6569 (2000)). In addition, for example, a virus can be produced using a host cell in which the F gene is integrated into the chromosome. When these proteins are supplied from the outside, the amino acid sequence is not the virus-derived sequence as it is, but if the activity in introducing the nucleic acid is equal to or higher than that of the wild type, a mutation is introduced or other Substituting with the homologous gene of the virus.
[0014] 本発明では、上述したようなベクターが繰返し投与されることが前提である力 投与 毎に全く同一のベクターが用いられる場合は当然のことながら、また、投与毎に同種 のベクターの野生型と改変型とを違えて、もしくは異なる改変型を違えて用いる場合  [0014] In the present invention, it is a matter of course that the same vector is used for each administration, which is based on the premise that the above-described vector is repeatedly administered. When using different types with different types or different types
、さらには、異種のベクターであっても互いにクロスリアクティビティ一があるベクター 間で繰返し投与される場合も本発明の適用対象に包含される。いずれも宿主免疫系 により同じ抗原として認識され得る限り、本発明の方法は有効である。 In addition, even when the vectors are heterogeneous, they may be repeatedly administered between vectors having the same cross-reactivity. As long as both can be recognized as the same antigen by the host immune system, the method of the present invention is effective.
[0015] これらベクターの宿主への投与方法は、特に限定はなぐベクターの種類に応じて 適した方法で投与される。投与経路は適宜選択することができるが、例えば経皮、鼻 腔内、経気管支、筋内、腹腔内、静脈内、関節内、脊髄腔内、または皮下等に行わ れ得るがそれらに限定されない。また局所あるいは全身に投与し得る。投与されるべ クタ一量は一般的には約 105 ClU/mlから約 1011 CIU/mU好ましくは約 107 CIU/mlから 約 109 CIU/ml,より好ましくは約 1 X 108 CIU/mlから約 5 X 108 CIU/mlの範囲内の量を 薬学上容認可能な担体中で投与される。ヒトにおいては 1回当たりの投与量は 2 X 10 5 CIU— 2 X 1010 CIUが好ましぐ投与回数は、 1回または臨床上容認可能な副作用 の範囲で複数回可能であり、 1日の投与回数についても同様である。 [0015] The method for administering these vectors to the host is not particularly limited, and is administered by a method suitable for the type of vector. The administration route can be selected as appropriate, but it can be performed, for example, transdermally, intranasally, transbronchially, intramuscularly, intraperitoneally, intravenously, intraarticularly, intrathecally, or subcutaneously, but is not limited thereto. . It can also be administered locally or systemically. The amount of vector administered is generally about 10 5 ClU / ml to about 10 11 CIU / mU, preferably about 10 7 CIU / ml to about 10 9 CIU / ml, more preferably about 1 X 10 8 CIU. An amount in the range of from / ml to about 5 × 10 8 CIU / ml is administered in a pharmaceutically acceptable carrier. In humans, the dose per dose is 2 X 10 5 CIU — 2 X 10 10 CIU is preferred, and can be given once or multiple times within the range of clinically acceptable side effects. The same applies to the number of administrations.
[0016] 本発明は、上述したような繰返し投与型ベクターを介して宿主内に導入された遺伝 子を継続的に発現させるために、補体抑制剤を用いる。  [0016] In the present invention, a complement inhibitor is used in order to continuously express a gene introduced into a host via a repeated administration type vector as described above.
補体とは生体に侵入した病原体などの異物を侵入初期に認識して排除する自然免 疫系に属する一群のタンパク質である。過去の感染などによって生体内にすでに異 物に対する抗体が存在する場合は、異物を抗体が捕らえることによって形成される抗 原抗体結合物が補体系の一部によって形成される抗原抗体結合物が補体系の一連 の反応を活性ィ匕し、抗体による異物排除を補強する。補体系成分は 20種類ほど存在 し、そのなかで補体成分と呼ばれるもには 9つの成分、 C1から C9がある。反応の経路 は古典経路と 2次経路 (又は、第 2経路、別経路、副経路)の二つがある。補体抑制 剤も、古典経路、代替経路あるいは両経路を抑制するものがあり（Makrides SC, Pharmacol Reviews 1998 50(l :59-87, bahu A et al. Immunopharmacology 2000 49:133-148)、本発明において使用し得る補体抑制剤は、繰返し投与型ベクターを 介して宿主内に導入された遺伝子を継続的に発現させ得るものであれば、特に制限 はない。 Complement is a group of proteins belonging to a natural immune system that recognizes and eliminates foreign substances such as pathogens that have invaded a living body. When antibodies against foreign substances already exist in the living body due to past infection or the like, the antigen-antibody conjugate formed by capturing the foreign body with the antibody is complemented by the antigen-antibody conjugate formed by a part of the complement system. A series of systems This enhances the elimination of foreign substances by antibodies. There are about 20 types of complement system components, of which nine are called complement components, C1 to C9. There are two reaction routes: the classical route and the secondary route (or the second route, alternative route, and sub route). Some complement inhibitors also inhibit the classical pathway, alternative pathway, or both pathways (Makrides SC, Pharmacol Reviews 1998 50 (l: 59-87, bahu A et al. Immunopharmacology 2000 49: 133-148). The complement inhibitor that can be used in the invention is not particularly limited as long as it can continuously express a gene introduced into a host via a repeated administration vector.
古典経路を抑制する補体抑制剤 Complement inhibitors that suppress the classical pathway
• タンパク質因子： C1INH (C1抑制、 Davis AE, Annu Rev Immunol. 1988; 6:  • Protein factor: C1INH (C1 inhibitor, Davis AE, Annu Rev Immunol. 1988; 6:
595-628) (595-628)
• ペプチド型抑制剤： 2J (C1抑制、 Roos A, et al. J Immunol. 2001 167(12):7052-9) 、 H17 (C2受容体抑制、 Inal JM, et al. J Immunol 2003 170(8):4310-7)  • Peptide type inhibitors: 2J (C1 inhibition, Roos A, et al. J Immunol. 2001 167 (12): 7052-9), H17 (C2 receptor inhibition, Inal JM, et al. J Immunol 2003 170 (8 ): 4310-7)
代替経路を抑制する補体抑制剤 Complement inhibitors that suppress alternative pathways
• 低分子合成剤： BCX-1470 (Factor Dおよび Cls抑制、 Szalai AJ et al. J Immunol. • Small molecule synthesis agent: BCX-1470 (Factor D and Cls inhibition, Szalai AJ et al. J Immunol.
2000 164(1): 463-8) 2000 164 (1): 463-8)
両経路に共通する補体抑制剤 Complement inhibitors common to both pathways
• 抗 C5抗体製剤 pexelizumab (Alexion Pharmaceuticals Inc)などのネ甫体系成分に対 する抗体製剤  • Anti-C5 antibody formulation Antibody formulation against necrotic components such as pexelizumab (Alexion Pharmaceuticals Inc)
• 可溶化タンパク質因子： TP10, TP20などの可溶化 Clq受容体（Clq抑制、 Marsh Hし and Ryan Ub, 1997, Xenotransplantation: The Transplantation of Organs ana Tissues Between Species (Cooper DKC, Kemp E, Piatt JL and White DJG, eds) 2nd ed, pp 437-455, Springer, Berlin)、可溶化 CD59 (MAC複合体形成抑制、 Suzuki H, et al. 1996 FEBS Lett 399:272- 276)、可溶化 DAF (C3/C5Convertase阻害、 Moran P et al. 1992 J Immunol 149:1736- 1743)および可溶化 MCP(C3/C5Convertase阻害 、 Christiansen D et al. 1996 Eur J Immunol 26:578-585)  • Solubilized protein factors: Solubilized Clq receptors such as TP10, TP20 (Clq inhibition, Marsh H and Ryan Ub, 1997, Xenotransplantation: The Transplantation of Organs ana Tissues Between Species (Cooper DKC, Kemp E, Piatt JL and White DJG, eds) 2nd ed, pp 437-455, Springer, Berlin), solubilized CD59 (Suppression of MAC complex formation, Suzuki H, et al. 1996 FEBS Lett 399: 272-276), solubilized DAF (C3 / C5Convertase Inhibition, Moran P et al. 1992 J Immunol 149: 1736-1743) and solubilized MCP (C3 / C5 Convertase inhibition, Christiansen D et al. 1996 Eur J Immunol 26: 578-585)
• ペプチド型抑制剤： C089 (C5受容体アンタゴ-スト、 Konteatis ZD et al. 1994 J Immunol 153:4200- 4205)、 Compstatin (C3抑制、 Sahu A, et al. J Immunol 1996 157(2):884-91) • Peptide inhibitors: C089 (C5 receptor antagonist, Konteatis ZD et al. 1994 J Immunol 153: 4200-4205), Compstatin (C3 inhibitor, Sahu A, et al. J Immunol 1996 157 (2): 884-91)
• 低分子合成剤： nafamostat mesilate (Fujii S, Hitomi Y, Biochim Biophys Acta 1981 661(2):342-5)、 K76および K76誘導体（K76COOHなど）（C5抑制、 Miyazaki W et al. 1980 Microbiol Immunol 24: 1091-1108、 Sindelar RD et al. 1996 U.S. Patent Number 5,506,247)、 PR226 (C5受容体アンタゴ-スト、 Baranyi L. et al. 1996 Nat Med 1:894-901)、 Complestatin (Momota K et al. 1991 Biochem Biophys Res Commun 179:243- 250)、 CGS32359 (C5受容体アンタゴ-スト、 Pellas TC et al. 1998 J Immunol 160:5616- 5621)、 A8 (C5受容体アンタゴ-スト、 de Vries B et al. 2003 J Immunol 170: 3883-3889)、 AcF-[OPdChaWR] (C5受容体アンタゴ-スト、 Strachan AJ et al. 2000 J Immunol 164: 6560-6565)、 W54011 (C5受容体アンタゴ-スト、 Sumichika H et al. 2002 J Bio Chem 277: 49403-49407)  • Small molecule synthesis agents: nafamostat mesilate (Fujii S, Hitomi Y, Biochim Biophys Acta 1981 661 (2): 342-5), K76 and K76 derivatives (such as K76COOH) (C5 inhibitor, Miyazaki W et al. 1980 Microbiol Immunol 24 : 1091-1108, Sindelar RD et al. 1996 US Patent Number 5,506,247), PR226 (C5 receptor antagonist, Baranyi L. et al. 1996 Nat Med 1: 894-901), Complestatin (Momota K et al. 1991 Biochem Biophys Res Commun 179: 243-250), CGS32359 (C5 receptor antagonist, Pellas TC et al. 1998 J Immunol 160: 5616- 5621), A8 (C5 receptor antagonist, de Vries B et al. 2003 J Immunol 170: 3883-3889), AcF- [OPdChaWR] (C5 receptor antagonist, Strachan AJ et al. 2000 J Immunol 164: 6560-6565), W54011 (C5 receptor antagonist, Sumichika H et al. 2002 J Bio Chem 277: 49403-49407)
上述したように、補体抑制剤は繰返し投与型ベクターに対する中和活性を増強する 補体を抑制し、ベクター力 の発現を継続させ得るもであれば、本発明の補体抑制 剤として使用し得る。好適な例としては、補体のうちでも Cほたは C5を抑制し得る補 体抑制剤が挙げられる。 C1を抑制し得る補体抑制剤としては、上記に列挙した補体 抑制剤中、「C1抑制」と記載されたものの他、抗 C1抗体、 C1受容体アンタゴ-ストが 含まれる。 C5を抑制し得る補体抑制剤としては、上記に列挙した補体抑制剤中、「C5 抑制」、「抗 C5抗体」、「C5受容体アンタゴニスト」、 「C3/C5Convertase阻害」と記載さ れたものあるいはこれらと同等の機能を有するものが含まれる。 As described above, a complement inhibitor can be used as a complement inhibitor of the present invention as long as it suppresses complement that enhances neutralizing activity against a repetitive administration type vector and can continue expression of vector force. obtain. Preferable examples include complement inhibitors that can suppress C or C5 among complements. Complement inhibitors that can suppress C1 include those listed as “C1 suppression” in the complement inhibitors listed above, as well as anti-C1 antibodies and C1 receptor antagonists. Complement inhibitors that can suppress C5 are described as “C5 suppression”, “anti-C5 antibody”, “C5 receptor antagonist”, “C3 / C5 convertase inhibition” among the complement inhibitors listed above. Or those having functions equivalent to these.
繰返し投与型ベクターからの遺伝子発現を継続させるための補体抑制剤の使用タ イミングは、ベクター投与時、好ましくは、初回にベクターを投与した後に再びべクタ 一が投与される繰返し投与時である。繰返し投与時は、 2回目以降に繰り返してベタ ターが投与される度であることが好まし 、。ここで「ベクター投与時」または「繰返し投 与時」における「投与時」とは、ベクター溶液に混合して同時に宿主に投与すること、 または、ベクター溶液とは別にベクター投与とタイミングを前後して、あるいは経路を 違えて投与することのいずれでもよい。補体抑制剤の投与方法は、ベクターの投与 方法、投与部位とは必ずしも同じである必要はなぐ一例を挙げれば、ベクターが静 脈投与に対して補体抑制剤は経口投与などのように、別の投与経路あるいは形態な どでも、ベクターからの遺伝子発現を継続できる投与方法であれば、本発明に包含さ れる。 The timing of the use of a complement suppressor to continue gene expression from a repeated dose vector is at the time of vector administration, preferably at the repeated dose where the vector is administered again after the first administration of the vector. . During repeated administration, it is preferable that the beta be administered repeatedly after the second dose. Here, “during administration” in “vector administration” or “repetitive administration” means mixing into a vector solution and administering it to the host at the same time, or separately from the vector solution and before and after vector administration. Or administration by different routes. The method of administration of the complement inhibitor is not necessarily the same as the method of administration of the vector and the administration site, and as an example, the complement inhibitor is administered orally to the intravenous administration of the vector. Another route or form of administration Any administration method capable of continuing gene expression from the vector is included in the present invention.
[0018] また、補体抑制剤は一種類の使用に限定されず、複数種を混合して用いてもよぐ また、ベクター投与毎にその種類を変更して用いてもよい。また、補体抑制剤の投与 量は一定であってもよぐまたベクター投与毎に変更してもよい。  [0018] Further, the complement inhibitor is not limited to one type of use, and a plurality of types may be used in combination. Alternatively, the type may be changed for each vector administration. Further, the dosage of the complement inhibitor may be constant or may be changed for each vector administration.
[0019] 上記補体抑制剤を用いることにより、繰返し導入されたベクター力 の遺伝子発現 の低下が抑制され、遺伝子の発現が継続される。本書において発現の継続とは、繰 返し投与されたベクターからの遺伝子発現を可能あるいは促進させることを意味する 。また、この発現継続の結果としての繰返し投与時の発現レベルは、初回のベクター 導入後の発現レベルと比較して、同程度であっても、それ以上であってもよぐまた、 それ以下であっても従来の補体抑制剤を投与しない場合に比べて有意に高い発現 を維持できる場合の ヽずれをも含めることができる。  [0019] By using the above complement suppressor, a decrease in gene expression due to repeatedly introduced vector force is suppressed, and gene expression is continued. In this document, continuation of expression means enabling or facilitating gene expression from repeatedly administered vectors. In addition, the expression level after repeated administration as a result of this continuation of expression may be the same level or higher than the expression level after the first vector introduction, or less. Even in this case, it is possible to include a deviation in the case where a significantly higher expression can be maintained as compared with the case where a conventional complement inhibitor is not administered.
[0020] 上記の通り、本発明によれば、補体抑制剤を用いることにより、繰返し投与型べクタ 一における繰返し投与されるベクターからの遺伝子発現を可能とし、これによつて、 補体抑制剤を用いない場合に比して有意に高い遺伝子発現を維持することが可能と なる。そのため、ベクターに搭載された遺伝子機能を宿主に継続して付与することが 可能となる。よって、本発明により、遺伝子治療などの医療分野に活用した場合には 遺伝子治療の治療効果の改善をもたらし、また、トランスジエニック動物の創生などの 研究分野に活用した場合には、より安定した表現形を提供する。  [0020] As described above, according to the present invention, the use of a complement suppressor enables gene expression from a repeatedly administered vector in a repeat-administration vector, thereby complement suppression. Significantly higher gene expression can be maintained than when no agent is used. Therefore, it is possible to continuously impart the gene function loaded on the vector to the host. Therefore, according to the present invention, the therapeutic effect of gene therapy is improved when used in the medical field such as gene therapy, and more stable when used in the research field such as the creation of transgenic animals. To provide a representation.
[0021] 本発明の別の態様としては、補体抑制剤を有効成分とするベクター発現継続剤に 関する。上述した通り、補体抑制剤は繰返し投与型ベクターを繰返し投与する際に 用いることにより、ベクターに搭載された遺伝子発現を継続させることができる。その ため、この補体抑制剤は、ベクター発現継続剤として利用することができる。ここで、 ベクター発現継続剤とは、一般に繰返し投与されるベクターにおいて見られる 2回目 以降の発現低下を抑制し、発現を継続させるための試薬を意味する。本ベクター発 現継続剤は、上記ベクターからの遺伝子発現を継続させ得る補体抑制剤を含有する  [0021] Another aspect of the present invention relates to a vector expression continuation agent comprising a complement inhibitor as an active ingredient. As described above, the complement suppressor can be used in the repeated administration of a repeated administration type vector to continue the expression of the gene loaded on the vector. Therefore, this complement inhibitor can be used as a vector expression continuation agent. Here, the vector expression continuation agent means a reagent for suppressing the second and subsequent expression reduction generally observed in vectors repeatedly administered and continuing the expression. This vector expression continuation agent contains a complement inhibitor capable of continuing gene expression from the vector.
[0022] 補体抑制剤力 ベクター力 の遺伝子の発現を継続させ得るか否かは、実施例に 示した方法を用いて評価することができる。すなわち、マウスなどの実験動物に初回 のベクターを導入後、 2回目のベクターを導入する際に候補となる補体抑制剤を投与 する。 2回目以降のベクター導入後に搭載されている遺伝子の発現をレポーター遺 伝子の発現を基に評価する。そして、薬剤を投与していない宿主内での搭載遺伝子 の発現レベルと、補体抑制剤を投与した宿主内での搭載遺伝子の発現レベルとを比 較して、補体抑制剤投与群のほうが、発現レベルが高い場合に、その補体抑制剤は ベクター発現継続剤として利用することができると評価される。 Whether or not the expression of the gene of complement inhibitory vector power can be continued is described in the Examples. It can be evaluated using the method shown. That is, after introducing the first vector into a laboratory animal such as a mouse, a candidate complement inhibitor is administered when the second vector is introduced. Evaluate the expression of the loaded gene after the second and subsequent vector introductions based on the expression of the reporter gene. Then, the expression level of the loaded gene in the host to which the drug is not administered is compared with the expression level of the loaded gene in the host to which the complement inhibitor has been administered. When the expression level is high, it is evaluated that the complement inhibitor can be used as a vector expression continuation agent.
[0023] この補体抑制剤をベクター発現継続剤として使用する場合、一種類の補体抑制剤 力も構成しても、複数種の補体抑制剤を混合して構成してもよい。なお、ベクター発 現継続剤として使用し得る補体抑制剤は上述した通りであり、ここでの説明は省略す る。また、補体抑制剤以外に他の成分を含有させることもできる。例えば、抗炎症剤（ コルチゾール、デキサメタゾン、プレドニゾロン、トリアムシノロンなど）や免疫抑制剤（ シクロフォスフアミド、ァザチォプリン、 6-メルカプトプリン、メトトレキサート、ミゾリビン、 シクロスポリン、タクロリムス、 OKT- 3、 Baslliximab、 Zenapax、 Remicadeなど）を混合し てもよい。さらに、必要に応じて、保存剤などを添加してもよぐ生理食塩水や緩衝剤 で希釈してもよい。  [0023] When this complement inhibitor is used as a vector expression continuation agent, it may be composed of one type of complement inhibitor force or a mixture of plural types of complement inhibitors. Note that complement inhibitors that can be used as a vector expression continuation agent are as described above, and a description thereof is omitted here. Moreover, other components can be contained in addition to the complement inhibitor. For example, anti-inflammatory drugs (cortisol, dexamethasone, prednisolone, triamcinolone, etc.) and immunosuppressants (cyclophosphamide, azathioprine, 6-mercaptopurine, methotrexate, mizoribine, cyclosporine, tacrolimus, OKT-3, Baslliximab, Zenapax, Remicade ) May be mixed. Furthermore, if necessary, it may be diluted with a physiological saline or a buffer that may contain a preservative or the like.
[0024] 上記本発明のベクター発現継続剤は、トランスジエニック動物を作製する際の試薬 として利用することができる。ベクター発現継続剤をトランスジエニック動物作製に利 用した場合、ベクターを介して導入された外来遺伝子の発現を継続させることができ ることから、安定した表現形を付与することができる。  [0024] The vector expression continuation agent of the present invention can be used as a reagent for producing a transgenic animal. When a vector expression continuation agent is used for the production of a transgenic animal, the expression of a foreign gene introduced through the vector can be continued, so that a stable phenotype can be imparted.
[0025] 本発明のさらなる別の形態は、補体抑制剤を有効成分とする遺伝子治療用薬剤で ある。上述した通り、補体抑制剤は繰返し投与型ベクターを繰返し投与する際に用い ることにより、ベクターに搭載された遺伝子発現を継続させることができるため、こうし た繰返し投与型のベクターを利用した遺伝子治療とともに併用される医薬品として利 用することができる。すなわち、本発明の補体抑制剤を有効成分とする遺伝子治療 用薬剤は、そのものが遺伝子治療の効果を発揮するものではなぐ患者に付与した い機能を備えた遺伝子を搭載させた繰返し投与型ベクターを繰返し投与する際に、 本発明の薬剤を併用することにより、ベクターに搭載させた遺伝子の発現を継続させ る効果を有する。 [0025] Yet another embodiment of the present invention is a gene therapy drug comprising a complement inhibitor as an active ingredient. As described above, the complement suppressor can be used for repeated administration of a repeated-dose vector, so that the expression of the gene loaded on the vector can be continued. It can be used as a drug used in combination with gene therapy. That is, the drug for gene therapy comprising the complement suppressant of the present invention as an active ingredient is a repetitive administration vector carrying a gene having a function desired to be given to a patient that does not itself exert the effect of gene therapy. In combination with the agent of the present invention, the expression of the gene loaded on the vector is continued. It has an effect.
[0026] 補体抑制剤を上記遺伝子治療用の補助的な薬剤として調製するには、必要に応じ て薬理学的に許容される所望の担体または媒体と組み合わせて医薬品とすることが できる。また、この担体または媒体は、補体抑制剤によるベクター上の搭載遺伝子の 発現を継続させ得る活性を有意に阻害しない材料の範囲であれば、懸濁剤、界面活 性剤、安定剤、殺生物剤、保存剤、その他の添加剤を添加することができる。  [0026] In order to prepare a complement inhibitor as an auxiliary drug for the above gene therapy, a pharmaceutical can be combined with a desired pharmacologically acceptable carrier or vehicle as necessary. In addition, this carrier or medium is a suspending agent, surfactant, stabilizer, killing agent as long as it is within the range of materials that do not significantly inhibit the activity that can continue the expression of the onboard gene on the vector by the complement inhibitor. Biological agents, preservatives, and other additives can be added.
[0027] 上記遺伝子治療用薬剤は、繰返し投与型ベクターを用いた遺伝子治療の補助薬 として、ヒトおよびヒト以外の動物の治療に利用することができる。本発明の薬剤を用 いること〖こより、繰返し投与型ベクターからの遺伝子発現を継続させ、治療効果を改 善することができる。本薬剤は、ベクターを繰返し投与する際に用いることが主要な 用法となる力 初回のベクター投与時に使用してもよい。例えば、繰返し投与型べク ターによる遺伝子治療を行う以前に、該ベクターと同種または異種であるが交差し得 る中和活性を惹起し得るウィルスに自然感染している場合には、 1回目のベクター投 与時であってもベクターからの遺伝子発現が抑制される場合がある。そのため、患者 の血清中に治療に用いるベクターと交差する中和活性が検出された場合には、一回 目のベクター導入時に本遺伝子治療用薬剤を使用し、搭載遺伝子の発現を可能と してちよい。 [0027] The gene therapy drug can be used for the treatment of humans and animals other than humans as an adjunct to gene therapy using a repetitive administration type vector. By using the drug of the present invention, gene expression from the repeated administration vector can be continued and the therapeutic effect can be improved. This drug can be used at the time of the first administration of the vector. For example, if you are naturally infected with a virus that can induce neutralizing activity that is the same or different from the vector but crossed before gene therapy with a repeated dose vector, Even at the time of vector administration, gene expression from the vector may be suppressed. Therefore, if neutralization activity that crosses the vector used for treatment is detected in the patient's serum, this gene therapy drug can be used at the first vector introduction to allow expression of the on-board gene. It ’s good.
[0028] また、本発明は、補体抑制剤と繰返し投与型ベクターとを含む、繰返し投与べクタ 一システムとして提供することもできる。このようにシステムとして、繰返し投与型べクタ 一と共に、このベクターからの発現を継続させるための補体抑制剤を提供することに より、トランスジェニック動物作製などの研究分野、遺伝子治療などの医療分野にお いて本発明を一層利用し易くすることができる。  [0028] The present invention can also be provided as a repeated administration vector system comprising a complement inhibitor and a repeated administration vector. In this way, as a system, together with a repetitive administration vector, a complement inhibitor for continuing expression from this vector is provided, so that research fields such as the production of transgenic animals, medical fields such as gene therapy, etc. Thus, the present invention can be made easier to use.
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に 組み入れられる。  All prior art documents cited in the present specification are incorporated herein by reference.
実施例  Example
[0029] 以下、参考例を交えて、本発明の実施例を示すが、本発明はここに示す実施例に 限定されるものではない。  Hereinafter, examples of the present invention will be described with reference examples. However, the present invention is not limited to the examples shown here.
[0030] [参考例 1] SeVベクター 2回目投与時の搭載遺伝子発現量低下 [0030] [Reference Example 1] Decreased gene expression at the second administration of SeV vector
SeVベクターの初回投与で誘導された特異的抗体存在下で、 SeVベクターの 2回目 投与を行なった場合のベクター搭載遺伝子の発現量を検討した。本検討を行うに当 たって、 F遺伝子欠失型 SeVベクターにレポーター遺伝子として firefly luciferase遺伝 子を搭載した。この SeVベクター（5xl06 CIU/マウス）を Balb/cAマウスに第一回目とし て経鼻投与し、その 8日後に第二回目として同ベクター lxlO8 CIUを大腿筋肉中に投 与した。 2回目投与後 2日目に筋肉を採取、ホモジネートし、そのホモジネート中の Luciferase活'性 Luciferase Assay System (Promega社) 用 ヽ H U定した。 In the presence of the specific antibody induced by the first administration of the SeV vector, the expression level of the vector-borne gene was examined when the second administration of the SeV vector was performed. In conducting this study, the Ffly-deficient SeV vector was loaded with the firefly luciferase gene as a reporter gene. This SeV vector (5xl0 6 CIU / mouse) was intranasally administered to Balb / cA mice as the first, and eight days later, the same vector lxlO 8 CIU was administered into the thigh muscle. On day 2 after the second administration, muscles were collected and homogenized, and luciferase activity in the homogenate was determined for the Luciferase Assay System (Promega).
[0031] その結果、図 1に示すように、初回と比較して 2回目投与時のルシフェラーゼ活性は 、ほぼバックグランドレベルに低下した。このことから、複数回の投与により宿主が SeV ベクターの中和活性を獲得したことを示す。  As a result, as shown in FIG. 1, the luciferase activity at the second administration was almost reduced to the background level as compared with the first time. This indicates that the host has acquired the neutralizing activity of the SeV vector by multiple administrations.
[0032] [参考例 2]  [0032] [Reference Example 2]
抗 SeV血清中の SeV中和活性の補体による増強  Complement enhancement of SeV neutralization activity in anti-SeV serum
上記 SeV中和活性が補体により増強されるか否かを検討した。本検討にあたって、 抗 SeV血清、非免疫血清、補体を準備した。抗 SeV血清の調製のために、 Balb/cAマ ウス（60頭）に SeVベクター（2xl07 CIU/マウス）を腹腔内投与後、 14日目と 21日目に 同量のベクターを追加投与した。初回投与から 30日目に全頭について全血採取し、 これをプールして抗 SeV血清とした。また、対照として用いた非免疫血清は非投与マ ウス 30頭分の血清をプールして用いた。なお、中和活性の測定に使用した抗 SeV血 清に混在する内在性補体を非働化するために、血清は予め 56°C、 30分加熱処理し た。補体はモルモット補体 (CedarLane社 CL5000-1)を用い、実験系にお 、て最終 濃度 1%で使用した。 It was investigated whether the SeV neutralization activity was enhanced by complement. In this study, anti-SeV serum, non-immune serum, and complement were prepared. For the preparation of anti-SeV serum, Seb vector (2xl0 7 CIU / mouse) was intraperitoneally administered to Balb / cA mice (60 mice), and the same amount of vector was additionally administered on days 14 and 21. . On the 30th day after the first administration, whole blood was collected from all the heads and pooled to obtain anti-SeV serum. The non-immune serum used as a control was pooled from the serum of 30 untreated mice. In order to inactivate the endogenous complement mixed in the anti-SeV serum used for measuring the neutralizing activity, the serum was previously heated at 56 ° C for 30 minutes. Guinea pig complement (CedarLane CL5000-1) was used as the complement, and was used at a final concentration of 1% in the experimental system.
[0033] 中和活性の測定は既知の方法（中和試験、 pp261-274、改訂二版 ウィルス実験学 総論 国立予防衛生研究所学友会編 1973丸善)を改変して行なった。まず抗 SeV血清あるいは非免疫血清の希釈系列を作製した。この血清の希釈系列に、必要 に応じて補体を添加し、その後、一定量の GFP遺伝子搭載 F遺伝子欠失型 SeVベタ ターと混合した。この混合溶液を 37°Cで 1時間保温した。保温後、混合液を 96 well中 の LLC- MK細胞（約 2xl05 cells/well)〖こ添加し、細胞を SeVに感染させた。 COイン キュベータ一中でさらに 37°Cで 1時間保温した後、ベクタ一一抗血清混合液を除去、 細胞を一度洗浄した後培養液 (DMEM)を加えて 3日間培養した。培養後、感染細胞 を測定するために、細胞内での GFP発現量を Microplate用 Fluorometerで測定した（ 図 2)。 [0033] Neutralization activity was measured by modifying a known method (neutralization test, pp261-274, revised second edition, Virus Experimental Studies, General Review, National Institute of Preventive Hygiene, Alumni Association, 1973 Maruzen). First, a dilution series of anti-SeV serum or non-immune serum was prepared. Complement was added to this serum dilution series as necessary, and then mixed with a certain amount of the GFP gene-loaded F gene-deficient SeV betater. This mixed solution was kept at 37 ° C. for 1 hour. After incubation the mixture was added LLC-MK cells (about 2xl0 5 cells / well) 〖this in 96 well, cells were infected with SeV. CO in After further incubating at 37 ° C for 1 hour in the incubator, the vector monoantiserum mixture was removed, the cells were washed once, and then the medium (DMEM) was added and cultured for 3 days. After incubation, in order to measure infected cells, the amount of GFP expression in the cells was measured with a Fluorometer for Microplate (Figure 2).
[0034] 図 2において、横軸に血清希釈率を、縦軸に中和活性を示す。また中和活性は、 ベクター非添加 wellの蛍光量を 0%、ベクター添加 -抗血清非添加 wellの蛍光量を 100%として相対的に示した。グラフでは曲線が右側へシフトするほど中和活性が強い ことになる。  In FIG. 2, the horizontal axis represents the serum dilution rate, and the vertical axis represents the neutralizing activity. Neutralizing activity was shown relative to 0% of the fluorescence in wells without vector addition and 100% of the fluorescence in wells without vector addition-antiserum. In the graph, the more the curve is shifted to the right, the stronger the neutralization activity.
[0035] 図 2Aに示す通り、補体存在下では非存在下の場合に比べて約 100倍の中和活性 が観察された。このことは SeVに対する中和活性に補体が関与することを強く示唆す る。  [0035] As shown in FIG. 2A, neutralization activity about 100 times greater in the presence of complement than in the absence of complement was observed. This strongly suggests that complement is involved in the neutralizing activity against SeV.
[0036] [実施例 1]  [Example 1]
In vitroにおける nafamostat mesilateのネ 体抑巿 ij作用  In vitro inhibition of nafamostat mesilate by ij action
上述の通り、補体存在下で中和活性が上昇したことから、補体抑制剤が SeVに対す る中和活性に与える影響を調べた。 Compstatin (Sahu A, et al. J Immunol 1996 157(2):884-91) , H17 (lnal JM, et al. J Immunol 2003 170(8):4310- 7)等多くの補体 抑制剤がこれまでに開発されているが（Makrides SC, Pharmacol Reviews 1998 50(1):59- 87, Sahu A et al. Immunopharmacology 2000 49: 133- 148)、その中でまず 既に臨沐で使用されてい onafamostat mesilate (Fujn S, Hitomi Y, Biochim Biophys Acta 1981 661(2):342- 5)を本実施例で用いた。  As described above, since neutralizing activity increased in the presence of complement, the effect of complement inhibitors on neutralizing activity against SeV was investigated. Many complement inhibitors such as Compstatin (Sahu A, et al. J Immunol 1996 157 (2): 884-91) and H17 (lnal JM, et al. J Immunol 2003 170 (8): 4310-7) (Makrides SC, Pharmacol Reviews 1998 50 (1): 59-87, Sahu A et al. Immunopharmacology 2000 49: 133-148), but it has already been used in the first onafamostat mesilate (Fujn S, Hitomi Y, Biochim Biophys Acta 1981 661 (2): 342-5) was used in this example.
[0037] 中和活性測定は補体抑制剤を添加する点を除き上記参考例 2に示した方法と同様 に実施した。 nafamostat mesilate (注射用コアヒビター 10、清水製薬）は最終濃度 0.1mg/mほたは O.Olmg/mlとなるように、上記参考例 2における抗血清と補体との混 合液 (または抗血清のみ）に添加し、その後、この混合液にベクター溶液を添加した。  [0037] Neutralization activity was measured in the same manner as described in Reference Example 2 except that a complement inhibitor was added. nafamostat mesilate (Core Inhibitor for Injection 10, Shimizu Pharmaceutical) is a mixture of antiserum and complement (or antiserum) in Reference Example 2 above so that the final concentration is 0.1 mg / m or O.Olmg / ml. Only) and then the vector solution was added to this mixture.
[0038] 図 3Aに示すように、 nafamostat mesilateを添カ卩することにより補体存在下で見られ た中和活性増強が図 3Bに示す補体非添カ卩時のレベルまで完全に抑制された。  [0038] As shown in Fig. 3A, by adding nafamostat mesilate, the enhancement of neutralization activity observed in the presence of complement was completely suppressed to the level when complement was not added as shown in Fig. 3B. It was.
[0039] [実施例 2]  [0039] [Example 2]
補体抑制 2Jの投与による SeVベクター 2回目投与時の搭載遺伝子発現量上昇 次にマウスを用いて in vivoにおける補体抑制剤の効果を調べた。本実施例では補 体抑制剤として、古典経路の C1を抑制するペプチド型補体抑制剤 2J (Roos A, et al. J Immunol. 2001 167(12):7052-9)を用いた。なお、本実施例で用いた 2Jペプチドは Roos Aらの文献掲載のアミノ酸配列をもとに合成して用いた。 Complement suppression 2J administration increases SeV vector expression at the second administration Next, the effect of complement inhibitors in vivo was examined using mice. In this example, a peptide-type complement inhibitor 2J (Roos A, et al. J Immunol. 2001 167 (12): 7052-9) that suppresses C1 of the classical pathway was used as a complement inhibitor. The 2J peptide used in this example was synthesized based on the amino acid sequence published in the literature of Roos A et al.
[0040] 2回投与群 (n=3)ではまず 1回目に GFP遺伝子搭載 F遺伝子欠失型 SeVベクターを Balb/cAマウス 1頭あたり 5xl06 CIU経鼻投与した。 1回目のベクター投与から 14日目 に 1頭あたり 250 g (100 1の DPBS (-)に溶解)の 2Jペプチドを尾静注し、その後 15 分以内に 2回目として Luciferase遺伝子搭載 F遺伝子欠失型 SeVベクター（5xl06 CIU/マウス)を 2Jペプチド（1.25 /z g)と混合し耳介投与した。 2回目投与後 2日目に 耳介を切断採取、ホモジネート中の Luciferase活性を Luciferase Assay System (Promega社）を用いて測定した。なお、対照実験として、 1回投与群 (n=4)を準備した 。一回投与群は初回の GFP搭載 SeV/dFベクター投与を行なわな 、こと以外は 2回投 与群と同じ操作で作製した。また、二回投与群および一回投与群のそれぞれに、 2J に代えて DPBS (-)を投与した対照実験も並行させた。 [0040] In the twice-administered group (n = 3), the GFP gene-loaded F gene-deficient SeV vector was first administered intranasally at 5xl0 6 CIU per Balb / cA mouse. On the 14th day after the first administration of the vector, 250 g of each 2J peptide (dissolved in 100 1 DPBS (-)) was intravenously injected into the tail. Type SeV vector (5xl0 6 CIU / mouse) was mixed with 2J peptide (1.25 / zg) and administered by auricle. On the second day after the second administration, the pinna was cut and collected, and the Luciferase activity in the homogenate was measured using the Luciferase Assay System (Promega). As a control experiment, a single administration group (n = 4) was prepared. The single-dose group was prepared in the same manner as the double-dose group except that the first administration of GFP-loaded SeV / dF vector was not performed. In addition, a control experiment in which DPBS (−) was administered in place of 2J in each of the twice-administered group and the once-administered group was performed in parallel.
[0041] 一回目投与群では、 2Jある!/、は DPBS投与の!/、ずれも高 ^Luciferase活性が示さ れたが、 2回目投与群において、 DPBS投与では 1回目投与時の約 100分の 1に低下 していたが、 2J投与により 10分の 1程度に発現量の低下が抑制された。即ち、 2Jの同 時投与により発現量を約 10倍上昇させることが可能になった。  [0041] In the first administration group, there were 2J! /, DPBS administration! /, Which showed high ^ Luciferase activity, but in the second administration group, DPBS administration was about 100 minutes after the first administration. However, the 2J administration suppressed the decrease in the expression level to about one-tenth. In other words, the simultaneous expression of 2J made it possible to increase the expression level by about 10 times.
[0042] [実施例 3]  [0042] [Example 3]
SeVベクター 2回目投与時の抗 SeV抗体価  Anti-SeV antibody titer at the second administration of SeV vector
上記実施例 2において、 14日目の 2回目投与直前に採取した血漿中の抗 SeV抗体 価を ELISA法 (プレザィム生研 HVJ、デンカ生研社）によって測定した。なお、 1回投 与群においては、 1回目ベクター導入前の抗体価の測定となる。  In Example 2 above, the anti-SeV antibody titer in the plasma collected just before the second administration on the 14th day was measured by ELISA (Presym Seika HVJ, Denka Seken). In the single dose group, the antibody titer is measured before the first vector introduction.
[0043] 図 5に示すように 1回投与群では抗体が生成されていないが、 2回投与群では 2J投 与予定群と非投与予定群との間に抗体価の差はなカゝつた。抗 SeV抗体存在下に SeV ベクターは補体による溶解、不活ィ匕作用を受けると予想されるが、 2Jの同時投与はこ の作用を抑制したものと考えられ、 SeVベクター 2回目投与における補体系抑制の有 効性が示された。 [0044] [実施例 4] [0043] As shown in Fig. 5, no antibody was generated in the single administration group, but in the two administration group, there was no difference in antibody titer between the 2J administration group and the non-administration group. . In the presence of anti-SeV antibody, the SeV vector is expected to be lysed and inactivated by complement, but simultaneous administration of 2J is considered to suppress this effect. The effectiveness of system control was shown. [0044] [Example 4]
In vitroにおける C1INHの補体抑制作用  Complement inhibitory action of C1INH in vitro
C1INHの補体抑制作用の検討は、 nafamostat mesilateに代えて補体抑制剤 C1INH である「ベリナ一ト!3」（アベンテイスファーマ社)を用いた点を除き、実施例 1に示した 中和活性測定により同様に行った。「ベリナ一ト!3」を最終濃度が 0、 3、 6、 12倍濃縮 血漿に相当となるように添加した。 The complement inhibitory action of C1INH was determined in Example 1, except that instead of nafamostat mesilate, the complement inhibitor C1INH “Belinaito! 3 ” (Aventis Pharma) was used. It carried out similarly by activity measurement. “Berinaito! 3 ” was added so that the final concentration was equivalent to 0, 3, 6, 12-fold concentrated plasma.
図 5 (A)はモルモット補体を最終濃度 1%で添加した実験群、 (b)は補体非添加の 実験群を示す。図 5 (A)と (B)におけるペリナート P(0、非添加）のグラフを比較すると、 補体の添カ卩により SeVに対する中和活性が高まることが示されている力 この補体に より中和活性の増強は濃度依存的に抑制された。「ベリナ一 1 P」（12倍濃縮血漿に 相当）添加では、補体非添加まで SeV中和活性が抑制された。  Figure 5 (A) shows the experimental group with guinea pig complement added at a final concentration of 1%, and (b) shows the experimental group with no complement added. Figure 5 Comparing the graphs of perinate P (0, no addition) in (A) and (B) shows that neutralization activity against SeV is increased by the addition of complement. The enhancement of neutralizing activity was suppressed in a concentration-dependent manner. Addition of “Berina 1 P” (equivalent to 12-fold concentrated plasma) suppressed SeV neutralization activity until no complement was added.
産業上の利用の可能性  Industrial applicability
[0045] 従来、ある種のベクターでは搭載遺伝子を継続して発現させるためにはベクターの 繰返し投与が必要とされながら、ベクターの免疫原性のためその繰返し投与が困難 であり、仮に繰返し投与を行ったとしても、宿主免疫系により排除されてしまうという問 題があつたが、本発明によれば、補体抑制剤より宿主免疫系によるベクター排除機 能を抑制され、繰返し投与が必要な多くのベクターにおいて、その繰り返し投与を行 うことを可能とすることができる。ベクターの繰返し投与が可能となることにより、遺伝 子治療効果の改善が期待されるとともに、こうした有用なベクターを用いた遺伝子治 療の適用疾患が拡大される。 [0045] Conventionally, with certain vectors, repeated administration of the vector is required for the continued expression of the loaded gene. However, repeated administration is difficult due to the immunogenicity of the vector. However, according to the present invention, the vector elimination function by the host immune system is suppressed by the complement suppressor, and many repeated administrations are required. This vector can be administered repeatedly. The ability to repeatedly administer the vector is expected to improve the effect of gene therapy, and the applicable diseases for gene therapy using such useful vectors will be expanded.

Claims

請求の範囲 The scope of the claims
[1] 所望の遺伝子を担持したベクターを繰返し投与することにより遺伝子を継続的に発 現させる方法において、ベクター投与時に補体抑制剤が投与される、方法。  [1] A method in which a complement inhibitor is administered at the time of administration of a vector in a method of continuously expressing a gene by repeatedly administering a vector carrying a desired gene.
[2] ベクター投与時がベクターが繰返し投与される時である、請求項 1記載の方法。  [2] The method according to claim 1, wherein the vector administration is a time when the vector is repeatedly administered.
[3] ベクター投与時が 2回目以降にベクターが投与される時である、請求項 1記載の方法  [3] The method according to claim 1, wherein the vector is administered at the second time or later.
[4] 補体抑制剤が補体成分 Cほたは C5を抑制し得る、請求項 1記載の方法。 [4] The method according to claim 1, wherein the complement inhibitor can inhibit complement component C or C5.
[5] 補体抑制剤を有効成分とする、ベクター発現継続剤。 [5] A vector expression continuation agent comprising a complement inhibitor as an active ingredient.
[6] 補体抑制剤が補体成分 Cほたは C5を抑制し得る、請求項 5記載のベクター発現継 続剤。  6. The vector expression continuing agent according to claim 5, wherein the complement inhibitor can inhibit complement component C or C5.
[7] 補体抑制剤を有効成分とする、遺伝子治療用薬剤。  [7] A gene therapy drug comprising a complement inhibitor as an active ingredient.
[8] 補体抑制剤が補体成分 Cほたは C5を抑制し得る、請求項 7記載の遺伝子治療用薬 剤。  [8] The gene therapy drug according to claim 7, wherein the complement inhibitor can inhibit complement components C and C5.
[9] 補体抑制剤と繰返し投与型ベクターとを含む、繰返し投与ベクターシステム。  [9] A repeated administration vector system comprising a complement inhibitor and a repeated administration vector.
[10] 補体抑制剤が補体成分 C1または C5を抑制し得る、請求項 9記載の繰返し投与べク ターシステム。 10. The repeated administration vector system according to claim 9, wherein the complement inhibitor can suppress complement component C1 or C5.
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