WO2002031139A1 - Vecteur d'expression de transporteur compulsif pour infection organique - Google Patents

Vecteur d'expression de transporteur compulsif pour infection organique Download PDF

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WO2002031139A1
WO2002031139A1 PCT/JP2001/008894 JP0108894W WO0231139A1 WO 2002031139 A1 WO2002031139 A1 WO 2002031139A1 JP 0108894 W JP0108894 W JP 0108894W WO 0231139 A1 WO0231139 A1 WO 0231139A1
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organ
transporter
gene
expression vector
transporter gene
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PCT/JP2001/008894
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Japanese (ja)
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Akira Tsuji
Yoshimichi Sai
Ikumi Tamai
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Japan Science And Technology Corporation
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a vector for forced expression of a transporter for organ infection containing a transporter gene and its use, and more specifically, to forcibly express the transporter gene in an organ in which the transporter gene is not originally expressed.
  • the present invention relates to a vector for compulsory expression of a transposon for organ infection used in a plant and a method for promoting substance transport in an organ by using the vector for compulsory expression of a transposon for organ infection.
  • a membrane protein that transports substances inside and outside the cell is called a transporter.
  • a transporter When a specific molecule binds to a transporter embedded in a lipid bilayer, the conformation changes and the substance is taken up. Alternatively, it is known to be discharged and transported.
  • transporters for example, organic anion transporters such as OAT 1 that transport organic anionic substances, organic cation transports such as CT 1 that transport organic cationic substances, and peptide substances that transport peptide substances
  • Genes such as peptide transporters such as PEPT 1 have been successively isolated and identified. Some of these transporter genes are localized in normal tissues and organs throughout the body, but others are known to be localized in specific tissues and organs such as kidney, liver, and brain.
  • organic anionic substances are distributed in the basolateral membrane by transposers localized in hepatocytes and tubular cells of the liver and kidney, which play important roles in the metabolism and excretion of xenobiotics and drugs. It is taken up into the liver and kidney via, and organic anionic substances produced by intracellular metabolism are excreted.
  • Tables 1 to 3 show the types, names, and accessions of gene banks that have been previously reported.
  • oral administration is useful because of its simplicity and low pain, and it is important to develop pharmaceuticals that can achieve oral effects.
  • absorption in the small intestine is an important factor.
  • cephradine, cephalexin, cefixime, and ceftibuten which are orally available drugs among the 3-lactam antibiotics, are involved in the efficient absorption of oligopeptides in the brush border membrane of the small intestine. 1 (Fei et al., Expression cloning of mammalian proton-coupled oligopeptide transporter.
  • transportable proteins are expressed in the small intestine to enable oral administration of these poorly-absorbable i3-lactam-based substances, it can be expected to improve gastrointestinal absorption.
  • OAT1 organic anion transporter expressed in the kidney is known (Tsuji et al., In vivo evidence for carrier). -mediatea uptake of beta-lactam antibiotics through organic anion transport systems In rat kidney and liver.J. Phamacol.
  • OAT1 is expressed in p-aminonippurate, cyclic AMP, cGMP, prostaglandin E2, urate, -ketoglutarate, methotrexate, etc. when expressed in Xenopus laevis oocytes, an exogenous gene expression system. Is transported and inhibited by various organic anions (Sekine et al., Expression cloning and characterization of a novel multispecific Organic Anion Transporter. J. Biol.
  • OAT 1 is expressed on the basement membrane side of the proximal tubule in the kidney, and is considered to play a central role in the excretion of anion drugs from the kidney with broad substrate recognition.
  • TJwai et a ⁇ . Functional characterization of the rat multispesi fic organic anion transporter OATl mediating basolateral uptake of anionic drugs in kidney.FEBS Letter, 438: 321-324 (1998), Tojo et al., Immunohistochemical localization of multispecific renal organic anion transporter 1 In rat kidney. J. Am. Soc.
  • Expression of renal transporter AT1 in the small intestine can be expected to promote absorption of poorly absorbable) 3-lactam antibiotics.
  • OAT1 is expressed in the small intestine, there is a problem whether it acts on the transport in the uptake direction or the transport in the excretion direction.
  • ⁇ AT1 is expressed on the basement membrane side in the kidney and is responsible for the transport of organic anionic substances in the direction of excretion.
  • OAT1 is expressed on the brush border membrane side, since OAT1 is an exchange transporter for organic anion and dicarboxylic acid, if the concentration of glutarate in the small intestinal epithelial cells is high, exchange transport with glutarate occurs and organic It is also conceivable that an anionic substance may work in the uptake direction.
  • the OAT1 gene delivery system is used to efficiently express OAT1 in the small intestine where the AT1 gene is not originally expressed, the direction of absorption or excretion of organic anionic substances in the small intestine It can be expected to provide directional transport activity.
  • the brain eliminates unnecessary metabolites in the brain into the systemic circulation via the capillary endothelial cells that form the blood-brain barrier.Strictly controls the exchange of substances between the brain and the systemic circulation by blocking the entry of xenobiotics. This makes brain delivery of drugs with central target sites very difficult.
  • the blood-brain barrier also has the function of actively ingesting substances necessary for maintaining the central function Z from the systemic circulation.
  • An object of the present invention is to impart a substance transporting function not originally possessed by various organs to the organ, to promote uptake and transport of useful substances such as drugs and nutrients to the organ, or to excrete substances from the organ.
  • Transformers for organ infections used to promote the transport of waste An object of the present invention is to provide a method for promoting substance transport in various organs using a vector for forced expression of a port and a vector for forced expression of a transposon for infection of an organ.
  • Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and forcibly expressing the target transporter gene in an organ in which the target transporter gene is not originally expressed.
  • the present invention relates to an expression vector containing a target transporter gene, which is used for forcibly expressing the target transporter gene in an organ in which the target transporter gene is not originally expressed.
  • the transporter forced expression vector for organ infection characterized by the above feature (Claim 1), and the transporter gene is an organic anion transporter gene.
  • Transporter for organ infection one forced expression base click evening one (claim 4) or Expression base Kuta one containing transporter gene, transporter gene and Ma one
  • Claim 6 characterized in that the transporter forced expression vector for organ infection according to any one of claims 1 to 6 (Claim 7) and the organ in which the transporter gene is not originally expressed is the small intestine. 8.
  • the vector for compulsory expression of a transposon gene for infecting an organ according to any one of claims 7 to 7 (claim 8), and an organ in which the transport gene is not originally expressed is the large intestine.
  • the transporter forced expression vector for organ infection according to any one of claims 1 to 7 (Claim 9), and an organ in which a transporter gene is not originally expressed is a blood-brain barrier.
  • the present invention also provides and promotes a substance transporting ability in an organ characterized by forcibly expressing an expression vector containing a target transporter gene in an organ in which the target transporter gene is not originally expressed.
  • the transporter gene is an organic anion transporter overnight gene (claim 11). 2. The method according to claim 11, wherein the transporter gene is an organic cation transport gene. 3. The method according to claim 11, wherein the transporter gene is an organic cation transport gene. 11. The method according to claim 11, wherein the gene is a peptide transposable overnight gene. Item 14), and the expression vector containing the transporter gene is an expression vector containing a transporter gene and a marker gene.
  • a method for imparting / promoting a substance transport ability in an organ according to any one of claims 11 to 16 (Claim 17), and a method for determining that an organ in which a transpoi gene is not originally expressed is the small intestine.
  • a method for imparting / promoting a substance transport ability in an organ according to any one of claims 11 to 17 (claim 18), and an organ in which a transporter gene is not originally expressed is used in the large intestine.
  • the method for imparting / promoting a substance transport ability in an organ according to any one of 1 to 17 (claim 20), and the substance transport in the organ is controlled uptake transport of the substance in the organ.
  • the method for imparting / promoting a substance transport ability in an organ according to any one of claims 11 to 2'0 (claim 21), and the substance transport in an organ is carried out by controlling the discharge of the substance in the organ.
  • the present invention relates to an artificial organ (Claim 23) in which a substance transporting ability is imparted and promoted by using an infectious transporter and a forced expression vector.
  • FIG. 1 is a diagram showing a recombinant adenovirus construction system of the present invention.
  • FIG. 2 is a diagram showing a shuttle vector and an adenovirus backpoon vector according to the present invention.
  • FIG. 3 shows the recombination of the invention by homologous recombination in E. coli
  • FIG. 3 shows the construction of a mid.
  • FIG. 4 is a diagram showing the results of electrophoresis of pAd rack-CMV-OAT1 used for homologous recombination in constructing the expression vector of the present invention.
  • FIG. 5 is a diagram showing the results of electrophoresis of linearized DNA of pAdTrack-CMV-OAT1 used for homologous recombination for constructing the expression vector of the present invention.
  • FIG. 6 shows the results of homologous recombination of rat OAT1 into pAdEasy-1 vector.
  • FIG. 7 shows the results of electrophoresis of the expression vector pAd-OAT1 of the present invention.
  • FIG. 8 shows the results of PCR using rat OAT1-specific primers.
  • FIG. 9 shows the results of transfection of the expression vector pAd-OATl of the present invention into HEK293 cells.
  • FIG. 10 is a diagram showing the results of transfection of the expression vector pAd-OAT1 of the present invention into Caco-2 cells.
  • FIG. 11 shows the results of [ 3 H] PAH uptake in OAT1-expressing Caco-2 cells.
  • FIG. 12 shows the results of the permeability of [ 3 H] PAH in OAT1-expressing Caco-2 cells.
  • FIG. 13 shows the results of the permeability of [ 3 H] PAH in the presence of penicillin G in OAT1-expressing Caco-2 cells.
  • FIG. 14 is a view showing the results of incorporation of [ 3 H] dipeptide into PEPT1-expressing RBEC1 cells.
  • the forced expression vector for a transporter for organ infection of the present invention is an expression vector containing a transporter gene of interest, and an organ in which the transporter gene of interest is not originally expressed.
  • Force expression of transporter gene The vector is not particularly limited as long as it is a vector used for the purpose of the present invention, and the method of imparting / promoting the substance transport ability in the organ of the present invention includes the expression vector containing the target transposon gene.
  • the method is not particularly limited as long as it is a method for forcibly expressing the target transporter gene in an organ in which the target transporter gene is not naturally expressed.
  • the organ refers to the brain (blood-brain barrier and brain parenchyma), , Liver, heart, stomach, small intestine, large intestine, lung, lentin, ovary, uterus, placenta, skeletal muscle, thyroid and other organs and tissues.
  • the transporter gene used in the present invention is not particularly limited as long as it encodes a transporter protein having a substance transporting ability.
  • Organic cation transporter genes encoding OCT1, OCT2, OCT2, OCT3, OCTN1, OCTN2, OCTN3, etc .; peptide transport genes encoding PEPT1, PEPT2, etc .; and amino acid transporters.
  • the MDR1, MRP2, etc., and the ABC transposon gene is a transporter that can transport many organic anions with different chemical structures, and also transports various anionic drugs. Very little expression is seen.
  • AT1 can be suitably exemplified.
  • the origin of these transporter genes is not particularly limited, and includes, for example, humans, dogs, puppies, pumas, goats, sheep, monkeys, monkeys, puppies, puppies, rats, mice, etc. be able to.
  • the expression vector containing the transporter gene in the present invention can contain a marker gene in addition to the transporter gene, and the expression vector containing the transporter gene and the marker gene is used.
  • Such a marker gene may be any gene that can easily confirm that various organs have been infected with the above-mentioned transporter forced expression vector for organ infection.
  • j3-galactosidase DNA encoding the enzyme such as ze, peroxidase, alkaline phosphatase, alkaline phosphatase, urease, lipase, ⁇ -darcopenidase, horseradish peroxidase, DNA encoding the Fc region of an antibody, and GFP DNA and encoding a fluorescence protein (green fluorescent protein) or the like, the neomycin resistance gene, Pew port hygromycin resistance gene, hygromycin resistance gene, the diphtheria toxin resistance gene, a 1 and a neo fusion gene of R (J3- geo )) And other specific genes such as DNA encoding a fluorescent protein such as the GFP gene is preferred in view of ease of infection confirmation.
  • GFPs include derivatives having different fluorescence wavelengths such as EGFP (Enhanced GFP), EYFP (Enhanced Yellow Fluorescent Protein), E-P (enhanced CYAN fluorescent protein) (red) and DsRed (red). Also, multiple labels can be performed.
  • EGFP Enhanced GFP
  • EYFP Enhanced Yellow Fluorescent Protein
  • E-P enhanced CYAN fluorescent protein
  • DsRed red
  • multiple labels can be performed.
  • Examples of the expression vector used in the present invention include, for example, an adenovirus vector (Science, 252, 431-434, 1991) used for transient expression in all cells including non-segregated cells (other than blood cells). ), Retrovirus vectors used for long-term expression in dividing cells (Microbiology and Immunology, 158, 1-23, 1992), and non-pathogenic, non-dividing cells can be introduced and used for long-term expression Specific examples include an adeno-associated virus vector (Curr. Top. Microbiol. Immunol., 158, 97-129, 1992) and liposomes, but are not limited thereto.
  • adenovirus vectors which are capable of expressing a gene directly in a living body without establishing a cell line orally and capable of gene expression in a cell line or a living organ with high efficiency, are particularly preferable.
  • the transporter gene to these expression vectors The expression vector can be introduced by a conventional method.
  • an expression vector can be constructed by inserting a transport gene or the like downstream of a suitable motor in these expression vectors.
  • the organ in which the target transporter gene is not originally expressed is not particularly limited.
  • the target transporter gene may be an organic substance localized in renal cells such as AT1.
  • AT1 the anion transporter overnight gene
  • the small intestine, large intestine, rectum and the like can be preferably exemplified. In this case, it can be expected to improve the ability to take up and transport useful substances such as drugs and nutrients in the small intestine, large intestine, and rectum, and the ability to excrete and transport excrement.
  • small intestine, large intestine, and rectum in patients with renal failure Can be expected to replace kidney function.
  • the blood brain A barrier (brain capillary endothelial cell) and the like can be preferably exemplified.
  • efficient delivery of an anticancer drug to a brain tumor and efficient delivery of a dipeptide derivative of L-dopa, a therapeutic agent for Parkinson's disease, to the brain can be expected.
  • an expression vector containing the target transporter gene is forcibly applied to an organ in which the target transporter gene is not originally expressed.
  • a method for imparting controlled transport of useful substances to such organs and a method for controlling the discharge of excreted substances, a method for promoting the ability to transport those substances, and the like can be specifically mentioned.
  • a method for forcibly expressing an expression vector containing the target transporter gene in an organ in which the target transporter gene is not originally expressed can be used.
  • the small intestine or large intestine can be infected by orally administering a ribosome containing an expression vector, or the expression vector can be contained.
  • Four Infection can be achieved by using an expression vector into which the transport gene has been inserted under one control.
  • the origin and type of the artificial organs are not particularly limited as long as the organs can be obtained by using the above-mentioned organ infection transport expression vector.
  • hybrid artificial organs Pulthophysiology (1990), Vol. 9, No. ll, p. 925-927
  • artificial organs induced by in vitro using ES cells and ES cells
  • Example 1 [Subcloning of OAT1 gene into adenovirus vector]
  • the OAT1 gene was used as the desired transpo- tional gene, and the adenovirus vector was used as a method for expressing the AT1 gene in living organs.
  • the adenovirus vector used was developed by He et al. (He, TC et al., A simulated system lor generating recombinant adenoviruses. Proc. Natl. Acad. Sci., 95: 2509-2514 (1998)). It can be prepared by two-stage subcloning (see FIGS. 1 and 2). In this method using an adenovirus vector, homologous recombination is performed in E.
  • the desired AT1 gene is integrated into the shuttle vector, and homologous recombination with the vector containing most of the adenovirus genes is performed using the homologous portion of the shuttle vector with the adenovirus vector.
  • a recombinant adenovirus vector was made.
  • This shuttle vector has a GFP (green fluorescent protein) gene and can express the GFP protein from a promoter separate from the OAT1 gene.
  • GFP green fluorescent protein
  • Example 11 (Preparation of pAd rack-CMV-OATl)
  • the plasmid pSPORTl-OATl in which the rat OAT1 gene was integrated and the shuttle vector pAdTrack-CMV were incubated with two restriction enzymes Kpnl and HindIII, respectively, at 37 ° C for 2 hours (Table 4).
  • the mixture was subjected to electrophoresis, and the inserted portion of AT1 and DNA of the shuttle vector were purified from the agarose gel using the QIAQuick Gel Extraction Kit and extracted into 30 L of Buffer PE.
  • the cells were transferred to a 500 xL LB medium (ryptone 10 g, yeast extract 5 g, NaClOg, 5N NaOH 0.2 M1, Add water to 100 mL) placed in a culture tube, and cultured with shaking for 1 hour. Colonies were planted at a rate of 250 / iL on LB plate medium (containing kanamycin). A colony of pAdTrack-CMV-OAT1 was stirred in 2 mL of LB medium, shake-cultured at 37 ° C, and the plasmid was purified using QIAprep Spin Miniprep Kit and extracted with 50 L of Buffer EB.
  • LB medium ryptone 10 g, yeast extract 5 g, NaClOg, 5N NaOH 0.2 M1
  • Add water 100 mL
  • Colonies were planted at a rate of 250 / iL on LB plate medium (containing kanamycin).
  • a colony of pAdTrack-CMV-OAT1
  • FIG. 4 shows the results for the pAdTrack-CMV-OAT1 construct. Expected fragment sizes are 9.2 and 2.2 kb.
  • PAdTrack-CMV-0AT1 (100 ng / ⁇ ) 2 ⁇ 200 ng
  • Hind III (Takara 12 U / JL / L) 1 23 jLL
  • FIG. 3 shows the production of a recombinant adenovirus plasmid by homologous recombination in E. coli.
  • the OAT1 gene used was integrated into the SalI and NotI sites of pSPORTl, cut at the Kpnl and HindIII sites at both ends of the ⁇ AT1 gene, separated by electrophoresis, and the OAT1 gene was isolated. Purified. Similarly, shuttle shuttles treated with restriction enzymes Kpn I and Hind III
  • the pAd rack-CMV-OAT1 obtained in Example 1-1 by restriction enzyme treatment (Table 7) was converted into a single strand with the restriction enzyme PmeI, and electrophoresed (100 V, 30 min, 1.0% agarose I TAE, Marker: ⁇ ). / Hind III) (see Figure 5).
  • 50 L of phenol: chloroform: isoamyl alcohol 25: 25: 1 was added and vortexed, and the mixture was centrifuged for 2 minutes.
  • the upper layer was taken, 50 ⁇ L of cloper form was added and vortexed, and the mixture was centrifuged for 2 minutes.
  • ethanol precipitation treatment (leaving for 5 minutes, centrifugation for 30 minutes) was performed, and the pellet was washed with 75% ethanol, ethanol was removed at room temperature, and the obtained pellet was dissolved in 12 / L of distilled water.
  • Fig. 6 shows the results. Based on the migration speed, clones in rows 1-8, 10, and 11 were considered potentially effective recombinants.
  • FIG. 7 shows the results of electrophoresis of pAdEasy-1 and pAd-OATl using representative restriction enzymes, and the expected fragment sizes.
  • the closed circular pAdTrack-CMV-OAT1 was treated with the restriction enzyme PmeI, and cut into linear form at only one site. At this stage, it was confirmed by electrophoresis that A signal appeared at 11.4 kb of the full length, confirming that it was linear (see Fig. 5).
  • pAdTrack-Homologous recombination between the PmeI digest of CMV-OAT1 and the adenovirus vector pAdEasy-1 was performed. Homologous recombination was performed by transformation into E. coli (B J5183). B J5183 is susceptible to homologous recombination in E. coli Rec BCD mutants.
  • This Escherichia coli was spread on an LB plate medium containing kanamycin and cultured. Since pAdTrack-CMV has a kanamycin resistance gene, it acquires resistance when it becomes a closed circular plasmid by homologous recombination, and forms a colony even in a kanamycin-containing medium. Since pAdEasy-1 which does not have kanamycin resistance and pAdTrack-CMV-OAT1 which has resistance does not form a colony, a desired pAd-OATl colony was selectively obtained. Next, the plasmid was purified from E. coli using the QIAprep Spin Miniprep Kit.
  • pAd-OAT1 was confirmed by restriction enzyme treatment.
  • pAd-OATl was treated with a restriction enzyme (BamH I, Pac I, Spe I), and the size was confirmed by electrophoresis (see FIG. 7).
  • BamH I, Pac I, Spe I a restriction enzyme
  • the expected number of bands were detected from the restriction enzyme sites.
  • the expected fragment size was almost the expected size when cut with BamHI.
  • the adenovirus vector used in Example 1 is a non-proliferative vector in which a portion required for expression of the adenovirus gene E1 is deleted.
  • E1 is required for adenovirus gene expression, and lacking E1 does not produce viral proteins. Therefore, the virus was grown in human embryonic kidney HEK293 cells, which continuously express the E1 gene, concentrated to a high-titer virus solution, and the virus titer was calculated.
  • this virus solution is used to infect cultured cells or animal tissues other than HEK293 cells, the virus genome is introduced into the nucleus at a high rate due to infectivity, but the E1 gene to be expressed first is deleted. Therefore, the virus expresses only the target gene without being replicated. Therefore, it is necessary to confirm whether cells other than the packaging cells are infectious. Therefore, it was examined whether GFP is expressed in LLC-1 PK1 cells derived from Buyu kidney.
  • Example 2-1 Preparation of pAd-OATl
  • Endotoxin-free pAd-OATl for transfection to HEK293 was prepared in large quantities as follows using the Endo Free Plasmid Maxi kit. First, colonies of Escherichia coli infected with pAd-OATl were collected in 5 mL of LB medium, cultured at ⁇ 37 ° C with shaking, and the grown E. coli was further added to 50 OmL of LB medium in two Sakaguchi flasks. The cells were cultured at 37 ° C with shaking. After cooling, the tube was divided into two 50 OmL centrifuge tubes, and centrifuged at .2500 g for 15 minutes.
  • Example 2-2 Culture of HEK293 cells derived from human fetal kidney
  • HEK293 cells were diluted 2-fold into one 25T flask and cultured until the cells reached 50-70% confluence. Add 500 L of serum-free medium (opti-mem-I) to each of the recombinant adenovirus vector pAd-OATl4 ⁇ g (20 / iL) and 20 L of lipofectamine, and add 15 to 30 at room temperature. Left for a minute. These two solutions were mixed and left at room temperature for 15 to 40 minutes to form a complex of DNA and liposome. The culture solution of HEK293 cells was discarded, and the cells were washed with 4 mL of PBS (1).
  • serum-free medium optical-mem-I
  • Fig. 9 shows the observation result using only the fluorescence
  • Fig. 9B shows the observation result combining both the phase contrast image and the fluorescence.
  • Example 2-5 (Adenovirus infection of HEK293 cells; preparation of secondary virus solution)
  • HEK293 cells were diluted 2-fold into one 25T flask and cultured until the cells reached 50-70% confluence.
  • the primary virus solution was added to HEK293 cells in a volume of 1 mL each in two 25 T flasks to infect the cells with the virus.
  • the state of GFP expression was observed with a fluorescence microscope.
  • the cells were peeled off, collected in a single centrifuge tube, and centrifuged. Thereafter, a secondary virus solution was obtained in the same manner as in Example 2-4.
  • Figure 9C shows the observation results combining both the phase contrast image and the fluorescence.
  • Example 2-6 Infection of HEK293 cells with adenovirus; preparation of tertiary virus solution
  • the virus solution was added to HEK293 in a volume of 1 mL each in four 25 T flasks (one 75 T, one 25 T) to infect the cells with the secondary virus. Two days later, the expression of GFP was observed with a fluorescence microscope. The results are shown in FIG. 9D. Same as Example 2-4 The cells were detached and centrifuged, and the medium was discarded. After adding 12 mL of opti-nenl, a tertiary virus solution was obtained in the same manner as in Example 2-4. FIG. 9D shows both the phase contrast image and the fluorescence, and it can be seen from FIG. 9D that the virus solution could be concentrated until almost 100% infection.
  • Example 2-7 Measurement of tertiary virus titer
  • HEK293 cells were spread on two 6-well dishes. When cells were confluent, the tertiary virus was serially diluted and applied to the cells. Two days later, the medium was replaced and the virus solution was removed. After 2 days and 5 days, the expression of GFP was confirmed by a fluorescence microscope, the ratio of the number of cells emitting fluorescence per unit area was determined, and the tertiary virus titer was determined by the following calculation. The percentage of infected cells when the tertiary virus was infected to HEK293 cells was calculated based on the number of cells that showed GFP fluorescence. 1 (i L / 9.
  • Virus infection of Caco-2 cells was examined as follows. C aco— 2 cells were plated on two 4-well dishes. The dishes used were collagen-coated dishes in advance. When cells are confluent, tertiary will Was serially diluted to MOI (multiplicity of Infection) 75, 15, 4. The density of Konfurue cement a C aco- 2 cells assuming 1 X 10 7 cells / 75 cm 2, and the tertiary virus amount shown in Table 10 were infected over the cells. Two days after infection, the medium was removed to remove the virus. Two and five days later, GFP expression was observed with a fluorescence microscope. FIG. 10 is a diagram showing the appearance of GFP expression.
  • the culture medium in the dish in the dish prepared in Example 3-1 was aspirated with a vacuum pump, and the cell surface was washed three times with a medium lm 1 warmed to 37 ° C without damaging the cells. The added medium was removed as cleanly as possible.
  • As a control for OAT1-expressing Caco-2 cells (AdOATl), normal Caco-2 cells (Normal) were similarly prepared in the dish. Two sets of each cell were prepared, and one group was pre-incubated with ImM's daltaric acid solution at 37 ° C for 30 minutes. The dish was maintained at 37 ° C, 210 L of 0.16 M [ 3 H] PAH solution was added to the wells, incubated, and sampled over time. To solubilize the cells, 5?
  • Figure 12 shows the results of these transmission experiments.
  • the cup method More increased OAT 1 basolateral membrane side (BL) in C aco- 2 cells expressing the lumen side (AP) in the direction [3 H] PAH the permeation amount significantly and the ratio is about 1. 6 times.
  • the speed was 4.5 times faster than the permeation rate of mannitol, a cell gap marker.
  • “Hachicho 1 expression” &. 0-2 The transport direction in cells is not from the luminal side (AP) to the basolateral membrane (BL), but from the basolateral side (BL) to the luminal side (AP). was found to have a transport action.
  • Example 1 In the same manner as in Example 4 [PEPT 1 [3 H] incorporation of a dipeptide in the expression RBEC 1 cells] Example 1, was constructed that incorporates a transponder Isseki PEPT 1 gene originating current adenovirus vector (AdhPEPTl-EYFP) The cultured rat brain capillary endothelial cells (RBEC 1) were infected. Next, the uptake of [ 3 H] GlySar into PEPT1-expressing RBEC1 cells by the dish method was examined according to Example 3-3 described above. The results are shown in FIG. As shown in Figure 14, PEPT1-expressing RBEC1 cells infected with the expression vector (AdhPEPTl-EYFP) showed higher uptake of dipeptide than control uninfected RBEC1 cells. Increased significantly. Industrial applicability

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Abstract

L'invention concerne un vecteur d'expression de transporteur compulsif pour infection organique, pouvant être utilisé pour ajouter une fonction de transport à un organe ne présentant pas cette fonction, de manière à faciliter l'incorporation / le transport d'une substance utile (médicaments, nutriments, etc.) dans l'organe, ou encore, à faciliter l'excrétion / le transport des excréments, etc depuis ledit organe. L'invention concerne également un procédé utilisant le vecteur susmentionné pour faciliter le transport dans plusieurs organes. Un vecteur adénovirus, permettant l'expression directe et transitoire d'un gène in vitro dans diverses espèces animales, y compris l'être humain et la souris par administration orale, est utilisé comme vecteur d'expression pour un gène transporteur (OAT1, PEPT1, etc.), et des cellules modèles intestinales sont transformées. Les résultats de l'étude du transport des médicaments à l'aide des cellules modèles ont permis d'observer que le transport et la diffusion de médicament étaient facilitét.
PCT/JP2001/008894 2000-10-10 2001-10-10 Vecteur d'expression de transporteur compulsif pour infection organique WO2002031139A1 (fr)

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JP2000308880A JP2002112769A (ja) 2000-10-10 2000-10-10 臓器感染用トランスポーター強制発現ベクター

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KR102269946B1 (ko) * 2012-09-11 2021-06-29 코닝 인코포레이티드 약물 트랜스포터 단백질(들) 및/또는 약물대사 효소(들)를 인코딩하는 유전자(들)를 일시적으로 과발현하는 소모성 냉동보존 세포
CN114736871A (zh) * 2022-04-27 2022-07-12 中山康晟生物技术有限公司 一种腺病毒包装wayne293 lvpro动物细胞培养方法

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TAKASHI SEKINE ET AL.: "Expression cloning and characterization of a novel multispecific organic anion transporter", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 272, no. 30, July 1997 (1997-07-01), pages 18526 - 18529, XP002097953 *
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