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WO1996029422A1 - Vecteur d'acide nucleique - Google Patents

Vecteur d'acide nucleique Download PDF

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Publication number
WO1996029422A1
WO1996029422A1 PCT/JP1996/000654 JP9600654W WO9629422A1 WO 1996029422 A1 WO1996029422 A1 WO 1996029422A1 JP 9600654 W JP9600654 W JP 9600654W WO 9629422 A1 WO9629422 A1 WO 9629422A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
toxin
gene
cells
carrier
Prior art date
Application number
PCT/JP1996/000654
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English (en)
Japanese (ja)
Inventor
Takuma KUSUNOKI
Osamu Iijima
Yousuke Suzuki
Original Assignee
Hisamitsu Pharmaceutical Co., 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 Hisamitsu Pharmaceutical Co., Inc. filed Critical Hisamitsu Pharmaceutical Co., Inc.
Priority to AU49546/96A priority Critical patent/AU4954696A/en
Priority to JP08528276A priority patent/JP3095248B2/ja
Publication of WO1996029422A1 publication Critical patent/WO1996029422A1/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

Definitions

  • the present invention relates to a novel carrier for introducing a nucleic acid into a cell. More specifically, the present invention relates to a carrier containing a toxin B chain protein and a nucleic acid-compatible substance for efficiently and safely introducing a nucleic acid into cells.
  • the present invention further relates to a regulator for promoting the introduction of a nucleic acid into cells, comprising the carrier and the nucleic acid.
  • augmentation gene therapy in which the abnormal (pathogenic) gene is left intact and a new (normal) gene is added
  • Replacement Gene Therapy replacement gene therapy
  • viral vector a recombinant virus
  • a recombinant virus was developed as a carrier to efficiently introduce foreign genes into target cells, and the first clinical application of gene therapy became possible.
  • viral vectors There are several types of viral vectors currently being considered for use in gene therapy, as shown below. However, these methods have not been generalized because their production methods are very complicated and at the same time, methods for ensuring their safety have not been established.
  • the virus vector currently receiving the most attention as a virus vector that can be used for gene therapy is a retrovirus vector derived from the mouse leukemia virus (Mo MLV: Moloney Murine Leukemia Virus). It takes advantage of the style. Retroviruses are enveloped RNA viruses that enter the cell by binding their envelope proteins to receptors on the host cell side. After invasion, the single-stranded virus RNA is converted to double-stranded DNA by reverse transcriptase and integrated into the infected cell genome DNA. However, for such integration to occur, the cells must be dividing and proliferating. Therefore, the biggest problem in practice is the inability to transfer genes to non-dividing cells.
  • Hematopoietic stem cells, hepatocytes, and muscle cells, which are the target cells for gene therapy, as well as nerve cells, are usually in the stationary phase, and therefore have low gene transfer efficiency. Cells that have been removed from the body are treated to promote division to increase gene transfer efficiency, but it is considered difficult to transfer genes into these cells in vivo. .
  • adenovirus vectors have recently attracted attention as being able to introduce genes into non-dividing cells.
  • adenovirus vectors do not integrate foreign genes into the genomic DNA of target cells, the genes can take several weeks to several months. The effect of introduction is lost. For this reason, gene transfer must be repeated frequently, which causes problems such as an increase in physical and physical burden on patients and a decrease in gene transfer efficiency due to the production of anti-adenovirus antibodies. .
  • clinical trials have been started to administer the adenovirus vector transbronchially to the lungs for the treatment of cystic fibrosis, but inflammation that appears to be due to the immunogenicity and cytotoxicity of adenovirus particles It is said that a reaction occurred.
  • Herpes virus vector is expected to be a vector that can introduce foreign genes into nerve cells, but it is highly cytotoxic and has a small genome size of the virus itself.
  • the HIV vector was developed as a vector that allows specific transfer of arrested genes to CD4 + T lymphocytes due to the host characteristics of the virus itself (Shimada T., et al., J. Clin. Invest. 88, 1043 (1991)).
  • One of the biggest drawbacks of HIV vectors is the possibility of contamination with wild strains.
  • AAV Addeno-Associated Virus
  • nuclear proteins derived from living organisms which are unlikely to be recognized as foreign substances, are also being studied. Since a nucleoprotein has a property of specifically binding to a nucleic acid and its derivative, there is a possibility that a nuclear protein can be used as a vector for gene transfer by forming an electrostatic complex.
  • histone proteins which are nuclear proteins, as a carrier for plasmid DNA (Yasufumi Kaneda, et al., SCIENCE 243, 375-378 (1899), Mirjam Breeuwer and David S. Goldfarb, Cell 60. 999-1008 (1990), JIAN CHEN, et al., Hunan Gene Therapy 5, 429-435 (1994)).
  • histone proteins which are nuclear proteins, as a carrier for plasmid DNA
  • the present inventors have focused on the effect of toxin protein being introduced into the cytoplasm by binding to a receptor present on the cell surface, and as a result of diligent studies, they have completed the present invention.
  • the carrier of the present invention is characterized by containing a toxin B chain protein and a nucleic acid binding substance.
  • the present invention provides a novel carrier (carrier) for introducing a nucleic acid into a cell.
  • C The present invention further provides a regulator for promoting the introduction of a nucleic acid into a cell, comprising the carrier and the nucleic acid.
  • FIG. 1 shows the incorporation of oligonucleotides into HeLa cells.
  • FIG. 2 shows the incorporation of plasmid DNA into He La cells.
  • FIG. 3 shows the uptake of plasmid DNA containing the galactosidase gene into HeLa cells and gene expression.
  • Figure 4 shows the construction of plasmid PHSX2.
  • FIG. 5 shows the construction of plasmid pUC—Neo2.
  • FIG. 6 shows the construction of plasmid pHS—Ne02.
  • FIG. 7 shows the construction of plasmid pHS—Luci.
  • FIG. 8 shows the expression of the luciferase gene introduced using the carrier of the present invention by thermotherapy. Best mode for carrying out the invention
  • Toxin protein is composed of two types of parts: A chain, which actually exerts toxicity in the cytoplasm, and B chain, which acts to introduce A chain into cells by binding to a receptor present on the cell surface.
  • a chain which actually exerts toxicity in the cytoplasm
  • B chain which acts to introduce A chain into cells by binding to a receptor present on the cell surface.
  • the B chain has little toxicity, and the portion responsible for the toxicity of the toxin molecule can be said to be the A chain.
  • the A chain and the B chain often form a conjugate, and do not show toxicity when used alone. This indicates that the A chain can be introduced into the cell only after the A chain and the B chain form a conjugate.
  • the carrier 1 of the present invention utilizes such a property that the B chain of the toxin protein introduces a high molecular weight substance into the cytoplasm.
  • Toxin B chain protein binds to sugar sialic acid on the cell surface. Therefore, the introduction of the nucleic acid into cells is promoted.
  • Glucose sialic acid is widely present in many cells in a living body, and therefore, according to the carrier of the present invention, a nucleic acid can be introduced into various cells. Therefore, it is possible to introduce a nucleic acid into non-dividing cells such as nerve cells, and even into cells taken out of a living body.
  • the toxin B chain protein has almost no toxicity, and has the advantage that, when used as a carrier together with a nucleic acid fusion substance, the nucleic acid can be safely introduced into cells.
  • the carrier of the present invention containing a toxin B chain protein and a nucleic acid binding substance has an advantage that it can be easily constructed and used as compared with conventional virus vectors and the like.
  • the toxin B chain protein that can be used as the carrier of the present invention is not particularly limited as long as it has a property of introducing a high-molecular-weight substance into the cytoplasm, but bacterial or vegetable proteins are preferable.
  • ricin or a related toxin such as abrin, diphtheria toxin, exotoxin, enterotoxin, cholera toxin, pertussis toxin, tetanus toxin, and B-chain protein of an attenuated strain of botulinum toxin can be used.
  • toxin B chain protein a protein obtained by a known method from a naturally expressed protein can be used.
  • the amino acid sequence and the gene sequence of these proteins are known, and can be produced by gene engineering techniques established based on these sequences.
  • the toxin B chain protein may or may not have a sugar chain bound, if desired.
  • Toxin B chain protein produced by the gene recombination technique is commercially available, and some are readily available.
  • ricin B chain protein is commercially available from Vector, and Used in
  • Toxin B chain protein also introduces high-molecular-weight substances into the cytoplasm, even if one or more amino acid residues in the amino acid sequence of the natural protein are mutated by substitution, deletion, insertion, etc. Any material having the following properties can be used as a component of the carrier of the present invention.
  • the mutation may be naturally occurring or may have been subjected to genetic engineering techniques. One of skill in the art would readily be able to produce such a mutant protein by conventional methods.
  • the carrier of the present invention further contains a nucleic acid binding substance.
  • Nucleic acid binding substances In the state of coexisting with or binding to the toxin B chain protein, those that can bind to nucleic acid introduced into cells can be used. By binding a nucleic acid to such a nucleic acid binding substance, it is possible to efficiently introduce the nucleic acid into the cytoplasm. Examples of such nucleic acid binding substances include, but are not limited to, various cationic lipids, polyamino acid derivatives, histone proteins.
  • cationic lipid examples include lipofectin (Lipoffect)-ribofectase (LipoffectAc): and lipofectamine (Lipoffectamine).
  • polyamino acid derivative examples include poly-L-lysine, poly-D-lysine, poly-L-orditin, poly-D-orutin, poly-L-lysine-L-serine copolymer, poly-D- Lysine-D-Serine Copolymer, Poly-L-Ordinine-L-Serine Copolymer, Poly-D-Ordinine-D-Serine Copolymer, Poly-L-Lysine Polyethylene Glycol (PEG) Block Copolymer, Poly-D- Lysine PEG Block Copolymer, Poly-L-Orditin PEG Block Copolymer, Poly-D-Orditin PEG Block Copolymer, Poly-L-Lysine-L-Serine PEG Block Copolymer, Poly-D-Lysine-D-Serine PEG Block Copolymer , Poly-L-orditin-L-serine PEG block copolymer, poly-D-
  • polyamino acid derivatives can be obtained by converting ⁇ -carbobenzoic acid L-lysine mono-carboxylic acid anhydride and benzylic L-serine ⁇ -carbonic acid anhydride into polyethylene oxide having a single amino group at one terminal (molecular weight 200- 25 0000), and the like.
  • the molecular weight of the polyamino acid moiety in the polyethylene oxide polyamino acid block copolymer can vary from 200 to more than 50,000.
  • the carrier 1 includes, but is not particularly limited to, a toxin II chain protein and a nucleic acid binding substance in a molar ratio of about 1: 1 to 1:10, preferably 1: 1 to 1: 5.
  • the toxin ⁇ chain protein and the nucleic acid binding substance may exist in a free state, or may be bound by an S—S bond or the like.
  • the size and type of nucleic acid that can be introduced into cells by the carrier of the present invention are particularly Not limited.
  • Examples of the type of nucleic acid include linear double-stranded DNA, circular double-stranded DNA, oligonucleotide, and RNA.
  • a structural gene encoding a useful protein can be introduced into cells to express the gene.
  • the gene expression is very high as shown in Example 9 below.
  • the expression of a specific gene can be controlled by introducing antisense.
  • it can be used as a carrier for Ribozyme, Triplex, Abata and others.
  • the nucleic acid also includes a derivative such as a phosphothioate nucleotide in which a phosphonate bond is replaced with a phosphothioate bond.
  • a carrier of about 0.1 to 1000 / zmo 1 is used for nucleic acid 1 / mo 1 without limitation.
  • the present invention further provides a regulator for promoting the introduction of a nucleic acid into a cell, comprising the above-mentioned carrier.
  • the modulator of the present invention can be used for autologous gene therapy (ex vivo gene therapy) in which target cells are first taken out of a patient, the target gene is introduced, and then the cells are returned to the patient.
  • ⁇ ⁇ ⁇ Can also be used for gene therapy in which genes are directly administered to patients (in vivo gene therapy).
  • gene therapy there is a method of adding a new (normal) gene while leaving the abnormal (cause) gene intact (Augmentation Gene Therapy) and a method of replacing the abnormal gene with a normal gene (Replacement Gene Therapy). ), But can be used for both.
  • the administration of the preparation of the present invention is not limited, it is generally performed parenterally, and can be preferably performed by, for example, injection.
  • the amount used in the present invention varies depending on the method of use, purpose of use, and the like.For example, when used as a carrier containing a ricin B-chain protein by injection, for example, a daily dose of about 0.1 lgZk It is preferable to administer 1 g Omg / kg, and more preferably, 1 mg / kg—1 mgZkg.
  • a gene designed to express various marker genes and therapeutic genes by temperature stimulation is introduced, and then a temperature stimulus such as hyperthermia is applied to thereby provide gene stimulation.
  • a temperature stimulus such as hyperthermia
  • Site-specific gene expression in E. coli The carrier containing the toxin B chain protein and the nucleic acid-compatible substance of the present invention forms a complex with a desired nucleic acid, and enables safe and efficient introduction of the nucleic acid into cells.
  • gene expression occurs with extremely high efficiency.
  • One-end methoxy One-end amino group polyethylene oxide (molecular weight 5000: Nippon Oil & Fats Co.) Dissolve 4.0 g in a 15-ml form of black-mouthed form and dilute the solution with ⁇ -carbobenzoxy-L-lysine- ⁇ -Rubonic anhydride and benzyl-L-serine Add to ⁇ -Rubonic anhydride solution. After 26 hours, the reaction mixture was dropped into 330 ml of getyl ether, and the precipitated polymer was collected by filtration, washed with getyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), dried in vacuo, and treated with hydrobromic acid.
  • S-PLPJ N-terminal S-introduced poly-L-lysine PEG block copolymer
  • S-PL SP N-terminal S-introduced poly-L-lysine-L-serine PEG block copolymer
  • S-PLLJ N-terminal S-introduced poly-L-lysine
  • Oligonucleotides were synthesized using a DNA synthesizer (Type 392: manufactured by Abu Biosystems). Poly L-lysine (PLL: Sigma), PLP, PLSP, Lipofectamine (Gibco) was used as a vector. The amount of oligonucleotide uptake was calculated by measuring the radiation dose of cells in each well after 2 hours. Figure 1 shows the results. The carrier synthesized this time shows much higher oligonucleotide uptake properties than lipofectamine, a cationic liposome that has been conventionally used as a carrier, and is effective as a carrier for various oligonucleotides. It became clear.
  • Carriers include poly-L-lysine (PLL: Sigma), PLP, PLSP, lipofectamine (giving Co.) was used.
  • the gene uptake was calculated by measuring the radiation dose of the cells in each cell 2 hours later.
  • Figure 2 shows the results.
  • the carrier synthesized this time showed much higher gene uptake properties than lipofectamine, a cationic liposome used conventionally as a carrier, and was found to be effective as a carrier.
  • HeLa cells were seeded on a 6-well cell culture dish at 4 ⁇ 10 5 cells and cultured in 10% FCS DMEM (manufactured by Gibco) for 18 hours. Thereafter, model plasmid DNA (pSV ⁇ -ga lactosidase: manufactured by Promega) was added to a concentration of 1 gZ / ml, in which case the various carriers were added in the same amount by weight as that of the plasmid DNA. And mixed. 48 hours later, the cells were stained with X-gal (manufactured by Gibco) to evaluate the gene transfer efficiency. The transgene introduction efficiency was compared by setting the staining degree when Lipofectamine (manufactured by Gibco) was 100 to 100. Figure 3 shows the results. The carrier synthesized this time showed much higher gene expression than ribofectamine, which is a cationic ribosome conventionally used as a carrier, and was found to be effective as a carrier.
  • model plasmid DNA pSV
  • Plasmid pBR322 (Pharmacia) was digested with Sa1I (Takara Shuzo) and Hind III (Takara Shuzo), and an approximately 3.74 kb DNA fragment containing the ampicillin-resistant gene and the replication origin was cut. Prepared. Next, plasmid P 1730R (manufactured by Stress Gen) containing the promoter of heat shock protein 70B (HSP70B) was cut with XhoI (manufactured by Takara Shuzo) and HindIII, and the HSP70B promoter region was cut.
  • Plasmid pHSXl was constructed by ligating these two DNA fragments with T4 DNA ligase (Takara Shuzo). This plasmid was digested with Pvu II (Takara Shuzo) and BamHI (Takara Shuzo), and the HSP70B promoter region was cut. An about 2.80 kb fragment containing about 0.47 kb at the 3 'end was prepared. The protruding end of this fragment was blunt-ended with Mung Bean N c 1 ease (Takara Shuzo) and K1 enow Fragment (Takara Shuzo), religated with T4 DNA ligation, and mixed with plasmid. PHSX2 was built.
  • Figure 5 shows all the general methods used to construct the prototype plasmid pUC-Neo2 containing the neomycin resistance gene.
  • Plasmid PUC119 (Takara Shuzo) was cut with Smal (Takara Shuzo) and then cut with BamHI to prepare an approximately 3.15 kb DNA fragment containing the ampicillin resistance gene and the replication origin.
  • the plasmid HXN2 (provided by Nippon Medical School) containing the neomycin resistance gene was cut with XhoI to prepare a DNA fragment of about 1.2 kb containing the neomycin resistance gene.
  • the protruding end of the DNA fragment was blunt-ended with K1newFrament and then cut with BamHI.
  • Plasmid pUC—Neo 2 was constructed with the ATG codon deleted at the I site.
  • the general method used to construct the prototype plasmid pHS-Ne0 containing the heat shock promoter and the neomycin resistance gene is shown in FIG.
  • the plasmid pHSX2 constructed in Example 10 was cleaved with EcoRI (Takara Shuzo) and HindIII to prepare a DNA fragment of about 2.77 kb.
  • the plasmid pUC-Neo2 constructed in Example 11 was digested with EcoRI and HindIII to prepare a DNA fragment of about 1.25 kb. These two DNA fragments were ligated in a T4 DNA case to construct pHS-Ne0.
  • Figure 7 shows the general method used to construct the plasmid pHS-Luci with the luciferase gene ligated downstream of the heat shock promoter.
  • Example 1 The plasmid pHS-Neo constructed in step 2 was digested with HindII and Sa1I to prepare a DNA of about 4.01 kb.
  • a plasmid pGV-P manufactured by Futaba Gene
  • a DNA fragment of about 2.69 kb was digested with HindIII and Sa1I to prepare a DNA fragment of about 2.69 kb.
  • HeLa cells were seeded on a 6-well cell culture dish with 4 ⁇ 10 5 nodules and cultured in 10% FCS DMEM (manufactured by Gibco) for 18 hours at 37 ° C, 5% carbon dioxide concentration. . Then, pHS-Luci was added to a concentration of 1 / gZ, and various carriers were mixed with plasmid DNA in the same amount by weight. After 24 hours, a part of the sample was cultured at 42 ° C for 3 hours, and then returned to the original environment and further cultured for 24 hours. Thereafter, the luciferase expressed in the cells was quantified using Norecyclase 'Atsushi System (Pitka Gene: manufactured by Toyo Ink Co., Ltd.).
  • Figure 8 shows the results. Since the expression of luciferase is higher when cultured at 42 ° C than when cultured at 37 ° C, the regulation of gene expression by hyperthermia can be controlled by introducing a therapeutic gene downstream of the heat shock promoter. It has been shown to be possible.

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Abstract

Nouveau vecteur permettant d'introduire un acide nucléique dans des cellules, régulateur renfermant ce vecteur et l'acide nucléique pour faciliter l'introduction de ce dernier dans les cellules. Ce vecteur comprend une protéine de chaîne B de toxine et une substance capable de se lier à l'acide nucléique.
PCT/JP1996/000654 1995-03-17 1996-03-15 Vecteur d'acide nucleique WO1996029422A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU49546/96A AU4954696A (en) 1995-03-17 1996-03-15 Nucleic acid carrier
JP08528276A JP3095248B2 (ja) 1995-03-17 1996-03-15 核酸運搬体

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP7/59194 1995-03-17
JP5919495 1995-03-17

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WO1996029422A1 true WO1996029422A1 (fr) 1996-09-26

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06303987A (ja) * 1991-11-27 1994-11-01 Boehringer Mannheim Gmbh 細胞への核酸運搬方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06303987A (ja) * 1991-11-27 1994-11-01 Boehringer Mannheim Gmbh 細胞への核酸運搬方法

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JP3095248B2 (ja) 2000-10-03
AU4954696A (en) 1996-10-08

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