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WO2004078213A1 - Composition pour introduire une substance cible et procede pour introduire une substance cible - Google Patents

Composition pour introduire une substance cible et procede pour introduire une substance cible Download PDF

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Publication number
WO2004078213A1
WO2004078213A1 PCT/JP2004/002816 JP2004002816W WO2004078213A1 WO 2004078213 A1 WO2004078213 A1 WO 2004078213A1 JP 2004002816 W JP2004002816 W JP 2004002816W WO 2004078213 A1 WO2004078213 A1 WO 2004078213A1
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WO
WIPO (PCT)
Prior art keywords
composition
target substance
introducing
nucleic acid
acid
Prior art date
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Ceased
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PCT/JP2004/002816
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English (en)
Japanese (ja)
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WO2004078213A9 (fr
Inventor
Chikako Nishigori
Yoshiki Miyachi
Yaeno Arima
Yoshiro Otsu
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Individual
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Individual
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Priority to JP2005503122A priority Critical patent/JPWO2004078213A1/ja
Publication of WO2004078213A1 publication Critical patent/WO2004078213A1/fr
Publication of WO2004078213A9 publication Critical patent/WO2004078213A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid

Definitions

  • the present invention relates to a target substance-introducing composition for introducing a target substance such as a peptide, a peptide hormone, a protein, a nucleoprotein, a nucleic acid, a derivative of a nucleic acid, an essential amino acid, an essential fatty acid, a vitamin or a low molecular drug, and the like. And a method for introducing a target substance using the method.
  • the target substance introduction composition and the target substance can be efficiently used for the treatment of diseases that develop over a wide range such as systemic diseases, symptom improvement, and study of the mechanism of the disease. It relates to the method for introducing target substances. Background art
  • the target substance When introducing the target substance, if the substance has a small molecular weight, a method such as dissolving the target substance itself in a dissolution solution is adopted.
  • the target substance can be introduced into adherent cultured cells by electoral poration, calcium phosphate method, DEAE dextran method, etc. In such a case, it is difficult to introduce the target substance into cells simply by dissolving the target substance itself in a lysis solution, so a carrier that efficiently introduces the transgene is required.
  • Viral vectors and non-viral vectors are known as carriers for introducing a target substance.
  • Viral vectors substitute a foreign gene for expressing a part of the viral genome and introduce it into cells.Therefore, there is a possibility of carcinogenesis, a possibility of inducing an immune / inflammatory reaction, a cytotoxicity, etc. An indication has been made.
  • Ribosomes and HVJ artificial viruses are known as non-viral vectors, but many of them have a lower transduction efficiency than viral vectors. HVJ people 004/002816
  • the virus is highly safe, with no decrease in activity due to serum components.
  • ribosomes are difficult to introduce large-sized proteins and DNAs
  • HVJ artificial viruses have the characteristic that large-sized proteins and DNAs can be introduced.
  • the target substance can be introduced into cells without a carrier if the amount is extremely small.However, the target substance introduced into cells is introduced to an amount that can actually function. In order to increase efficiency, it is necessary to perform non-physiological treatments such as performing chemical hair removal, applying ultrasonic waves, and performing tape stripping. With damage.
  • a transdermal gene transfer method using a viral vector is described, for example, in Japanese Patent Application Laid-Open No. 2001-112475 / Japanese Patent Application Laid-Open No. 2000-286282.
  • a method for transdermally introducing a target substance using ribosomes as a carrier is described in, for example, Japanese Patent Application Laid-Open No. 2002-515856.
  • the transdermal introduction of HV J ribosome has been announced, for example, at the 26th Annual Scientific Meeting of the Japanese Society of Dermatology.
  • the use of a biocompatible composition or the introduction of a target substance into a tissue using the HVJ envelope vector as a carrier has not yet been known.
  • the present invention relates to a composition for introducing a target substance, comprising a target substance and a biocompatible composition, and a method for introducing a target substance.
  • a composition for introducing a target substance comprising a target substance and a biocompatible composition
  • a method for introducing a target substance Efficient and easy conversion of target substances to compositions for introducing target substances and a wide range of target sites that can be used for treatment of diseases that develop over a wide range, such as systemic diseases, improvement of symptoms, and study of disease mechanisms, etc. It relates to the method of introducing the target substance that can be introduced well.
  • xeroderma pigmentosum is an autosomal recessive inherited disease characterized by a deficiency in the function of nucleoside excision and repair enzymes. Increases, skin atrophy, multiple actinic keratosis, etc. Moreover, in the xeroderma pigmentosum, malignant tumors occur at a high rate at the exposed site.
  • resulting t invention of not only performed resection like symptomatic treatment of skin cancer at present are peptides in a wide range of areas, peptidase de hormones, proteins, nuclear proteins, nucleic acids It is an object of the present invention to provide a means for efficiently introducing a substance to be introduced such as a derivative of a nucleic acid, an essential amino acid, an essential fatty acid, vitamin, and a low molecular weight drug.
  • a first object of the present invention is to provide a composition for introducing a target substance which can be used for treatment of a widespread disease such as a systemic disease, symptom improvement, and study of the mechanism of the disease. is there.
  • a composition for introducing a target substance that can efficiently introduce the target substance into a wide range of target sites for introduction specifically, a gene, an enzyme, an antibody, a cytokine (signal transmitting substance), It is a composition for introduction containing factors, low molecular weight pharmaceuticals and the like.
  • a second object of the present invention is to provide a method for introducing a target substance that can easily and efficiently introduce a target substance into a wide range of target sites. Disclosure of the invention
  • the basis of the present invention is a composition for introducing a target substance comprising a target substance and a biocompatible composition, and a method for introducing the target substance.
  • the target substance is a peptide, a peptide hormone, a protein, a nucleoprotein, a nucleic acid, a derivative of a nucleic acid, an essential amino acid, an essential fatty acid, vitamin, or a small molecule drug to be introduced
  • the composition for introducing the target substance is And the target substance and the biocompatible composition.
  • the carrier contains the HVj envelope vector as a carrier, and further contains the HVJ envelope vector enclosing the target substance.
  • the gist of the present invention is to introduce peptides to be introduced, peptide hormones, At least one selected from the group consisting of proteins, nucleoproteins, nucleic acids, derivatives of nucleic acids, essential amino acids, essential fatty acids, vitamins and low-molecular-weight drugs, and the aforementioned peptides, peptide hormones, proteins, nucleoproteins, nucleic acids, A subject containing a biocompatible composition capable of retaining, or retaining and further introducing at least one selected from the group consisting of a derivative of a nucleic acid, an essential amino acid, an essential fatty acid, vitamin, and a low molecular weight drug.
  • a composition for introducing a substance which is at least selected from the group consisting of peptides, peptide hormones, proteins, nucleoproteins, nucleic acids, nucleic acid derivatives, essential amino acids, essential fatty acids, vitamins and low molecular weight drugs to be introduced.
  • HVJ envelope vector and the peptides, peptide hormones, proteins, nucleoproteins, nucleic acids, nucleic acids A biocompatible composition capable of retaining at least one selected from the group consisting of a derivative, an essential amino acid, an essential fatty acid, vitamin, a low molecular weight drug and an HVJ envelope vector in a tissue, or holding and further introducing the composition.
  • HVJ envelope containing at least one selected from the group consisting of peptides, peptide hormones, proteins, nucleoproteins, nucleic acids, nucleic acid derivatives, essential amino acids, essential fatty acids, vitamins, and low molecular weight drugs to be introduced
  • a vector selected from the group consisting of the vector, the peptide, the peptide homolemon, a protein, a nucleoprotein, a nucleic acid, a derivative of a nucleic acid, an essential amino acid, an essential fatty acid, vitamin, a low-molecular-weight drug, and an HVJ envelope vector.
  • a target substance-introducing composition containing a biocompatible composition that can be retained or retained and further introduced.
  • target substances such as “peptides, peptide hormones, proteins, nucleoproteins, nucleic acids, nucleic acid derivatives, essential amino acids, essential fatty acids, vitamins, and low-molecular-weight drugs” are abbreviated as (T) below for convenience of explanation. .
  • the biocompatible composition may contain mucopolysaccharide or a salt thereof.
  • Mucopolysaccharides include, specifically, hyaluronic acid, chitin, colominic acid, chondroitin, dermatan sulfate, chondroitin sulfate such as chondroitin 4-sulfate and chondroitin 6-sulfate, heparin, Keratan sulphate, heparan sulphate, tycronic acid and the like can be suitably used.
  • the salts of mucopolysaccharides include, specifically, hyaluronic acid, chitin, colominic acid, chondroitin, de / rematan sulfate, chondroitin 4- Salts such as chondroitin sulfate such as sulphate-chondroitin 6-sulfate, heparin, keratan sulfate, heparan sulfate and tiecronic acid can be suitably used.
  • the pH is preferably pH 3 to 9.0.
  • the nucleic acid or a derivative of the nucleic acid can be at least one selected from the group consisting of DNA, RNA, peptide nucleic acid, nucleic acid containing a nucleotide analog, and the like, and a plasmid vector incorporating these nucleic acids.
  • the present invention provides a method for introducing a target substance into tissues or cells by administering the composition for introducing a target substance described above.
  • the composition for introducing a target substance of the present invention is a composition for introducing a target substance, comprising at least one of the target substances (T) and a biocompatible composition.
  • HVJ envelope vector containing at least one target substance (T) which contains at least one target substance (T) and contains a carrier such as an HVJ envelope vector and a biocompatible composition.
  • This is a target substance-introducing composition containing a first-class carrier and a biocompatible composition.
  • nucleic acid or nucleic acid derivative to be introduced is DNA, RNA, peptide nucleic acid, nucleotide analog-containing nucleic acid, etc., or a plasmid vector incorporating these nucleic acids, etc., as the aforementioned “peptide to be introduced”.
  • peptide to be introduced is DNA, RNA, peptide nucleic acid, nucleotide analog-containing nucleic acid, etc., or a plasmid vector incorporating these nucleic acids, etc., as the aforementioned “peptide to be introduced”.
  • peptide to be introduced are long-chain peptides, non-natural amino acid-containing peptides, phosphorylated peptides, intramolecular S--S-linked peptides, polyantigenic peptides
  • MAP Carrier protein binding peptide
  • Kallikrein Substance P
  • Bradykinin Kallidin
  • Cholesis tokinin Neuropeptide
  • Neuropeptide Gramidine
  • Colistin Polymyxin
  • Oxytocin Vasopressin
  • Secretin Gastrointestinal peptide
  • Antibacterial Defensin hepcidin: Liver-Expressed Antimicrobial Peptide 1
  • cell death antagonists Humanin, etc.
  • vasoconstrictor peptides Endothelin: Endothelin-I, ⁇ rotincin: Urotensin- ⁇ , etc.
  • endogenous growth hormone Secretion-promoting peptides active Ghrelin, etc.
  • the “proteins” to be introduced include XPA gene products, various DNA damage repair enzymes, superoxide dismutase, catalase, glutathione, peroxidase, and peroxidase.
  • Enzymes such as xyredoxin, antibodies such as b12, IFN-a, IFN- ⁇ , IFN-] 3, site force-ins such as IL-12 and their antibodies, growth factors such as EGF and NGF,
  • the essential amino acids to be introduced are methionine, threonine, valine, tryptophan, phenylalanine, leucine, and isoleucine. , Lysine, histidine, etc.
  • the “essential fatty acids” to be introduced are ⁇ -linolenic acid, eicosapentaenoic acid ( ⁇ ⁇ ⁇ ), docosahexaenoic acid (DHA), linoleic acid, ⁇ -linolenic acid (GLA).
  • vitamin A1 retinol
  • vitamin A2 3-dehydrodrethino
  • vitamin D2 ergocalcium
  • Ferol vitamin D3 (cholecalciferol)
  • vitamins tocopherolone
  • vitamin F vitamin F
  • vitamin F vitamin F
  • vitamin F vitamin F
  • vitamin K1 phytoquinone
  • vitamin K2 vitamin K2
  • Fat-soluble vitamins such as vitamin U and derivatives thereof
  • vitamin B1 thiamine
  • vitamin B2 riboflavin
  • vitamin B6 pyridoxine
  • nicotinic acid nicotinic acid Mid, pantothenic acid
  • vitamin H biotin
  • folic acid vitamin B122 (cyanopparamine), choline
  • inosit vitamin L1, vitamin L2, vitamin B13, vitamin BT (carnitine), lipoic acid
  • Water soluble vitamins and their derivatives such as (butyric acid), vitamin B14, vitamin B
  • the biocompatible composition retains the target substance in the tissue and can be introduced into cells.
  • hyanoreonic acid chitin, colominic acid, chondroitin, dermatan sulfate, chondroitin sulfate (chondroitin 4-monosulfate, chondroitin 6-sulfate, etc.)
  • mucopolysaccharides such as heparin, keratan sulfate, heparan sulfate and tycronic acid, and salts of these mucopolysaccharides.
  • the present invention provides a method of applying a sodium hyaluronate solution containing an HVJ envelope vector enclosing a plasmid vector containing an X ⁇ gene or a plasmid vector containing an XPA gene to the skin of an XPA mouse.
  • the XPA gene is introduced into mouse cells, and the function of the gene is expressed. That is, even under UVB irradiation, the generation of sunburn cells is suppressed, and DNA repair ability is restored, and furthermore, ultraviolet light is restored.
  • the target substance which is the central substance of the composition for introducing a target substance of the present invention, is a peptide, peptide hormone, protein, nucleoprotein, nucleic acid, derivative of nucleic acid, essential amino acid, essential fatty acid, vitamin or small molecule drug to be introduced. And so on.
  • This target substance can be used in the form of peptides, peptide hormones, proteins, nucleoproteins, nucleic acids, derivatives of nucleic acids, essential amino acids, essential fatty acids, vitamins or low molecular weight drugs themselves.
  • HVJ envelope vectors Proteins, nucleoproteins, nucleic acids, derivatives of nucleic acids, essential amino acids, essential fatty acids, vitamins or low molecular weight pharmaceuticals can be mixed with HVJ envelope vectors. Furthermore, a HVJ envelope vector containing peptides, peptide hormones, proteins, nucleoproteins, nucleic acids, derivatives of nucleic acids, essential amino acids, essential fatty acids, vitamins or low molecular weight drugs can also be used. These target substances may be dissolved in an appropriate buffer solution or the like.
  • the target site for introducing the composition for introducing a target substance of the present invention includes, for example, skin, mucous membrane, liver, kidney, lung, knee, brain, digestive tract, blood vessel, bladder, child, ovary, spleen, thymus, adjuvant Thyroid gland, thyroid gland, salivary gland, parotid gland, submandibular gland, lymph node and the like.
  • Application methods include skin, mucous membranes (oral cavity, eyes, gastrointestinal tract, nose, respiratory tract, anus), external use, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, and catheters. Local administration, application to the surgical site, and the like.
  • FIG. 1 is a view showing the situation of a low dose of UV-induced skin tumor when a composition containing a target substance but not containing a carrier and a biocompatible composition is administered to mice by skin application.
  • Fig. 2 shows a low-dose UV-induced dose when a composition containing the target substance, sodium hyaluronate as a biocompatible composition and HVJ envelope vector as a carrier was applied to mice by skin application. It is a figure showing the situation of a skin tumor. No.
  • FIG. 3 shows the situation of low-dose UV-induced skin tumors when a composition containing sodium hyaluronate but not a carrier as a target substance and a biocompatible composition was applied to mice by skin application.
  • FIG. Fig. 4 shows the situation of low-dose UV-induced skin tumors when a target substance, a composition containing sodium hyaluronate as a biocompatible composition and HVJ envelope vector as a carrier were administered to mice by subcutaneous injection.
  • FIG. Fig. 5 shows the situation of a moderate amount of UV-induced skin tumor when a composition containing sodium hyaluronate but not a carrier as a target substance and a biocompatible composition was applied to mice by skin application.
  • FIG. Fig. 6 shows the biocompatible composition containing the target substance.
  • FIG. 2 is a view showing the situation of a moderate amount of UV-induced skin tumor when a composition containing no substance and a carrier is applied to mice by skin application.
  • nucleic acid to be introduced is not particularly limited, but a nucleic acid encoding the “protein to be introduced”, a ribozyme, a tumor suppressor gene such as p53, Rb, PTCH, a gene-deficient disease or gene
  • the gene include a causative gene of a disease caused by a mutation, a site force gene such as IFN- ⁇ , IF IF-, IF ⁇ , and IL_2.
  • Dystrophy epidermolysis bullosa (Dystrophic EB): COL7A1, etc.
  • Junctional EB (Junctional EB): LAMA3, LAMB3, LAMC2, etc.
  • GBEB Systemic atrophic benign epidermolysis bullosa
  • EB-PA Epidermolysis bullosa—pyloric atresia
  • PA-JEA Congenital pyloric atresia—junctional EB syndrome
  • EB-MD Epidermolysis bullosa monomuscular dystrophy
  • Ectodermal dysplasia Skin fragility PLP1, etc.
  • Epidermolytic hyperkeratosis KRT1, KRTIO, etc.
  • Non-epidermolytic palmar keratoderma (Nonepidermolytic PPK): KRT16, etc., Bohwinkel's syndrome: LOR, GJB2, etc., Ichthyosis bullosa Siemens: KRT2e, etc.
  • Type 1 and Type 2 congenital nail disease (Pachonychia congenita type 1 and 2): KRT 6a, 16, 17 etc.
  • Lamellar ichthyosis TGM1, etc.
  • Waardenburg syndrome PAX 3, etc.
  • albinism different forms: TYR, TYRP-1, OCA2, OA1, etc.
  • Erythropoietic protoporphyria FECH, etc.
  • Familial porphyria cutanea tarda Familial porphyria cutanea tarda: URO-D, etc.
  • Xeroderma pigmentosum XPA, XPB, XPC, XPD, XPE, XPG, XPF, XPV, etc.
  • Cockayne syndrome CSA, CSB, etc.
  • necrotic basal cell carcinoma syndrome necrotic basal cell carcinoma syndrome: PTCH, etc.
  • Pewtz-Jeghers syndrome STK11 / LKB1, etc.
  • Cowden syndrome PTEN, etc.
  • Bannayan-Zonana svndrome group PTEN, etc.
  • Capillary diastolic ataxia (Ataxia telangiectasia): Hereditary hemorrhagic telangiectasia: ENG, AL ⁇ 1 etc.
  • nucleic acid or nucleic acid derivative to be introduced is at least one kind selected from DNA, RNA, peptide nucleic acid, nucleotide analog-containing nucleic acid and the like and a plasmid vector incorporating these nucleic acids.
  • the plasmid vector for incorporating the nucleic acid can be appropriately selected depending on the site to be introduced and the like.
  • pCAGGS pcDNA3, Escherichia coli plasmid (for example, pUC18, PUC1) 9, pBR322, pBluescript II (registered trademark; manufactured by Stratagene), pET, pCAL, pBluescript Am P (registered trademark; manufactured by Stratagene)], vector for eukaryotic cells (PC MV-Script (registered trademark; manufactured by Stratagene)), pCMV—Tag, pBK—CMV, pBK—RSV, pi—RED1, pESP, pESC), cosmid (p AT5, pWE15) and the like.
  • PC MV-Script registered trademark; manufactured by Stratagene
  • the “peptide to be introduced” is not particularly limited. For example, long-chain peptides, non-natural amino acid-containing peptides, phosphorylated peptides, intramolecular S—S-bonded peptides, multi-peptides, etc.
  • Antigenic peptide MAP
  • Carrier monoprotein binding peptide Calycrain
  • Substance P Bradykinin
  • Calidine Cholestatic tokinin
  • Neuropeptide Gramidine
  • Cristine Polymyxin
  • Oxytocin Gastrointestinal peptides such as nosopressin and secretin
  • antimicrobial peptides defense : Defensin, hepcidin: Liver-Expressed Antimicrobial Peptide 1 etc.
  • cell death antagonist Haumanin: Humanin etc.
  • vasoconstrictor peptide Endothelin-I
  • perotinsin Urotensin- ⁇
  • endogenous Growth hormone secretion promoting peptide active ghrelin: Active Ghrelin, etc.
  • the “protein to be introduced” is not particularly limited, and examples thereof include XPA gene products, various DNA damage repair enzymes, superoxide dismutase (SOD), catalase, glutathione, peroxidase, peroxyredoxin, b12 and the like. And cytokines such as IFN-a, IFN-y, IFN-j3 and IL-12 and their antibodies, and growth factors such as EGF and NGF.
  • Low-molecular-weight drugs include antioxidants such as polyphenols such as kytecin, tannin, anthocyanin, quercetin, isoflavone, and rutin; antioxidants such as glycyrrhetinic acid and glutylretinic acid; anthelmintics such as kainic acid; and tannins.
  • Astringents such as acids, analgesics such as acetaminophenone, inotropic diuretics such as caffeine, local anesthetics such as lidocaine, sleeping pills such as parpital, antipyretics such as aspirin and antipyrine, analgesics, Antibiotics such as vancomycin, S-galactosidase (penicillium) and methicillin, antibacterials such as hinokitiol, metabolic drugs such as ratatulose, cardiac glycosides such as sigitoxin, betamethasone valerate, and hydrocortisone butyrate Anti-inflammatory agents such as cortisone acetate, prednisolone acetate, etc.
  • Anti-parkinsonian drugs such as carbidopa
  • anti-rheumatic drugs such as sodium gold thiomalate and glucocorticoid
  • prostaglandins such as PGE1 and PGE2 ⁇
  • epinefilin Sympathetic stimulants such as quinine sulfate, antimalarial drugs such as quinine sulfate, chlorophyll such as black-mouth fil, triothyronine (III), thyroxine (III)
  • hormone-like substances such as y-oryzanol.
  • nucleoprotein to be introduced is not particularly limited, and examples thereof include RNA nucleoprotein, acidic nucleoprotein, soluble nucleoprotein, and Fos protein (cancer 2004/002816
  • pigment cell stimulating hormone a MSH As peptide hormones, pigment cell stimulating hormone a MSH
  • Vitamin A 1 retinol
  • Vitamin A 2 3-dehydrodrenotinol
  • Vitamin A3 Cold-dehydrodrenotinol
  • Vitamin D2 Ergocalci feronore
  • Vitamin D3 Cold-decanolecipherone
  • Vitamin E tocopherol
  • Vitamin F Vitamin F
  • Vitamin F Vitamin F
  • Vitamin K1 Physicalloquinone
  • Vitamin K2 Vitamin K2 (Phlanoquinone)
  • Fat-soluble vitamins such as vitamin U and derivatives thereof, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B6 (pyridoxine), nicotinic acid, nicotinamide, pantothenic acid, vitamin H ( Biotin), folic acid, vitamin B12 (cyanopparamine), cho
  • Esters such as sodium chloride, hydrochloric acid, palmitic acid, and rhodanic acid, salts such as sodium, potassium, calcium, magnesium, arginine, lysine, hydrochloric acid, phosphoric acid, nitric acid, cetyl sulfate, naphthalenedisulfonic acid, etc.
  • Ascorbic acid 2- And stable vitamin C such as darcoside.
  • the content of the target substance in the composition for introducing a target substance of the present invention may be within a range in which the function of the target substance can be exhibited at the site of introduction.
  • the target substance may be a nucleic acid or a derivative of a nucleic acid or
  • the content may be 1.0% in the composition for introducing a target substance according to the present invention.
  • X 1 0- 9% to 9 9.9 wt%, good Mashiku is, 1. 0 X 1 0- 6% to 9 9 wt%, more preferably, 1.
  • the target substance if an HV J envelope base click coater encapsulating peptides or peptide in the subject material for introducing the compositions of the present invention, 1. 0 X 1 0- 9% to 9 9.9 wt%, preferably, 1. 0 X 1 0- 6% to 9 9 weight 0/0, more preferably, 1. 0 X 1 0- 5 wt% to 9 5% by weight it is preferable to blend.
  • the composition for introducing a target substance of the present invention contains 1.0 X 10 to 8 % by weight. % ⁇ 9 9.9 wt%, preferably, 1. 0 X 1 0- 5 wt% to 9 9 wt%, more preferably, 1. 0 X 1 0 _ 5 wt% to 9 0 to wt% blending Is good.
  • the target substance is an HVJ envelope vector containing a low-molecular-weight drug, a protein or a nucleoprotein
  • the content is 1.0 X 10 to 8 % by weight to 99% of the total amount of the composition. .
  • the substance when it is HV J envelope vector encapsulating the substance or substance other than the above other than the above, examples of content, the total composition weight, 1. 0 X 1 0- 9 wt 0 / . 1-9 9.9 wt 0/0, preferably, 1. 0 X 1 0- 6 weight 0 /. 1-9 9 weight 0/0, more preferably, 1. 0 X 1 0 one 5% to 9 5% by weight it is preferable to blend.
  • the content of the target substance is not limited to the range exemplified above when the function of the target substance is exhibited. Incidentally, the composition for introducing a target substance of the present invention, It may be diluted at the time of use.
  • the target substance such as (T) to be introduced can be retained in a tissue, or can be retained and further introduced into cells.
  • Any biocompatible composition may be used, for example, hyaluronic acid, chitin, colominic acid, chondroitin, chondroitin 4-monosulfate, dermatan sulfate, chondroitin 6-sulfate, heparin, keratan sulfate, heparan sulfate, tycronic acid, etc. Mucopolysaccharides and their salts.
  • the salts of mucopolysaccharide include inorganic salts such as alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as magnesium salts, metal salts such as copper salts and zinc salts, and diethanolamine. Salt, 2-amino-2-ethyl-1,3-propanediol salt, alkananolamine salt such as triethanolamine salt, morpholine salt, heterocyclic salt such as piperidine salt, arginine salt, lysine salt, Organic acid salts such as basic amino acid salts such as histidine salts; and inorganic acids or organic acid salts such as sulfuric acid, hydrochloric acid, acetic acid, citric acid, and lactic acid.
  • the proportion of the salt-forming mucopolysaccharide and salt is not particularly limited, and may be any as long as it is within the pH range of the biocompatible composition. Les ,.
  • the “biocompatibility” means that the substance does not adversely affect the living body and does not give a strong stimulus for a long period of time, and the original function of the target substance and the target substance are retained in the tissue, or retained and further introduced and stored. It means the property of a material that can coexist with organisms while exhibiting its properties.
  • the ⁇ retention property exhibited by retaining or further introducing and retaining the target substance in the tissue '' is such that the target substance remains in the tissue when the composition for introducing a target substance of the present invention is administered to the tissue.
  • the target substance can be retained on the skin or can be retained and further introduced.
  • pH is not particularly limited. Any range is possible as long as the target substance can be held stably, that is, in a range where the physiological function of the target substance can be exhibited. Is preferably 4.0 or more, more preferably 4.5 or more, 9.0 or less, preferably 8.5 or less, more preferably 8.0 or less. Desirably. Further, the pH of the biocompatible composition may be in a range suitable for a living body.
  • the biocompatible composition may be selected according to the use, conditions at the time of use, and the like.
  • the composition for introducing a target substance of the present invention can be used as a therapeutic agent in the treatment of a disease or the like.
  • the biocompatible composition is selected from the viewpoint of the feeling of use in the individual to be administered. May be.
  • the content of the biocompatible composition in the target substance-introducing composition of the present invention varies depending on the biocompatible composition to be used, but the range in which the target substance can be retained in the tissue and further introduced to exhibit storage properties. Should be fine.
  • the content of the biocompatible composition in the target substance-introducing composition of the present invention is 0.001% by weight or more, and preferably 0.001% by weight or more. And more preferably 0.1% by weight or more, 99.999% by weight or less, preferably 99.9.99% by weight or less, more preferably 99.9% by weight or less.
  • the content and the like are mentioned in the range of weight%.
  • the biocompatible composition of the present invention may be used after being diluted at the time of use, and at the time of use, a 100% pure lyophilized powder or the like may be appropriately diluted with a buffer solution or the like at the time of use to obtain the above-mentioned concentration. May be used in
  • the composition for introducing a target substance of the present invention includes a bactericide, a metal-sequestering agent, a preservative, an antioxidant, a humectant, an oily component, a stabilizer, a conventional buffer for stably retaining the target substance. , Water, etc., as appropriate.
  • the target substance is (T), ( ⁇ ⁇ ⁇ ⁇ ) and an HVJ envelope vector, or an HVJ envelope vector enclosing (T)
  • the composition for introducing a target substance of the present invention includes the decomposition of (T). May contain a pharmaceutically acceptable substance for suppressing lipase.
  • the components other than the target substance and the biocompatible composition include the above-mentioned bactericide, sequestering agent, preservative, antioxidant, humectant, oil component, and oil component. It may be a stabilizing agent, a conventional buffer, water or the like.
  • the pH is not particularly limited and varies depending on the application.
  • the lower limit is 3 or more. Yes, preferably at least 4.0, more preferably at least 4.5, and the upper limit is at most 9.0, preferably at most 8.5, more preferably 8.0 or less.
  • the lower limit is 5 or more, preferably 6.0 or more, more preferably 6.5 or more, and the upper limit is 9 or more. 0.0 or less, preferably 8.5 or less, and more preferably 8.0 or less.
  • the lower limit is 3 or more, preferably 4.0 or more, more preferably 4.5 or more, and the upper limit. Is not more than 9.0, preferably not more than 8.5, more preferably not more than 8.0.
  • the lower limit is 4 or more, preferably 4.3 or more, more preferably 4.5 or more, and the upper limit is It is 9.0 or less, preferably 8.0 or less, and more preferably 7.0 or less.
  • the lower limit is 3 or more, preferably 4.0 or more, more preferably 4.5 or more, and the upper limit. Is 9.0 or less, preferably 8.5 or less, and more preferably 8.0 or less.
  • the content of the target substance is not limited to the range exemplified above when the function of the target substance is exhibited.
  • the application site of the target substance-introducing composition can be applied to the whole tissue.
  • the method of application is skin, mucous membranes (oral, ocular, gastrointestinal tract, nose, respiratory tract, anus), etc., clothing, intravenous injection, intramuscular injection, subcutaneous injection, force Local administration to tissues, application to surgical sites, etc. can be performed using auxiliary devices such as catheters.
  • compositions for introducing a target substance of the present invention include, for example,
  • Dystrophic epidermolysis bullosa dystrophic epidermolysis bullosa
  • Junctional epidermolysis bullosa junctional epidermolysis bullosa
  • GBEB systemic atrophic benign epidermolysis bullosa
  • PA-JEB congenital pyloric atresia-junction EB syndrome
  • simple epidermolysis bullosa with muscular dystrophy EBS / MD
  • simple epidermolysis bullosa EB-simplex
  • outside Dermal dysplasia / skin fragility EDA / skin fragility
  • epidermolytic hyperkeratosis epidermolytic PPK, non-epidermal palmar keratoderma ( Nonepidermolytic PPK), Vohwinkel's syndrome, Ichthyosis bullosa Siemens, Ichthyosis bullosa Siemens, Pachonychia congenita type
  • the composition for introducing a target substance of the present invention may be used for auxiliary devices such as skin, mucous membranes (oral cavity, eyes, gastrointestinal tract, nose, respiratory tract, and anus), internal use, intravenous injection, intramuscular injection, subcutaneous injection, catheter, etc. It can be used by local administration to tissues using, application to a surgical site, and the like. From the viewpoints of simplicity and efficiency in use, use by application is particularly preferable.
  • auxiliary devices such as skin, mucous membranes (oral cavity, eyes, gastrointestinal tract, nose, respiratory tract, and anus), internal use, intravenous injection, intramuscular injection, subcutaneous injection, catheter, etc. It can be used by local administration to tissues using, application to a surgical site, and the like. From the viewpoints of simplicity and efficiency in use, use by application is particularly preferable.
  • the form of the container or preparation is not particularly limited, and may be a liquid, a gel, a cream, an ointment, an eye ointment, a buccal tablet, a troche (sublingual tablet), an eye drop, a nasal drop, a gargle, Inhalants, compresses, poultices, suppositories, vaginal suppositories, enemas, sprays, packs, foam aerosols and the like can be mentioned.
  • the composition for introducing a target substance of the present invention can be produced, for example, as follows. That is, when the target substance is a nucleic acid, the nucleic acid to be introduced is incorporated into a conventional plasmid vector by a conventional method to obtain a recombinant plasmid vector.
  • the obtained recombinant plasmid vector may be used as it is as naked-DNA or by a method of resentment, for example, encapsulated in an HVJ envelope vector to obtain a recombinant plasmid vector-containing HVJ envelope vector.
  • a biocompatible composition for example, sodium hyaluronate is swollen to a concentration of 5% by weight in 100 ml of Dulbecco's PBS (I) solution to give 5% by weight of sodium hyaluronate. A solution (pH 7.4) is obtained.
  • the above Dulbecco's PBS (-) solution is, for example, commercially available Dulbecco's PBS (-) powder (9.6 g) was dissolved in distilled water to make a volume of 1 liter. (15 minutes by high-pressure steam sterilization.
  • the target solution is then mixed with a sodium solution to obtain a composition for introducing the target substance.
  • the gene transfer method of the present invention comprises the steps of (T) and ( ⁇ ) to be introduced and the HVJ envelope vector or ( ⁇ ).
  • the HVJ envelope vector thus obtained and (T), (() and the HVJ envelope vector containing the HVJ envelope vector or (T) are retained in a tissue, or a biocompatible composition capable of retaining and further introducing the HVJ envelope vector.
  • the composition containing the target substance introduced is administered to the tissue, whereby the target substance (T), ( ⁇ ) and the HVJ envelope vector or the HVJ envelope vector containing (T) are retained in the tissue. Further, the target substance is introduced into cells, and the area of the gene transfer composition applied to the skin is (T), ( ⁇ ) and ⁇ .
  • Any area may be sufficient as long as it contains the site required for the VJ envelope vector or the HVJ envelope vector containing (T) to exhibit its effect. Further, the number of times and timing of applying the composition for introduction to the skin can be appropriately selected according to the intended use.
  • the coating amount of that is not particularly limited, for example, as the target material, unit area, for example, per 1 cm 2, lng ⁇ lmg, preferably , 1 ⁇ g to 100 ⁇ g.
  • the administration of the composition for gene transfer into the blood may be performed at any site where (T), ( ⁇ ) and the HVJ envelope vector or the HVJ envelope vector containing (T) can exert its effect. Further, the number of times and timing of administration of the composition for introduction into blood can be appropriately selected depending on the intended use.
  • the dose when the target substance-introducing composition is administered into blood, the dose is not particularly limited.
  • the dose may be, for example, from 1 pg to: L mg, preferably from 1 ng to 100 ⁇ g per mL.
  • the method for oral ingestion of the composition for gene transfer may be any method as long as (T), ( ⁇ ) and the HVJ envelope vector or the HVJ envelope vector containing ( ⁇ ) express the effect.
  • the number and timing of oral ingestion of the composition for introduction can be appropriately selected depending on the intended use.
  • the amount of intake is not particularly limited.
  • the daily intake for example, per body weight (kg), lng to 10 Omg, preferably 1 ng to 10 mg.
  • the method of local administration of the composition for gene transfer to a tissue using a catheter or by surgery or the like is as follows: (T), ( ⁇ ) and the HVJ envelope vector or the HVJ envelope vector containing (T). Any method can be used as long as the effect is exhibited. Further, the number of times and timing of insertion of the composition for introduction can be appropriately selected depending on the application.
  • the amount of intake is not particularly limited.
  • it may be lpg to lmg, preferably lng to 100g, per body weight (kg).
  • Xeroderma pigmentosum has eight subgroups: Group A, Group B, Group C, Group D, Group E, Group F, Group G and Group V.
  • group A causative gene XPA gene
  • XPA group A causative gene
  • the HVJ envelope vector containing the XPA gene obtained by enclosing the recombinant plasmid vector in which the XPA gene is incorporated into a conventional plasmid vector such as pCAGGS or pcDNA3 into the HVJ envelope vector is also used.
  • a conventional plasmid vector such as pCAGGS or pcDNA3 into the HVJ envelope vector.
  • sodium hyaluronate sodium hyaluronate.
  • a composition of an HVJ envelope vector containing sodium hyaluronate at a final concentration of 1% by weight and XPA gene (50 g in terms of nucleic acid amount) can be used.
  • the composition for gene transfer is applied to the entire skin for a time sufficient for the nucleic acid to be introduced and expressed in the living body, for example, 48 hours before sun exposure by going out. This can be expected to restore DNA repair ability, suppress epidermal damage, and suppress the formation of samban cells.
  • HVJ Envelope Vector for XPA Gene Transfer Human XPA gene (Genebank Accession No .: 14) was added to pCAGGS, which has a cytomegaloy / les-enhancer and ⁇ -actin promoter. 5 3 3; provided by Dr. Kajiyo Tanaka) to obtain p CAGG S—XPA (provided by Dr. Kajiyo Tanaka (Osaka University, Center for Cell Biotechnology)).
  • pcHA-XPA obtained by incorporating the human XPA gene into the EcoRI recognition site of pcDNA3 (manufactured by InVitrogen) and adding DNA encoding a hemagglutinin epitope as a label was obtained. Obtained. What's the result?
  • Each of 11-PA was encapsulated in an HVJ envelope vector. As a result, an HVJ envelope vector for XPA gene introduction was obtained.
  • the Darbecco PBS (-) solution may be prepared, for example, by dissolving 9.6 g of Darbecco PBS (-) powder manufactured by Wako Pure Chemical Industries, Ltd. in distilled water to make a volume of 1 liter. The obtained solution was obtained by autoclaving the obtained solution at 121 ° C for 15 minutes.
  • the mouse was applied to the back (about 4.5 cm 2 ) of the ⁇ ⁇ ⁇ model mouse so as to be 75 ⁇ l + HVJ envelope vector solution (300 ⁇ l).
  • the skin (4.5 cm 2 ) of the UVB-irradiated part was excised, and the excised skin was fixed with a 10% neutral formalin solution overnight. After fixing, the skin is embedded in paraffin.
  • Raffin-embedded samples were sliced to a thickness of 4 to 5 ⁇ , and the obtained samples were deparaffinized and washed with 5% cooled TC II. The sample was then immersed in an emulsion containing silver particles (photographic emulsion) and stored at 4 ° C in a dark room.
  • the sample was developed, fixed and washed with water, and stained with Giemsa. For the stained sample, the number of darkened particles on the cells was counted under a microscope.
  • the control gene transfection composition (C1-1) containing no XPA gene or the control gene transfection composition (C1-1) containing no sodium hyaluronate was measured.
  • the number of blackening particles (grain number) of most cells was 0 to 5
  • the skin application group of the gene transfer composition (E 1) many cells having a grain number of 15 to 20 per nucleus were found, indicating that the repair ability was restored. That is, surprisingly, it was found that the nucleic acid to be introduced was introduced into cells from the skin, and that the gene in the nucleic acid to be introduced was expressed over a wide area of the skin.
  • the obtained sample was stained with hematoxylin and eosin (HE) to obtain an HE-stained specimen.
  • the morphology of the HE-stained specimen was observed, and the difference between the three groups of mice (1) was determined for sunburn cells (epidermal cells that had undergone nucleus enrichment and epidermal necrosis) as indicators of epidermal cell damage.
  • the observation of the sunburn cells was performed using the ratio of the number of sunburn cells to the total number of epidermal cells in the section as an index. Further, the skin pieces of the three groups of mice (1) into which pc HA-XPA was introduced were frozen, HA-tag was stained, and the localization of the introduced gene was observed by staining intensity.
  • the number of sunburn cells was 20/100 cells after 24 hours of UV irradiation, whereas
  • the mouse to which the control gene transfection composition (C1-2) containing no sodium hyaluronate as a control had 35/100 cells, and the nucleic acid encoding the control XP gene was The number of cells in the mouse to which the control gene-introducing composition (C 1-1) was not applied was 55 cells / 100 cells.
  • the group to which the gene transfer composition was applied it was clarified that the damage to the epidermis and the formation of sunburn cells were suppressed compared to the two groups.
  • the hyaluronic acid sodium, Dulbecco PBS (-) solution 1 0 0 m 1 to swell at 2 wt 0/0 concentration 2 wt 0/0 hyaluronic A sodium acid solution (pH 7.4) was obtained.
  • the Dulbecco's PBS (-) solution is obtained, for example, by dissolving 9.6 g of Dulbecco's PBS (-) powder manufactured by Wako Pure Chemical Industries, Ltd. in distilled water to a volume of 1 liter. The solution was obtained by autoclaving at 121 ° C. for 15 minutes.
  • the nucleic acid for introduction ( ⁇ ) 50 ⁇ g DNA obtained in the above (1) and the sodium hyaluronate solution 100% ⁇ 1 of 2% by weight obtained in the above (2) are combined. By mixing, a composition for gene introduction was obtained. The concentration of hyaluronic acid in the composition for introducing a gene is 1% by weight.
  • Test Example 2 Effect of introduction of XPA gene into XP mice
  • the composition for gene transfer (E2) obtained in Example 2 was applied to the back of three XPA model mice in the same manner as in (1) of Test Example 1, and UVB was applied to the back.
  • UVB was applied to the back.
  • a composition for control gene transfer (C2-1) using pCAGGS or pcDNA3 containing no XPA gene or sodium hyaluronate was contained.
  • UVB irradiation 24 hours after, and 1 week after UVB irradiation, the degree of erythema, edema, etc. was classified into three grades: mild, moderate, and advanced, and compared with the control group for evaluation. Long-term irradiation test, irradiation once / week was repeated 15 times and observed.
  • mice to which the gene transfer composition (E 2) was applied to the skin mild erythema was observed immediately after UVB irradiation, mild erythema was observed 24 hours later, and one week after 1 week. However, only moderate erythema was observed in the two mice.
  • the control group transfection composition (C2-1) containing the plasmid vector (pCAGGS or pcDNA3) which does not contain the nucleic acid encoding the control XP gene all of the 3 Three of them had severe erythema and edema.
  • control gene transfer composition (C2-1) containing (pCAGGS or pcDNA3) or control gene transfer composition (C2-2) containing no sodium hyaluronate
  • UV-induced skin tumors were observed. From these results, it was found that the restoration ability was restored. That is, it was surprisingly shown that the nucleic acid to be introduced was introduced into cells from the skin. It was also found that the gene in the nucleic acid to be introduced was expressed over a wide area of the skin.
  • Example 1 (1) Preparation of HVJ Envelope Vector for XPA Gene Transfer
  • pc HA—XPA was constructed.
  • the obtained pc HA—XPA was encapsulated in an HVJ envelope vector.
  • an HVJ envelope vector for XPA gene transfer was obtained.
  • the DNA amount in each HVJ envelope vector for XPA gene transfer was 508 mice.
  • a chondroitin sulfate sodium ⁇ beam, Dulbecco PBS (-) solution 1 m 1 to swell at 2 wt 0/0 concentration, 2% by weight chondroitin sulfate sodium (PH 7.4) was obtained.
  • the above Dulbecco's PBS (-) solution was prepared by dissolving 9.6 g of Dulbecco's PBS (I) powder (manufactured by Wako Pure Chemical Industries, Ltd.) in distilled water to a volume of 1 liter. The obtained solution was obtained by autoclaving at 121 ° C. for 15 minutes.
  • the nucleic acid for introduction 200 ⁇ l (50 ⁇ g DNA) obtained in the above (1) was mixed with 50 ⁇ l of the 2% by weight sodium chondroitin sulfate solution obtained in the above (2).
  • a composition for gene transfer ( ⁇ 3) was obtained.
  • the concentration of sodium chondroitin sulfate in the composition for gene transfer was 0.4% by weight.
  • Example 3 Skin application group of gene transfer composition
  • the composition for gene transfer (E 3) obtained in Example 3 was applied to the back of five XPA model mice in the same manner as in (1) of Test Example 1, and UVB was applied to the back for 4 k. J Zm 2 irradiation.
  • a control gene transfer composition (c3-1) using pcDNA3 containing no nucleic acid encoding the XPA gene or a control containing no chondroitin sulfate sodium The composition for gene transfer (C3-2) was applied to each of five mice, and the same operation was performed thereafter.
  • the degree of erythema, edema, etc. was classified into three grades: mild, moderate, and advanced, and compared with the control group for evaluation.
  • the skin of the mouse (1) was excised to obtain each skin piece, and an HE-stained specimen was obtained from the skin piece.
  • the morphology of the HE-stained specimen was observed, and formation of damage to the epidermis and suppression of formation of samban cells were evaluated.
  • human EGF for example, Epidermal Growt Facter, Human, recombinant manufactured by Wako Pure Chemical Industries, Ltd. was purchased and used. This was adjusted to S mgZm 1 in a Talbecco PBS (-) solution. For human EGF, 0.9 mg was used per mouse.
  • the 5% by weight sodium hyaluronate solution (pH 7.4) is obtained by swelling sodium hyaluronate to a concentration of 5% by weight in 100 ml of Dulbecco's PBS (-) solution.
  • the Darbecco PBS (-) solution is prepared, for example, by dissolving 9.6 g of Darbecco PBS (-) powder manufactured by Wako Pure Chemical Industries, Ltd. in distilled water to a volume of 1 liter. And the resulting solution was subjected to high pressure steam sterilization with 121 for 15 minutes.
  • the protein-introducing composition (E 4) obtained in Example 4 was used as a protein amount in 0.1 mg SmZ mouse (total amount: 0.375 ml: sodium hyaluronate 0.075 m The solution was applied to the back (approximately 4.5 cm 2 ) of C57 mice so that 1 ml of the protein solution was 0.3 ml).
  • a composition for protein introduction without human EGF (C4-1) or a composition for protein introduction without sodium hyaluronate (C4-2) was applied.
  • C4-1 human EGF
  • C4-2 composition for protein introduction without sodium hyaluronate
  • the mouse skin (4.5 cm 2 ) of (1) was excised, and the excised skin was embedded in OTC and then frozen and fixed.
  • the frozen sections were sliced to a thickness of 4 to 5 ⁇ m, and the obtained sections were stained with a FITC-labeled anti-human EGF antibody.
  • A—XPA was encapsulated in an HVJ envelope vector.
  • an HVJ envelope vector for XPA gene introduction was obtained.
  • the amount of DNA in the HVJ envelope vector for XPA gene transfer was 50 / ig / mouse.
  • Pc HA—XPA was constructed in the same manner as in Example 1.
  • the obtained pc HA—XP A was encapsulated in HV J ribosome.
  • HV J ribosome for XPA gene transfer was obtained.
  • the amount of DNA in the HVJ ribosome for introducing the XPA gene was 50 ⁇ g / mouse.
  • the hyaluronic acid sodium, Dal Beck co p BS (-) in solution 1 0 0 m 1 is swelled such that 2 weight 0/0 concentration 2 weight % Sodium hyaluronate solution (PH 7.4) was obtained.
  • the Dulbecco's PBS (-) solution is, for example, distilled 9.6 g of Dulbecco's PBS (-) powder manufactured by Wako Pure Chemical Industries, Ltd. The volume was adjusted to 1 liter by dissolving in water, and the resulting solution was obtained by autoclaving at 121 ° C for 15 minutes.
  • the HVJ envelope vector-containing gene transfer composition (E4) obtained in the above (4) was applied to the back of five XPA model mice in the same manner as in the above Test Example 1 (1). The back was irradiated with 0.5 kj / m 2 of UVB. As a control, instead of the HV J enveloping vector-containing composition for gene transfer obtained in (4), the HV J ribosome-containing control gene transfer composition (C 4) obtained in (5) above was used. It was applied to the back of five XPA model mice, and the same operation was performed thereafter.
  • mice to which the gene transfer composition was applied to the skin E4
  • only mild erythema was observed immediately after UVB irradiation, 24 hours, and 1 week later.
  • control group (C4) containing HVJ ribosome for XPA gene introduction 3 out of 5 mice showed mild erythema and 2 mice showed moderate erythema. That is, it was found that the gene transfer composition containing the HVJ envelope vector was more effective than the gene transfer composition containing the HVJ ribosome.
  • the skin in the mice of the two groups in Test Example 4 (1) was excised, and each skin piece was obtained, and an HE-stained specimen was obtained from the skin piece.
  • the morphology of the HE-stained specimen was observed, and the formation of epidermal damage and the suppression of formation of sunburn cells were evaluated.
  • mice coated with the gene transfer composition (E4) containing the HVJ envelope vector showed 20/1/24 sunburn cells 24 hours after UV irradiation.
  • the gene transfer composition it was found that the formation of epidermal damage and the formation of sunburn cells were suppressed as compared with the control.
  • HV J Envelope Vector for XPA Gene Transfer pc HA-XPA was constructed in the same manner as in Example 1. The obtained pc HA—XPA was sealed in an HV J envelope probe. This As a result, an HV J envelope vector for XPA gene introduction was obtained. Here, the DNA amount in the HV Jembello ⁇ "vector for XPA gene transfer was 50 gZ mouse.
  • the Dulbecco's PBS (-) solution is obtained, for example, by dissolving 9.6 g of Dulbecco's PBS (-) powder manufactured by Wako Pure Chemical Industries, Ltd. in distilled water to a volume of 1 liter. The solution was obtained by autoclaving at 121 ° C. for 15 minutes.
  • the HVJ envelope vector for XPA gene transfer obtained in (1) above was mixed with 225 ⁇ l of the HVJ envelope vector and the 2.5 wt% sodium hyaluronate solution obtained in (2) was mixed with 15 ⁇ l.
  • a composition for gene introduction was obtained.
  • hyaluronic acid sodium Me concentrations in the transgenic composition is 1 wt 0/0.
  • Test Example 5 Effect of introduction of X X gene into X ⁇ mouse (1) Skin application group of gene transfer composition
  • Example 5 The composition for gene transfer (E5) obtained in Example 5 was applied to the back of an XPA model mouse in the same manner as in (1) of Test Example 1 described above. After 8 hours, anesthetize with N embuta 1 ® by intraperitoneal injection, and place TOSH I BA fluorescent S unlam FL 20 SE (peak wavelength: 305 nm, 280 to 3) on the back of the XPA model mouse. UVB was irradiated at 400 JZ m 2 times once a week for 20 weeks. The amount of ultraviolet rays was measured with a UVR-305Z365D ultraviolet dosimeter.
  • HVJ envelope vector containing only the XPA gene and the HVJ envelope vector and sodium hyaluronate were used instead of the HVJ envelope vector for XPA gene transfer.
  • a control composition not containing was applied in the same manner, and the same operation was performed.
  • Example 6 Five months after the end of UV irradiation, UV-induced skin tumors were visually observed. As a result, as shown in FIG. 1, the skin application group of the control composition containing only the XPA gene but not containing HV J. Envelope vector and sodium hyaluronate coincided with the ultraviolet irradiation site. In contrast, a fast-growing tumor was observed, whereas in the group to which the composition for gene transfer (E5) of Example 5 was applied to the skin, as shown in FIG. Only papules were observed, but no obvious malignant tumor. That is, it was surprisingly shown that the nucleic acid to be introduced was introduced into cells from the skin. In addition, it was found that the gene in the nucleic acid to be introduced was expressed over a wide area of the skin.
  • E5 the composition for gene transfer
  • PcHA-XPA was constructed in the same manner as in Example 2.
  • the amount of DNA in the nucleic acid for XPA gene introduction was 50 g / mouse.
  • the hyaluronic acid sodium, Dal Beck co p BS (-) was Rise moistened so that the solution 1 m 1 to 5% strength by weight, 5 wt 0/0 Sodium hyaluronate (pH 7.4) was obtained.
  • the above Dulbecco's PBS (-) solution is obtained, for example, by dissolving 9.6 g of Dulbecco's PBS (-) powder manufactured by Wako Pure Chemical Industries, Ltd. in distilled water to adjust the volume to 1 liter.
  • the solution was obtained by autoclaving at 121 ° C. for 15 minutes.
  • the concentration of hyaluronic port phosphate sodium ⁇ beam in said transgenic composition is 2 wt 0/0.
  • Test Example 6 Effect of introduction of X ⁇ gene into X ⁇ ⁇ mouse
  • Example 6 The composition for gene transfer (E 6) obtained in Example 6 was applied to the back of an XPA model mouse in the same manner as in Test Example 1 (1). After 48 hours, anesthetize with N embuta 1 ® by intraperitoneal injection, and place TOSHIBA fluorescent S unlamp FL 20 SE (peak wavelengths of 305 nm, 280 to 360 nm) on the back of the XPA model mouse. ) was used to irradiate UVB at a dose of 400 J / m 2 once a week for 20 weeks. The amount of ultraviolet rays was measured with a UVR-305 / 365D ultraviolet dosimeter.
  • control gene transfer composition (c6-1) using pcDNA3 containing no XPA gene or a control gene transfer composition not containing sodium hyaluronate (C6-2) was applied, and the same operation was performed as follows.
  • the hyaluronic acid sodium, Dulbecco PBS (-) solution 1 ml of swollen so that 0.3 wt% concentration, 0.3 wt 0/0 hyaluronate Sodium (pH 7.4) was obtained.
  • the Dulbecco's PBS (-) solution is obtained, for example, by dissolving 9.6 g of Dulbecco's PBS (-) powder manufactured by Wako Pure Chemical Industries, Ltd. in distilled water to a volume of 1 liter. The solution was obtained by autoclaving at 121 ° C. for 15 minutes.
  • Example 7 The composition for gene transfer ( ⁇ 7) obtained in Example 7 was subcutaneously injected into the back of a ⁇ model mouse in four to five parts. Twenty-four hours later, anesthesia was performed by intraperitoneal injection using N embuta 1 ®, and TOSH I BA fluorescent S using un 1 # 038 FL 2 0 SE (peak wavelength 3 0 5 nm, 2 8 0 ⁇ 3 6 0 nm), at 4 0 0 J Roh m 2 Bruno times weekly to UVB, were irradiated 2 0 weeks . The amount of ultraviolet rays was measured with a UVR-305 / 365D ultraviolet dosimeter.
  • control gene introduction composition (C7-2) containing no sodium hyaluronate, and the same operation was performed.
  • PCAGG S—XPA was constructed in the same manner as in Example 2. here,
  • DNA amount in the XPA gene transfer for nucleic acid was a 5 0 ⁇ ⁇ _ mice.
  • chondroitin sulfate was added at a concentration of 0.5% by weight to 1 ml of Dulbecco's PBS (-) solution. Swelling was performed to obtain 0.5% by weight of sodium chondroitin sulfate (pH 7.4).
  • the Dulbecco's PBS (-) solution is prepared, for example, by dissolving 9.6 g of Dulbecco's PBS (-) powder manufactured by Wako Pure Chemical Industries, Ltd. in distilled water to a volume of 1 liter. The solution obtained was obtained by autoclaving at 121 for 15 minutes.
  • the XPA gene transfer nucleic acid 25 ⁇ l obtained in the above (1) was mixed with the 0.5 wt% sodium chondroitin sulfate solution 50 ⁇ l obtained in the above (2) to prepare a gene transfer composition. Obtained. The concentration of sodium chondroitin sulfate in the composition for gene transfer is 0.083% by weight.
  • Test Example 8 Effect of introduction of X ⁇ gene into X ⁇ ⁇ mouse
  • composition for gene transfer (E 8) obtained in Example 6 was subcutaneously injected into the back of an XPA model mouse in the same manner as in (1) of Test Example 7, and 400 B of UVB was applied to the back. j Zm 2 / time was irradiated once a week for 20 weeks.
  • a composition for control gene transfer using pCAGGS containing no XPA gene (C8-1) or a composition for control gene transfer containing no chondroitin sulfate sodium (C 8-2) was applied, and the same operation was performed thereafter.
  • pc HA—XPA was constructed.
  • the amount of DNA in the nucleic acid for XPA gene transfer was S Og / mouse.
  • the hyaluronic acid sodium, Dal Beck co p BS (-) solution 1 m 1 to 5 weight 0 /. It was moistened Rise at a concentration to give a 5 weight 0/0 hyaluronate sodium (p H 7. 4).
  • the above Dulbecco's PBS (-) solution is obtained, for example, by dissolving 9.6 g of Dulbecco's PBS (-) powder manufactured by Wako Pure Chemical Industries, Ltd. in distilled water to adjust the volume to 1 liter. The resulting solution was obtained by autoclaving at 121 for 15 minutes.
  • the nucleic acid composition for gene introduction was prepared by mixing 300 ⁇ l of the nucleic acid for XPA gene introduction obtained in the above (1) with 75 ⁇ l of the 5% by weight sodium hyaluronate solution obtained in the above (2). I got something. The concentration of sodium hyaluronate in the composition for gene introduction was 1% by weight.
  • Test Example 9 Effect of introducing X X gene into X X mouse (1) Skin application group of gene transfer composition
  • the gene transfer composition (E 9) obtained in Example 6 was applied to the back of an XPA model mouse in the same manner as in Test Example 1 (1). 4 8 hours later, anesthetized by intraperitoneal injection using N embuta 1®, and TO SHI BA fluorescent S unlamp FL 20 SE (peak wavelength of 300 n m, 280-360 nm) and UVB was irradiated 1500 times three times a week for 5 weeks. The amount of ultraviolet rays was measured with a UVR-305 / 365D ultraviolet dosimeter.
  • control gene transfer composition (C9-1) using pC DNA3 not containing the XPA gene or a control gene transfer without sodium hyaluronate
  • the composition for application (C9-2) was applied, and the same operation was performed thereafter.
  • HV J envelope vector for human EGF introduction Human EGF is, for example, Epidermal manufactured by Wako Pure Chemical Industries, Ltd.
  • HVJ envelope vector Human and recombinant were purchased and used. This was encapsulated in an HVJ envelope vector to obtain an HVJ envelope vector for human EGF gene transfer.
  • the Darbecco PBS (-) solution may be prepared, for example, by dissolving 9.6 g of Darbecco PBS (-) powder manufactured by Wako Pure Chemical Industries, Ltd. in distilled water to a volume of 1 liter. The resulting solution was prepared by autoclaving the resulting solution at 121 with high pressure steam for 15 minutes.
  • the human EGF solution 300 ⁇ l (500 g human EGF) obtained in the above (1) was mixed with 75 ⁇ l of the 5% by weight sodium hyaluronate solution obtained in the above (2). Thus, a composition for protein introduction was obtained.
  • the concentration of sodium hyaluronate in the composition for protein introduction is 1% by weight.
  • Test Example 10 Effect of introduction of human EGF into 0 C57 mouse (1) Skin application group of protein-introducing composition
  • the protein-introducing composition (E 10) obtained in Example 10 was used to obtain a protein mass of 500 ⁇ g / mouse at the back of a C57 mouse (about 4.5 cm). 2 ) was applied.
  • a composition for protein introduction without human EGF (C10-1) or a composition for protein introduction without sodium hyaluronate (C10-2) was applied, and The operation was performed.
  • the mouse skin (4.5 cm 2 ) of (1) was excised, and the excised skin was embedded in OTC and then frozen and fixed.
  • the frozen sections were sliced to a thickness of 4 to 5 ⁇ m, and the obtained sections were stained with a FITC-labeled anti-human EGF antibody.
  • the composition for introducing a target substance according to the present invention can efficiently and efficiently introduce the target substance in the treatment of a widespread disease such as a systemic disease, the improvement of symptoms, and the study of the mechanism of the disease. It has excellent effects.
  • the wide-range gene introduction means of the present invention has an excellent effect that a gene can be easily and efficiently introduced into a wide range of target sites. That is, the present invention has an effect that the target substance can be introduced effectively and under physiological conditions, and the introduced substance surely functions.
  • composition for introducing a target substance of the present invention can effectively introduce a target substance under physiological conditions, and has an excellent effect that the introduced substance functions reliably. It is useful for the treatment of disease that develops over a wide range, improvement of symptoms, and study of the mechanism of the disease.

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Abstract

La présente invention concerne une composition pour introduire une substance cible, qui comprend une substance cible, telle qu'un peptide, une hormone peptidique, une protéine, une nucléoprotéine, un acide nucléique, un dérivé d'acide nucléique, un acide aminé essentiel, un acide gras essentiel, une vitamine ou un médicament à faible poids moléculaire, ou la substance cible avec un vecteur d'enveloppe HVJ, ainsi qu'une composition biocompatible de mucopolysaccharide, un sel de celle-ci, etc., capable de la maintenir sur des tissus. Afin de maintenir la substance cible sur des tissus, puis de l'introduire dans des cellules, ladite composition est administrée au niveau des tissus.
PCT/JP2004/002816 2003-03-06 2004-03-05 Composition pour introduire une substance cible et procede pour introduire une substance cible Ceased WO2004078213A1 (fr)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
JP2023528333A (ja) * 2020-05-28 2023-07-04 ロンザ リミテッド ウイルスベクター用製剤

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WO2001080902A2 (fr) * 2000-04-25 2001-11-01 Biovector Therapeutics, S.A. Complexe particulaire permettant d'administrer un acide nucleique dans une cellule
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Publication number Priority date Publication date Assignee Title
JP2023528333A (ja) * 2020-05-28 2023-07-04 ロンザ リミテッド ウイルスベクター用製剤

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