WO1996029422A1

WO1996029422A1 - Nucleic acid carrier

Info

Publication number: WO1996029422A1
Application number: PCT/JP1996/000654
Authority: WO
Inventors: Takuma KUSUNOKI; Osamu Iijima; Yousuke Suzuki
Original assignee: Hisamitsu Pharmaceutical Co., Inc.
Priority date: 1995-03-17
Filing date: 1996-03-15
Publication date: 1996-09-26
Also published as: AU4954696A; JP3095248B2

Abstract

A novel carrier for the introduction of a nucleic acid into cells and a regulator containing this carrier and the nucleic acid for promoting the introduction of the nucleic acid into the cells. This carrier comprises a toxin B chain protein and a substance capable of binding to the nucleic acid.

Description

Description Nucleic acid carrier Technical field

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. Background art

With the rapid development of genetic engineering, various molecular biological techniques have been developed. Along with this, 解析 remarkable progress has been made in the analysis of gene information and the elucidation of gene functions, and many attempts have been made to transfer the results obtained therefrom to actual treatment sites. Among them, one of the most remarkable fields is the gene therapy field. The genes responsible for various genetic diseases have been discovered and deciphered, and gene therapy is developing from the stage of basic experiments to the stage of actual clinical application. For example, in the United States, 54 gene therapy protocols were approved by the NIH Recombinant DNA Committee (RAC) by July 1994, and congenital immunodeficiency disorders, familial high cholesterol Clinical trials have already been conducted on more than 20 G patients for hereditary diseases such as hematosis, cystic fibrosis, and various types of cancer (Experimental Medicine Vol. 12—N o 31 9 9 4 pp. 03-page 307). As described above, gene therapy has been attracting attention as a method of treating diseases from the root of the cell level.However, one of the major technical issues in clinical application is how to efficiently and safely target foreign genes to target cells. Or introduce it to There are two types of gene therapy: augmentation gene therapy (Augmentation Gene Therapy), in which the abnormal (pathogenic) gene is left intact and a new (normal) gene is added, and replacement gene therapy (Replacement Gene Therapy), in which the abnormal gene is replaced with the normal gene. Therapy) Although roughly classified, any therapy requires a method for efficiently and safely introducing normal telegenes into target cells.

Attempts to apply physical techniques such as microinjection were made in the early 1980s, but the efficiency of gene transfer was low, and it was not possible to stably guide humans. It did not lead to practical use.

Later, a recombinant virus (viral vector) was developed as a carrier to efficiently introduce foreign genes into target cells, and the first clinical application of gene therapy became possible. 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.

For example, 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. Therefore, gene repair of nerve cells, which is a problem in many congenital metabolic disorders, cannot be performed. 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. .

In addition, adenovirus vectors have recently attracted attention as being able to introduce genes into non-dividing cells. However, since 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. . Currently, 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.

Development is not progressing because it is very large at 150 kb.

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.

The wild type of the AAV (Adeno-Associated Virus) vector was found to be integrated at a specific location on chromosome 19, and was noted as a vector that could target the location of the gene integration. However, recent studies indicate that recombinant AV vectors have lost this property, and that foreign genes are integrated into non-specific locations on the chromosome. Furthermore, AAV vectors have a limitation that the size of foreign genes that can be introduced is limited, and that only genes of 5 kb or less can be packaged in vectors.

Many attempts have been made to perform gene therapy using various artificial gene transfer systems other than virus vectors. For example, gene-lipid complexes with positively charged lipids have been developed as non-viral carriers for gene therapy. However, it has been pointed out that these carriers have high cytotoxicity when used in large amounts (Bioconjugate Chem. 3, 323-327 (1992), Proc. Natl. Acad. Sci. USA 89, 7934-7938 (1992), J. Biol. Chem. 269, 12918-12924 (1994), Tokuhei 6-505980, Tokuhei 6-507158).

In addition, by utilizing the fact that nucleic acids and their derivatives have a negative charge, an electrostatic complex is formed with a synthetic polymer derivative having a positive charge, thereby forming a target cell. Many research reports have attempted to deliver genes into cells or cells

(Bioconjugate Chem. 3, 323-327 (1992), Proc. Natl. Acad. Sci. USA 89, 7 934-7938 (1992), J. Biol. Chem. 269, 12918-12924 (1994), Tokuheihei 6-505980, Tokuhyohei 6-507158). However, it has long been pointed out that a synthetic polymer having a positive charge has high cytotoxicity when used alone (Bio-conjugate Chem. 1, 149-153 (1990)). At the same time, when these synthetic polymer derivatives are injected into a living body, they are recognized as foreign substances, and there is a concern that they may affect the immune system such as anaphylactic shock.

On the other hand, attempts to use 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. There are examples of studies using 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)). In these examples, only methods for incorporating genes into cells have been studied, and are not necessarily aimed at improving gene expression efficiency in many cell types. Therefore, there is no example designed as a vector for efficiently introducing a gene into the cytoplasm in many cell types.

As described above, in gene therapy, a highly efficient and safe method for introducing a foreign gene into cells was required. Disclosure of the invention

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. provide. BRIEF DESCRIPTION OF THE FIGURES

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

The configuration and preferred embodiments of the present invention will be described in detail below.

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. There are many. In general, 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. In addition, it is known that 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. In addition, 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. Furthermore, 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. For example, 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.

As the 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.ト Toxin B chain protein produced by the gene recombination technique is commercially available, and some are readily available.c For example, 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.

Examples of the cationic lipid include lipofectin (Lipoffect)-ribofectase (LipoffectAc): and lipofectamine (Lipoffectamine).

Examples of the polyamino acid derivative 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-orditin-D-serine PEG Click copolymers thereof. These polyamino acid derivatives can be obtained by converting ε-carbobenzoic acid L-lysine mono-carboxylic acid anhydride and benzylic L-serine Ν-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. In addition, the toxin Β 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. For example, by using the carrier of the present invention, a structural gene encoding a useful protein can be introduced into cells to express the gene. When a structural gene is introduced, the gene expression is very high as shown in Example 9 below. In addition, the expression of a specific gene can be controlled by introducing antisense. In addition, it can be used as a carrier for Ribozyme, Triplex, Abata and others. Further, the nucleic acid also includes a derivative such as a phosphothioate nucleotide in which a phosphonate bond is replaced with a phosphothioate bond. In addition, 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.できる Can also be used for gene therapy in which genes are directly administered to patients (in vivo gene therapy). In 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.

Although 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.

Furthermore, by utilizing the present invention, 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. 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. Furthermore, when a gene is introduced by the carrier of the present invention, gene expression occurs with extremely high efficiency. Example

Hereinafter, the present invention will be described more specifically with reference to examples. Note that the present invention is not limited by these examples.

Example 1:

Dissolve 2.0 g of e-carbobenzoxy-L-lysine-N-carboxylic anhydride (manufactured by Sigma) in 30 ml of N, N-dimethylformamide (DMF) (manufactured by Wako Pure Chemical) Add 15 ml of black mouth form (manufactured by Wako Pure Chemical Industries). One-end methoxide One-end amino group polyethylene oxide (molecular weight 5000, manufactured by Nippon Oil & Fats) 4.0 g is dissolved in 15 ml of a form of black mouth, and the solution is added to the solution. Add to the anhydride solution. After 26 hours, the reaction mixture was dropped into 330 ml of getyl ether, and the precipitated polymer was recovered by filtration, washed with getyl ether (manufactured by Wako Pure Chemical Industries, Ltd.), dried in vacuo, and treated with a hydrogen bromide acetic acid solution. (Wako Pure Chemical Industries, Ltd.) to obtain a poly-L-lysine PEG block copolymer (hereinafter referred to as “PLP”). Yield 5.0 g.

Example 2:

1.0 g of ε-carbobenzoxy-L-lysine-N-carboxylic acid anhydride (manufactured by Sigma) and 1.0 g of Benziru L-serine-N-carboxylic acid anhydride (manufactured by Sigma) were Dissolve in 30 ml of DMF (manufactured by Wako Pure Chemical Industries), and add 15 ml of black mouth form (manufactured by Wako Pure Chemical Industries). 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 ε-carbobenzoxy-L-lysine-力 -Rubonic anhydride and benzyl-L-serine Add to 力 -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. Dissolution Deprotection with a liquid (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain poly-L-lysine-L-serine PEG block copolymer (hereinafter referred to as “PLSP”). Yield 4.6 g.

Example 3:

30 mg of undeprotected poly-L-lysine PEG block copolymer obtained in Example 1, N-succimidyl 3- (2-pyridyldithio) probionate (hereinafter SPDP) (manufactured by Pierce) 7 mg, triethanolamine (Wako Pure Chemical Industries) 52 21 is dissolved in 5 ml of DMFO and stirred at room temperature for 16 hours. The reaction product is dropped into 1 Oml of distilled water, and the precipitated polymer is recovered by filtration and freeze-dried. Thereafter, deprotection is performed with a hydrobromic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain an N-terminal S-introduced poly-L-lysine PEG block copolymer (hereinafter referred to as “S-PLPJ”).

Example 4:

Dissolve 30 mg of undeprotected poly-L-lysine-L-serine PEG block copolymer 30 mg, SPDP 7 mg, and triethanolamine (manufactured by Wako Pure Chemical Industries) 51 obtained in Example 1 in 5 ml of DMFO. Stir at room temperature for 16 hours. The reaction product is dropped into 10 ml of distilled water, and the precipitated polymer is recovered by filtration and lyophilized. Thereafter, deprotection is performed with a hydrogen bromide acetic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain an N-terminal S-introduced poly-L-lysine-L-serine PEG block copolymer (hereinafter, referred to as “S-PL SP”). Yield 16mg.

Example 5:

Dissolve 30 mg of poly ε-potency L-lysine L-lysine (manufactured by Sigma), 7 mg of SPDP, and triethanolamine (manufactured by Wako Pure Chemical Industries) 51 in 0.5 ml of DMF and stir at room temperature for 16 hours. The reaction product is dropped into 1 Oml of distilled water, and the precipitated polymer is recovered by filtration and freeze-dried. After that, deprotection is performed with hydrogen bromide acetic acid solution (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain N-terminal S-introduced poly-L-lysine (hereinafter referred to as “S-PLLJ”) Yield 18 mg.

Example 6:

1 Omg of the N-terminal S-introduced polyamino acid and 1 Omg of lysine B chain (manufactured by Vector) obtained in Examples 3, 4 and 5 were dissolved in 2 ml of a buffer solution, and dissolved at 4 ° C. Stir for 6 hours. The product was gel-purified using Sephadex G-75 (Pharmacia). The desired fraction was desalted and concentrated by ultrafiltration to obtain the desired product. An aqueous solution of ToxB-PLP (14 mg / m1), ToxB-PLSP (15 mg / m1), and ToxB-PLL (16 mg / m1) was obtained. Example 7:

24 © Le dish cell culture, 10 ⁵ Z Uyuru with H e L a cell (Dainippon Pharmaceutical) seeding cultured for 18 h in DMEM (Gibco) containing 10% FCS (Gibco) and Was done. Then, the model oligonucleotide (2 Omer) labeled with 32 P with the DNA 5 'end labeled kit (Takara) was diluted with the unlabeled model oligonucleotide to a final concentration of 10 μmo 1/1. added. At that time, the various carriers for gene transfer synthesized this time were mixed with the oligonucleotide at the same ratio by weight. 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.

Example 8:

24 Ueru dish cell culture, a 18-hour culture in DMEM (Gibco) containing 10 ⁵ Z Ueru with H e L a cell (Dainippon Pharmaceutical) were seeded 10% FCS (Gibco) went. Thereafter, nick translation labeled kit in part by (Amersham) ³² P-labeled model plus Mi de DNA: Non to a concentration of 0. l mo 1 1 at a final concentration of (PGV- C manufactured by Toyo Ink) Diluted with labeled plasmid DNA was added. At that time, various gene transfer carriers were mixed with the plasmid DNA at the same ratio by weight. 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.

Example 9:

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.

Example 10: Construction of recombinant plasmid pHSX2

The general method used to construct the prototype plasmid pHSX2, including the heat shock promoter (he atsock promoter), is shown in FIG. 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. Approximately 2.57 kb DNA fragment was prepared. 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.

Example 11: Construction of recombinant plasmid pUC—Ne02

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. Next, 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. These two DNA fragments were ligated with T4 DNA ligation to construct plasmid pUC-Neo. This plasmid was cut with SphI (Takara Shuzo), the protruding end was blunted with Mung Bean Nuclease and Klenow Fragment, religated with T4 DNA ligation, and spliced. Plasmid pUC—Neo 2 was constructed with the ATG codon deleted at the I site.

Example 12: Construction of recombinant plasmid pHS-Neo

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. Next, 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.

Example 13: Construction of recombinant plasmid pHS—Luci

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. Next, a plasmid pGV-P (manufactured by Futaba Gene) containing the luciferase gene was digested with HindIII and Sa1I to prepare a DNA fragment of about 2.69 kb. These two DNA fragments were ligated with T4 DN ligase to construct plasmid pHS-Luci.

Example 14:

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.

Claims

The scope of the claims

1. Carriers for promoting the transfer of nucleic acids, including toxin B chain proteins and nucleic acid binding substances, into cells.

2. The carrier according to claim 1, wherein the toxin B chain protein is a bacterial or plant toxin B chain protein.

3. One or more amino acid residues in the amino acid sequence of naturally occurring toxin B chain protein have been substituted, deleted or inserted in the toxin B chain protein. 3. The carrier according to claim 1, which has an activity of introducing a nucleic acid binding substance and a nucleic acid into cells.

4. The carrier according to any one of claims 1 to 3, wherein the toxin B chain protein is a ricin B chain protein.

5. The carrier according to any one of claims 14 to 14, wherein the nucleic acid binding substance is selected from the group consisting of a positively charged lipid, a polyamino acid or a derivative thereof, and a nucleoprotein or a derivative thereof.

6. The carrier according to claim 5, wherein the nucleic acid binding substance is a poly-L-lysine PEG block copolymer or a poly-L-lysine-L-serine PEG block copolymer.

7. The carrier according to claim 16, wherein the toxin B chain protein and the nucleic acid binding substance are bound by an SS bond.

8. A regulator for promoting the introduction of a nucleic acid into a cell, comprising the carrier according to any one of claims 17 to 17 and a nucleic acid.