JP2009081997A - Method for utilizing botulinus toxin component ha as carrier for intracellular introduction of nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a safe and simple system for introducing a nucleic acid into a living body with a non-viral vector, usable as a nucleic acid medicine and having good introduction efficiency and high convenience. <P>SOLUTION: There are provided a method for using a hemagglutinin derived from a bacterium of the genus Clostridium as a carrier for intracellular introduction of the nucleic acid, and a medicinal composition composed of the protein and an optional nucleic acid to be introduced into a cell. The hemagglutinin comprises preferably an amino acid sequence represented by Asn-X-Asn-X-Ile-Lys-Ile-Ile-Glu (sequence number 1) or Asp-X-X-Gly-Gly-Gln-Thr-X-X-Gly-Thr (sequence number 2) (wherein, X is an optional protein constituent amino acid). Gene expression cassettes in which genes encoding substances selected from among the group consisting of protective antigens in microbial infections, physiologically active substances, enzyme inhibitors, receptor inhibitors, carcinogenesis inhibitors, apoptosis promoters, apoptosis inhibitors, cell regeneration promoters, immunoreaction promoters, and immunoreaction inhibitors are incorporated, and functional genes of ribozymes and anti-sense genes are cited as the nucleic acid to be introduced into the cell. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to the use of hemagglutinating protein produced by Clostridium bacteria, especially Clostridium botulinum. More specifically, the present invention relates to a method of using a hemagglutination active protein, which is one of the components of botulinum toxin, as a carrier for introducing a nucleic acid into a cell, and to introduce the hemagglutination activity protein and the cell into the cell. The present invention relates to a pharmaceutical composition comprising any nucleic acid.

  Attempts to use nucleic acids for pharmaceuticals have been ongoing for about 40 years, but the nucleic acid pharmaceuticals for human use on the market so far are two products, antisense and aptamers. The reason why nucleic acid drugs are difficult to commercialize is that no method for introducing a safe and highly efficient nucleic acid into a cell that can withstand clinical trials has been found. As a method for introducing a nucleic acid into a cell, there are roughly two methods, a method using a virus as a vector and a method using a non-viral vector.

  Studies using viruses as vectors have been conducted with adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, Sendai virus, and the like. When a viral vector is used, it is said that high introduction efficiency into a cell is obtained and long-term expression of an administered nucleic acid is possible. On the other hand, 1) antibodies against viral vectors are produced and cannot be administered continuously, 2) there is a risk of side effects due to the immune reaction, and 3) mutations occur when incorporated into host genes. There is a risk of causing cancer, 4) toxicity to cells, 5) risk of mutation of vector virus itself, 6) difficult to control expression level, and safety is required. There are many challenges for pharmaceuticals that must be solved.

  Methods using non-viral vectors include methods using liposomes, methods using cationic proteins and peptides, methods using saccharides, methods using virus shells, methods using nanoparticles, and binding to gold particles. A number of methods have been reported, including a method of driving them with a gene gun. Non-viral vectors are superior in terms of safety. However, when intramuscular administration, intravenous administration, nasal administration, oral administration, transdermal administration, etc. are performed, 1) efficiency of introduction into cells is improved. There are problems such as low, 2) transient expression, and 3) high production cost.

  In addition to these, there is a report on a method for introducing a nucleic acid into a cell by using a nucleic acid carrier in which a toxin B chain protein and a nucleic acid are chemically bound with a binder (see, for example, Patent Document 1 and Patent Document 2). . This was done by paying attention to the property that a toxin protein produced by bacteria has an action of binding to a cell surface receptor and introducing the toxin protein into the cell. However, this report only shows that the nucleic acid was introduced into the cells in an in vitro experimental system using cultured cells, and does not disclose the results when administered to an individual animal. The effectiveness has not been revealed.

  In addition, since a peptide having a membrane fusion activity derived from the hemagglutination activity protein of influenza virus is positively charged and binds to a gene, the peptide, a plasmid containing the gene of interest, and polylysine 3 to which transferrin is chemically bound are combined. A method has been reported in which a person is electrostatically bound and introduced into a cell (for example, see Non-Patent Document 1). This electrostatically bound complex binds to the cell's transferrin receptor, is taken into the cell, the peptide causes membrane fusion at acidic pH in the cytoplasm, destroys the membrane, and releases the plasmid into the cytoplasm. Can be made. The peptide is used to increase the introduction efficiency, but the effect of this method when administered to an animal individual is not known, and its effectiveness has not been clarified. Many non-viral vectors have a cell introduction effect in in vitro experiments, but most of them are practically insufficient or not recognized in vivo.

Furthermore, as a method with high gene introduction efficiency, an electroporation method has been developed in which a plasmid is directly incorporated by opening a hole in a cell with an electric pulse. However, this method has been pointed out with problems such as 1) necrosis of administered local cells with animal cells, and 2) low introduction efficiency in large animals such as rabbits and monkeys. Nucleic acid transport within an animal is subject to inhibition by binding to blood components and body fluid components and degradation by enzymes, and if it does not bind to a receptor, it will not be taken up into cells, and its transfer to the nucleus is a high hurdle. For administration to animals, the know-how of nucleic acid delivery to cultured cells is not valid. Therefore, development of a nucleic acid introduction system into a living body that can be used as a nucleic acid pharmaceutical, is safe, has good introduction efficiency, is simple and convenient is desired.
Patent No. 3095248 International Publication WO96 / 29422 Proc. Natl. Acad. Sci. 89, 7934-7938, 1992

  When a gene is used together with a non-viral vector, it can be prepared so as not to be incorporated into the host gene without worrying about self-replication, and thus the gene may have a dangerous impact on humans and the environment. It can be made as small as possible. Therefore, a non-toxic vector carrying a gene can be used as a safe nucleic acid drug as long as it can be administered to a living body and can be expressed even if its medicinal effect is recognized. However, there are few reports on non-viral vectors that can express a desired biological activity in animals.

  Therefore, the present inventors have found that a hemagglutinating protein (Botulinum Toxin Hemagglutinin; hereinafter abbreviated as BoHA) produced by Clostridium botulinum passes through the small intestine, nasal cavity, and mucosal cells of the eye to the neurotoxin that is a toxic component of botulinum toxin. Focusing on the fact that it plays a major role when it is delivered into the living body, a method for delivering the drug into the living body using this action has been intensively studied. As a result, it was found that BoHA associates with nucleic acid and that this BoHA / nucleic acid aggregate is effectively introduced into the cell without damaging cultured cells or living cells. Furthermore, the present inventors have also found that there is a specific amino acid sequence capable of associating with a nucleic acid in BoHA, and have completed the present invention.

  An object of the present invention is to provide a method of using BoHA as a carrier for introducing a nucleic acid into cultured cells or living cells. A further object of the present invention is to provide a pharmaceutical composition comprising BoHA and an arbitrary nucleic acid to be introduced into cells.

That is, the present invention is as follows.
1. A method of using a hemagglutination activity protein derived from Clostridium spp. As a carrier for introducing nucleic acid into cells.
2. 2. The method according to 1 above, wherein the genus Clostridium is Clostridium botulinum.
3. 2. The method according to 2 above, wherein the Clostridium botulinum is a serotype selected from the group consisting of A type, B type, C type, D type and G type.
4). 4. The method according to any one of 1 to 3 above, wherein the hemagglutination active protein is HA1 or HA3.
5). Asn-X-Asn-X-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 1) or Asp-XX-Gly-Gly-Gln-Thr-XX-Gly-Thr (SEQ ID NO: 2) (X is any (5) The method according to any one of (1) to (4) above, wherein the protein is a hemagglutination active protein comprising an amino acid sequence represented by (protein-constituting amino acid).
6). Asn-Ser-Asn-Tyr-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 3) or Asp-Leu-Tyr-Gly-Gly-Gln-Thr-Ala-Asp-Gly-Thr (SEQ ID NO: 4) 5. The method according to any one of 1 to 4 above, which is a hemagglutination active protein comprising the amino acid sequence shown.
7). 7. The method according to any one of 1 to 6 above, which is a hemagglutination active protein obtained by a gene recombination technique.
8). A pharmaceutical composition comprising a hemagglutinating protein derived from a genus Clostridium and an arbitrary nucleic acid to be introduced into cells.
9. 8. The pharmaceutical composition according to 8 above, wherein the Clostridium bacterium is Clostridium botulinum.
10. 9. The pharmaceutical composition according to 9 above, wherein the Clostridium botulinum is a serotype selected from the group consisting of A type, B type, C type, D type and G type.
11. The pharmaceutical composition according to any one of 8 to 10 above, wherein the hemagglutination active protein is HA1 or HA3.
12 Asn-X-Asn-X-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 1) or Asp-XX-Gly-Gly-Gln-Thr-XX-Gly-Thr (SEQ ID NO: 2) (X is any (8) The pharmaceutical composition according to any one of (8) to (11) above, which is a hemagglutination active protein comprising an amino acid sequence represented by (Protein constituent amino acid).
13. Asn-Ser-Asn-Tyr-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 3) or Asp-Leu-Tyr-Gly-Gly-Gln-Thr-Ala-Asp-Gly-Thr (SEQ ID NO: 4) The pharmaceutical composition according to any one of 8 to 11 above, which is a hemagglutination active protein comprising the amino acid sequence shown.
14 14. The pharmaceutical composition according to any one of 8 to 13 above, which is a hemagglutination active protein obtained by a gene recombination technique.
15. The nucleic acid introduced into the cell is a protective antigen in microbial infection, a physiologically active substance, an enzyme inhibitor, a receptor inhibitor, a carcinogen, an apoptosis promoter, an apoptosis inhibitor, a cell regeneration promoter, an immune response promoter And the pharmaceutical composition according to any one of 8 to 14 above, which is a gene expression cassette in which a gene encoding a substance selected from the group consisting of an immune reaction suppressing substance is incorporated.
16. 15. The pharmaceutical composition according to any one of 8 to 14 above, wherein the nucleic acid to be introduced into the cell is a gene expression cassette in which an erythropoietin gene is incorporated.
17. 15. The pharmaceutical composition according to any one of 8 to 14 above, wherein the nucleic acid introduced into the cell is a functional nucleic acid selected from the group consisting of a ribozyme, an antisense gene, and an inhibitory ribonucleic acid.

  According to the present invention, there is provided a method of using a hemagglutination active protein that binds to any nucleic acid as a carrier for introducing the nucleic acid into a cell. By using such a hemagglutination active protein according to the method of the present invention, it is not necessary to use a binding agent that binds the hemagglutination activity protein and the nucleic acid, and the nucleic acid can be easily transferred in vitro and in vivo simply by mixing the two. In any system, it can be introduced into cells.

  In particular, hemagglutinating protein (BoHA) produced by Clostridium botulinum is known to bind to sugar chains such as galactose and sialic acid on the cell surface (Inoue et al., “Microbiology” Vol. 145, p. 2533-2542, 1999; Fujinaga et al., “Microbiology”, Volume 150, p.1529-1538, 2004), BoHA that specifically binds to nucleic acid to form a BoHA-nucleic acid aggregate is a receptor for BoHA. The aggregate bound to the sugar chain on the cell surface and bound to the sugar chain is taken into the cell by endocytosis, and thus the nucleic acid can be introduced into the cell.

  The present invention provides a method of using a hemagglutination active protein (hereinafter sometimes referred to as “HA”) having an ability to bind to any nucleic acid as a carrier for introducing nucleic acid into a cell, and has a specific function with the HA. Characterized by a pharmaceutical composition comprising a nucleic acid.

  HA is not particularly limited as long as it has an ability to bind to any nucleic acid. Examples of such HA include HA produced by Clostridium bacteria, and hemagglutinating activity protein (BoHA) produced by Clostridium botulinum is particularly preferable. Clostridium botulinum is classified into A type, B type, C type, D type, E type, F type, and G type depending on the reactivity with antiserum. Of these, all botulinum bacteria except E and F produce BoHA. BoHA is classified into HA-33 (HA1), HA-17 (HA2), and HA-70 (HA3). HA-70 (HA3) is further classified into HA-20 (HA3a) and HA-50 (HA3b). There are two categories. In the present invention, it is preferable to use HA-33 (HA1), HA-70 (HA3), especially HA-50 (HA3b).

  The above-mentioned BoHA is involved in binding to Asn-X-Asn-X-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 1) and DNA involved in sugar chain binding by homology search and similarity search. It is presumed that an amino acid sequence represented by Asp-XX-Gly-Gly-Gln-Thr-XX-Gly-Thr (SEQ ID NO: 2) (X is an arbitrary protein-constituting amino acid) exists. More specifically, Asn-Ser-Asn-Tyr-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 3) and Asp-Leu-Tyr-Gly-Gly-Gln-Thr-Ala-Asp-Gly- There is an amino acid sequence represented by Thr (SEQ ID NO: 4). These amino acid sequence regions play a central role when nucleic acids are introduced into cells. That is, by mixing BoHA of the present invention with an arbitrary nucleic acid, the nucleic acid binds at the amino acid sequence region represented by Asp-XX-Gly-Gly-Gln-Thr-XX-Gly-Thr (SEQ ID NO: 2) of BoHA. To do. By contacting this BoHA-nucleic acid conjugate with a cell, the BoHA-nucleic acid conjugate is converted into an amino acid sequence represented by BoHA Asn-X-Asn-X-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 1). It binds to sugar chains on the cell surface in the region and is thought to be taken up by endocytosis. As a result, the nucleic acid is introduced into the cell without damaging the cell. According to such a mechanism, it is suggested that the HA protein containing the above two types of amino acid sequence regions is the minimum unit that functions as a carrier for introducing nucleic acid into cells. Therefore, the method of the present invention is not limited to BoHA, but may use other HA containing the above amino acid sequence or HA artificially inserted with the above amino acid sequence (for example, by a gene recombination technique). It seems possible. Examples of such HA candidates include influenza virus Hemagglutinin (FuHA), pertussis filamentous hemagglutinin (FHA), and Concanavalin A (Con A).

  Further, it is a peptide or polypeptide obtained by partial degradation of the HA of the present invention with a proteolytic enzyme or a chemical reagent (for example, cyanogen bromide), which contains the above-mentioned specific amino acid sequence and retains hemagglutination activity. Peptides or polypeptides can also be used. In the method of the present invention, these hemagglutination active proteins, peptides or polypeptides (hereinafter sometimes simply referred to as “derivatives”) can be used alone or in combination.

  When the HA of the present invention is extracted / purified from bacterial cells or bacterial cultures, the following method is used. When the bacteria are grown, a medium suitably containing caseinate hydrolyzate, casein peptone, starch digest, yeast extract, potassium dihydrogen phosphate, sodium chloride and the like is used. For example, KI broth, reflel medium (Kyokuto Pharmaceutical), normal bouillon medium (Nippon Pharmaceutical), Muller's casein hydrolyzed medium (Kyokuto Pharmaceutical), Bordet-Gengou agar medium (Kyokuto Vital Media), methylated β cyclodextrin added Stainer and Examples include Scholte's modified liquid medium and Cohen-Willer medium, which may be appropriately selected according to the purpose, such as separation of bacteria, preparation of master seed, and mass culture. The culture conditions are usually performed at a temperature of 34 to 38 ° C. and a period of 1 to 5 days, but may be appropriately adjusted according to the purpose of use, the culture form, the amount of planted bacteria, the medium scale, and the like. When collecting and purifying the target HA from the cells or the culture solution of the cells, purification methods generally used in protein chemistry, such as centrifugation, salting out, ultrafiltration, isoelectric precipitation, electrolysis, A combination of methods such as electrophoresis, ion exchange chromatography, gel filtration, affinity chromatography, hydrophobic chromatography, hydroxyapatite chromatography, and CS resin chromatography are used. The amount of the obtained protein is measured using a protein measurement reagent such as BCA Protein Assay Reagent Kit (Pierce Biotechnology, Inc) or Protein Assay Kit (BIO-RAD, Inc).

  When the HA of the present invention is obtained by a gene recombination technique, the following method is taken. The gene encoding HA is a general gene recombination technique described by Sambrook et al. (Molecular Cloning, A Laboratory Manual, Second Edition, Cold, starting from total RNA, mRNA or genomic DNA extracted from bacterial cells. Spring Harbor Laboratory Press, NY, 1989). In practice, commercially available kits are used. For example, for RNA extraction, reagents such as TRIzol reagent (Invitrogen), ISOGEN (Nippon Gene), StrataPrep Total RNA Purification Kit (Toyobo), for extraction of chromosomal DNA, ISOPLANT (Wako Pure Chemical Industries), mRNA purification Includes mRNA Purification Kit (Amersham Biosciences), Poly (A) Quick mRNA Isolation Kit (Toyobo), mRNA Separator Kit (Clontech), and SuperScript plasmid system for cDNA synthesis and Plasmid cloning (Invitrogen), cDNA Synthesis Kit (Takara Shuzo), SMART PCR cDNA Synthesis & Library Construction Kits (Clontech), Directional cDNA Library Construction systems (Novagen), GeneAmp PCR Gold (Applied Biosystems) are used. The The base sequence of the obtained HA gene was once cloned into a plasmid such as pBluescript II SK + (Stratagene) or pCR2.1-TOPO (Invitrogen) and then determined by a DNA sequencer (ABI Prism 377 Applied Biosystems). Is done.

  As a method for introducing a mutation into a part of the HA gene, the site-directed mutagenesis method is generally used. In fact, Takara's Site-Directed Mutagenesis System (Mutan-Super Express Km, Mutan-Express Km, Mutan-K, etc.), Stratagene's QuickChange Multi Site-Directed Mutagenesis Kit, QuickChange XL Site- A commercially available kit such as Directed Mutagenesis Kit or GeneTailor Site-Directed Mutagenesis System of Invitrogen is used according to the attached protocol.

  The HA gene or its modified gene is expressed by incorporating the thus obtained HA gene or the modified HA gene into an appropriate expression vector and introducing the expression vector into a host. Bacteria, yeast, animal cells, plant cells, insect cells and the like are commonly used as hosts for expressing foreign proteins, but may be selected according to the purpose. For example, when E. coli used in the examples of the present invention is used as a host, various expression vectors for E. coli having trp promoter, T7 promoter, and cspA promoter have been developed and marketed. And use it. An appropriate Escherichia coli such as BL21, HMS174, DH5α, HB101, JM109 or the like is selected as a host according to the expression vector. In order to facilitate purification of the target protein, it may be expressed in a form fused with another polypeptide or peptide. GST fusion protein purification system (Amersham Pharmacia) capable of producing a fusion protein with glutathione S transferase (GST) as a vector for expressing such a fusion protein, HAT protein expression / addition of oligohistidine Examples thereof include a purification system (Clonetech Inc) and a MagneHis Protein Purification System (Promega Inc). For example, as expressed in the examples of the present invention, after being expressed as a fusion protein with glutathione S-transferase (GST), BoHA-GST is collected using glutathione-sepharose, and then proteolytic enzyme of human rhinovirus BoHA and GST are separated using (Amersham), and BoHA is purified. The purification method described above is used for the purification of BoHA. BoHA is detected by a method based on a molecular size such as SDS-PAGE or gel filtration, or a method based on an antigen-antibody reaction such as ELISA, Western blot, or dot blot. Both are general methods for detecting foreign proteins, and may be selected as appropriate according to the purpose.

  The HA and derivatives thereof thus obtained are used as carriers for introducing any nucleic acid into cells. The size and type of the nucleic acid introduced into the cell are not particularly limited. For example, linear double-stranded DNA, circular double-stranded DNA, linear double-stranded RNA, linear single-stranded RNA, oligonucleotide, siRNA, miRNA and the like can be used. More specifically, infection protective antigens in microbial infections, physiologically active substances, enzyme inhibitors, receptor inhibitors, carcinogenesis inhibitors, apoptosis promoting substances, apoptosis inhibiting substances, cell regeneration promoting substances, immune reaction promoting substances, and Examples include a nucleic acid having a function such as a gene expression cassette in which a gene encoding a substance such as an immune reaction inhibitor is incorporated, a ribozyme or antisense gene, and a suppressive ribonucleic acid. Here, the gene expression cassette means an expression vector appropriately constructed so that a foreign gene is expressed in a cell.

  As a buffer solution when the HA of the present invention or a derivative thereof (hereinafter also referred to as “carrier”) is mixed with a nucleic acid, physiological saline, phosphate buffer, phosphate buffer, citrate buffer The liquid is not particularly limited as long as it does not adversely affect cells and living bodies. The pH of the buffer solution can be used in the range of pH 6.0 to pH 8.5, but is preferably in the range of pH 7.0 to pH 8.0. The salt concentration is 0 to 10%, preferably 0.7% to 1.1%. As the salt, sodium chloride, potassium chloride, magnesium chloride and the like can be used, and sodium chloride is preferable. Proteins and saccharides may be allowed to coexist in a mixed solution of carrier and nucleic acid. The mixing ratio of the carrier and the nucleic acid can be 1/100 to 10,000 times that of the nucleic acid 1 in terms of molar ratio, but is preferably 1/10 to 1000 times. For example, the carrier may be used in an amount of 0.01 to 10000 mol, preferably 0.1 to 1000 mol, relative to 1.0 mol of nucleic acid. The composition comprising the carrier and nucleic acid of the present invention can be administered to animals by any route such as nasal administration, intravenous administration, intramuscular administration, and oral administration. The carrier of the present invention can introduce a desired nucleic acid into a cell simply by mixing the carrier and the nucleic acid without using a binder that binds the carrier and the nucleic acid. It can be used as an agent or vaccine.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

In vitro evaluation of BoHA transduction ability
(1) Preparation of BoHA and GFP plasmids Botulinum type A HA1 (AHA1), type A HA3b (AHA3b), type B HA1 (BHA1) and type B HA3b (BHA3b) BoHA and glutathione S transferase (GST) Recombinant E. coli producing the fusion protein (BoHA-GST) was provided by Prof. Okuma of Okayama University (Fujinaga et al (2000) FEBS Lett 467, 179-183). These BoHA-producing E. coli were sonicated, centrifuged at 15,000 G for 60 minutes, and the supernatant was applied to glutathione-sepharose (Amersham) and eluted with 50 mM Tris (pH 7.5) containing 10 mM glutathione. BoHA-GST Was isolated. This was digested with human rhinovirus proteolytic enzyme (Amersham) to separate BoHA and GST, and GST and BoHA-GST were adsorbed on glutathione-Sepharose 4B (Amersham) to collect BoHA passing through. The obtained BoHA was dialyzed against 0.1M PBS (pH 7.4) and stored at -20 ° C. The GFP plasmid used as a marker was cloned into the pCAG-mcs-PolyA plasmid, and the GFP plasmid used as a marker was purchased from the pEGFP-N1 vector (Clontech). E. coli TOP10 was transformed into the pCAGmcs-polyA vector by (BamHI / HindIII) treatment and ligase reaction. A plasmid of a clone confirmed to be completely coincident with the target sequence was purified with a plasmid purification kit (QIAGEN). The purified plasmid was stored at -20 ° C.

(2) Intracellular introduction of GFP plasmid using BoHA Human colon epithelial cell line T-84 cell, human lung epithelial cell line NCI-H727 cell, human liver cell line, and mouse lung epithelial cell line Each was plated on a 6-well cell culture plate (Corning, 9.5 cm ² / well) and cultured until 70-80% confluent.

Each of the cells obtained by aspirating the medium was mixed with equal amounts of 20 μg protein / mL of each BoHA (AHA1, AHA3b, BHA1, BHA3b) prepared in (1) and 4 μg / mL of GFP plasmid (shown as DNA in Table 1). 1.0 mL of the mixed solution was added to each well and cultured in a CO ₂ furan vessel at 37 ° C. for 20 hours. The number of cells expressing GFP was counted under a fluorescence microscope. As controls, a mixture of each BoHA-GST (AHA1-GST, AHA3b-GST, BHA1-GST, BHA3b-GST) and a GFP plasmid, a commercially available mixture of Lipofectamin and GFP plasmid, or a solution containing only a GFP plasmid was used. The results are shown in Table 1.

  Expression of GFP was observed in all cells to which the BoHA-GFP plasmid was added. In addition, no cell detachment or increase in dead cells was observed with the addition of the aggregate, and no toxicity to cells was observed. In the case of using Lipofectamin, the number of cells into which the gene was introduced was larger than that of BoHA-GFP, but an increase in dead cells was observed. On the other hand, no GFP expression was observed in cells to which only the BoHA-GST / GFP plasmid and the GFP plasmid were added. “Many” in Table 1 indicates that measurement was not possible.

In vivo evaluation of the ability of BoHA to be introduced into cells Using the human erythropoietin (EPO) gene, the ability of BoHA to be introduced into cells in vivo was examined. For the EPO gene, a human EPO clone was purchased from Invitrogen. A plasmid was extracted from the purchased EPO clone, and the target region was amplified by PCR. This PCR product was incorporated into pCAGmcs-polyA vector by restriction enzyme (BamHI / HindIII) treatment and ligase reaction, and E. coli TOP10 was transformed. A plasmid of a clone confirmed to be completely coincident with the target sequence was purified with a plasmid purification kit (QIAGEN). The purified plasmid was prepared with a 20% glycerol solution so as to have a concentration of 1.0 mg / mL, and stored at -20 ° C.

(1) Evaluation by intravenous and intramuscular injection of AHA1 AHA1 40 μg protein / mL obtained in Example 1- (1) and 4, 20, 100 μg / mL of EPO plasmids were mixed in an equal amount. Weekly female, ICR mice (5 / group) were injected into the tail vein (200 μL / mouse; n = 5) and intramuscularly (200 μL / mouse; n = 5) before administration (day 0). The red blood cell count after administration (10 days) was examined. The number of red blood cells was measured using a fully automatic blood cell counter for animals (Nihon Kohden). As a result, a significant increase was observed in the group injected intravenously with “AHA1: EPO = 20 μg protein: 50 μg” and the group injected intramuscularly with “AHA1: EPO = 20 μg protein: 50 μg” compared to day 0. (P <0.05; t-test with pairing) (FIG. 1). In these administration groups, EPO was expressed, and it seems that the number of red blood cells was increased. AHA1 was found to be able to introduce genes into cells even in vivo.

(2) Evaluation by intravenous injection and nasal administration of BHA3b Nasal administration was performed as follows. BHA3b 1 mg / mL obtained in Example 1- (1) and EPO plasmid 1 mg / mL were mixed in equal amounts, and 20 μL was dropped into the nasal cavity of 6-week-old female, ICR mice (9 mice / group) (n = 9).

  In addition, intravenous administration was performed as follows. BHA3b 400 μg / mL obtained in Example 1- (1) and EPO plasmid 200 μg / mL and 1 mg / mL were mixed in equal amounts, and each mixture was added to a 6-week-old female, 100 μL into the tail vein of ICR mice. Injection (n = 9).

  Blood was collected on the 10th day after the inoculation, and the hematocrit value (HCT) was measured using a fully automatic blood cell counter for animals (Nihon Kohden). As a result, a significant increase in HCT was observed in the nasal administration group and the group to which “BHA3b / EPO plasmid = 20 μg protein: 50 μg” was intravenously administered compared to day 0 (P <0.01; corresponding) Case t-test) (Figure 2). BHA3b was found to be able to introduce the EPO gene into cells by intranasal and intravenous administration to mice.

  EPO gene to mice using influenza virus Hemagglutinin (FuHA), pertussis filamentous hemagglutinin protein (Filamentous hemagglutinin: FHA), Concanavalin A (Con A), and BoHA obtained in Example 1- (1) We compared the effects of introducing. The mice were 6 week old female, ICR mice (10 / group). FuHA inoculated sperm eggs with A / New Caledonia // 0621 strain and allowed to grow at 35 ° C. for 2 days, and then collected 600 mL of urinary cavity fluid. A 30% amount of PEG6000 was added to the chorioallantoic fluid to collect the virus as a precipitate and purified by sucrose gradient centrifugation. TritonX100, which is 3 times the protein concentration of the virus solution, was added and stirred at room temperature for 1 hour, and then FuHA was purified by Sephacryl S300 gel chromatography. FHA is gonococcus pertussis Higashihama strain in a modified medium of Stainer and Scholte, and cultured for 35 days at 35 ° C, 35% ammonium sulfate is salted out, and 0.1M PB (pH8.0) buffer solution is used. Gel filtration was performed using an equilibrated Sepharose CL 6B column, and then purification was performed using a CS resin column. Con A used a Sigma product.

The nucleic acid used was the EPO plasmid (EPO-pCAG-mcs-polyA) obtained in Example 1- (1). Intravenous administration may be taken into the liver by the injection pressure at the time of inoculation, and intramuscular injection may be taken up from injured cells because muscle cells are damaged by inoculation. We decided to compare with intranasal administration. For this reason, BoHA decided to use HA3b. Using a 6-week-old female ICR mouse, a specimen prepared by preparing HA: EPO plasmid at 2.5 μM: 5 μg was inoculated into 20 μL / mouse nasal cavity. Inoculation was carried out twice for 2 consecutive days. Blood was collected before inoculation (day 0) and on day 10 after inoculation, and HCT was measured with a fully automatic blood cell counter for animals (Nihon Kohden).
In the BHA3b / EPO plasmid administration group, a significant increase in HCT was observed compared to day 0 (P <0.05; t-test when there was a correspondence), but FuHA / EPO plasmid, Con A / EPO plasmid, No significant increase was observed in the groups administered with FHA / EPO plasmid and EPO plasmid. BHA3b was found to have a stronger nucleic acid introduction effect in vivo than other proteins with hemagglutination activity.

  Since BoHA for introducing a nucleic acid in the present invention into a cell can bind to a nucleic acid, a gene expression cassette or an antisense gene that expresses a gene that encodes an infection protective antigen of a physiologically active substance or a pathogenic microorganism, When administered in vivo together with a functional nucleic acid such as a ribozyme, it can be used for the prevention and treatment of diseases involving the substance.

FIG. 1 shows the red blood cell count (RBD) at day 0 and day 10 when a mixture of AHA1 and EPO plasmids was injected into ICR mice intracaudally (iv) or intramuscularly (im). Comparison of RBD on day 0 and day 10 used the paired t-test. 20 in (20:10) represents the amount of protein (μg protein n) administered to AHA1 mice, and 10 represents the amount of EPO plasmid (μg).

FIG. 2 shows the hematocrit values (HCT) on day 0 and day 10 when a mixture of BHA3b and EPO plasmid was inoculated intranasally (in) or iv into ICR mice. Comparison of HCT on day 0 and day 10 used the t-test with a pair.

FIG. 3 shows the hematocrit values (HCT) at day 0 and day 10 when ICR mice were inoculated in a mixture of various hemagglutinating proteins and EPO plasmids. A t-test was used to compare the HCT on day 0 and day 10. FuHA is Infuluenza virus hemagglutinin, Con A is Concanavalin A, FHA is Bordetella pertussis Filamentous Hemagglutinin, and EPO is Erythropoietin plasmid.

Claims (17)

  A method of using a hemagglutination activity protein derived from Clostridium spp. As a carrier for introducing nucleic acid into cells.   The method according to claim 1, wherein the Clostridium bacterium is Clostridium botulinum.   The method according to claim 2, wherein the Clostridium botulinum is a serotype selected from the group consisting of type A, type B, type C, type D and type G.   The method according to any one of claims 1 to 3, wherein the hemagglutination active protein is HA1 or HA3.   Asn-X-Asn-X-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 1) or Asp-XX-Gly-Gly-Gln-Thr-XX-Gly-Thr (SEQ ID NO: 2) (X is any The method according to any one of claims 1 to 4, wherein the protein is a hemagglutination active protein comprising an amino acid sequence represented by (protein-constituting amino acid).   Asn-Ser-Asn-Tyr-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 3) or Asp-Leu-Tyr-Gly-Gly-Gln-Thr-Ala-Asp-Gly-Thr (SEQ ID NO: 4) The method according to any one of claims 1 to 4, wherein the protein is a hemagglutination active protein comprising the amino acid sequence shown.   The method according to any one of claims 1 to 6, which is a hemagglutination active protein obtained by a gene recombination technique.   A pharmaceutical composition comprising a hemagglutinating protein derived from a genus Clostridium and an arbitrary nucleic acid to be introduced into cells.   9. The pharmaceutical composition according to claim 8, wherein the genus Clostridium is Clostridium botulinum.   10. The pharmaceutical composition according to claim 9, wherein the Clostridium botulinum is a serotype selected from the group consisting of A type, B type, C type, D type and G type.   The pharmaceutical composition according to any one of claims 8 to 10, wherein the hemagglutination active protein is HA1 or HA3.   Asn-X-Asn-X-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 1) or Asp-XX-Gly-Gly-Gln-Thr-XX-Gly-Thr (SEQ ID NO: 2) (X is any The pharmaceutical composition according to any one of claims 8 to 11, which is a hemagglutination active protein comprising an amino acid sequence represented by (protein-constituting amino acid).   Asn-Ser-Asn-Tyr-Ile-Lys-Ile-Ile-Glu (SEQ ID NO: 3) or Asp-Leu-Tyr-Gly-Gly-Gln-Thr-Ala-Asp-Gly-Thr (SEQ ID NO: 4) The pharmaceutical composition according to any one of claims 8 to 11, which is a hemagglutination active protein comprising the amino acid sequence shown.   The pharmaceutical composition according to any one of claims 8 to 13, which is a hemagglutination active protein obtained by a gene recombination technique.   The nucleic acid introduced into the cell is a protective antigen in microbial infection, a physiologically active substance, an enzyme inhibitor, a receptor inhibitor, a carcinogen, an apoptosis promoter, an apoptosis inhibitor, a cell regeneration promoter, an immune response promoter The pharmaceutical composition according to any one of claims 8 to 14, which is a gene expression cassette into which a gene encoding a substance selected from the group consisting of an immune reaction suppressing substance is incorporated.   The pharmaceutical composition according to any one of claims 8 to 14, wherein the nucleic acid to be introduced into the cell is a gene expression cassette in which an erythropoietin gene is incorporated.   The pharmaceutical composition according to any one of claims 8 to 14, wherein the nucleic acid to be introduced into the cell is a functional nucleic acid selected from the group consisting of a ribozyme, an antisense gene, and an inhibitory ribonucleic acid.

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