WO2004078213A1 - Composition for target substance introduction and method of target substance introduction - Google Patents

Abstract

A composition for target substance introduction comprising a target substance, such as peptide, peptide hormone, protein, nucleoprotein, nucleic acid, nucleic acid derivative, essential amino acid, essential fatty acid, vitamin or low-molecular drug, or the target substance together with HVJ envelope vector, and a biocompatible composition of mucopolysaccharide, salt thereof, etc. capable of holding them on tissues. The target substance can be held on tissues and further introduced in cells by administration of this composition to tissues.

Description

 Detail

Target substance introduction composition and target substance introduction method

 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. For details, 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

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. In the case of a gene, 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

Compared to ribosomes, the virus is highly safe, with no decrease in activity due to serum components. In addition, ribosomes are difficult to introduce large-sized proteins and DNAs, whereas HVJ artificial viruses have the characteristic that large-sized proteins and DNAs can be introduced. In addition, 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. Also, 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. However, 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. 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.

For example, with regard to systemic diseases, xeroderma pigmentosum (XP) 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. At present, it is theoretically optimal to replace the defective gene as a treatment for xeroderma pigmentosum, but at present, development of a method suitable for such treatment has not progressed, and fundamental treatment law and said ones is no shading, 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. Another object of the present invention is to provide means for treating a disease or the like that develops over a wide range, such as a systemic disease, improving the symptoms, and studying the mechanism of the disease. Specifically, 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. In other words, 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. Further, 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. Here, 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, and 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. That is, 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. Contains the target substance

 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. For convenience of explanation, 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. Further, 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.

 Further, 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.

 The “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”. 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, 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, ゥ rotincin: Urotensin-Π, etc.), endogenous growth hormone Secretion-promoting peptides (Active Ghrelin, etc.), etc., and 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-γ, 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 α-linolenic acid, eicosapentaenoic acid (Ε Ρ 、), docosahexaenoic acid (DHA), linoleic acid, γ-linolenic acid (GLA). ), Arachidonic acid, etc. The "vitamins" to be introduced include vitamin A1 (retinol), vitamin A2 (3-dehydrodrethino), vitamin A3, and vitamin D2 (ergocalcium). Ferol), vitamin D3 (cholecalciferol), vitamins (tocopherolone), vitamin F (linoleic acid, linolenic acid), vitamin K1 (phyloquinone), vitamin K2 (fuarquinone), 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 B15, paraaminobenzoic acid, vitamin C (ascorbic acid), and vitamin P,] 3 carotene, ergosterol, Provitamins such as dehydrocholesterol and their derivatives, vitamins, and derivatives of provitamins include methyl alcohol, ethyl alcohol , Phosphoric acid, acetic acid, hydrochloric acid, palmitic acid, rhodanic acid, etc., sodium, potassium, potassium, magnesium, arginine, lysine, hydrochloric acid, phosphoric acid, nitric acid, cetyl sulfate, naphthalenedisulfonic acid, etc. And stable vitamin C such as ascorbic acid 2-darcoside.

 The biocompatible composition retains the target substance in the tissue and can be introduced into cells. In addition, hyanoreonic acid, chitin, colominic acid, chondroitin, dermatan sulfate, chondroitin sulfate (chondroitin 4-monosulfate, chondroitin 6-sulfate, etc.) And 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 XΡ gene or a plasmid vector containing an XPA gene to the skin of an XPA mouse. As a result, 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. Based on the inventors' surprising finding that the formation of induced skin tumors is suppressed. Furthermore, it is based on the finding that the same effect was obtained with sodium hyaluronate solution containing the XPA gene and HV J. Envelop vector.

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. 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. BRIEF DESCRIPTION OF THE FIGURES

 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.

Figure 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. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described based on embodiments. However, the present invention is not limited to such an embodiment.

 The “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 Examples of the gene include a causative gene of a disease caused by a mutation, a site force gene such as IFN-α, IF IF-, IF β, and IL_2.

 As the “gene-deficient disease or disease caused by gene mutation” and the causative gene, for example,

Dystrophy epidermolysis bullosa (Dystrophic EB): COL7A1, etc.

Junctional EB (Junctional EB): LAMA3, LAMB3, LAMC2, etc.

Systemic atrophic benign epidermolysis bullosa (GABEB): COL17A1, etc.

Epidermolysis bullosa—pyloric atresia (EB-PA): ITGA6, ITGB4, etc.

Congenital pyloric atresia—junctional EB syndrome (PA-JEA): ITG a6, ITG j3 4, etc.

Epidermolysis bullosa monomuscular dystrophy (EB-MD): PLEC1, etc.

Epidermolysis bullosa simplex (EB-simplex): KRT5, KRT14, etc.

Ectodermal dysplasia Skin fragility (EDA / skin fragility): PLP1, etc. Epidermolytic hyperkeratosis: KRT1, KRTIO, etc.

Epidermolytic PPK: KRT9, 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.

X-linked ichthyosis: STS, etc.

Lamellar ichthyosis: TGM1, etc.

Deafness associated with palmar keratoderma (PPK with deafness): GJB2, etc.

Erythrokeratoderma variabilis: GJB3, etc., Darier's disease: ATP2A2, etc.

Striate PPK: DSP, etc.

Striate keratoderma: DSG1, etc.

Congenital atrichia: HR, etc.

Monilethrix: hHBl, hHB6, etc.

Waardenburg syndrome: PAX 3, etc., albinism (different forms): TYR, TYRP-1, OCA2, OA1, etc.

Tipz syndrome: MITE, etc.

Hermansky-Pudlak syndrome: HPS, etc.

Erythropoietic protoporphyria: FECH, etc.

Congenital erythropoietic porphyria: UR0S, etc.

Familial porphyria cutanea tarda (familial porphyria cutanea tarda): URO-D, etc.

Variegate porphyria: PPO, 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.

No, Bannayan-Zonana svndrome group: PTEN, etc.

Sulfur-deficient hair growth disorders (Trichothiodystrophy): XPB, XPD, etc., Fabry's disease: GLA, etc.

Capillary diastolic ataxia (Ataxia telangiectasia): Hereditary hemorrhagic telangiectasia: ENG, ALΚ1 etc.

And a plasmid vector incorporating these genes.

 The “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. For example, 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.

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 (Hyumanin: Humanin etc.), vasoconstrictor peptide (Endoselin: Endothelin-I, perotinsin: Urotensin-Π), endogenous Growth hormone secretion promoting peptide (active ghrelin: Active Ghrelin, etc.) and the like.

 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 PGE2α, and epinefilin Sympathetic stimulants such as quinine sulfate, antimalarial drugs such as quinine sulfate, chlorophyll such as black-mouth fil, triothyronine (III), thyroxine (III)

4) etc., and hormone-like substances such as y-oryzanol.

The “nucleoprotein to be introduced” is not particularly limited, and examples thereof include RNA nucleoprotein, acidic nucleoprotein, soluble nucleoprotein, and Fos protein (cancer 2004/002816

 13 genes), PQBP-1 (binding to polyglutamine sequence), NP95, PCNA, topoisomerase I, RNA polyisomerase I, UBF (upstream binding factor), fibrarin, nucleon, nucleophosmin, etc. .

 As peptide hormones, pigment cell stimulating hormone a MSH

(melanocyte stimulating hormon, melanotropin) Strength is raised, and essential amino acids include methionine, threonine, palin, tryptophan, fenii / lealanine, leucine, isoleucine, lysine, histidine and the like. Include linolenic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid, _V- linolenic acid (GLA), and arachidonic acid; Vitamin A 1 (retinol), Vitamin A 2 (3-dehydrodrenotinol), Vitamin A3, Vitamin D2 (Ergocalci feronore), Vitamin D3 (Colecanolecipherone), Vitamin E (tocopherol) ), Vitamin F (linoleic acid, linolenic acid), Vitamin K1 (Phyloquinone), 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), choline, inosit, vitamins: L1, vitamin L2, vitamin B13, vitamin BT (carnitine), lipoic acid (cholic acid), Water-soluble vitamins such as vitamin B14, vitamin B15, para-aminobenzoic acid, vitamin C (ascorbic acid) and vitamin P and derivatives thereof, provitamins such as β-carotene, ergosterol, 7-dehydrocholesterol and the like誘導 体 Derivatives of these derivatives, vitamins and provitamins include methyl alcohol, ethyl alcohol, phosphoric acid, and acetic acid. 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.For example, the target substance may be a nucleic acid or a derivative of a nucleic acid or In the case of an HVJ envelope vector enclosing a nucleic acid or a derivative of a nucleic acid, for example, 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. 0 X 1 0- ⁵ wt% It is better to mix up to 95% by weight. Further, 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. When the target substance is a protein or nucleoprotein or an HVJ envelope vector enclosing a protein or nucleoprotein, 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. When 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. . 9% by weight, is preferred properly, 1. 0 X 1 0- ⁵ wt ⁰ /) - 9 9 weight _^0/0, more preferably, 1 · 0 X 1 0- ⁵ wt. /. 990% by weight is preferred. Moreover, 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. Further, 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.

 As the biocompatible composition used in the composition for introducing a target substance of the present invention, 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. Examples of 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. In the biocompatible composition, 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 ,.

 In addition, 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. For example, it means that the target substance can be retained on the skin or can be retained and further introduced.

In the biocompatible composition, 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. For example, 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.In this case, 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. For example, 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. For example, when the target substance is (T), (ベ ク タ ー) 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. In the target substance-introducing composition of the present invention, 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.

 In the target substance-introducing composition of the present invention, the pH is not particularly limited and varies depending on the application.For example, when the target substance-introducing composition is used for external use, 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. When the target substance-introducing composition is used for internal use, 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. When the target substance-introducing composition is used for injection, infusion, or the like, 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. When the target substance-introducing composition is used as an eye drop, 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. When the target substance-introducing composition is used for other purposes, 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. Further, 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. For example, skin, mucous membrane, liver, kidney, lung, kidney, brain, digestive tract, bladder, uterus, ovary, spleen, thymus, parathyroid, thyroid, salivary gland, parotid, submandibular, lymph nodes, blood vessels, blood vessels, Joints, thoracic cavity, abdominal cavity, etc. 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. By administering the composition for introducing a target substance of the present invention, a gene or a target substance can be simply and efficiently introduced into a wide range of sites.

 Applications of the composition for introducing a target substance of the present invention include, for example,

• Treatment or improvement of symptoms of a disease that develops over a long period of time, and study of the mechanism of the disease (eg, production of disease model animals, etc., and examination of therapeutic effects, etc.).

Diseases to which the composition for introducing a target substance of the present invention can be applied include, for example, dystrophic epidermolysis bullosa (Dystrophic EB), junctional epidermolysis bullosa (Junctional EB), systemic atrophic benign epidermolysis bullosa (GABEB), congenital pyloric atresia-junction EB syndrome (PA-JEB), 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 1 and 2 and Xanthia ichthyosis (X- linked ichthyosis), ichthyosis foliaceus (Lamellar ichthyosis), palmar keratoderma with deafness (PPK with deafness), variability Erythrokeratoderma variabilis, Darier's disease, striated palm, striate PPK, striate keratoderma, congenital atrichia, tanju hair Monilethrix), Wa Rudenpurugu syndrome (Waardenburg syndrome eight white bark ¾E [Albinism (different forms)] , tee one wand syndrome (Tierz syndrome) _N Hermann Ski - Padorakku syndrome (Hermansky-Pudlak syndrome), myeloid pro Toporufi phosphorus diseases (Erythropoietic protoporphyria), Congenital erythropoietic porphyria, familial development te skin porphyria (Familial porphyria cutanea tarda), atypical ponorefilin Syndrome (Variegate porphyria), chromogenic xeroderma pigmentosum (Xeroderma pigmentosum), Corden syndrome, basal cell nevus syndrome, Peutz-Eye Garz syndrome group (Peutz- Jeghers Syndrome, Cowden Syndrome Non-Ton-Zonana Syndrome (Bannayan-Zonana Syndrome, Sulfur Deficiency Trichothiodystrophy), Fabry's Disease, Ataxia-telangiectasia (Ataxia telang iectasia), genetic diseases such as hereditary hemorrhagic telangiectasia, and malignant tumors such as malignant melanoma and malignant endothelial cell tumor.

 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. 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. As 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 obtained recombinant plasmid vector-containing HVJ envelope vector or recombinant plasmid vector naked—a type selected from the group consisting of DNA and the hyaluronic acid 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 (Τ) to be introduced and the HVJ envelope vector or (τ). 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), (Τ) 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), (Τ) and Η.

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.

For example, when applying the target material for introducing the composition on the skin, 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 μg to 100 μg.

 The administration of the composition for gene transfer into the blood may be performed at any site where (T), (Τ) 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.

For example, 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 μg per mL.

 The method for oral ingestion of the composition for gene transfer may be any method as long as (T), (Τ) and the HVJ envelope vector or the HVJ envelope vector containing (τ) express the effect. In addition, the number and timing of oral ingestion of the composition for introduction can be appropriately selected depending on the intended use.

 For example, when the target substance-introducing composition is orally ingested as a food or an internal medicine, the amount of intake is not particularly limited. For example, as the target substance, 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), (Τ) 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.

 For example, when the above-mentioned composition for introducing a target substance is locally administered to a tissue using a catheter or by a surgical operation, the amount of intake is not particularly limited. For example, it may be lpg to lmg, preferably lng to 100g, per body weight (kg).

Hereinafter, the application to xeroderma pigmentosum as a therapeutic agent for a disease will be described. Xeroderma pigmentosum (XP) has eight subgroups: Group A, Group B, Group C, Group D, Group E, Group F, Group G and Group V. The introduction of a group A causative gene (XPA gene) into xeroderma pigmentosum group A (XPA) mice is illustratively described. As the nucleic acid, for example, an XPA gene can be used. Such a nucleic acid is directly mixed with a biocompatible composition, for example, sodium hyaluronate as naked-DNA incorporated in a conventional plasmid vector such as pCAGGS, pcDNA3, etc. May be combined. 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. And sodium hyaluronate.

 As the target substance-introducing composition, for example, 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. Example 1

 Preparation of composition for gene transfer

 (1) Preparation of 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 β-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)). In addition, 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.

 (2) Preparation of sodium hyaluronate solution

By swelling sodium hyaluronate in 100 ml of Dulbecco's PBS (-) solution to a concentration of 5% by weight, 2816

23 A sodium sodium alluronate solution (pH 7.4) 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.

 (3) Preparation of composition for gene transfer

Wherein (1) mixing the resulting XPA gene transfer HVJ envelope base Kuta one 3 0 0 mu 1 and the 5 and weight _^0/0 hyaluronic Sanna tri © arm solution 7 5 mu 1 obtained in (2) As a result, a composition for gene introduction was obtained. The concentration of sodium hyaluronate in the composition for gene transfer was 1% by weight. Test Example 1 Effect of introduction of X X gene into XΡ mouse (1) Skin application group of gene transfer composition

Example 1 obtained in transgenic for composition (E 1), as D NA amount, 5 0 mu § / mouse (total volume 3 7 5 μ 1: 5 by weight _^0/0 hyaluronic Nsan'na Application Benefits © The mouse was applied to the back (about 4.5 cm ² ) of the Χ Ρ Α model mouse so as to be 75 μl + HVJ envelope vector solution (300 μl). After 24 hours or 48 hours, anesthetize with N embuta 1 R by intraperitoneal injection, and place TOSHIBA fluorescent S unlamp FL 20 SE (peak wavelength of 3 0 5 nm, 2 8 0 ~ 3 6 0 nm) was used to 4 k J Zm ² irradiated with UV B. The amount of ultraviolet rays was measured with a UVR-305-365D ultraviolet dosimeter. As a control, instead of the XPA gene-introducing HVJ envelope vector, a control gene-introducing composition (C-11) containing a HVJ envelope vector enclosing pCAGGS or pcDNA3 containing no XPA gene was used. ) Or a control gene transfer composition (C-12) containing no sodium hyaluronate was applied, and the same operation was performed.

(2) Detection of unscheduled DNA synthesis (UDS) Out

Using the UDS as a guide, we examined whether the repair ability could be improved by introducing the XPA gene that repairs UV DNA damage. Wherein the UVB irradiation site scissors in Shitakonko, methyl ^{3 H- d T hd (1 0} 0 C i / m 1) containing saline solution 0. 5 ml in three groups of mice (1), said radiation The site was injected subcutaneously. After placing the mouse in an incubator at 35 ° C for 60 minutes, the tongue was removed. After a further 3 hours, the mice were sacrificed.

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 μπι, 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.

 One week later, 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.

 As a result, when UDS was measured in epidermal cells, 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. In the skin application group of 2), the number of blackening particles (grain number) of most cells was 0 to 5, whereas in the skin application group of the gene transfer composition (E 1), However, 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.

 (3) Morphological changes in the UVB irradiation area

The skin in each of the mice described in (1) above was excised, and each skin piece was obtained. Each of the obtained skin pieces was fixed with a 10% neutral formalin solution. T 脑 Ken 2816

25 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.

 According to the observation results of the HE stained specimen, in the mouse coated with the composition for introducing a gene (E1), 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. In 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.

 This is a result of the gene being efficiently introduced by the HVJ envelope vector containing the gene of the present invention, and its function being expressed under physiological conditions. These effects are due to the fact that the HVJ envelope vector containing the gene, for example, fuses with the cell membrane of epidermal cells or hair follicle cells in the presence of mucopolysaccharides by passing HVJ through the stratum corneum. However, it is considered that the content was expressed as a result of being taken into cells. Example 2

 Preparation of composition for gene transfer

 (1) Preparation of nucleic acid for introduction

In the same manner as in Example 1, p CAGG S—XPA was obtained. In the same manner as in Example 1, pc HA—XPA was obtained. Where nake 2004/002816

The DNA amount of 26 d—DNA, p CAGG S—XPA or pc HA—XPA, was 50 μg / mouse each.

 (2) Preparation of sodium hyaluronate solution

In the same manner as (2) of Example 1, 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.

 (3) Preparation of composition for gene transfer

 The nucleic acid for introduction (ΙμΟ) (50 μg 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

 (1) Skin application group of gene transfer composition

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. Was irradiated with 0.5 kJ m ² . As a control, instead of the composition for gene transfer, a composition for control gene transfer (C2-1) using pCAGGS or pcDNA3 containing no XPA gene or sodium hyaluronate was contained. The control gene-introducing composition (C2-2), which was not used, was applied to the backs of three mice, respectively, and the same operation was performed.

 (2) Observation of skin symptoms at UVB irradiation area

Immediately after 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.

 As a result, in the 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. In 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. In the group to which the control gene-introducing composition (C2-2) containing no sodium hyaluronate was applied, one out of three mice exhibited moderate erythema and two exhibited severe erythema and edema. Was. In the long-term irradiation test, 4 months after the end of ultraviolet irradiation, the skin application group of the gene transfer composition (E2) showed only skin thickening and red papules at the site of ultraviolet irradiation in the group to which the gene transfer composition (E2) was applied. Plasmid vector containing no nucleic acid encoding the XP gene

Skin of control gene transfer composition (C2-1) containing (pCAGGS or pcDNA3) or control gene transfer composition (C2-2) containing no sodium hyaluronate In the applied group, 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.

 (3) Morphological changes in the UVB-irradiated part ′ The skins of the mice in the three groups of the test example 2 (1) were excised, and each skin piece was obtained, and an HE-stained specimen was obtained from the skin piece. As in (3) of Test Example 1, 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.

As a result, from the observation results of the HE stained specimens, it was revealed that in the group to which the gene transfer composition was applied, epidermal damage and formation of sunburn cells were suppressed as compared with the two control groups. Example 3

 Preparation of composition for gene transfer

 (1) Preparation of HVJ Envelope Vector for XPA Gene Transfer As in Example 1, pc HA—XPA was constructed. The obtained pc HA—XPA was encapsulated in an HVJ envelope vector. As a result, an HVJ envelope vector for XPA gene transfer was obtained. Here, the DNA amount in each HVJ envelope vector for XPA gene transfer was 508 mice.

 (2) Preparation of sodium chondroitin sulfate solution

In the same manner as (2) of Example 1, 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.

 (3) Preparation of composition for gene transfer

 The nucleic acid for introduction 200 μl (50 μg DNA) obtained in the above (1) was mixed with 50 μl of the 2% by weight sodium chondroitin sulfate solution obtained in the above (2). Thus, a composition for gene transfer (Ε3) was obtained. The concentration of sodium chondroitin sulfate in the composition for gene transfer was 0.4% by weight. As a control, instead of 対 照 Α Α for gene transfer Η VJ envelope vector ^ ”, a control gene transfer composition using HVJ envelope vector containing pcDNA3 that does not contain nucleic acid encoding XPA gene Example 3 XPA to XP mice was applied by applying a control gene transfection composition (C3-2) containing no product (C3-1) or sodium chondroitin sulfate. Effects of gene transfer

(1) 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. As a control, instead of the gene transfer composition, 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.

 (2) Observation of skin symptoms

 Immediately after 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.

 As a result, in the mouse to which the composition for gene transfer (E3) of (1) was applied, only mild erythema was observed immediately after UVB irradiation, 24 hours or 1 week later. However, in the group to which the control gene transfer composition (C3-1) was applied, 5 out of 5 animals showed severe erythema and edema, and the control gene transfer composition (C3-2) was applied to the skin. In the group, one out of five had moderate and four had severe erythema and edema. It was found that the mouse to which the composition for gene introduction (E 3) was applied had recovered repairability.

 (3) Morphological change of UVB irradiation part

 The skin of the mouse (1) was excised to obtain each skin piece, and an HE-stained specimen was obtained from the skin piece. In the same manner as in (3) of Test Example 1, 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.

As a result, according to the observation results of the HE stained specimen, the mice to which the composition for gene transfer (E3) described in (1) above was applied, 24 hours after UV irradiation, compared with the two groups of the control, the epidermal damage and the sunburn cell It was clarified that the formation of was suppressed. Example 4

 Preparation of composition for protein introduction

 (1) Preparation of human EGF

 As 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.

 (2) Preparation of sodium hyaluronate solution

 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. Got. 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.

 (3) Preparation of composition for protein introduction

Wherein (1) obtained in human EGF solution 0. 3 m 1 and said (2) sodium 5 weight _^0/0 hyaluronic acid obtained in © beam solution 0. 0 7 5 m 1 were mixed with protein introduced A composition for use was obtained. The concentration of sodium hyaluronate in the composition for introducing a protein was 1% by weight. Test Example 4 Effect of introducing human EGF into C57 mice

 (1) Skin application group of protein-introducing composition

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). As a control, a composition for protein introduction without human EGF (C4-1) or a composition for protein introduction without sodium hyaluronate (C4-2) was applied. Was done. (2) Detection of human EGF

Six hours later, 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 μm, and the obtained sections were stained with a FITC-labeled anti-human EGF antibody.

 As a result, fluorescence was observed in the epidermal tissue only in the mice of the group to which the one prepared in Example 4 was applied. That is, it was surprisingly shown that the protein to be introduced was introduced into cells from the skin. Comparative Example 1

 Preparation of composition for gene transfer

 (1) Preparation of HVJ Envelope Vector for XPA Gene Transfer pcHA-XPA was obtained in the same manner as in Example 1. Obtained c H

A—XPA was encapsulated in an HVJ envelope vector. As a result, an HVJ envelope vector for XPA gene introduction was obtained. Here, the amount of DNA in the HVJ envelope vector for XPA gene transfer was 50 / ig / mouse.

 (2) Preparation of HV J ribosome for XPA gene transfer

 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. Thus, HV J ribosome for XPA gene transfer was obtained. Here, the amount of DNA in the HVJ ribosome for introducing the XPA gene was 50 μg / mouse.

 (3) Preparation of sodium hyaluronate solution

In the same manner as (2) of Example 1, 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.

 (4) Preparation of a composition for gene transfer containing HV J envelope vector for XPA gene transfer

 The HVJ envelope vector for XPA transfection obtained in the above (1) 10-101 (50 μg DΝ) and the sodium bicarbonate solution of the double weight% hyaluronate obtained in the above (3) The mixture was mixed with 100 μl each to obtain a composition for gene transfer (E4) containing HVJ envelope vector for XΡ gene transfer. The concentration of sodium hyaluronate in the composition for gene introduction was 1% by weight.

 (5) Preparation of control composition containing HVJ liposome for XPA gene transfer

Wherein (2) XPA obtained by gene transfer HV J ribosome 1 0 0 μ 1 (50 μ g DNA) and the 2 wt _^0/0 arsenide Al port phosphate obtained in (3) sodium © beam solution The control composition (C 4) containing HV J ribosome for XPA gene introduction was obtained by mixing each with 100 / i 1. The concentration of sodium hyaluronate in the composition for gene introduction was 1% by weight. Comparative Test Example 1 Effect of introduction of XP A gene into XP mouse

(1) Skin application group of gene transfer composition

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.

(2) Observation of skin symptoms at the UVB irradiation area Immediately after UVB irradiation, 24 hours later, and 1 week later, the extent of erythema, edema, etc. was classified into three grades of mild, moderate, and severe, and compared with the control group.

 As a result, in the 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. In the 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.

 (3) Morphological change of UVB irradiation part

 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. As in (3) of Test Example 1, 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.

 As a result, based on the observation results of the HE stained specimen, the mice coated with the gene transfer composition (E4) containing the HVJ envelope vector showed 20/1/24 sunburn cells 24 hours after UV irradiation. There were 30 cells and 100 cells in the mice to which the control composition containing the HVJ ribosome was applied, compared to 100 cells. In the group to which the gene transfer composition was applied, it was found that the formation of epidermal damage and the formation of sunburn cells were suppressed as compared with the control.

Example 5

 Preparation of composition for gene transfer

(1) Preparation of 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.

 (2) Preparation of sodium hyaluronate solution

In the same manner as (2) of Example 1, the hyaluronic acid sodium, Dulbecco PBS (-) solution lml 2. swelled to be 5% by weight concentration, 2.5 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.

 (3) Preparation of composition for gene transfer

The HVJ envelope vector for XPA gene transfer obtained in (1) above was mixed with 225 μl of the HVJ envelope vector and the 2.5 wt% sodium hyaluronate solution obtained in (2) was mixed with 15 μl. As a result, a composition for gene introduction was obtained. Incidentally, 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 XΡ mouse (1) Skin application group of gene transfer composition

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. As a control, the 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.

 (2) Observation of skin symptoms

 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. Example 6

 Preparation of composition for gene transfer

 (1) Preparation of nucleic acid for gene transfer

 PcHA-XPA was constructed in the same manner as in Example 2. Here, the amount of DNA in the nucleic acid for XPA gene introduction was 50 g / mouse.

 (2) Preparation of sodium hyaluronate solution

In the same manner of (2) as in Example 1, 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.

 (3) Preparation of composition for gene transfer

The XPA gene transfer nucleic acid (225 μl) obtained in (1) above and The 5% ⁰ / o sodium hyaluronate solution (150 μl) obtained in (2) was mixed to obtain a composition for gene transfer. 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 Ρ gene into X Ρ Α mouse

(1) Skin application group of gene transfer composition

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. As a control, instead of the composition for gene transfer, a 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.

 (2) Observation of skin symptoms

 Five months after the end of UV irradiation, UV-induced skin tumors were visually observed. As a result, in the group to which the gene transfer composition (E 6) was applied to the skin, as shown in FIG. In contrast, the composition for transfection of a control gene containing no XPA gene (C6-1) or the composition for transfection of a control gene not containing sodium hyaluronate

In the (C 6 -2) skin-applied group, tumors that grew rapidly were observed at the site of UV irradiation. That is, it was surprisingly shown that the nucleic acid to be introduced was introduced into the 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 7

 Preparation of composition for gene transfer

(1) Preparation of HV J Envelope Vector for XPA Gene Transfer In the same manner as in Example 1, pCAGG S—XPA was constructed. The resulting CAGG S- X PA encapsulated in HV J Enbe rope downy compactors _c This gave HVJ envelope vector for XPA gene transfer. Here, the amount of DNA in the HVJ envelope vector for XPA gene transfer was 50 μg Z mouse.

 (2) Preparation of sodium hyaluronate solution

In the same manner as (2) of Example 1, 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.

 (3) Preparation of composition for gene transfer

Wherein (1) obtained in XPA gene transfer HV J envelope base Kuta one 2 5 0 mu 1 and the (2) obtained in 0.3 wt _^0/0 hyaluronic acid sodium © beam solution 5 0 mu 1 Was mixed to obtain a composition for gene introduction. The concentration of sodium hyaluronate in the composition for gene introduction is 0.05% by weight. Test Example 7 Effect of introduction of ΡΑ ΡΑ gene into ΡΑ ΡΑ mouse

 (1) Subcutaneous injection group of the composition for gene transfer

The composition for gene transfer (Ε7) obtained in Example 7 was subcutaneously injected into the back of a Χ 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. As a control, a composition for transfection of a control gene (C 7-1) containing an HVJ envelope vector enclosing pCAGGS containing no XPA gene instead of the envelope vector instead of the envelope vector ) Or a control gene introduction composition (C7-2) containing no sodium hyaluronate, and the same operation was performed.

 (2) Observation of skin symptoms

 Five months after the end of UV irradiation, UV-induced skin tumors were visually observed. As a result, in the group to which the control gene introduction composition containing no XPA gene (C5-1) or the control gene introduction composition containing no sodium hyaluronate (C5-2) was applied, the skin was irradiated with ultraviolet light. In accordance with the site, a rapidly growing tumor was observed, whereas in the subcutaneous injection group of the gene transfer composition (E7), as shown in FIG. And a red papule, 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. Example 8

 Preparation of composition for gene transfer

 (1) Preparation of nucleic acid for gene transfer

 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 0μ _§ _ mice.

 (2) Preparation of sodium chondroitin sulfate solution

In the same manner as in (2) of Example 1 described above, 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.

 (3) Preparation of composition for gene transfer

 The XPA gene transfer nucleic acid 25 μl obtained in the above (1) was mixed with the 0.5 wt% sodium chondroitin sulfate solution 50 μl 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 Ρ gene into X Ρ Α mouse

(1) Subcutaneous injection group of the composition for gene transfer

The 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. As a control, instead of the composition for gene transfer, 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.

 (2) Observation of skin symptoms

Five months after the end of UV irradiation, UV-induced skin tumors were visually observed. As a result, in the group to which the gene-introducing composition (E 8) was applied, only skin thickening and red papules were observed at the site irradiated with ultraviolet light, and no obvious malignant tumor was observed. In the group to which the control gene transfer composition (C8-1) containing no or the control gene transfer composition not containing sodium chondroitin sulfate (C8-2) was applied to the skin, the area corresponding to the UV-irradiated site was matched. A fast growing tumor was observed. That is, surprisingly, the nucleic acid to be introduced is introduced into cells from the skin. It was shown that. It was also found that the gene in the nucleic acid to be introduced was expressed over a wide area of the skin. Example 9

 Preparation of composition for gene transfer

 (1) Preparation of nucleic acid for gene transfer

 In the same manner as in Example 2, pc HA—XPA was constructed. Here, the amount of DNA in the nucleic acid for XPA gene transfer was S Og / mouse.

 (2) Preparation of sodium hyaluronate solution

In the same manner as (2) of Example 1, 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.

 (3) Preparation of composition for gene transfer

 The nucleic acid composition for gene introduction was prepared by mixing 300 μl of the nucleic acid for XPA gene introduction obtained in the above (1) with 75 μl 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. As a control, instead of the composition for gene transfer, a 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.

 (2) Observation of skin symptoms

 One or two weeks after the end of the UV irradiation, UV-induced skin tumors were visually observed. As a result, in the group to which the gene transfer composition (E9) was applied to the skin, as shown in Fig. 5, only skin thickening and red papules were observed at the site irradiated with ultraviolet light, and Only hyperplasia and red papule were observed, and no UV-induced skin tumor was observed. In contrast, a control gene transfer composition (C9-1) containing no XPA gene or sodium hyaluronate was used. In the group to which the control gene-introducing composition (C9-2) containing no skin was applied, as shown in FIG. 6, a rapidly growing tumor was observed in accordance with the ultraviolet irradiation position. 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 10

 Preparation of composition for protein introduction

 (1) Preparation of HV J envelope vector for human EGF introduction Human EGF is, for example, Epidermal manufactured by Wako Pure Chemical Industries, Ltd.

Growth Facter, 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.

 (2) Preparation of sodium hyaluronate solution

Add sodium hyaluronate to Dulbecco's PBS (-) solution 100 m By swelling to a concentration of 5% by weight, a 5% by weight sodium hyaluronate solution (pH 7.4) 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 a volume of 1 liter. The resulting solution was prepared by autoclaving the resulting solution at 121 with high pressure steam for 15 minutes.

 (3) Preparation of composition for protein introduction

 The human EGF solution 300 μl (500 g human EGF) obtained in the above (1) was mixed with 75 μl 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 μg / mouse at the back of a C57 mouse (about 4.5 cm). ² ) was applied. As a control, 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.

 (2) Detection of human EGF

Six hours later, 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 μm, and the obtained sections were stained with a FITC-labeled anti-human EGF antibody.

As a result, fluorescence was observed in the epidermal tissue only in the mice of the group to which the one prepared in Example 10 was applied. That is, it was surprisingly shown that the protein to be introduced was introduced into cells from the skin. 4 002816

43

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. In addition, 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. Industrial applicability

 INDUSTRIAL APPLICABILITY The 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.

Claims

The scope of the claims 1. 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 small molecule drugs to be introduced, as well as the aforementioned peptides and peptide hormones A biocompatible composition capable of retaining 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 in tissues A composition for introducing a target substance. 2. At least one HVJ envelope vector selected from the group consisting of peptides, peptide hormones, proteins, nucleoproteins, nucleic acids, nucleic acid derivatives, essential amino acids, essential fatty acids, vitamins and small molecule drugs to be introduced. And at least one selected from the group consisting of the peptides, peptide hormones, proteins, nucleoproteins, nucleic acids, nucleic acid derivatives, essential amino acids, essential fatty acids, vitamins, low molecular weight drugs and HVJ envelope vectors is retained in the tissue. A composition for introducing a target substance, comprising a biocompatible composition that can be used. 3. 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. Tissue, 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 small molecule drug, and a HVJ envelope vector. A composition for introducing a target substance, which comprises a biocompatible composition that can be retained in the composition. 4. The composition for introducing a target substance according to any one of claims 1 to 3, wherein the biocompatible composition contains mucopolysaccharide or a salt thereof. 5. The composition for introducing a target substance according to any one of claims 1 to 4, wherein the pH of the biocompatible composition is pH 3.0 to 9.0. 6. The derivative of the nucleic acid according to claim 1, wherein the derivative of the nucleic acid is at least one selected from the group consisting of DNA, RNA, peptide nucleic acid, nucleic acid containing a nucleotide analog, and a plasmid vector incorporating these nucleic acids. 6. The composition for introducing a target substance according to any one of 5. 7. The mucopolysaccharide is selected from the group consisting of hyaluronic acid, chitin, colominic acid, chondroitin, dermatan sulfate, chondroitin sulfate such as chondroitin 4-monosulfate and chondroitin 6-sulfate, heparin, keratan sulfate, heparan sulfate, and dye mouth. 5. The composition for introducing a target substance according to claim 4, wherein the composition is at least one selected from the group consisting of an acid. 8. The salts of the mucopolysaccharide include hyaluronic acid, chitin, colominic acid, chondroitin, dermatan sulfate, chondroitin sulfate such as chondroitin 4-sulfate and chondroitin 6-sulfate, heparin, keratan sulfate, heparan sulfate and the like. 5. The composition for introducing a target substance according to claim 4, wherein the composition is at least one kind of salt selected from the group consisting of tycolate. 9. A method for introducing a target substance into a tissue, which comprises administering the composition for introducing a target substance according to any one of claims 1 to 8 to the target substance.