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WO1997017844A1 - Composition pour l'introduction de genes et procede d'introduction de genes utilisant cette composition - Google Patents

Composition pour l'introduction de genes et procede d'introduction de genes utilisant cette composition Download PDF

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Publication number
WO1997017844A1
WO1997017844A1 PCT/JP1996/003360 JP9603360W WO9717844A1 WO 1997017844 A1 WO1997017844 A1 WO 1997017844A1 JP 9603360 W JP9603360 W JP 9603360W WO 9717844 A1 WO9717844 A1 WO 9717844A1
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WO
WIPO (PCT)
Prior art keywords
gene
composition
cells
epidermis
vector
Prior art date
Application number
PCT/JP1996/003360
Other languages
English (en)
Japanese (ja)
Inventor
Ryuichi Morishita
Yasushi Kaneda
Toshio Ogiwara
Tsuneaki Sakata
Mamoru Hasegawa
Original Assignee
Dnavec Research Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dnavec Research Inc. filed Critical Dnavec Research Inc.
Priority to AU11312/97A priority Critical patent/AU1131297A/en
Publication of WO1997017844A1 publication Critical patent/WO1997017844A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • composition according to (1) or (2) which comprises an inactivated Sendai virus (HVJ).
  • HVJ Sendai virus
  • the "compound having an affinity for both DNA and cells” in the “composition for gene transfer” of the present invention includes ribosome, which is an artificially produced membrane structure composed of phospholipid / glycose glycolipid And cationic ribosomes and complexes composed of cationic phospholipids (PL Feigner et al., Proc. Natl. Acad. Sci. USA, 84, 7413 (1987), CM Corma et al., Mol. Cell. Biol., 2, 1044 (1982), N. Haga, K. Yagi, J. Clin. Biochem. Nutr., 7, 175 (1989), JR Neuman et al., Biotechniques 12, 643 (1987).
  • ribosome is an artificially produced membrane structure composed of phospholipid / glycose glycolipid And cationic ribosomes and complexes composed of cationic phospholipids
  • surviving epidermis cells in the present invention includes any living epidermis that is not keratinized, and includes, for example, skin epithelial cells and skin fibroblasts.
  • gene therapy for skin hereditary disorders such as fish scales and scalp abnormalities can be suitably performed.
  • a gene in a “stage-specific” manner a gene can be placed at a specific site in the embryo that forms the epidermis at that stage, and the gene expression can be controlled regardless of the vector and the motor used. It can be controlled in a tissue-specific manner and can be applied to the treatment of genetic diseases.
  • examples of the gene introduced by the “gene transfer vector” of the present invention include all types of genes such as development stage-specific genes, organ-specific genes, particularly skin-specific genes, and genes for treating various diseases. Can be
  • composition for gene transfer of the present invention can be applied to the treatment of genetic diseases in general, but it can be inferred that it is particularly effective for skin disorders such as fish and abnormalities of the skin such as abnormalities of the scalp.
  • FIG. 1 (A) -FIG. 1 (B) are photomicrographs showing the distribution of FITC-labeled 0DN introduced into fetal rat amniotic fluid by direct injection at 2 hours. (A) was observed at 40x and (B) at 100x.
  • Figure 2 is a micrograph showing the distribution of FITC-labeled 0DN introduced into fetal rat amniotic fluid by direct injection 5 days later.
  • FIG. 3 is a diagram showing the structure of a plasmid pSV40Gal that expresses a / 3-galactosidase gene.
  • FIG. 4 (A) -FIG. 4 (C) are photographs of rat fetal skin 5 days after transfection.
  • (A) and (B) are the three-galactosidase vector and control vector, respectively.
  • (C) relates to the untreated rat.
  • Figure 6 (A)- Figure 6 (C) are photographs showing histological analysis of rats 10 days after transfusion.
  • (A) and (B) relate to a rat transfected with a 3-galactosidase vector and a control vector, respectively, and (C) relates to an untreated rat.
  • Phosphatidylserine, phosphatidylcholine, and cholesterol were mixed at a weight ratio of 1: 4.8: 2.
  • the dried lipid was suspended in a 200 1 BSS solution (137 mM NaCl, 5.4 mM KC1, 10 mM Tris-HCl, pH 7.6) containing plasmid MA or FITC-labeled oligonucleotide (ODN).
  • Ribosomes were prepared by shaking and ultrasound (see "The Manual of Ribosome Z Experiments in Life Sciences", Springer-Fairark Tokyo (1992), p. 282-287).
  • HVJ Z strain
  • the ribosome solution described above (10 mg ribosome / 0.5 ml solution) was mixed with 10,000 hemagglutinating units of HVJ and diluted with BSS solution to a final volume of 4 ml. The mixture was kept at 4 for 5 minutes and then gently shaken at 37 for 30 minutes. Unreacted HVJ and ribosome were removed from HVJ-ribosome by a sucrose density gradient.
  • the oligonucleotide (0DN) was labeled with FITC at the 5 'end and 3' end (Antisense Res. Develop. Vol. 2, 27-39 (1992)).
  • the HVJ ribosome containing the labeled ODN was prepared according to Example 1. Prepared by the method. The final concentration of FITC-labeled ODN to be injected into amniotic fluid (0DN relative to the total ribosome solution) was used. HVJ-ribosome 101 containing labeled 0DN prepared by the method of Example 1 was injected into the amniotic fluid of a Sprague-Dawley rat on the day of pregnancy through a 30-gauge injection needle.
  • HVJ-ribosome containing no FITC-labeled 0DN prepared by the method of Example 1 was injected. All rats were sacrificed after 2 hours or 5 days, and the extirpated tissues were fixed in 4% paraformaldehyde solution according to the standard method, and the sections were stained with erythrochrome black and observed with a fluorescence microscope. did.
  • PSV40Gal a plasmid that expresses the / S-galactosidase gene
  • Plasmid DNApAct-c-inyb containing a 370 base pair 5'-promoter region and a 900 base pair first exon region of the chicken ⁇ actin promoter was restricted to remove the C-myb region from the restriction enzymes Ncol and Xbal. Then, the Sail linker was ligated. 3.
  • the 1-kilobase pair E. coli 3-galactosidase gene was excised from plasmid DN ApMC1871 using the restriction enzyme Sail and ligated to the Sail site of the above plasmid DNA ( Figure 3).
  • Escherichia coli; a plasmid DMpAct-CV containing no S-galactosidase gene was simultaneously prepared.
  • HVJ-ribosomes containing the ⁇ -ii root gene plasmid pSV40Gal or the control plasmid DNA pAct-CV were prepared.
  • HMG-1 protein was simultaneously encapsulated in HVJ-ribosomes.
  • Hit the syringe directly into the amniotic fluid from the uterine wall Then, 10 / il of HVJ-ribosome prepared by the method of Example 2 was injected. Rats were sacrificed 5 days and 10 days after injection, and the removed tissue was fixed in a 1% paraformaldehyde solution according to a standard method, and the tissue was removed. Staining solution.
  • X-gal chromadin buffer solution 49 mg / ml 5-promo-4-chloro-3-indolinole-yS-D-galactoside: 1 ramol Mg 2 and 3 mmol K 3 Fe ( CN) 6 : l: 99
  • X-gal chromadin buffer solution 49 mg / ml 5-promo-4-chloro-3-indolinole-yS-D-galactoside: 1 ramol Mg 2 and 3 mmol K 3 Fe ( CN) 6 : l: 99
  • the cells were observed with a microscope.
  • HVJ-liposome containing plasmid DNA containing no E. coli galactosidase gene was injected.

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physics & Mathematics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un procédé d'introduction efficace d'un gène spécifiquement dans l'épiderme. Ce procédé comprend la mise en contact direct avec l'épiderme d'un mammifère, mettant en jeu des cellules épidermiques vitales, d'une composition contenant un composé qui a des affinités à la fois pour l'ADN et les cellules, et un vecteur d'introduction de gènes.
PCT/JP1996/003360 1995-11-16 1996-11-15 Composition pour l'introduction de genes et procede d'introduction de genes utilisant cette composition WO1997017844A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU11312/97A AU1131297A (en) 1995-11-16 1996-11-15 Composition for gene introduction and method for gene introduction by using the composition

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP29871495 1995-11-16
JP7/298714 1995-11-16
JP8/114253 1996-04-11
JP11425396 1996-04-11

Publications (1)

Publication Number Publication Date
WO1997017844A1 true WO1997017844A1 (fr) 1997-05-22

Family

ID=26453048

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP1996/003360 WO1997017844A1 (fr) 1995-11-16 1996-11-15 Composition pour l'introduction de genes et procede d'introduction de genes utilisant cette composition

Country Status (2)

Country Link
AU (1) AU1131297A (fr)
WO (1) WO1997017844A1 (fr)

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BLOOD, Vol. 78(4), 1991, WADE CLAPP et al., pages 1132-1139. *
GENE THERAPY, Vol. 2, July 1995, PITT et al., pages 344-350. *
GENE, Vol. 149, 1994, MORISHITA et al., pages 13-19. *
J. BIOL. CHEM., Vol. 264, 1989, KANEDA et al., pages 12126-12129. *
J. CLIN. INVEST., Vol. 95, June 1995, McCRAY et al., pages 2620-2632. *
NATURE SCIENCE, Vol. 1(11), 10 November 1995, HARMANJATINDER et al., pages 1201-1203. *

Also Published As

Publication number Publication date
AU1131297A (en) 1997-06-05

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