JPH1121255A - Functional molecule transporter and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject transporter capable of efficiently releasing functional molecules such as an anti-sense chain nucleic acid, and useful for e.g. gene therapy, by mixing H1-free subunits among histone subunits in a specific order. SOLUTION: For H1-free histone subunits consisting of H2a, H2b, H3 and H4, among histone subunits, H2a and H2b are first mixed with each other followed by adding H3 and then H4 to the mixture to form the final mixture to obtain the objective functional molecule transporter, which is capable of forming a complex with a functional molecule consisting of a nucleic acid such as anti-sense oligonucleotide, phosphorothioate-type anti-sense oligonucleotide, ribozyme or plasmid, transporting the complex, and efficiently releasing the functional molecule, therefore, useful for e.g. gene therapy.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel functional molecule transporter for introducing a functional molecule into cells, a method for producing the same, a complex of the functional molecule and a functional molecule transporter, and a complex thereof. It relates to a manufacturing method. More specifically, the present invention is useful mainly for gene transfer such as gene therapy,
Histone protein-derived functional molecule transporter for efficiently and safely introducing functional molecules such as nucleic acids into cells and a method for producing the same, and functional molecule such as nucleic acids and histone protein-derived functional molecule transport The present invention relates to a complex with a body and a method for producing the same.

[0002]

2. Description of the Related Art Gene therapy is a completely new treatment method for treating a disease by introducing and expressing a foreign gene acting as a drug (hereinafter referred to as "drug gene") in the body. Diseases that can be expected to have therapeutic effects by gene therapy include all diseases caused by genetic abnormalities, whether congenital or acquired, but are particularly fatal and have no established treatment. It is considered to be a very useful treatment for AIDS.

[0003] In gene therapy, an additional gene therapy (Augmentation Gene Th) in which an abnormal (causal) gene is left as it is and a new (normal) gene is added.
erapy) and replacement gene therapy (Replacement Gene) which replaces abnormal genes with normal genes.
Therapy).

[0004] Clinical applications of gene therapy include 1989
Since the first gene therapy clinical trial in the United States in 2000, clinical trials have already started in Italy, the Netherlands, France, the United Kingdom, and China. However, in clinical applications of gene therapy, development of optimal gene forms and methods for efficiently and safely introducing drug genes into target cells has become one of the major technical issues.

[0005] In the early 1980's, application of physical techniques such as microinjection was attempted, but it was not possible to stably and efficiently introduce a drug gene required for treating a disease, so that clinical application was not achieved. Was.

[0006] Subsequently, a recombinant virus (virus vector) as a carrier for efficiently introducing a foreign gene into cells was developed, and the clinical application of gene therapy became possible for the first time (Miller, AD Human Ge.).
ne Therapy, 1, 5-14, 1990). However, it is necessary to culture a large amount of cells in order to produce the amount of viral vector required for treatment, which is very expensive compared to the production cost of general drugs. In some cases, cytotoxicity, immunogenicity, etc. are confirmed, and there are still many improvements.

[0007] As a conventional technique relating to a non-viral transporter, the viewpoint has been narrowed down to a method via a receptor attached to a cell surface or a method via a cationic lipid. Examples of oligonucleotide introduction via a receptor include transferrin and asialoglycop (asialoglycop).
There is a report that attempts to transport this into a cell using a complex of a ligand and DNA, such as protein (protein). ((1) Wagner, E., Proc. Na
tl. Acad. Sci. , USA 88, 425
5-4259 (1991), (2) Wilson,
J. M. , J. et al. Biol. Chem. , 267,
11483-11489 (1992)) In addition, many systems using liposomes, which are structures having a lipid bilayer membrane structure similar to biological membranes, are often found. For example, there is a report that an oligonucleotide drug is encapsulated in a liposome to improve the stability, and at the same time, the affinity for a target cell is increased by chemically modifying the liposome. ((1) Felgner, PL.
, Proc. Natl. Acad. Sci. ,
USA 84, 7413-7417 (1987),
(2) Malone, R .; W. , Proc. N
atl. Acad. Sci. , USA 86, 60
77-6087 (1989), (3) Hawley
-Nelson, P .; , Focus 15, 73-7
9 (1993)) Further, utilizing the fact that a nucleic acid has a negative charge, an attempt is made to form a complex with a synthetic polymer having a positive charge, thereby delivering the complex to a target cell or a cell. There are also research reports. ((1) Nature, 2
71, 130-135 (1978), (2)
Biol. Chem. , 262, 4429-443
2 (1987), (3) Biol. Che
m. , 263, 14621-1462 (1988),
(4) Proc. Natl. Acad. Sc
i. , USA 87, 3410-3414 (199).
0)) In studies focusing on the artificial synthetic polymers listed,
Use of these transporters has ensured stable transport of nucleic acids, efficient introduction into cells, and an increase in targeting functions. However, release of the transported nucleic acid from the transporter is in the midst of progress.

On the other hand, there is also an attempt to use a natural protein derived from a living body as a transporter. For example, in the following literature, studies have been conducted using a histone or the like as a nuclear protein and using it as a transporter for plasmid DNA.
((A) Masaru Yamaizumi, Nat
ure, 273, 782-784 (1978). ,
(B) Yasufumi Kaneda, SCIEN
CE, 243, 375-378 (1989), (C)
Mirror Jam and David
S. Goldfarb, Cell, 60, 999-
1008 (1990). , (D) Jian Che
n. , Human Gene Therapy, 5, 4
29-435 (1994)) In the literature (A), it is shown that the transfer of histones to the cell nucleus occurs very smoothly, and in the literature (B),
By introducing a nucleoprotein into a liposome encapsulating plasmid DNA, the uptake into cultured cells is improved. In the literature (C), the uptake into the cell nucleus is improved by introducing cytochrome C into histone H1. In the literature (D), the incorporation of the plasmid DNA into a galactosylated histone is examined to improve the uptake into the cell and the expression of the oligonucleotide.

[0009] The liposome transporter has a major drawback in that it is difficult to release the oligonucleotide contained therein. In addition, it has been previously pointed out that a polymer having a positive charge such as a synthetic amino acid transporter has high cytotoxicity (Bio-conjugate Chem).
m. , 1, 149-153 (1990)). At the same time, when these synthetic polymers are administered into a living body, they are recognized as foreign substances, and there is a concern about the influence on the immune system such as anaphylactic shock, which has not been realized yet.

[0010] In eukaryotes, chromosomal DNA binds to histones to form a complex called chromatin. Histones are usually H1, H2a, H2b, H3,
It is known that it consists of five subunits of H4 and has a very high content of basic amino acids. The fact that histones have many positive charges is one key feature of this molecule. Therefore, it is possible to interact with a substance having a negative charge such as DNA. The primary structure of histone subunits other than H1 is very well conserved even if the species is different, and it is known that histone itself is reconstituted even when the histone subunits of different species are combined. It is also widely known that histones do not have special enzymatic activity despite being a type of protein, which is an advantageous feature in using histones as functional molecular transporters. it is conceivable that. Furthermore, the histone subunits H2a and H
It has been reported that an amino acid sequence having a function as a cell nuclear transport signal exists in the primary structure of 2b (Robert B. Moreland, Molec).
ullar and Cellular Biology
, 7, 4048-4057 (1987)). Utilizing the properties of these histones, the histone subunit is converted to H2a
There has been disclosed a method for producing a histone transporter for oligonucleotides, which comprises reconstituting histones by mixing in the order of → H2b → H3 → H4 → H1 (Japanese Unexamined Patent Publication No. Hei 8-
301791).

However, although the stability of the encapsulated nucleic acid in the functional molecule transporter has been enhanced, the release of nucleic acid is low, and the properties of the functional transporter cannot be said to be sufficient.

[0012] In particular, a transporter that efficiently releases once bound or encapsulated nucleic acid has not yet been developed.
This remains an important issue to be solved in developing a functional molecular transporter.

[0013]

DISCLOSURE OF THE INVENTION The object of the present invention is based on the above-mentioned prior art, and the present inventors have made various studies and found that the polypeptide can be stabilized by constructing a polypeptide by regular mixing of histone subunits. To provide a functional molecule transporter capable of efficiently releasing a functional molecule contained therein, and a method for producing the same. In addition, in the process of constructing the polypeptide, the functional molecule and the It is an object of the present invention to provide a complex with an acidic molecule transporter and a method for producing the same.

[0014]

Means for Solving the Problems The present inventors have conducted intensive studies in order to achieve the above-mentioned object, and as a result, have found a transporter that efficiently releases contained functional molecules.

That is, the present invention provides a functional molecule transporter that does not contain H1 among histone subunits.

The present invention further provides a method for producing a functional molecule transporter comprising mixing histone subunits in the order of H2a → H2b → H3 → H4; and a functional molecule transporter obtained by this method. I will provide a. The functional molecule transporter obtained here belongs to the functional molecule transporter not containing the histone subunit H1.

Further, the present invention provides a complex of a functional molecule and a functional molecule transporter; and a histone subunit.
In a process for producing a functional molecule transporter, which comprises mixing in the order of H2a → H2b → H3 → H4, there is provided a method for producing a complex, characterized by coexisting a functional molecule.

[0018] Here, comparing the method of the present invention with the method of the above-mentioned Japanese Patent Application Laid-Open No. Hei 8-301793, which is a prior application of the present invention, it shows that the histone subunit is converted from H2a → H
In contrast to a method for producing a histone transporter for oligonucleotides, which comprises reconstituting a histone by mixing in the order of 2b → H3 → H4 → H1, the present invention provides a method for producing a histone subunit of H2a → H2b → This is a method for producing a functional molecule transporter, which comprises mixing in the order of H3 → H4.

In short, in the present invention, by eliminating the step of mixing the H1 subunit, a transporter capable of efficiently releasing the encapsulated functional molecules could be obtained.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The method for producing a functional molecule transporter according to the present invention is characterized in that the mixing order of histone subunits and the H1 subunit are not used. As described above, this functional molecule transporter is composed of H2a, H2b, H3, and H4 except for the histone subunit H1, and is reconstructed by mixing in the order of H2a → H2b → H3 → H4. However, the order of H2a and H2b may be reversed, but it is necessary to first create a mixture of H2a and H2b. The subunits are then added to the mixture in the order H3, H4. As a method of mixing the subunits, a factorial of 4, that is, 24 ways can be considered. However, in the process of constructing the functional molecular transporter, when mixed in an order other than the method of the present invention, cloudiness or precipitation occurs and re-generation occurs. No build histone formation was observed. The method for producing the functional molecule transporter of the present invention is schematically shown in FIG.

Examples of subunits used for constructing the functional molecule transporter of the present invention include Histone H2a, Histone H2b and Histone H manufactured by Boehringer Mannheim.
3, histone H4.

In order to produce a functional molecule transporter by the method of the present invention, first, each histone subunit is treated with a phosphate buffer (pH 7) having an ionic strength of 0.01 to 0.05, preferably 0.02. After dissolving in 2), each concentration is 5 × 10 ⁻⁵ M to 1 × 10 ⁻⁴ M, preferably 1 × 10 ⁻⁵ M.
Adjust to M. Histone reconstruction is H2a
After mixing the solution and the H2b solution, the H3 solution is applied thereto, and finally the H4 solution is added. When mixing these subunit solutions, it is necessary to prepare them in advance so that each subunit has an equimolar number. The reconstituted histone obtained according to the present invention is useful as a functional molecular transporter.

Further, in order to produce a complex of the functional molecule transporter of the present invention and a functional molecule, a functional molecule is added in the process of producing the functional molecule transporter described above. The functional molecule may be added at any time, but preferably after mixing H2a and H2b, more preferably H2a and H2b.
It is preferable to add the functional molecules to the mixture of H2a and H2b after adding a and H2b and before adding H4, and more preferably to add the subunits in the order of H3 and H4.

The functional molecule to be included is a nucleic acid, and the size and type of the molecule are not limited. Examples of the type of the nucleic acid include linear double-stranded DNA, circular double-stranded DNA, oligonucleotide, RNA, and the like.
It is selected from antisense nucleotides, phosphorothioate antisense nucleotides, ribozymes and plasmids.

[0025]

EXAMPLES The present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. In the following examples, a series of operations were performed at around room temperature.

Synthesis and labeling of a model oligonucleotide which can be used as a functional molecule were performed by the following method.

[Synthesis of Model Oligonucleotide] FIG.
Were synthesized using a DNA synthesizer (type 380B or type 392, manufactured by Applied Biosystems). All amidite reagents used were those manufactured by Millipore. In FIG. 2, adenosine nucleotide is A, guanosine nucleotide is G,
C for cytosine nucleotides and T for thymidine nucleotides
It is described. These are phosphoramidite methods using a t-butyldimethylsilyl group as a protecting group for a 2′-hydroxyl group (Nucleic Acid Res., Vo.
l. 17, 7059-7071 (1989)). Oligonucleotide purification is described in the literature (Nucl
eic Acid Res. , Vol. 19,5125-
5130 (1991)).

[Labeling of Oligonucleotides] The ³² P labeling of oligonucleotides is performed by using oligonucleotides (1.8
3OD, 260 units, 5 pmol) 27.3 μl,
Autoclaved distilled water 15.7μl, γ- ³² P-A
1 μl of TP, 10 parts of T4-polynucleotide kinase kit (Takara Shuzo Co., Ltd., Code No. 2030)
× After mixing 1 μl each of kinase buffer and kinase, 3
The reaction was performed at 7 ° C. for 1 hour.

H2a → H2b → H used in the following examples
The reconstituted histone constructed by enclosing the oligonucleotide in the order of 3 → H4 → H1 is the same as the reconstituted histone of JP-A-8-301791.

Example 1 <Measurement of Absorbance and Formation of Precipitation> Four kinds of histone subunits were dissolved in a phosphate buffer (pH 7.2) having an ionic strength of 0.02, and each concentration was 1 × 10 ⁻⁵ M. Adjust so that Each subunit and oligonucleotide solution (about 5 pmol / μl) were mixed in the order as shown in FIG.

The turbidity and precipitation of the resulting product were reduced to 360
Spectroscopically by measuring absorbance in nm.

FIG. 3 shows the results of the absorbance measurement. The vertical axis shows the mixing order of the histone subunits, and the horizontal axis shows the absorbance at 360 nm, that is, the turbidity of the system. As a method of mixing the subunits, a factorial of 4, that is, 24 ways can be considered. In the construction of the functional molecule transporter,
When mixed in the order of H2a → H2b → H3 → H4, the system became cloudy. On the other hand, in the case of mixing based on the above regularity, no formation of cloudiness or precipitate was observed in the system. After adding the oligonucleotide having the above concentration to a mixed solution of H2a and H2b, H3, H2
An oligonucleotide / functional molecule transporter complex was prepared by mixing the subunits in the order of 4, but no turbidity or formation of a precipitate was observed in the system. From these results, a functional molecular transporter was obtained by the regular mixing of the present invention.

[Example 2] <Measurement of circular dichroism (CD)> The three-dimensional structure of a histone protein in a solution was examined by CD measurement. J-720 type from JASCO Corporation was used as a CD measuring device, and a cylindrical cell having an optical path length of 1 mm was used as a cell. Scanning was performed four times in the wavelength range of 200 to 250 nm at 25 ° C. The spectrum was determined.

FIG. 4 shows natural histones (Histone, trade name, manufactured by Boehringer Mannheim) (1) purchased from Boehringer, and histones (2), H2 of Boehringer manufactured by mixing oligonucleotides.
Reconstructed histones (3) constructed by enclosing oligonucleotides in the order of a → H2b → H3 → H4 → H1, and functional molecule transporters (4) of the present invention constructed in the order of H2a → H2b → H3 → H4 3 shows a CD spectrum of a functional molecule transporter (5) of the present invention containing an oligonucleotide. The five CD spectra were perfectly consistent within the accuracy of the instrument.

From these results, it is found that the functional molecule transporter of the present invention (4), the functional molecule transporter of the present invention containing an oligonucleotide (5), and a mixture of a natural histone and an oligonucleotide ( 2) was shown to have the same steric structure as the natural histone (1). This result suggests that the functional molecule transporter of the present invention has a function close to a natural histone, at least at the level of the physiological activity of the protein.

Example 3 The amount of oligonucleotide released from the functional molecule transporter of the present invention was examined by the following method.

First, the oligonucleotide was labeled with ³² P by the above-described method, and then a natural histone containing the oligonucleotide, a reconstructed histone containing H1, and a functional molecule transporter were prepared. That is, using natural histones, reconstituted histones, and functional molecule transporter subunits of the present invention, oligonucleotides labeled with ³² P-ATP were used as drugs, and three types of oligonucleotides / functional A molecular transporter complex solution was prepared.

An ultrafiltration tube having the ability to fractionate only the oligonucleotide without passing through the natural histone, the reconstituted histone, and the functional molecule transporter subunit of the present invention, was used for the three samples. (Ultra-free C3-GCUFC3TGCOO, manufactured by Nippon Millipore Limited), and the release of oligonucleotides from various complexes was examined by the following three methods.

(Method 1) A 0.01 N hydrochloric acid (Wako Pure Chemical Industries, Ltd., 075-2431) solution was added to a functional molecule transporter solution containing an oligonucleotide to adjust to an acidic pH.

(Method 2) Urea was added to a functional molecule transporter solution containing an oligonucleotide so as to have a final concentration of 7M.

(Method 3) The functional molecule transporter solution containing the oligonucleotide was heated to 85 ° C.

After all the operations, the mixture was allowed to stand still for 30 minutes,
Centrifuged at 00 rpm for 15 minutes. Only the free oligonucleotide released from the functional molecule transporter by this centrifugation was collected in the lower layer. The lower layer solution was subjected to electrophoresis on a 20% polyacrylamide gel, and the remaining oligonucleotide was quantified using a bioimaging analyzer (BAS2000) manufactured by Fuji Photo Film Co., Ltd. Then, the free oligonucleotide amount (%) was calculated from the quantitative value. These calculation results are shown in FIGS.

FIG. 5 shows the degree of oligonucleotide released by the acid denaturation of histone or a functional molecule transporter. No release of the oligonucleotide was observed in the case of the remodeled histone containing the natural histone and the H1 subunit from Behringer. On the other hand, the release of the oligonucleotide from the functional molecule transporter of the present invention started around pH 4.5 of the functional molecule transporter solution, and almost 100% of the oligonucleotide was released near pH 3. These results suggest that when the functional molecule transporter containing the oligonucleotide is taken into cells by the endocytosis route, the encapsulated oligonucleotide may be released by the acidic pH of the lysosome. is there.

FIG. 6 shows the observation of the degree of oligonucleotide released by urea denaturation of histone or a functional molecule transporter. No release of the oligonucleotide was observed in the case of the remodeled histone containing the natural histone and the H1 subunit from Behringer. On the other hand, the release of the oligonucleotide from the functional molecule transporter of the present invention is caused by the urea concentration of 5 M of the functional molecule transporter solution.
Approximately 80% of the oligonucleotides were released near the high concentration of urea, starting near. This result suggests that the histone subunit H1 strengthens the structure of histone, and that the functional molecule transporter lacking this subunit has a very flexible structure against external conditions.

FIG. 7 shows the degree of oligonucleotide released by thermal denaturation of histone or a functional molecule transporter. Due to thermal denaturation in the temperature range of 25 ° C. to 85 ° C., no release of the oligonucleotide was observed in the case of the reconstructed histone containing the natural histone and the H1 subunit from Boehringer. On the other hand, the release of the oligonucleotide from the functional molecule transporter of the present invention started around 50 ° C. of the functional molecule transporter solution, and almost 70% or more of the oligonucleotide was released at around 80 ° C. It can be seen that these results are also very flexible with regard to external energy effects.

[0046]

As described in detail above, the present invention provides a functional molecule transporter capable of efficiently releasing encapsulated functional molecules. Such functional molecule transporters can efficiently and safely introduce functional molecules such as nucleic acids into cells during gene therapy, and contribute to the development of gene transfer technology and other gene transfer technologies. It is.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a method for constructing a functional molecule transporter.

FIG. 2 shows the nucleotide sequence of an oligonucleotide used as a model drug.

FIG. 3 is a graph of absorbance measurement comparing the random mixing of histone units with the method of the present invention.

FIG. 4 is a graph comparing circular dichroism spectra of various histone substances.

FIG. 5 is a graph showing the results of oligonucleotide release tests at various pHs.

FIG. 6 is a graph showing the results of oligonucleotide release tests at various urea concentrations.

FIG. 7 is a graph showing the results of an oligonucleotide release test at various temperatures.

Claims (7)

[Claims] 1. A functional molecule transporter that does not contain H1 among histone subunits. 2. The histone subunit is changed from H2a to H2.
The functional molecule transporter according to claim 1, which is obtained by mixing in the order of b → H3 → H4. 3. The histone subunit is changed from H2a to H2.
b → H3 → H4 in the order of:
A method for producing a functional molecule transporter according to claim 1 or 2. 4. A complex of a functional molecule and the functional molecule transporter according to claim 1 or 2. 5. The complex according to claim 4, wherein the functional molecule is a nucleic acid selected from an antisense oligonucleotide, a phosphorothioate-type antisense oligonucleotide, a ribozyme and a plasmid. 6. The histone subunit is changed from H2a to H2.
5. The method for producing a complex according to claim 4, wherein the functional molecule is coexistent in the step of producing the functional molecule transporter, which comprises mixing in the order of b → H3 → H4. 7. The method according to claim 6, wherein the functional molecule is added after mixing H2a and H2b.