WO2004075924A1 - Promoteur de transfert genique et composition de transfert genique contenant ce promoteur - Google Patents

Promoteur de transfert genique et composition de transfert genique contenant ce promoteur Download PDF

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Publication number
WO2004075924A1
WO2004075924A1 PCT/JP2004/000253 JP2004000253W WO2004075924A1 WO 2004075924 A1 WO2004075924 A1 WO 2004075924A1 JP 2004000253 W JP2004000253 W JP 2004000253W WO 2004075924 A1 WO2004075924 A1 WO 2004075924A1
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Prior art keywords
gene
gene transfer
composition
cells
promoter
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PCT/JP2004/000253
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English (en)
Japanese (ja)
Inventor
Yoshiaki Hitomi
Hideki Ohno
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Japan Genome Solutions, Inc.
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Publication date
Application filed by Japan Genome Solutions, Inc. filed Critical Japan Genome Solutions, Inc.
Priority to JP2005502813A priority Critical patent/JP4499035B2/ja
Publication of WO2004075924A1 publication Critical patent/WO2004075924A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a gene transfer promoter and a composition for gene transfer containing the same.
  • a method of administering to a living body a recombinant vector containing a desired foreign gene to be transfected and capable of expressing the foreign gene in cells of an animal into which the gene is to be introduced is known.
  • an object of the present invention is to provide a gene transfer promoter and a composition for gene transfer containing the same, which can increase the efficiency of gene transfer without causing significant damage to cells or tissues. .
  • the present inventors have conducted intensive studies and found that a substance that captures calcium ions and / or magnesium ions has an effect of increasing gene transfer efficiency, and completed the present invention.
  • the present invention provides a gene transfer promoter comprising a substance that captures calcium ions and / or magnesium ions. Further, the present invention provides a recombinant vector containing a desired foreign gene to be transfected and capable of expressing the foreign gene in cells of an animal into which the gene is to be transfected; A composition for gene transfer, comprising: Further, the present invention relates to a method for introducing a foreign vector into a cell of an animal into which the gene is to be introduced, the recombinant vector being capable of expressing the foreign gene in a cell of the animal. A method for promoting gene transfer by performing gene transfer for introducing a gene into a cell in the presence of the gene transfer promoter of the present invention is provided.
  • the present invention provides a gene transfer method, comprising administering the above-described composition for gene transfer of the present invention to animal cells. Furthermore, the present invention provides the use of a substance that captures calcium ions and Z or magnesium ions for the production of a gene transfer promoter. Furthermore, the present invention includes a desired foreign gene to be transfected, Use of a composition comprising, in a medium, a recombinant vector capable of expressing the exogenous gene in animal cells and the above-described gene transfer promoter of the present invention for the production of a composition for gene transfer I will provide a.
  • a novel gene transfer promoter and a composition for gene transfer containing the same which can enhance the efficiency of gene transfer without causing significant damage to cells or tissues.
  • the efficiency of gene transfer into cells can be improved without causing significant damage to cells or tissues. Efficiency can be improved.
  • FIG. 1 is a diagram showing the relationship between the concentration of EDTA used in the gene transfer performed in Example 1 of the present invention and the number of skeletal muscle fibers expressing the introduced EGFP gene.
  • FIG. 2 is a graph showing the number of skeletal muscle fibers expressing the introduced EGFP gene when the same amount of calcium ion or magnesium ion as EDTA was added in the gene transfer performed in Example 1 of the present invention. It is.
  • FIG. 3 is a diagram showing the relationship between the concentration of EDTA used and the presence or absence of PBS and the number of skeletal muscle fibers expressing the introduced EGFP gene in the gene transfer performed in Example 1 of the present invention.
  • the gene transfer promoter of the present invention is composed of a substance that captures calcium and / or Z or magnesium, that is, a substance that captures one or both of calcium ion and magnesium ion.
  • a substance that captures calcium ion and magnesium ion As such a substance, a chelating agent of calcium ion and Z or magnesium ion is preferable.
  • EDTA is the most preferred because it causes minimal damage to cells and tissues and is already approved as a pharmaceutical.
  • a chelating agent is preferable, but it is not necessarily limited to a chelating agent, and any substance capable of capturing calcium ions and / or magnesium ions may be used. It may be a substance.
  • the gene transfer promoter of the present invention contains a desired foreign gene to be transfected, and is added to a tissue or a cell together with a recombinant vector capable of expressing the foreign gene in a cell of an animal into which the gene is to be introduced. Is administered. Therefore, the present invention also provides a recombinant vector containing a desired foreign gene to be transfected and capable of expressing the foreign gene in cells of an animal into which the gene is to be transfected; Also provided is a composition for gene transfer, comprising an agent and a medium in a medium.
  • the concentration of the gene transfer promoter in the gene transfer composition is not particularly limited, but is preferably 1 to 3 OmM, more preferably 2 to 2 OmM, and 3 to 1 Orn. More preferred.
  • an aqueous medium is preferable, and an aqueous buffer or water is more preferable. If the osmotic pressure of the composition is higher than the osmotic pressure in the cells, tissue and cells are damaged, and the gene transfer efficiency is reduced.
  • the composition is preferably isotonic or hypotonic. As specifically shown in the examples below, the composition is most preferably a hypotonic solution, since the gene transfer efficiency is increased in the case of hypotonicity.
  • the hypotonic solution can be easily prepared by using pure water as a medium or using a buffer having a lower concentration of a buffer and a salt than a physiological buffer.
  • the potassium ion concentration is important, and the potassium ion concentration in the buffer is not more than 5 O mM. And it is 160 mM, preferably 80 mM to 130 mM.
  • mM or 160 mM preferably 8 OmM to 130 mM potassium ion, 10 mM to 120 mM, preferably 20 mM to 70 mM sodium ion, 1 OmM to 8 OmMs, preferably 15 mM to 3 OmM
  • a potassium-rich phosphate buffer containing a phosphate ion and 9 OmM to 160 mM, preferably 11 OmM to 14 OmM chloride is preferred. It should be noted that such a rich buffer can be used as an isotonic solution, or the buffer can be diluted with water and used as a hypotonic solution. When diluting with water, the dilution ratio is preferably 10 times or less.
  • Monosaccharides and oligosaccharides such as sucrose and sucrose, which are sometimes used as osmotic pressure regulators, should not be used when the composition is injected intramuscularly, since it reduces the gene transfer promoting effect of the present invention. Is preferred.
  • the recombinant vector contained in the gene transfer composition is not particularly limited as long as it contains a desired foreign gene to be transfected and can express the foreign gene in cells of the animal into which the gene is to be introduced. Instead, a gene obtained by inserting a desired foreign gene to be introduced into an expression vector for gene expression, which has been conventionally used for gene therapy, can be used.
  • An expression vector for gene therapy is usually located downstream of an origin of replication enabling replication in Escherichia coli, a promoter derived from a virus or a promoter suitable for expression of a target gene in a target tissue, and one of the promoters, A multicloning site containing various restriction enzyme sites, a virus-derived terminator located downstream of the multicloning site, and a selection marker such as drug resistance, auxotrophy or temperature sensitivity are provided.
  • the green fluorescent protein (GFP) gene persif: c-lase gene is incorporated, and these genes are expressed together with the desired foreign gene to achieve green fluorescence.
  • the expression vector may be a plasmid vector or a viral vector such as a retrovirus or an adenovirus, but a plasmid vector is preferred from the viewpoint of safety.
  • Expression vectors used for gene therapy are well known in the art, and various types are commercially available. And commercially available vectors can be used as they are.
  • the concentration of the recombinant vector in the composition for gene introduction is not particularly limited and may be appropriately selected, but is usually preferably about 1 mg / ml to 10 mg / ml.
  • the composition for gene transfer of the present invention can be used in the same manner as a conventional composition for gene transfer. That is, it can be directly administered to a living body by intramuscular injection, subcutaneous injection, or the like.
  • the dose is not particularly limited and is appropriately selected depending on the purpose of the treatment, the transduction efficiency, and the like, but is usually about 101 to 100 I / kg body weight of the living body.
  • the composition for gene transfer of the present invention can also be used for gene transfer into fibroblasts and lymphocytes in vitro. If the cells are damaged when taken out of the body and transfected, it is thought that the engraftment rate will be reduced when the cells are re-transfected into the living body. The survival rate is improved.
  • the amount of the composition used in this case is not particularly limited, and is appropriately selected.
  • a desired foreign gene is introduced into a living body by administering the gene introducing composition of the present invention to a living body, or by transplanting a gene-transfected cell into the living body by causing the gene introducing composition of the present invention to act. can do.
  • the desired foreign gene is not limited at all, and may be a conventionally known IL-5, erythropoietin, proinsulin or the like.
  • replacement of a biological agent factor VIII gene for hemophilia A, Factor IX gene for hemophilia B
  • DNA vaccines malaria, tuberculosis, cedar pollen, vaccinia, virus infections such as HBV, etc.
  • in vitro gene therapy genes introduced into cells taken out of the body and returned to the living body, genes for ADA deficiency, etc. Treatment, etc.
  • DNA vaccines for animals for example
  • the present invention is not limited to these.
  • the present invention will be described more specifically based on examples.
  • the present invention is not limited to the following examples.
  • EGFP Enhanced green fluorescence protein
  • PEGFP-G1 was introduced into Escherichia coli (XL-1Blue competent cells), seeded on LB agar plates containing kanamycin, and cultured at 37 ° C for about 12 hours. From this plate, a single E. coli colony was selected, and the E. coli was shake-cultured in a kanamycin-containing Superbroth culture solution for about 20 hours. Plasmids were extracted from the grown Escherichia coli by a real force lysis method and prepared by two rounds of GsGI 2 average density gradient centrifugation.
  • the anterior tibial muscle on the lateral front of the lower limb was selected because of the conditions, etc.
  • the composition of the sample is as follows.
  • the sample for intramuscular injection was placed in a sterilized 1.5 ml tube and cooled on ice. Immediately before use, return to room temperature and use an insulin self-injection syringe with a 29 gauge needle ( 29G 0.5ml myjector SS-05 2913, Terumo) was transferred to avoid air bubbles and intramuscular injection was performed.
  • 29G 0.5ml myjector SS-05 2913, Terumo 29G 0.5ml myjector SS-05 2913, Terumo
  • ICR mice (oss, 6 weeks old) purchased from Shizuoka Experimental Animals (SLG) were bred and used at 7 to 8 weeks of age.
  • dilute somnopentyl (pentobarbital) equivalent to 30 mg / kg body weight was administered intraperitoneally.
  • the lower limb was disinfected with 70% alcohol, and an approximately 3 mi incision was made in the skin corresponding to the anterior tibialis muscle.
  • the 29-gauge needle of the insulin self-injecting syringe was inserted at right angles to the running of the muscle, and the plasmid solution was gently injected. The needle was inserted into the anterior tibialis muscle for a while to prevent leakage.
  • the animals were sacrificed by cervical dislocation after injection.
  • the skin of the lower limb was peeled off and the tibialis anterior muscle was removed.
  • the anterior tibial muscle sample was immediately divided into four sections perpendicular to the muscle fiber running, embedded in a 0GT compound, and then rapidly frozen in isopentane sufficiently cooled with liquid nitrogen.
  • a serial frozen section of the tibialis anterior muscle sample was prepared according to the standard method, and the fluorescence microscopy (using a Nikon E600 fluorescence microscope B2-A filter) was used to identify specific EGFP expressed in skeletal muscle. The fluorescence was observed.
  • Example 2 Luciferase expressed in anterior tibialis muscle of IG mice
  • a 1.7 kb luciferase gene fragment (53-1742) was excised from the pGL3basic plasmid (Accession No. U47295) by digestion with the restriction enzymes Hindi 11 and Xbal.
  • This gene fragment was digested with HindlM / Xbal restriction enzyme and inserted into a multicloning site of expression vector pGR3 (manufactured by Invitrogen) into T4 DNA ligase.
  • Escherichia coli XL-1Blue was transformed using the ligation product and selected on an agar medium supplemented with ampicillin.
  • a recombinant plasmid containing a 1.7 kb gene fragment was selected by agarose electrophoresis, and the vector-specific primers GLprimer2 (ctttatgttttttttggcgtcttcc) and lucifera Clones containing the lysylperase-expressing plasmid PGMVIUG were selected by the PGR method using zeprimer IUGA (tcgtcatcgctgaatacatg).
  • the selected E. coli clone was cultured with shaking in Superbroth culture solution containing ampicillin for about 20 hours.
  • the plasmid was extracted by the alkaline lysis method of the grown colon bacillus and prepared by two rounds of GsGI 2 average density gradient centrifugation.
  • the composition of the sample is as follows.
  • the sample for intramuscular injection was placed in a sterilized 1.5 ml tube and cooled on ice. Immediately before use, return to room temperature and use a syringe for self-injecting insulin with a 29 gauge needle (
  • 29G 0.5 ml myjector SS-05 (3, Terumo) was transferred without bubbles and intramuscular injection was performed.
  • luciferase activity For protein quantification, a kit (BioRad) based on the Bradford method was used. For detection of luciferase activity, Dual-Luc TM erase Reporter Assay System manufactured by Promega was used. In advance, 50 I of luciferase Atsushi reagent was dispensed into a glass vial, placed in a luminometer (BLR-301, manufactured by Aroka), and 10 iI of skeletal muscle extract was added with stirring. The luminescence was integrated for 15 seconds, and the value corrected for the amount of protein was used for comparison between samples.
  • BLR-301 manufactured by Aroka
  • Example 1 As a result, as in Example 1 in which pEGFP-C1 was expressed, when 3 mM of so-called EDTA-2Na was added to the plasmid sample, the luciferase activity was increased by 10 times. It was found that the use of a hypotonic solution without the addition of dPBS further increased the expression of luciferase by about three times (Fig. 3).
  • a potassium-rich buffer solution hereinafter referred to as PBS ⁇ -PBSJ
  • a dPBS having the following composition, having the composition shown in Table 1 below, having an ion balance of the intracellular solution.
  • the same experiment as in Example 1 was performed.
  • the concentration of EDTA-2Na in the composition for gene transfer was 3 mM.
  • the preparation of K-PBS was specifically performed as follows.
  • the CI powder was weighed to a final concentration of 120 mM.
  • the weighed KGI was dissolved in an appropriate amount of distilled water, and sodium phosphate buffer was added to a final concentration of 25 mM, and the volumes were precisely adjusted to 120 mM KG to obtain a 25 mM sodium phosphate buffer.
  • the number of EGFP-expressing muscle fibers was clearly higher in the case of using K-PBS than in the case of using dPBS. It was confirmed that the gene transfer efficiency was increased by using -PBS.
  • 100% of a 0.1% Evans blue (dye) solution was injected from the tail vein one hour after the administration of the composition for gene transfer.
  • the anterior tibial muscle was collected, and frozen tissue sections were prepared according to a standard method. The dye incorporated in the muscle fibers was observed using a fluorescence microscope (G filter-1).
  • the gene transfer promoter can increase the efficiency of gene transfer without causing significant damage to cells or tissues.
  • the efficiency of gene transfer into cells can be improved without causing significant damage to cells or tissues. It is intended to provide a composition for gene transfer which can enhance the efficiency of gene transfer and is useful for gene therapy.

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Abstract

L'invention concerne un promoteur de transfert génique permettant d'augmenter l'efficacité du transfert génique sans causer de dommages majeurs aux cellules ou aux tissus, et une composition de transfert génique contenant ce promoteur. Ce promoteur de transfert génique contient une substance capturant un ion calcium et/ou un ion magnésium.
PCT/JP2004/000253 2003-01-17 2004-01-15 Promoteur de transfert genique et composition de transfert genique contenant ce promoteur WO2004075924A1 (fr)

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JP2005502813A JP4499035B2 (ja) 2003-01-17 2004-01-15 遺伝子導入促進剤及びそれを含む遺伝子導入用組成物

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000143548A (ja) * 1998-11-16 2000-05-23 Univ Of Iowa Research Foundation レトロウイルスの感染性を増大させるための方法および組成物
JP2000143549A (ja) * 1998-09-30 2000-05-23 Transgene Sa ポリヌクレオチドを細胞にトランスフェクトさせる治療用組成物の製造におけるマグネシウム（Ｍａ２＋）の使用、および遺伝子治療に有用な組成物
WO2001047563A1 (fr) * 1999-12-28 2001-07-05 Transgene S.A. Utilisation du lithium (li+) pour preparer une composition permettant la transfection d'un polynucleotide dans une cellule, et compositions utiles en therapie genique
WO2001092453A1 (fr) * 2000-06-02 2001-12-06 Novozymes A/S Inhibition de la redeposition ou de la recoloration au cours d'un procede de delavage a la pierre
JP2003104914A (ja) * 2001-09-28 2003-04-09 Yasuhiko Tabata Ｄｎａ−金属−水溶性高分子複合体

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2000143549A (ja) * 1998-09-30 2000-05-23 Transgene Sa ポリヌクレオチドを細胞にトランスフェクトさせる治療用組成物の製造におけるマグネシウム（Ｍａ２＋）の使用、および遺伝子治療に有用な組成物
JP2000143548A (ja) * 1998-11-16 2000-05-23 Univ Of Iowa Research Foundation レトロウイルスの感染性を増大させるための方法および組成物
WO2001047563A1 (fr) * 1999-12-28 2001-07-05 Transgene S.A. Utilisation du lithium (li+) pour preparer une composition permettant la transfection d'un polynucleotide dans une cellule, et compositions utiles en therapie genique
WO2001092453A1 (fr) * 2000-06-02 2001-12-06 Novozymes A/S Inhibition de la redeposition ou de la recoloration au cours d'un procede de delavage a la pierre
JP2003104914A (ja) * 2001-09-28 2003-04-09 Yasuhiko Tabata Ｄｎａ−金属−水溶性高分子複合体

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
CHU Q, ET AL: "EGTA enhancement of adenovirus-mediated gene transfer to mouse tracheal epithelium in vivo", HUM. GENE THER., vol. 12, no. 5, 2001, pages 455 - 467, XP002904813 *
CHU Q. ET AL.: "Binding and uptake of cationic lipid: PDNA complexes by polarized airway epithelial cells", HUMAN GENE THERAPY, vol. 10, 1999, pages 25 - 36, XP000952585 *
HOCHMAN J. ET AL.: "Mechanisms of absorption enhancement and tight junction regulation", JOURNAL OF CONTROLLED RELEASE, vol. 29, 1994, pages 253 - 267, XP002054267 *
JOHNSON L.G.: "Safety and effiency of modulating paracellular permeability to enhance airway epithelial gene transfer in vivo", HUM. GENE THER., vol. 14, no. 8, 2003, pages 729 - 747, XP002904811 *
MYLES, C.: "Augmentation of adenovirus mediated gene transfer in epithelial cells by calcium chelation", HUM. GENE THER., vol. 12, no. 5, 2001, pages 455 - 467, XP002904813 *
OSADA K. ET AL.: "Saibo eno DNA no donyu -baiyo honyurui dobutsu saibo eno donyu o chushin to shite-", PROTEIN, NUCLEIC ACID AND ENZYME, vol. 28, no. 14, 1983, pages 1569 - 1581, XP002904073 *
ROOS DE W.K. ET AL.: "Isolated organ perfusion for local gene delivery: efficient adenovirus mediated gene transfer into the liver", GENE THER., vol. 4, no. 1, 1997, pages 55 - 62, XP002904814 *
WALTERS R. ET AL.: "Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia", J. BIOL. CHEM., vol. 274, no. 15, 1999, pages 10219 - 10226, XP002149604 *
WANG G. ET AL.: "Influence of cell polarity on retrovirus-mediated gene transfer to differentiated human airway epithelia", J. VIROL., vol. 72, no. 12, 1998, pages 9818 - 9826, XP002904812 *
WANG, G. ET AL.: "Increasing epithelial junction permeability enhances gene transfer to airway epithelia in vivo", AM. J. RESPIR. CELL MOL. BIOL., vol. 22, no. 2, 2000, pages 129 - 138, XP002193193 *
YASUHIKO TAHATA: "Separate vol. gene & medicine drug delivery system DDS gijutsu no arata na tenkai to sono katsuyoho", KABUSHIKI KAISHA MEDICAL DO, 20 June 2003 (2003-06-20), pages 271 - 274, XP002904072 *

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JP4499035B2 (ja) 2010-07-07

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