JP2005176830A - Nucleic acid introduction carrier - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a carrier for introducing a nucleic acid, having specificity to a target cell and excellent in expression of the nucleic acid, for example, expression of a gene, and to obtain a composition for introducing the nucleic acid. <P>SOLUTION: This carrier for introducing the nucleic acid contains a cationic polymer material and hyaluronic acid or its salt and/or its negatively charged derivative in such a state that the components are not covalently bonded to each other. The composition contains the nucleic acid, the cationic polymer material, and the hyaluronic acid or its salt and/or its negatively charged derivative in such a state that the components are not covalently bonded to one another. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a carrier used for introducing a nucleic acid into a cell, and a composition for introducing a nucleic acid containing the nucleic acid together with the carrier.

  Currently, there are roughly two gene therapy methods in practical use. One is a treatment method for infectious genetic diseases such as congenital immune deficiency and inborn errors of metabolism, such that the missing gene is introduced from the outside, and the other is cancer cells and AIDS. It is a treatment method that introduces genes for targeting cells or viruses such as those that suppress their growth or kill them. In any of the above-described treatment methods, it is known that it is important to introduce and express a target gene into cells.

  However, since both DNA and cell membrane are anionic and electrically repelled, it is very difficult to introduce a gene (DNA) directly into a cell alone.

  Thus, various substances have been studied as DNA carriers so far, but there are problems in terms of safety and introduction efficiency, which hinders practical use. For example, a retrovirus vector, which is a typical viral vector, has the advantage of high introduction efficiency, but (1) cannot use large-sized DNA, (2) cannot be introduced into non-dividing cells, (3) expression There are disadvantages such as low level. Adenovirus vectors can also be introduced into non-dividing cells, but have the disadvantage of strong immunogenicity and the production of antibodies. Herpes virus vectors are excellent for introduction into neurons, but are cytotoxic. Has the disadvantage of being strong. In addition, cationic liposomes, lipids, polymers, and the like have been studied as carriers other than viral vectors, but there has been a problem that introduction efficiency and cell specificity are lowered.

  As a method using a polymer, Patent Document 1 discloses a carrier for gene introduction containing a polyethylene glycol derivative and a cationic polymer. This carrier achieved high gene expression by coating a complex composed of a gene and a cationic polymer with a polyethylene glycol derivative and administering it in vivo. This polyethylene glycol derivative has a sugar residue-containing side chain, but the sugar of the sugar residue is an oligosaccharide and is insufficient to specifically target any tissue.

  As a composition for introducing a gene product having target cell specificity, Patent Document 2 discloses a cationic liposome composition in which hyaluronic acid is covalently bound to the liposome surface. This cationic liposome formulation is described as characterized by bioadhesion to target cells, low immunogenicity, resistance to macrophage phagocytosis, and masking of cationic lipids on the liposome surface. However, there is a problem that gene expression efficiency is low.

  Patent Document 3 discloses a composition comprising DNA and hyaluronic acid or a derivative thereof and a pharmaceutically acceptable carrier. In this composition, the viral vector containing the gene may simply be mixed with or bound to hyaluronic acid. The examples show that hyaluronic acid increases the uptake of adenoviral vectors into target cells. However, the adenovirus vector has a drawback that it is highly immunogenic and can produce an antibody, which may hinder the use of a high dose or repeated administration.

  Hyaluronic acid has been shown to specifically target tissues where hyaluronic acid receptors are expressed at high levels. Hyaluronic acid levels have been shown to correlate with tumor invasion and may be indicative of tumor cell invasion characteristics. Hyaluronic acid-specific cell surface receptors have been identified, including the histocompatibility antigen CD44, hyaluronic acid-mediated motility (RHAMM), intercellular adhesion factor (ICAM), and receptors for similar proteins in the CD44 family.

  A carrier for gene transfer in which a cationic polymer and hyaluronic acid are covalently bonded can specifically target tissues, but gene expression efficiency is low and gene expression after being taken into cells is disturbed. there is a possibility.

Hyaluronic acid has the ability to bind to large amounts of water and becomes a viscous hydrous compound with viscoelasticity in vivo, so it is not only used for target cell specificity but also as a carrier for general drugs Has been. For example, Patent Document 4 discloses a pharmaceutical composition in which a drug is contained in a matrix formed of a polymer having biodegradability and biocompatibility and / or a polyvalent metal ion and hyaluronic acid. . This pharmaceutical composition is for controlling the release rate of a drug for treating diseases, and is not suitable for use as a carrier for gene transfer into cells.
JP 2003-231748 A Special table 2003-528131 gazette Special table 2003-507915 gazette JP-A-11-130697

  The problem to be solved by the present invention is to provide a carrier for introducing a nucleic acid which has no immunogenicity and is excellent in the efficiency of gene introduction into a target cell, and a composition for introducing a nucleic acid containing the carrier and a nucleic acid. .

The present inventors have used a nucleic acid, for example, a complex composed of a gene and a cationic polymer substance, hyaluronic acid (hereinafter referred to as “HA”), which is a negatively charged polymer substance, or a salt thereof, or negatively. The present invention has been completed by finding that excellent gene expression ability can be achieved by targeting cells having cell surface molecules such as HA receptors by coating with a charged HA derivative and in vivo administration.
That is, the present invention is as follows.

(1) A nucleic acid introduction carrier comprising a cationic polymer substance and hyaluronic acid or a salt thereof and / or a negatively charged derivative thereof in a state where they are not covalently bonded.
(2) The nucleic acid introduction carrier according to (1), wherein hyaluronic acid or a salt thereof or a derivative thereof is negatively charged and has a molecular weight of 10,000 or more.
(3) The carrier for nucleic acid introduction according to (2), wherein the cationic polymer substance is a protein, polypeptide, dendrimer, polyethyleneimine, chitosan, or dextran.
(4) The molar ratio of each charged group in the cationic polymer substance and hyaluronic acid or a salt thereof and / or a negatively charged derivative thereof is 1: 0.01 to 1: 200 according to (2) Nucleic acid introduction carrier.
(5) The carrier for introducing a nucleic acid according to (2), wherein the cationic polymer substance is chitosan. (6) A composition containing a nucleic acid, a cationic polymer substance, and hyaluronic acid or a salt thereof and / or a negatively charged derivative thereof in a state where they are not covalently bonded.
(7) The composition according to (6), wherein the molar ratio of each charged group in the nucleic acid and the cationic polymer substance is 1: 0.1 to 1:50.
(8) The composition according to (6), wherein the molar ratio of each charged group in the nucleic acid and hyaluronic acid or a salt thereof and / or a negatively charged derivative thereof is 1: 0.001 to 1: 1000.
(9) The composition according to any one of (6) to (8), wherein the nucleic acid is a gene.
(10) The composition according to (9), which is for gene therapy.

The carrier for introducing a nucleic acid according to the present invention is a composite of a nucleic acid and a cationic polymer substance without breaking the complex of the nucleic acid and the cationic polymer substance by using negatively charged HA without covalently bonding. Since the dispersibility of the body can be increased and the specificity to the target cell can be given, the activity of gene expression is high compared to the conventional gene-cationic polymer substance complex,
Since there is no immunogenicity like a viral vector, it is highly safe and cell-specific gene delivery can be achieved. Therefore, the composition containing a nucleic acid together with the nucleic acid introduction carrier of the present invention can efficiently carry out various gene therapies for humans and animals, or the production of experimental animals and cells into which specific genes have been introduced. It is extremely useful.

  Examples of the nucleic acid compatible with the nucleic acid introduction carrier of the present invention include nucleic acids and nucleic acid derivatives such as various plasmid DNAs, sense or antisense oligonucleotides, RNA, ribozymes, or mixtures thereof.

The cationic polymer substance is a positively charged, naturally-occurring or synthetic polymer substance having a molecular weight of about 1,000 to 500,000. For example, protamine, histone, HelΔ1, gelatin and other proteins and polypeptides, Polyamino acids such as L-L lysine, polyarginine and polyornithine, polyamidoamine dendrimers, dendrimers such as polylysine dendrimers, synthetic polymers such as polyethyleneimine, dextrans such as diethylaminoethyl-dextran, polysaccharides such as chitosan, or salts thereof, And combinations thereof.
The molecular weight of the protein or polypeptide is preferably about 1000 to 500,000, and examples of the salt of the protein or polypeptide include hydrochloride, phosphate, borate and the like.
The molecular weight of the dendrimer is preferably about 1,000 to 500,000, and examples of the salt of the dendrimer include hydrochloride, phosphate, borate and the like.
The molecular weight of polyethyleneimine, which is an example of a synthetic polymer, is preferably about 1,000 to 500,000, more preferably about 5,000 to 200,000, and most preferably about 10,000 to 100,000. , Hydrochloride, phosphate, borate and the like.
The molecular weight of chitosan is preferably about 1000 to 500,000, more preferably about 5000 to 200,000, and most preferably about 10,000 to 100,000. Salts of chitosan include hydrochloride, acetate, etc. Can be illustrated. Further, chitosan is usually produced by deacetylating raw material chitin, and the degree of deacetylation of chitosan used in the present invention is preferably 5 to 100%, more preferably 50 to 100%. Preferably it is 70 to 100%. Here, for example, the degree of deacetylation of chitosan being 70% means that the constituent units D-glucosamine and N-acetyl-D-glucosamine are actually present in a molar ratio of 7: 3. Means that. Chitosan can be obtained from a variety of sources including crustaceans, fungi and other substances.
Cationic polymers that are not normally positively charged can be used as long as they are positively charged by the introduction of a functional group such as an amino group. It may be modified.

HA or a salt thereof or a negatively charged derivative thereof may have a molecular weight of 10,000 or more, preferably 100,000 or more, and more preferably 100,000 to 50 million.
Examples of the salt of HA include sodium salt, potassium salt, ammonium salt and the like.
Examples of the negatively charged derivative include those obtained by introducing polyethylene glycol, peptides, sugars, proteins, iodic acid, antibodies or a part thereof into HA.

  The mixing ratio of nucleic acid and cationic polymer substance (molar ratio of each charged group, negative charge: positive charge ratio) varies depending on the type of target cell / nucleic acid / cationic polymer substance, but is 1: 0.1 to 1: It is good that it is 50, Preferably it is 1: 0.2 to 1:20, More preferably, it is 1: 1 to 1:10. The compounding ratio of the nucleic acid and the cationic polymer here is the molar ratio of each charged group, and specifically refers to the negative charge by the phosphate anion of the nucleic acid: the positive charge ratio of the cationic polymer.

The compounding ratio of nucleic acid and HA (or a salt thereof and / or a negatively charged derivative thereof) (molar ratio of each charged group, negative charge: negative charge ratio) varies depending on the type of target cell / nucleic acid / cationic polymer. However, it is good that it is 1: 0.001-1: 1000, Preferably it is 1: 0.1-1: 100, More preferably, it is 1: 1-1: 50. The compounding ratio of the nucleic acid and HA here is the molar ratio of each charged group, and specifically refers to the negative charge ratio of the nucleic acid phosphate anion to the negative charge ratio of the HA carboxylate anion.

  The compounding ratio (molar ratio of each charged group, positive charge: negative charge ratio) of the cationic polymer substance and HA (or a salt thereof, and / or a negatively charged derivative thereof) is 1: 0.01-1 : 200, preferably 1: 0.1 to 1:50, more preferably 1: 0.1 to 1:10.

The carrier for introducing a nucleic acid of the present invention is produced by mixing a cationic polymer substance and HA.
The nucleic acid introduction composition of the present invention is produced by mixing a nucleic acid introduction carrier with a nucleic acid. Or it can manufacture also by mixing a nucleic acid with a cationic polymer substance and HA in arbitrary orders.
The resulting nucleic acid introduction composition is composed of a nucleic acid, a cationic polymer substance, and HA that are loosely bonded by ionic bonds to form a complex, or such a complex depending on the composition of each component. An embodiment is formed in which the body structure is further covered by HA.

The nucleic acid introduction composition of the present invention can be used for various gene therapies for humans and animals, or for the production of experimental animals and cells into which specific genes have been introduced. For example, after introducing a gene (nucleic acid) into a target cell taken out of the body using the gene (nucleic acid) introduction composition of the present invention, the cell is returned to the living body to express the target gene ex vivo. And direct gene transfer methods such as in vivo and in situ methods.
Direct administration methods include intravenous, subcutaneous or intramuscular, intraperitoneal injection, inhalation into the nasal cavity, oral cavity, lung, etc., or direct administration into the affected tissue or nearby blood vessels, gels, sponges, etc. Any method of gene therapy technology is possible, for example, carrying it in a porous body or non-woven fabric.

  Various additives commonly used in pharmaceutics (excipients, diluents, thickeners, stabilizers, etc.) are used as necessary in order to obtain a dosage form suitable for administration or a sustained-release preparation. Preservatives, etc.) can be used.

  Nucleic acids such as genes and cationic polymer substances are electrostatically combined to form small folded complexes that are normally taken up into cells by endocytosis, so genes and cationic polymer substances Many of these complexes are degraded by enzymes in the endosomes that have become weakly acidic. On the other hand, the carrier of the present invention is considered to have a cell membrane destruction function under weak acidity because it is further combined with negatively charged hyaluronic acid. By coating the nucleic acid-cationic polymer substance complex with such anionic polymer substance, the endosomal membrane is broken before the nucleic acid is enzymatically degraded, and the nucleic acid is transferred to the cytoplasm, thereby improving gene expression efficiency. It is thought to let you.

  In addition, the nucleic acid-cationic polymer substance complex is likely to aggregate, and this aggregation prevents the complex from being taken into cells. By covering the complex with hyaluronic acid, the nucleic acid-cationic polymer complex It is considered that the aggregation of the substance complex is prevented. In addition, since the hyaluronic acid of the present invention is hydrophilic, it is considered that the complex is protected from interactions such as aggregation by serum proteins, blood cells, extracellular matrix and the like.

As described above, in the present invention, the nucleic acid, the cationic polymer substance, and the hyaluronic acid form a complex by ionic bonding without being covalently bonded to each other. In particular, since hyaluronic acid is not covalently bonded to the cationic polymer substance by covalent bond, the gene expression effect of the cationic polymer substance is not impaired after the transfer into the cell, and the gene expression efficiency Can be significantly improved.
Moreover, since the carrier for gene transfer according to the present invention has an adhesive ability to target cells, cells having cell surface molecules such as CD44 that specifically bind to hyaluronic acid can be used as targets.

  The carrier for gene transfer according to the present invention exhibits bioadhesiveness that improves transfection efficiency and reduces the amount of carrier used. Hyaluronic acid promotes the binding of the carrier to the cell membrane and promotes the transfer to the cell. This not only increases the carrier concentration in the target cells, but also improves the residence time in the target cells.

  The present invention will be described more specifically with reference to examples. In addition, these Examples are for demonstrating this invention, Comprising: The scope of the present invention is not limited at all.

Inhibitory effect of hyaluronic acid (hereinafter referred to as “HA”) on aggregation of albumin of protamine / plasmid complex by albumin A luciferase plasmid (pCMV-Luc) was prepared as follows. The luciferase cDNA of pGL3-control vector (Promega, Madison, WI, USA) was excised with restriction enzyme HindIII / Xbal and the fragment was incorporated into the multicloning site of pcDNA3 vector (Invitrogen, Carlsbad, CA, USA). This luciferase plasmid (pCMV-Luc) was grown in E. coli DH5alpha and purified with the Qiagen Plasmid Giga Kit (QIAGEN GmbH, Hilden, Germany).
The luciferase plasmid prepared as described above is labeled with the fluorescent dye DAPI (4,6-diamidino-2-phenylindole diacetate, manufactured by Sigma), and protamine sulfate (manufactured by Wako, derived from salmon sperm) is charged with a charge ratio plasmid: protamine A complex was prepared by adding 1: 2 (negative charge of phosphate anion of plasmid: molar ratio of arginine side chain guanidino group of protamine and positive charge of N-terminal amino group cation). (Luciferase plasmid concentration: 6 μM (with nucleobase concentration), protamine concentration: 2.32 μg / ml). When observed with a fluorescence microscope, a small globule-like complex was observed to be in Brownian motion. When albumin was added to a final concentration of 6 mg / ml, the complex instantly aggregated and precipitated.
On the other hand, HA sodium salt (manufactured by Nacalai Tesque, derived from microorganisms) is charged in advance in the luciferase plasmid / protamine complex. Protamine: HA = 2: 1 (Protamine arginine side chain guanidino group and N-terminal amino group cation positive charge: HA 6 mg / ml in a system (luciferase plasmid concentration: 6 μM (with nucleobase concentration), protamine concentration: 2.32 μg / ml, HA concentration: 9.62 μg / ml) added at a negative charge molar ratio of the carboxylate anion of Even after the addition of ml of albumin, the complex remained in a small globule state and continued browning.
From the above observations, it was confirmed that HA coats the DNA complex surface and has the ability to effectively block aggregation by serum proteins.

Gene expression by HelΔ1 / HA / Plasmid Complex Gene (Luciferase Plasmid), HelΔ1 (Amphipathic Cationic Peptide), and HA, a three-component complex, and this complex interacts with Chinese hamster ovary-derived cell CHO And the expression of the luciferase gene was confirmed.
Operating procedure
(1) On the day before the introduction of the complex, seed CHO cells derived from Chinese hamster ovary at a density of 5 × 10 ⁴ cells / well in a 24-well multiplate, DMEM (Dulbecco's Modified Eagle's Medium), Asahi Techno Glass Incubation was performed for 24 hours using the medium.
(2) 180 μl of DMEM (Dulbecco's Modified Eagle's Medium, manufactured by Asahi Techno Glass) solution containing 2.5 μg of luciferase plasmid prepared in the same manner as in Example 1, 0.52 μg / μl HelΔ1 solution (HelΔ1 is described in the literature) Therefore, the prepared (T. Niidome, K. Takaji, M. Urakawa, N. Ohmori, A. Wada, T. Hirayama, H. Aoyagi, Chain Length of Cationic a-Helical Peptide Sufficient for Gene Delivery into Cells, Bioconjugate Chem. , 10, 773-780 (1999)))) mixed with 10 μl, charge ratio plasmid: HelΔ1- = 1: 2 (negative charge of the phosphate anion of the plasmid: primary amino group in the side chain of the Lys residue of HelΔ1 The molar ratio of the positive charge of the cation) was left at room temperature for 5 minutes.
(3) Add 10 μl of HA solution (HA sodium salt, manufactured by Nacalai Tesque, from microorganism) with HA concentration of 1.12 μg / μl or 2.43 μg / μl, and charge ratio plasmid: HA = 1: 4 or 1: 8 (of plasmid The negative charge of the phosphate anion: the molar ratio of the negative charge of the carboxylate anion of HA) was further allowed to stand at room temperature for 10 minutes.
(4) 50 μl of DMEM was mixed with the prepared complex solution and added to the wells.
(5) Incubated for 3 hours at 37 ° C. under 5% CO ₂ -95% air.
(6) Remove the medium containing the complex, wash the cell surface 2-3 times with PBS (-) (Phosphate Buffered Salts, Dalbecco's Formula (Modified), Without Magnesium and Calcium, Takara), and add a new medium. It was.
(7) After 24 hours of incubation, the cell surface was washed 3 times with PBS (−), and then 200 μl of PicaGene cell lysate was added to each well. After leaving for about 20 minutes, the cells were detached and collected in an eppen.
(8) After centrifugation (15000 rpm, 1 min), Luciferase Assay was performed using the supernatant. Luciferase assay was performed according to the method of PicaGene Luminescence kit.
Note that this cell lysate was used as it was for the Protein Assay. Protein Assay was performed using a Bio-Rad Protein assay kit.
For comparison, gene expression was also examined for those to which HA was not added.
Results The expression activity was 2417% at 21417306.49 (RLU / mg protein) when the charge ratio plasmid: HA = 1: 4, compared with the case where HA was not added (Luciferase Activity 738244.48 (RLU / mg protein)). When the charge ratio plasmid: HA = 1: 8, the expression was increased by 450% with 3339644.80 (RLU / mg protein).

Gene expression by KG6 / HA / Plasmid Complex Experiments similar to Example 2 were performed according to the literature, instead of HelΔ1, KG6 (Lys 6th generation dendrimer, KG6 (M. Ohsaki, T. Okuda, A. Wada, T. Hirayama, T. Niidome, H. Aoyagi, In Vitro Gene Transfection Using Dendritic Poly (L-Lysine), Bioconjugate Chem., 13, 510-517 (2002). , Charge ratio plasmid: KG6 = 1: 4 (negative charge of the phosphate anion of the plasmid: molar ratio of the positive charge of the primary amino group cation of the outermost 6th generation Lys of KG6) Therefore, the luciferase plasmid・ When KG6 / HA ternary complex is formed, plasmid concentration is 0.0125μg / μl, KG6 concentration is 0.0191μg / μl, HA concentration is charge ratio plasmid: HA = 1: 4 (negative charge of phosphate anion of plasmid: (The molar ratio of negative charge of HA carboxylate anion) is 0.0608μg / μl, charge ratio plasmid: HA 1: I became 0.1215μg / μl at the time of 8.
Results The expression activity is 28035% with 1976351064.56 (RLU / mg protein) when the charge ratio plasmid is HA = 1: 4, compared with the case where HA is not added (Luciferase Activity 695554636.82 (RLU / mg protein)). When the charge ratio plasmid: HA = 1: 8, expression increased by 330% 294.0296 (RLU / mg protein) by 330%.

Gene expression in the presence of serum by HelΔ1 / HA / Plasmid Complex Experiments similar to those in Example 2 were carried out. Incubation of the gene complex with cells was performed with 10% serum (Fetal Bovine Serum, Cosmo Bio Co., Ltd.). Manufactured in a medium containing Therefore, when forming a ternary complex of luciferase plasmid, KG6, and HA, the plasmid concentration is 0.0125 μg / μl, the HelΔ1 concentration is 0.0262 μg / μl, and the HA concentration is the charge ratio plasmid: HA = 1: 4 (Phosphate Anion Negative charge: molar ratio of negative charge of the carboxylate anion of HA) was 0.0608 μg / μl, and when the charge ratio plasmid: HA = 1: 8, it was 0.1215 μg / μl.
Results Compared to the case where HA is not added (Luciferase Activity 47936.02 (RLU / mg protein)), when the charge ratio plasmid: HA = 1: 4, 167160.27 (RLU / mg protein) is 350%, When the charge ratio plasmid: HA = 1: 8, the expression increased by 210815.58 (RLU / mg protein) by 210%.

Gene expression by PEI / HA / Plasmid Complex Gene (Luciferase Plasmid), PEI (Polyethyleneimine, Linear, Molecular Weight approx. 25,000, manufactured by Polysciences), HA, 3 component complex, and this complex Chinese hamster ovary The luciferase gene expression was confirmed by interacting with the derived cell CHO.
Operating procedure
(1) The day before the introduction of the complex, Chinese hamster ovary-derived cells CHO were plated at 5 × 10 ⁴ cells / well on a 24-well multiplate and incubated in F12 medium (Ham's F12, manufactured by GibcoBRL) for 24 hours.
(2) Charge ratio plasmid was prepared by mixing 12.5 μl of an aqueous solution containing 2.5 μg of luciferase plasmid prepared in the same manner as in Example 1 with 25 μl of an aqueous solution of HA concentration 4.86 μg / μl (HA sodium salt, manufactured by Nacalai Tesque, microorganism). : HA = 1: 40 (negative charge of phosphate anion of plasmid: molar ratio of negative charge of carboxylate anion of HA).
(3) PEI 1.6: in an amount such that the charge ratio of plasmid: PEI = 1: 5 (negative charge of the phosphate anion of the plasmid: molar ratio of the positive charge of the secondary amino group cation in the PEI main chain) 12.5 μl of an aqueous solution containing μg was added.
(4) PBS (-) (Phosphate Buffered Salts, Dalbecco's Formula (Modified), Without Magnesium and Calcium. Takara) 50 μl was mixed with the prepared complex solution.
(5) The cells were added to 1 ml of F12 medium (Ham's F12, manufactured by GibcoBRL) and incubated for 3 hours at 37 ° C. under 5% CO ₂ -95% air.
(6) The medium containing the complex was removed and a new medium was added.
(7) After incubation for 40 hours, the cell surface was washed three times with PBS (−), and then 200 μl of PicaGene cell lysate was added to each well. After leaving for about 20 minutes, the cells were detached and collected in an eppen.
(8) After centrifugation (15000 rpm, 1 min), Luciferase Assay was performed using the supernatant. Luciferase assay was performed according to the method of PicaGene Luminescence kit.
Note that this cell lysate was used as it was for the Protein Assay. Protein Assay was performed using a Bio-Rad Protein assay kit.
For comparison, gene expression was also examined for those to which HA was not added.
Results The expression activity is 690% expressed in the case of 2360467027.23 (RLU / mg protein) when the charge ratio plasmid is HA = 1:40, compared with the case where HA is not added (Luciferase Activity 342273243.33 (RLU / mg protein)). Went up.

Gene expression in the presence of serum using protamine / HA / plasmid complex The same experiment as in Example 5 was performed using protamine (protamine sulfate, Wako, derived from salmon sperm) instead of PEI, and gene complexes and cells. Was incubated in a medium containing 10% serum (Fetal Bovine Serum, manufactured by Cosmo Bio Co., Ltd.).
However, charge ratio plasmid: protamine = 1: 2 (negative charge of phosphate anion of plasmid: molar ratio of positive charge of arginine side chain guanidino group and N-terminal amino group cation of protamine). Therefore, when forming a ternary complex of luciferase plasmid / KG6 / HA, the plasmid concentration is 0.05 μg / μl, the protamine concentration is 0.0585 μg / μl, and the HA concentration is charge ratio plasmid: HA = 1: 2 (phosphate anion of plasmid Negative charge: molar ratio of negative charge of the carboxylate anion of HA) was 0.1215 μg / μl, and when charge ratio plasmid: HA = 1: 16, it was 0.9721 μg / μl.
Results Compared to the case where HA is not added (Luciferase Activity 1100288.94 (RLU / mg protein)), 8908359.70 (RLU / mg protein) is 890% when the charge ratio plasmid is HA = 1: 2. When the charge ratio plasmid: HA = 1: 16, 60312.332 (RLU / mg protein) increased expression by 550%.

Gene expression in the presence of serum by chitosan / HA (derived from microorganism) / plasmid complex Gene (luciferase plasmid) chitosan (chitosan hydrochloride, molecular weight about 40,000, deacetylation degree 80%, manufactured by Yaizu Suisan Chemical Co., Ltd.) -A three-component complex of HA was prepared, and this complex was allowed to interact with Chinese hamster ovary-derived CHO cells to confirm the expression of the luciferase gene.
Operating procedure
(1) The luciferase plasmid was prepared as follows. pGL3-control vector (Promega, Madison, WI, USA) was propagated in E. coli (competent ceLL DH5, TOYOBO, Lot. 116600) and purified with Qiagen Plasmid Giga Kit (QIAGEN GmbH, Hilden, Germany).
(2) The day before introducing the complex, spread CHO-derived Chinese hamster ovary cell CHO cells to a 24-well multiplate at 5 × 10 ⁴ cells / well, 10% serum (Fetal Bovine Serum, JRH BOPSCIENCES ) Incubated for 24 hours in Ham F12 medium (Nissui Pharmaceutical Co., Ltd.) containing 10,000 units / mg penicillin (Meiji Seika Co., Ltd.) and 1 mg / ml streptomycin (Meiji Seika Co., Ltd.) .
(3) Serum-free ham containing 10 mM MOPS (Morpholinepropanesulfonic acid, Sigma (St. Louis, MO)) prepared in advance to pH 6.5 with 1 N HCl in 1 mg / ml luciferase plasmid solution dissolved in sterile water It was dissolved in F12 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) to 25 μg / ml and incubated for 30 minutes.
(4) Add 1.25 μl of a 4.87 μg / μl HA solution (HA sodium salt, manufactured by Nacalai Tesque, derived from microorganisms) previously dissolved in PBS (−) adjusted to pH 6.5 with 1N HCl, and charge ratio plasmid: HA = 1: 4 (negative charge of plasmid phosphate anion: molar ratio of negative charge of HA carboxylate anion) and incubation for 15 minutes.
(5) Add 1.25 μl of 3.16 μg / μl chitosan solution dissolved in PBS (−) (pH 6.5) to 48.75 μl of Ham F12 medium (10 mM MOPS, pH 6.5, serum-free) and incubate for 30 minutes The mixture was incubated for 15 minutes under the charge ratio plasmid: chitosan = 1: 5 (negative charge of the phosphate anion of the plasmid: molar ratio of the positive charge of the amino group cation of chitosan).
(6) The prepared complex solution was added to cells together with 398.75 μl of Ham's F12 medium with 10% serum (10 mM MOPS, pH 6.5) to make the volume of each well 500 μl, and 3 hours at 37 ° C., 5% CO ₂ -95 Incubated under% air.
(7) The medium containing the complex was removed, and 1 ml of fresh Ham F12 medium containing 10% serum was added.
(8) After 40 hours of incubation, the cell surface was washed 3 times with PBS (−), and then 100 μl of Cell Culture Lysis Reagent (Promega) was added to each well to lyse the cells and collected in an eppen.
(9) After centrifugation (4 ° C., 12000 rpm, 15 seconds), Luciferase Assay was performed using the supernatant. For Luciferase Assay, 20 μl of the supernatant and 100 μl of luciferin (Promega) were placed in a special tube (Promega), shaken gently, and then the absorbance was measured with a luminometer (TD-20 / 20 Luminometer, TURNER DESIGNS). The measurement time was 30 seconds.
Note that this cell lysate was used as it was for the Protein Assay. Protein Assay was performed using a Bio-Rad Protein assay kit.
For comparison, gene expression was also examined for those to which HA was not added.
Results The expression activity is 170% expressed in 302867 (RLU / mg protein) when the charge ratio plasmid is HA = 1: 4 compared to when HA is not added (Luciferase Activity 174570 (RLU / mg protein)) Went up.

Gene expression in the presence of serum by chitosan / HA (derived from Tosaka) / plasmid complex The same experiment as in Example 7 was performed using HA derived from Tosaka (HA sodium salt, manufactured by WAKO) instead of HA derived from microorganisms. And so on. However, the charge ratio plasmid: chitosan = 1: 5 (negative charge of phosphate anion of plasmid: molar ratio of positive charge of amino group cation of chitosan). Therefore, when the ternary complex of luciferase plasmid / chitosan / HA is added to the cells, the plasmid concentration is 2.5 μg / ml, the chitosan concentration is 7.9 μg / ml, and the HA concentration is the charge ratio plasmid: HA = 1: 4 (plasmid When the negative charge of the phosphate anion of (a molar ratio of the negative charge of the carboxylate anion of the HA) was 12.175 μg / ml.
The resulting expression activity is 280% expressed at 486480 (RLU / mg protein) when the charge ratio plasmid is HA = 1: 4 compared to when HA is not added (Luciferase Activity 174570 (RLU / mg protein)) Went up.

Claims (10)

  A nucleic acid introduction carrier comprising a cationic polymer substance and hyaluronic acid or a salt thereof and / or a negatively charged derivative thereof in a state where they are not covalently bonded. The carrier for nucleic acid introduction according to claim 1, wherein hyaluronic acid or a salt thereof or a derivative thereof is negatively charged and has a molecular weight of 10,000 or more.   The nucleic acid introduction carrier according to claim 2, wherein the cationic polymer substance is a protein, polypeptide, dendrimer, polyethyleneimine, chitosan or dextran.   The nucleic acid introduction method according to claim 2, wherein the molar ratio of each charged group in the cationic polymer substance and hyaluronic acid or a salt thereof and / or a negatively charged derivative thereof is 1: 0.01 to 1: 200. carrier.   The carrier for introducing a nucleic acid according to claim 2, wherein the cationic polymer substance is chitosan.   A composition comprising a nucleic acid, a cationic polymer substance, and hyaluronic acid or a salt thereof and / or a negatively charged derivative thereof in a state where they are not covalently bonded.   The composition according to claim 6, wherein the molar ratio of each charged group in the nucleic acid and the cationic polymer substance is 1: 0.1 to 1:50.   The composition according to claim 6, wherein the molar ratio of each charged group in the nucleic acid and hyaluronic acid or a salt thereof and / or a negatively charged derivative thereof is 1: 0.001 to 1: 1000.   The composition according to any one of claims 6 to 8, wherein the nucleic acid is a gene.   The composition according to claim 9, which is used for gene therapy.