WO1997031656A1 - Composition pour introduire de maniere stable des genes dans des cellules hepatiques et procede pour introduire des genes a l'aide de cette composition - Google Patents

Composition pour introduire de maniere stable des genes dans des cellules hepatiques et procede pour introduire des genes a l'aide de cette composition Download PDF

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Publication number
WO1997031656A1
WO1997031656A1 PCT/JP1997/000612 JP9700612W WO9731656A1 WO 1997031656 A1 WO1997031656 A1 WO 1997031656A1 JP 9700612 W JP9700612 W JP 9700612W WO 9731656 A1 WO9731656 A1 WO 9731656A1
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liver
gene
composition
stably
hepatocytes
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PCT/JP1997/000612
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English (en)
Japanese (ja)
Inventor
Yasufumi Kaneda
Toshio Ogiwara
Mamoru Hasegawa
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Dnavec Research Inc.
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Publication of WO1997031656A1 publication Critical patent/WO1997031656A1/fr

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    • 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
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention relates to a technique for gene transfer into cells or a technique for gene therapy.
  • the present invention particularly relates to a composition for gene transfer containing a vector used for gene transfer or gene therapy to the liver, and a gene transfer method using the composition.
  • the calcium phosphate method, the DEAE dextran method, the cationic ribosome method, and the electroporation method have been used to introduce foreign genes into various cells in vitro.
  • In vivo gene transfer methods include viral (retroviral, adenoviral, etc.) transfer, ribosome transfer, and direct injection.
  • viral retroviral, adenoviral, etc.
  • ribosome transfer ribosome transfer
  • direct injection retroviral, etc.
  • the retrovirus method can incorporate genes into the genome with high efficiency.However, 1) the insertion size is limited, 2) the efficiency is low in terminally differentiated cells, 3) wild-type virus infection and It has drawbacks such as the possibility of activation of oncogenes. Also adeno The virus method is widely used because of its efficiency in non-replicating differentiated cells.
  • HVJ Sendai virus
  • the HVJ method is characterized by forming a complex of exogenous DNA with ribosomes, nucleoproteins, and viral coat proteins that mediate fusion to the cell membrane.
  • This method has the advantages of 1) efficient, 2) safe, 3) easy to operate, 4) short incubation time, 5) unlimited size of inserted DNA, It has been used successfully for in vivo gene transfer into many tissues, including kidney, kidney, and vascular wall (Kaneda Y, Iwai K, Uchida TJ Biol Chem 1989; 264: 12126-12129, Kato K et al.
  • HVJ-liposome method it is also possible to use a nuclear protein called High Mobility Group 1 (HMG-1).
  • HMG-1 High Mobility Group 1
  • HMG-1 can translocate the fusion protein to the nucleus immediately after entry into the cytoplasm and contribute to maintaining the stability of the introduced foreign gene in cells (Kane da Y, Iwaki K, Uchida T Science 1989; 243: 375-378).
  • this method also has the disadvantage that the expression of the foreign gene is temporary.
  • human insulin ⁇ In the transfection of the gene and the human renin gene into adult liver,
  • the liver Since many proteins synthesized in the liver are secreted into the circulatory system, and the liver is a place where many metabolic diseases occur, the liver is a desirable target site for gene therapy for diseases. It is an important issue to carry out stable gene transfer to DNA. Disclosure of the invention
  • An object of the present invention is to provide a method for stably introducing a gene into a living body liver, and a composition applied to the method.
  • the present inventors thought that by introducing a gene into the liver of a newborn rat, the gene could be stably introduced.
  • the reason for this is that the neonatal liver is an actively replicating organ, so it is expected that foreign genes can be introduced into the replicating host genome.
  • the present inventors inject a composition containing a compound having an affinity for both a nucleic acid and a hepatocyte and a gene transfer vector into the liver of a live non-human lactating neonatal animal. It was confirmed through experiments that the gene could be efficiently and stably introduced into hepatocytes by the experiment, and the present invention was completed. More specifically, the present inventors use a composition containing a compound having affinity for both nucleic acids and hepatocytes and a gene transfer vector in the liver of a lactating neonatal animal. When the gene was introduced, it was found that continuous gene expression was achieved, and the present invention was completed.
  • the present invention includes the following.
  • composition according to (1) which comprises an inactivated Sendai virus
  • composition according to (1) which comprises a nuclear transport protein
  • composition or a lysate thereof according to any of (1) to (4) is applied to the liver of a lactating neonatal animal other than a human, and the gene is stably transferred to hepatocytes of the living organism. How to introduce.
  • composition of the present invention can be used as it is or suspended in a pharmaceutically acceptable liquid.
  • a method for applying the composition of the present invention or a lysate thereof to the liver of a lactating neonatal animal include a method of directly injecting into the liver through an abdominal wall using a syringe or the like, or a method of injecting into the portal vein.
  • the “compound having an affinity for both nucleic acids and hepatocytes” of the present invention includes liposomes and complexes that are artificially prepared membrane structures composed of phospholipids, and ribosomes containing HVJ-liposomes. (See “Ribosomal Experiments in Life Science Manual,” Springer's Fairlark Tokyo (1992), p. 282-288, etc.).
  • vector for introducing a gene basically, all vectors for introducing a gene are used.
  • a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector for example, a vector for gene introduction is used.
  • composition of the present invention can be applied to all mammals, for example, humans, mice, rabbits, rabbits, and monkeys.
  • compositions of the present invention may contain inactivated Sendai virus (HVJ).
  • HVJ Sendai virus
  • the nuclear transport protein refers to a protein that binds to DNA and facilitates nuclear transport of DNA. More specifically, High Mobility Group-1 (HMG-1), SV40 —Di-T antigen, poliovirus large ⁇ antigen, nucleoblastin and the like.
  • HMG-1 High Mobility Group-1
  • SV40 —Di-T antigen
  • poliovirus large ⁇ antigen poliovirus large ⁇ antigen
  • nucleoblastin and the like.
  • the composition of the present invention may contain these nuclear transport proteins.
  • the gene therapy according to the present invention can be applied to many metabolic disorders targeting hepatocytes, particularly amino acid metabolism disorders and organic acid metabolism disorders (see “Clinical Genetics [VI] Gene Therapy). And prevention ”, see Diagnosis and Therapeutics (1995) etc.). Specific examples of application include the following.
  • the most frequent form of congenital hyperammonemia is orditintranscarbamylase deficiency.
  • the ornithine transgenic rubamylase gene is located on the X chromosome, and many mutations have been reported so far.
  • Current treatments include plasmapheresis, physical removal of ammonia by peritoneal dialysis, drug treatment (sodium benzoate, L-carnitine, citrulline, etc.), and nutritional treatment (protein restriction and arginine supplementation)
  • current treatments have not been effective enough.
  • liver transplantation has been performed and has been effective. Therefore, a therapeutic method in which the orditin transcarbamylase gene is introduced into the liver by this method can be considered.
  • citrullinemia one of the urea cycle disorders, is similar to orditin transpotency rubamylase deficiency, and as a result, a decrease in enzyme activity in the liver is central to the pathology. Is thought to rise.
  • current treatment is not effective enough for severely ill dysfunction of rubamyl phosphate synthase.
  • This disease is considered to be a disease for which gene therapy can be performed with the same strategy as that of ornithine trans-active rubamylase deficiency.
  • Argininosuccinic aciduria Current treatments have not been effective enough.
  • the therapeutic effect of this disease has been confirmed by liver transplantation, and gene therapy targeting the liver is considered to be effective.
  • arginine basically, enzyme deficiency in the liver plays an important role in the onset of the disease, so if gene transfer into the liver is achieved, the therapeutic effect is expected to increase.
  • This disease has a high prevalence, and dietary therapy and pharmacotherapy have been used for heterozygous patients, but this therapy is ineffective for homozygous patients. Cis and liver transplants have been performed. However, none of the treatments has been effective enough. Treatment of this disease requires and is sufficient to restore LDL receptor function in the liver, confirmed by the fact that liver transplantation in homozygous patients corrected hypercholesterolemia Was done. In other words, the introduction and expression of the LDL receptor gene into the liver is a central issue in gene therapy for familial hypercholesterolemia, and the method can be used effectively.
  • Hemophilia B is a sex-linked recessive disorder caused by an inborn deficiency of coagulation factor IX made in the liver.
  • the only treatment is transfusion of clotting factor IX products made by blood donation or genetic engineering techniques.
  • an effective therapeutic method is expected to be established.
  • FIG. 1 is a diagram showing the constructed insulin vector.
  • FIG. 2 is a diagram showing RT-PCR analysis of human insulin mRNA expressed after injecting human insulin vector and a control vector into the liver.
  • FIG. 4 is a view showing the results of measuring the time-dependent change of middle insulin by radioimnoassy.
  • lipid was added to plasmid DNA or fluorescein isothiocyanate (FITC) -labeled oligonucleotide (0DN) in 200 1 BSS solution (137 mM NaCl, 5.4 # 1 KC1, 10 mM Tris- Hydrochloric acid / pH 7.6). Liposomes were prepared by shaking and sonication.
  • FITC fluorescein isothiocyanate
  • HVJ strain Z
  • the ribosome solution described above (10 mg ribosome / 0.5 ml solution) was mixed with 10,000 hemagglutination units of HVJ and diluted with BSS solution to a final volume of 4 ml. The mixture was kept at 4 ° C for 5 minutes and then gently shaken at 37 ° C for 30 minutes. Unreacted HVJ and liposomes were removed from HVJ-ribosomes by sucrose density gradient centrifugation.
  • Oligonucleotides (0DN) were prepared using a DM synthesizer (Applied Biosystems), purified by high performance liquid chromatography (HPLC), washed with 70% ethanol, dried, and treated with TE buffer (10 mM Tris). -Hydrochloric acid; pH 7.5, 1 mM ethylenediaminetetraacetic acid; pH 8.0). The 5 'end of 0DN was labeled with FITC, and the concentration of 0DN was determined with a spectrophotometer. This knowledge 0DN and 100 l of HVJ-ribosome prepared by the method of Example 1 were mixed and injected into the liver of Wistar rat 2 days after birth through the abdominal cavity wall using a 27-gauge injection needle.
  • FITC-labeled 0DN to be injected into the liver is the final concentration (0D relative to the total ribosome solution). N concentration).
  • HVJ-ribosome containing no FITC-labeled 0DN prepared by the method of Example 1 was injected. All the rats were sacrificed 2 hours later, and the tissues excised according to a conventional method were immersed in a 4% paraformaldehyde solution and fixed. The sections were treated with erythrochrome black T and observed with a fluorescence microscope.
  • fluorescence was observed in at least 50 to 60% of the hepatocytes 2 hours after the introduction. Fluorescence was not observed in tissues other than the liver, ie, kidney, heart, lung, and brain. No fluorescence was observed in rats injected or not injected with HVJ-liposomes without FITC-labeled 0DN.
  • the human insulin vector was constructed as follows. Human fetal fibroblast DNA was partially cut with the restriction enzyme EcoRI and inserted into lambda Charon 4A. Ri by to Haipuridzudo formed by 3 2 P- labeled probe of 5 'and 3' terminal region of the human insulin gene was screened files one Jipuraku containing human insulin gene. Lambda Charon 4A containing the human insulin gene was isolated, ligated with the BglII / BglI fragment (1.7 kb) using the BamHI linker, and then subcloned into plasmid PUC118.
  • the Nco I / Bam HI fragment of the human insulin gene (1.6 kb) was introduced into the Nco I / Bam HI site of the plasmid DNA pAct-c-myb containing the 5 'promoter region of the chicken actin gene and the first intron region. ) was inserted (Fig. 1).
  • Niwato Li - Akuchinpuromo Yuichi is, r anei-Ishii C Ishi i S., Nucleic Acids Res earch 1989;? 17; used as described in 1521-1536J
  • human insulin genomic DM is "Bell GI et al., Nature, 1980; 284; 26-32. " PAct without the human insulin gene was used as a control vector.
  • HVJ-ribosomes containing human insulin vector or control vector prepared .
  • High Mobility Group a non-histone protein, to improve transfection efficiency-1
  • HMG-1 was simultaneously encapsulated in HVJ-ribosome.
  • 1001 HVJ-ribosome prepared by the method of Example 2 was injected into the liver of a neonatal rat through the abdominal wall, and the human insulin mRNA expression in the liver was examined thereafter. Rats were sacrificed 2, 4, 6, and 8 weeks after injection, and mRNA was extracted from the liver as follows. Immediately at the time point described above, the liver was removed, frozen in liquid nitrogen, and removed until RM extraction. Saved in C. All MA was extracted from all livers by the guanidine thiosinate cesium monochloride method.
  • 0M primer complementary to the human insulin gene (5 'primer: 5, -TCA-CAC-CTG-GTC-GAA-GCT-CTC-TAC-CTA-GTG-3' / SEQ ID NO: 1, 3 'primer : 5, -GTT-TT T-TGC- AGT- AGT-TCT-CCA-CCT-TCG-TAG-AGG-GA-3 '/ SEQ ID NO: 2) 30 cycles for 0.5 mg of extracted RNA was performed.
  • a primer against rat-actin was used as a control.
  • the PCR product was subjected to electrophoresis on a 2% agarose gel and stained with B. bromide.
  • Radioimnoattsui was performed as follows. Rats were sacrificed 2 weeks, 4 weeks, 6 weeks, and 8 weeks after HVJ-liposome injection as described above, and blood was collected. It was measured Insurin in blood using 1 2 ⁇ - Insurin'ni antibody RIA kit (Shionogi).
  • Example 3 Separately from Example 3, at 3 weeks, 5 weeks, and 7 weeks after HVJ-ribosome injection, blood for radioimmunoassay was used for 4 rats (two of which received the human insulin vector). Rats, and the other two rats were transfected with the control vector). All four rats were sacrificed 7 weeks after HVJ-ribosome injection and tissues were immediately frozen in liquid nitrogen and stored at -70 ° C until analysis. Genomic DNA derived from nuclei of rat hepatocytes was extracted using a DM extraction kit (QIAGEN).
  • the extracted genomic DNA l ⁇ g was analyzed by performing a PCR amplification reaction of 30 cycles with a solution 51 cut with restriction enzymes EcoRI and HindII at 37 ° C in a total volume of 10 1 buffer solution.
  • the conditions for the PCR amplification reaction were the same as in the RT-PCR method described in Example 3.
  • the PCR product was subjected to electrophoresis on a 2% agarose gel, and stained with bromide reagent. A similar experiment was performed for a control experiment using a primer for rat actin.
  • the human insulin gene is present in the nucleus of rat liver cells after injection. Furthermore, PCR analysis of the human insulin vector in the host genome was performed. The presence of the human insulin gene was confirmed in the DNA of rat liver cells transfected with the human insulin vector, but no band was observed in the rat transfected with the control vector. On the other hand, in a control experiment using a primer for rat --actin, the / 5-actin band was easily detected using any of the vectors.
  • a composition comprising a compound having an affinity for both nucleic acid and hepatocytes and a gene transfer vector is applied to the liver of a lactating newborn animal, It has been found that the gene is stably introduced into hepatocytes.
  • the liver is a site for the synthesis of many proteins, and proteins that are stably secreted by the stable introduction of genes into hepatocytes circulate throughout the body through the bloodstream, making them more efficient against diseases. A therapeutic effect is expected.
  • the liver is also a place where many metabolic diseases occur, and it is expected that gene therapy for various diseases will be established by stably introducing and expressing genes into hepatocytes. Further, it is expected that the technique will be applicable to many other diseases.
  • Sequence type nucleic acid
  • Sequence type nucleic acid

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Abstract

On a trouvé que des gènes peuvent être introduits, de manière stable, dans des cellules hépatiques en appliquant une composition contenant un composé ayant une affinité aussi bien pour les acides nucléiques que les cellules hépatiques, et un vecteur pour l'introduction de gènes dans le foie d'un animal nouveau né en lactation. Comme le foie constitue le site de synthèse de différentes protéines et que les protéines sécrétées de manière très stable, grâce à l'introduction stable de gènes dans les cellules hépatiques circulent dans tout le corps par le circuit sanguin, on peut s'attendre à ce que cette méthode ait un effet thérapeutique marqué sur des maladies. Comme le foie est également le siège de différentes maladies métaboliques, l'introduction stable de gènes dans les cellules hépatiques et leur expression constituent des méthodes de thérapie génique pour différentes maladies.
PCT/JP1997/000612 1996-03-01 1997-02-28 Composition pour introduire de maniere stable des genes dans des cellules hepatiques et procede pour introduire des genes a l'aide de cette composition WO1997031656A1 (fr)

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JP8/45250 1996-03-01
JP4525096 1996-03-01

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000056368A1 (fr) * 1999-03-23 2000-09-28 Takara Shuzo Co., Ltd. Therapeutique genique
WO2002031138A1 (fr) * 2000-10-06 2002-04-18 Dnavec Research Inc. Vecteur de paramyxovirus permettant de transferer un gene etranger dans le muscle squelettique

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM., Vol. 266, No. 6, 1991, KATO K. et al., "Expression of Hepatitis B Virus Surface Antigen in Adult Rat Liver", p. 3361-3364. *
SCIENCE, Vol. 243, 1989, KANEDA Y. et al., "Increased Expression of DNA Cointroduced with Nuclear Protein in Adult Rat Liver", p. 375-378. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000056368A1 (fr) * 1999-03-23 2000-09-28 Takara Shuzo Co., Ltd. Therapeutique genique
WO2002031138A1 (fr) * 2000-10-06 2002-04-18 Dnavec Research Inc. Vecteur de paramyxovirus permettant de transferer un gene etranger dans le muscle squelettique

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