JPH09308484A - Carrier for carrying gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a safe and stable carrier for gene transfer formed of a polymer substance obtained by binding a hydrophilic polymer to a polymer containing a residue having a positive electron charge in a physiologically pH range and combining the polymer with a specific amount of gene in a cell or in vivo. SOLUTION: This carrier for carrying gene is formed of a polymer substance obtained by binding a hydrophilic polymer (e.g. polyethylene glycol) to a polymer (e.g. polylysine) such as a polyamino acid comprising a basic amino acid containing a residue having a positive electron charge in a physiological pH range. In the carrier, the polymer substance is combined with a carrying gene in a ratio of a value obtained by dividing weight parts of a polymer part having a positive electron charge in the polymer substance by molecular weight of the residue to 1/300 of weight parts of the carrying gene. The carrier for carrying gene is safe and stable in a cell or in vivo and is useful for gene therapy for carrying out treatment of diseases, means, etc., for obtaining organisms such as new bacteria or animals and plants which have extremely useful properties.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention has a novel therapeutic property for treating genetic and non-genetic diseases, and gene therapy for treating diseases, and has novel properties which are unprecedentedly useful in fermentation, agriculture, livestock, fisheries and the like. Vi as a means to obtain living organisms such as various bacteria and plants
The present invention relates to a gene delivery carrier for vo or in vitro gene transfer.

[0002]

2. Description of the Related Art A technique for directly introducing a gene into a cell or an individual and expressing it is a gene therapy for treating a disease, or a new organism such as a bacterium or plant having a useful property which has never been obtained. As a means, it is expanding rapidly. However, in the process of delivering a gene to a target tissue or cell, for example, when it is administered to an individual, it is cleaved by various nucleolytic enzymes during systemic circulation or is taken up by phagocytic cells. When a cell is taken out of the body and a gene is added to it, it is also cleaved by the nucleolytic enzyme in the serum added to the culture medium. That is, in order to efficiently perform such gene transfer, a gene carrier that is not subject to cleavage in serum and that can stably move in the systemic circulation until the gene can reach the target tissue is essential. However, a carrier sufficient for this purpose has not been developed yet, and this is the hindrance to the progress of gene transfer technology.

[0003]

In order to solve this problem, it is necessary to develop a carrier for gene delivery that is not easily attacked by nucleolytic enzymes and is not taken up by phagocytic cells. But what is known so far is
Nucleic acid-liposome complexes, etc., are particularly easily taken up by phagocytic cells, and have not escaped sufficiently from the attack of nucleolytic enzymes, and the use of viruses as carriers for gene delivery has Due to problems such as the above, there are many dangers when directly introduced into an individual. Therefore, the problem to be solved by the present invention is to provide a carrier for gene delivery, which enables efficient gene transfer to a target tissue or cell and is safe even when introduced into a living body.

[0004]

The present invention is a carrier used for the purpose of causing a gene to act on the body of an animal, a tissue, or a cell, in which a residue having a positive charge within a physiological pH range is introduced into the molecule. Formed by a polymer substance in which a hydrophilic polymer is bound to a polymer substance contained in, and the positive charge of the polymer substance among the polymer substance with respect to 1/300 of the weight part of the gene to be carried. Is a carrier for gene delivery, wherein the ratio of the value obtained by dividing the weight part of the polymer part having the formula by the molecular weight of the residue is 0.5 or more.

The polymer containing a residue having a positive charge in the physiological pH range in the molecule is not particularly limited as long as it protects the gene from the attack of nucleolytic enzymes, but it is a polyamino acid consisting of basic amino acids. Amino acids are also excellent in biocompatibility. Polymers such as lysine, histidine, and arginine are conceivable as such polyamino acids, but polylysine is preferable from the viewpoint of easy production.

The hydrophilic polymer to be bonded to such a polymer having a positive charge may be any one as long as it has a function of preventing the nucleic acid from being taken up by phagocytic cells, but polyethylene glycol, dextran, etc. The water-soluble polysaccharides mentioned above are listed, and polyethylene glycol is particularly preferable in terms of availability and selection of one having a suitable molecular weight distribution. Moreover, the molecular weight of the hydrophilic polymer is 1,
It is preferably from 0000 to 100,000, and it is preferable to bind in a range of 0.1 to 10 by weight ratio to a polymer containing a residue having a positive charge in a physiological pH range in the molecule, Even if a polymer containing a residue having a positive charge in a plurality of physiological pH ranges is bound to one hydrophilic polymer chain, or conversely, a residue having a positive charge in a physiological pH range remains. A plurality of hydrophilic macromolecules may be bonded to one macromolecule containing a group in the molecule, and it is possible to make an optimal combination depending on the structure and molecular weight of each macromolecule to be used.

The gene carried by such a carrier is typically a plasmid in which a gene encoding a specific protein and a promoter are bound, an antisense DNA which binds to the mRNA of the specific gene and suppresses its expression, and the like. However, it is not limited to being DNA or RNA. The ratio of such a polymer to a gene is a value obtained by dividing the weight part of the polymer part having the positive charge of the polymer by 1/300 of the gene weight part by the molecular weight of the residue. The ratio (hereinafter sometimes referred to as the “ratio”)
It is necessary to form a complex with 0.5 or more, preferably 1 or more. When polylysine is used as the polymer having the positive charge, the polylysine (as the molecular weight of lysine residue, ie, the molecular weight of lysine-18 = 124) of 1/300 parts by weight of DNA is used. 1 part by weight
A ratio of 1/24 to 0.5 or more, that is, 1 part by weight of DN
For A, the polymer was polylysine (hereinafter, this may be referred to as “polylysine / DNA ratio”), 600
More than 124 (about 0.2) parts by weight, preferably 300
It is necessary to use more than 124 (about 0.4) parts by weight.

[0008]

EXAMPLES Next, the present invention will be described more specifically by showing Examples and Test Examples, but it goes without saying that the scope of the present invention is not limited thereby. (1) Synthesis of polylysine-polyethylene glycol conjugate Polyethylene glycol (molecular weight =
2,4-bis (o-methoxypolyeth) in which two molecules of 5,000) are bound
yleneglycol) -6chloro-S-triazine (# 68 manufactured by Seikagaku Corporation)
0032) was dissolved to 37 mg / ml, and 0.33 ml thereof was similarly dissolved at a concentration of 25 mg / ml of sodium borate buffer (pH 9.0) Poly-L-Lysine (Sigma # p-7890) Added to 1 ml. After reacting at 37 ° C for 26 hours in the dark, 1 mM EDT
After adding 5 ml of 10 mM Tris-Cl buffer containing A, it was concentrated by centrifuging at Centriprep-30 at 1500 xg for 15 minutes. The concentrated solution was further concentrated in the same manner with Centriprep-30 to which 5 ml of 10 mM Tris-Cl buffer containing 1 mM EDTA was added, and finally 8.66 mg of the conjugate was obtained as Poly-L-Lysine.

(2) Preparation of sample solution (DNA / polylysine-polyethylene glycol conjugate complex) As DNA, plasmid pEF321-CAT (Kim, DW, Uetsk
i, T., Kaziro, Y., Yamaguchi, N. and Sugano, S. Use of
the human elongation factor 1α promoteras a vee
rsatile and eficient expression system. Gene 91 (19
90), 213-223.).
To 150 μg of DNA, 75 μl of 700 mM saline was added and dissolved. To this, the polylysine-polyethylene glycol conjugate prepared in (1) dissolved in 75 μl of 700 mM saline was added as polylysine with stirring at 30.9, 47.5, and 65.0 μg, respectively, and 5 M saline was added to give a final salt concentration of 1 M. And sample solutions 1, 2, 3 (ratio = about 0.5, about 0.8, about 1,
The polylysine / DNA ratio was 0.206, 0.317, 0.433).

(3) Preparation of Comparative Solution Comparative Solution 1: 30 μl of 5M saline was added to 120 μl of ultrapure water to prepare a 1M saline solution. Comparative solution 2: 120 μl of ultrapure water in 150 μg of DNA (pEF321-CAT)
And 30 μl of 5M saline were added to make a 1M saline solution. Comparative solution 3: DNA (pEF321-CAT) 150 μg was added to 700 mM saline 75
μL was added and dissolved, and 47.5 μg of polylysine dissolved in 75 μl of 700 mM saline was added with stirring to this, and 5 M of this was added.
Saline was added to bring the final salt concentration to 1M.

(4) Evaluation of stability in the body 1 100 μl of the sample solution 2 and the comparative solutions 1, 2, and 3 were Balb / c.
Administered from the tail vein of mice (4 weeks old, body weight about 25 g),
50 to 100 μl of blood was collected at 10, 20, and 60 minutes after administration. Equal amount of lysate (100mM TrisCl (pH7.5
~ 8.0), 100 mM, 2% SDS) was added and immediately dissolved by stirring. Proteinase K (Wako Pure Chemical Industries, Ltd. # 160-14001) was added to a final concentration of 200 μg / ml, left overnight at 37 ° C., and then DNA was extracted by a conventional method. The extracted DNA is Southe
rn Bloting method (Southern, EM: Detection of specifi
c sequences among DNA fragments separated by gel e
lectrophoresis. J. Mol. Biol. 98 (1975), 503-517.). PEF321-CAT was used as a probe.

(5) Evaluation of stability in the body 2 100 μl of the sample solutions 1, 2, 3 and the comparative solution 3 were administered through the tail vein of Balb / c mice (4 weeks old, body weight: about 25 g).
50 μl of blood was collected before and 10 and 20 minutes after administration, and the same amount of lysate (100 mM TrisCl (pH 7.5-8.
0), 100 mM, 2% SDS) was added and immediately dissolved by stirring. Thereafter, the same operation as in (4) was performed to analyze the DNA.

(6) Results of evaluation of internal stability The results are shown in FIGS. As shown in FIG. 1, the band of the administered DNA was observed only in the sample solution, and it was clear that this complex stably retained the DNA in the body. FIG. 2 shows that the stability of DNA is higher as the amount of complex is larger. That is,
When the polylysine / DNA ratio of the complex was 0.206, DNA was detected after 10 minutes.
Fragments were slightly observed, and at the same ratio of 0.317, 10 minutes later, and at the same ratio of 0.433, DNA of the same length as the plasmid was observed even after 20 minutes, showing that it was stably transported in blood. .

[0014]

As described above, when the DNA is administered as it is or with the polylysine alone, the DNA in the body is
Was very unstable and was not recovered from the blood.
However, the carrier of the present invention is formed by a polymer substance in which a hydrophilic polymer is bound to a polymer containing a residue having a positive charge in the physiological pH range, and the weight part of the gene to be carried is part by weight. The ratio to the value obtained by dividing the weight part of the polymer part having the positive charge in the polymer substance by the molecular weight of the residue is about 0.5, and the fragment is observed. Since the full length DNA was recovered after about 10 minutes at about 0.8 and after about 20 minutes in the complex with about 1, the carrier of the present invention shows that the carrier of the present invention is degraded by nucleolytic enzymes or phagocytic cells. It was shown that the gene can be protected from phagocytosis by the bacterium, and that the gene administered into the living body such as cells, tissues, or individuals can be stably carried in them.

[Brief description of drawings]

FIG. 1 is a diagram showing the in-vivo stability of a gene carried on the carrier for gene delivery of the present invention.

FIG. 2 is a diagram showing the in-vivo stability of a gene carried on the carrier for gene delivery of the present invention.

Claims (4)

[Claims] 1. A 300 parts by weight part of a gene formed by a polymer substance in which a hydrophilic polymer is bound to a polymer containing a residue having a positive charge in a physiological pH range and carried by the hydrophilic polymer.
The ratio of the value obtained by dividing the weight part of the polymer portion having the positive charge in the polymer substance by the molecular weight of the residue to
A carrier for gene delivery, characterized in that the macromolecular substance and the gene are combined at a ratio of 0.5 or more. 2. The carrier for gene delivery according to claim 1, wherein the polymer containing a residue having a positive charge in a physiological pH range in the molecule is a polyamino acid consisting of a basic amino acid. 3. The carrier for gene delivery according to claim 2, wherein the polyamino acid is polylysine. 4. The gene delivery carrier according to claim 1, wherein the hydrophilic polymer is polyethylene glycol.