JPH09107964A - Gene introduction and complex used therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject safe complex useful for introducing an extraneous gene into an animal cell including a human cell and having good gene introduction efficiency by binding three components of F type α2 -macroglobilin, a molecule having characteristics associating with DNA and DNA. SOLUTION: Three components of (A) F type α2 -macroglobilin as a ligand for gene introduction, (B) a molecule having characteristic associating with DNA and (C) DNA are bound to provide the objective complex for gene introduction. A method comprising associating, e.g. a bound body obtained by directly binding the molecule A to the molecule B or binding the molecule A through a cross-linking agent to the molecule B with the molecule C is adapted in order to obtain the complex.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for introducing a foreign gene into animal cells including human cells, and a complex for gene introduction used in this method.

[0002]

2. Description of the Related Art A technique for introducing a foreign gene into a cell, that is, a gene introduction technique is used to produce a foreign substance by constructing a recombinant cell, breeding an animal or plant by introducing a gene into a primordial germ cell or a totipotent cell, and further to human. It is one of the basic technologies of biotechnology that extends to gene therapy by introducing genes into cells.

Various gene transfer methods and gene transfer media have already been devised in order to introduce gene DNA into cells and express the gene function in the cells.

The gene transfer method according to the prior art is described in the textbook on gene manipulation, for example, "Maniatis, T., Fr.
itsch, E .; F. , Sambrook, J .; Mol
ecological Cloning. A laborato
ry Manual, 2nd Edition, 3: 1
6.30-16.55 (1989), Cold Spr.
ing Harbor Laboratory et al., or a review (PL Felgner, Laborato)
ry Invest, 68: 1-3 (1993)) and the like, but in summary, they are roughly classified into three types. The first is a method of introducing genetic DNA into cells by mechanical or physical means. The second is a method of introducing a gene DNA into a cell via a substance having an affinity in a non-chemical bond with a molecule on the cell surface. The third method is to use particles of microorganisms such as viruses that are infectious to cells.

The first method is typically a method in which a capillary tube is inserted into cells under a microscope and gene DNA is introduced through the capillary tube (microinjection method). In addition, in a solution containing DNA, There are a method of indiscriminately making holes in cells to introduce DNA (pricking method), a method of using electric pulses as a means for making holes in cells (electroporation method), and the like.

The representative of the second method is D in solution.
A gel formed from NA and calcium phosphate utilizes the property of being easily taken up by cells, and is called a calcium phosphate method, which is widely used as an in vitro gene transfer technique. As a similar method, DE
AE dextran polymer (McCutchan, Pag
ano, J .; Natl. Canc. Inst. , 41:
351 (1968)), polybrene (Kawai,
S. , Nishizawa, M .; , Mol. Cell.
Biol. , 4: 1172 (1984)), polyornithine (Dong, Y., Skoultchi, A .;
I. , Pollard, J .; W. , Nucleic A
clds Res. 21: 771-772 (199
3)) and other basic macromolecular substances and gene DNA are associated with each other, the aggregate has an electrostatic affinity with the cell surface substance,
A method that utilizes the fact that cells are easily taken up by the mechanism of endocytosis is used.
Examples include the liposome method and the like, in which A is encapsulated in an artificial lipid membrane vesicle (liposome), the liposome is fused with the cell membrane due to the affinity between the liposome and the cell membrane, and the encapsulated DNA is taken into the cell. To be A method of associating DNA with a molecule in which a basic polymer such as polylysine is combined with a positively charged lipid and bringing the DNA into contact with a cell (lipofectin method, Fagner, PL, Rin
gold, G .; M. , Naure, 337: 387-3.
88 (1989), X. Zhau, A .; L. Kliba
nov, L.N. Huang, Biochim. Bioph
ys. Acta, 1065: 8-14 (1991)) has a high gene transfer efficiency and considerably improves the drawbacks of the liposome method and the like.

Furthermore, a method has been devised in which DNA is introduced through a ligand molecule having a specific affinity with a receptor existing on the cell surface (ligand complex method, G. et al.
Y. Wu, C.I. H. Wu, J. Biol Chem. ,
263: 14621-14624 (1988)). In this method, as the target receptor, asialoglycoprotein receptor of hepatocytes (NR Chowdhury,
C. H. Wu, G .; Y. Wu, P.W. C. Jernen
i, V. R. Bomminenl, J. et al. R. Chowd
hury, J.M. Biol. Chem. , 268: 112
65-11271 (1993)), transferrin receptor (Wagner, E., Zenke, M., Cot.
ten, M. , Beug, H .; , Birnstiel,
M. L. Proc. Natl. Acad. Sci. ,
87: 3410-3414 (1990), E.I. Wagne
er, Zatalkal F .; Cotten, M .; K
irlappos, H .; Mechter, K .; , Cu
Riel, DT. Proc. Natl. Acad. ,
Sci. , 89: 6099-6103 (1992)),
Receptors for multimeric immune proteins (T. Ferkol,
C. S. Kaetzel, R.M. B. Davis, J .; C
lin. Invest. , 92: 2394-2400
(1993)) has been used.

The third method is a typical method using virus particles, in which the desired gene DNA has been introduced by a recombination technique and further unnecessary traits such as pathogenicity have been removed. Is produced, and the gene DNA is introduced by binding it to the cell via a receptor that is present on the surface layer of the cell and specifically binds to virus particles (Friedmann, T., Science).
e, 244: 1275-1281 (1989), And.
erson. W. F. , Science, 226: 40.
1-409 (1984)). A retrovirus, an adenovirus, a vaccinia virus, or the like is used as a medium virus.

[0009]

There is an extremely efficient gene transfer method among the above-mentioned methods, but it is an obstacle to cells, versatility of application to various cells, gene transfer efficiency, safety of medium, etc. Was not always a satisfactory method.

For example, the physical methods such as the pricking method and the electroporation method, which are the first method, require that the cells be brought into a physical device, so that the damage to the cells can be ignored. However, there is a limit to the use for medical purposes such as gene therapy.

The second method, which is a basic polymer association method such as the calcium phosphate method and the DEAE dextran method, is simple and most often used for gene transfer into cultured cells because no special device is required. It is used. However, these media are not always high in gene transfer efficiency and may cause damage to cells, and thus their use in gene therapy and the like is still limited. Also, including the lipofectin method, it has the property of indiscriminately binding to substances with a positive charge, so it is limited to use in isolated cells, and it is suitable for gene transfer in a state containing a large amount of biological substances. Is left with various problems.

Furthermore, the third method, which uses a viral vector, has high transfection efficiency, little cell damage, and gene transfer specific to cells even in the presence of a large amount of biological substances. From what you can do,
Most often used for gene therapy. However, not only is it extremely time-consuming to prepare, but since virus particles containing various heterologous genes other than the target gene or heterologous antigenic substance are to be introduced into cells, its safety and side effects must be carefully monitored. Although it has been considered, it is a general trend to avoid it if there is a way to avoid using it.

Among the second methods, the ligand complex method is highly safe in that it does not need to use a viral vector in spite of using a cell-specific receptor, and it will be used in the future including gene therapy. As described above, gene transfer via transferrin receptor, asialoglycoprotein receptor and immune protein receptor has already been attempted. However, in the ligand complex method, it is necessary to develop a gene transfer technique that makes use of the characteristics of the receptor to be used and that the cell type targeted for gene transfer differs depending on the receptor to be used. There are many problems.

On the other hand, α_Two-Macroglobulin is
A protein that is synthesized by cells and secreted into the blood.
Is a receptor for glutinous cells in liver parenchymal cells, liver, lungs and lymphoid tissues.
Cells that widely originate from mononuclear cells such as
Cells, astrocytes, gastrointestinal epithelial cells, smooth muscle cells,
Fibroblasts, ovarian granulosa cells, renal dendritic stromal cells, testis L
eydig cells also distributed (SK Moes
trup, J .; Glemann, G .; Palace
n, Cell & Tissue Res. , 269: 37
5-382 (1992)). α_Two-For macroglobulin
Is the natural type that does not bind to the receptor and the
There is an F type. Natural α_Two-Macroglobulin
Has the property of associating with proteolytic enzymes,
Is α_Two-Macroglobulin is in a form capable of binding to the receptor
Converted to (F type). Natural α _Two-Macrogloblis
By treatment with methylamine
F type α that can bind to the receptor_Two-Macroglobulin (hereinafter
"Fα_Two-Macroglobulin ")
Can be.

Therefore, it has already been proposed to use α ₂ -macroglobulin as a ligand molecule for gene transfer (Sy Cheng. G. T. Emrino &
I. H. Pastan, Nucleic Acid R
es 11: 659-669 (1983)), but it was merely a proposal, a concrete means for carrying out it was not shown, and it was unrealistic.

Therefore, an object of the present invention is to provide a safe and highly efficient gene transfer method using Fα ₂ -macroglobulin as a ligand molecule for gene transfer.

[0017]

In view of such circumstances, the present inventors have conducted diligent research and as a result, as a result, gene transfer in which three members of Fα ₂ -macroglobulin, a molecule having a property of associating with DNA, and DNA are bound. The present inventors have completed the present invention by discovering that the use of a complex for use can safely and efficiently introduce a gene into an animal cell.

That is, the present invention provides the first invention of Fα ₂ -macroglobulin, a molecule having the property of associating with DNA, and a complex for gene transfer to which DNA is bound. Furthermore, the present invention provides the gene transfer complex as F
The present invention provides a gene transfer method, which comprises contacting a human or animal cell having a receptor that specifically interacts with α ₂ -macroglobulin on the cell surface.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The α ₂ -macroglobulin, which is a raw material of the complex for gene transfer of the present invention, can be prepared by a known method (Arakawa, H., Osa).
da, T, Ikai, A .; Arch. Biochi
m. Biophys. 244: 447-453 (198
6)). In order to convert this into F type, a known method using methylamine may be used (Mitsuda,
S. , Nakagawa. T. , Osada, T .; , S
Himamoto, T .; , Nakazato, H .; , I
Kai, A .; , Biochem. Biophys. Re
s. Commun. , 194: 1155-1160 (1
993)).

In the present invention, the molecule having the property of associating with DNA is (A) a basic polymer,
Examples thereof include (B) an association of a basic polymer with a positively charged fatty acid or a derivative thereof, or (C) a complex of a virus fusion peptide and a basic polymer. Here, examples of the basic polymer include polylysine, polyornithine, polyamine, and DEAE dextran, and polylysine is preferable. Examples of the positively charged fatty acid or its derivative include dimethyloctadecyl ammonium bromide, and examples of the viral phage peptide include a peptide contained in the N-terminal of influenza virus hemagglutinin subunit HA-2. Further, a known auxiliary component having an affinity with a cell membrane may be added to these (A), (B) or (C). Furthermore, in the case of polylysine, SPDP (N-succinimidyl-3-
A state in which a reactive group such as an SH group is introduced by further reduction treatment using a known cross-linking agent such as (2-pyridyldithio) propionate), and Fα ₂ -macroglobulin having a pyridyl disulfide group introduced by SPDF can be bound. Can be

The DNA that can be introduced into cells in the present invention is not particularly limited.

The gene transfer complex of the present invention comprises Fα ₂ −
Macroglobulin and a molecule having a property of associating with DNA are allowed to associate with a conjugate bound directly or through a cross-linking agent (method 1), or an association of a molecule having a property of associating with DNA and a DNA To Fα ₂ -macroglobulin directly or through a cross-linking agent (method 2)
It can be manufactured by the following.

In the method 1, the Fα ₂ -macroglobulin is bound to a molecule having the property of associating with DNA by, for example, introducing a pyridyl disulfide group into Fα ₂ -macroglobulin by a crosslinking agent SPDP and associating with DNA. It can be carried out by a method in which an SH group previously introduced into a molecule having characteristics, such as polylysine, is bound by an SS exchange reaction, or the like. In order to associate the DNA with the conjugate thus obtained, electrostatic association may be achieved by mixing. In Method 2, in order to obtain a DNA-associated molecule having a property of associating with DNA, a method of electrostatically associating, for example, polylysine is charged as described above, and electrostatically interacting with DNA Because of the nature of association, there is a method of utilizing this. Further, in order to bind Fα ₂ -macroglobulin to this association through a cross-linking agent, for example, Fα ₂ -macroglobulin can be bound by SPDP as a cross-linking agent.
_{A method in} which a pyridyl disulfide group is introduced into _2- macroglobulin and the SH group introduced in advance into the association is bound by an SS exchange reaction or the like may be used.

The gene transfer complex thus obtained is brought into contact with a human or animal cell having a receptor capable of specifically interacting with α ₂ -macroglobulin on the cell surface to give the gene in the cell. Can be introduced. This effect is considered as follows.

Gene DN, which is one of the components of the complex
A molecule having the property of associating with A, for example, a basic polymer
Quality has the ability to electrostatically associate with genetic DNA
Therefore, the gene DNA is retained in the complex. Complex
Have a more electrostatic affinity for the negatively charged cell surface.
To get close to the cell surface. Where Fα in the complex _Two
-Macroglobulin is the α of the cell surface_Two-Macroglob
It binds to the phosphorous receptor with high affinity and allows cells to enter the complex
Promote dosiosis. Cells by endocytosis
The gene information of the gene DNA taken in is expressed
You.

[0026]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 (1) Addition of SH group to polylysine Poly-L-lysine hydrobromide (molecular weight: 591)
00, degree of polymerization 280) was obtained from Sigma. SPD
P was obtained from Pierce. 122 μM, 244 μl of 40 mM SPDP solution was added to 2 ml of polylysine aqueous solution,
Stir gently for 30 minutes. After that, the reaction solution was separated with Sephadex G- using Dulbecco's phosphate buffered saline.
Gel filtration was performed at 25. Gel filtration process OD280
The polylysine introduced with the pyridyl disulfide group at a recovery rate of 90% or more was separated and collected from the unreacted low molecular weight substance by monitoring with. Recovered polylysine having introduced pyridyl disulfide group (22.4 μM, 1
0 ml) to 50 mM dithiothreitol (DT)
T) An SH group-introduced polylysine was prepared by reduction with 400 μl of an aqueous solution, and purified by performing dialysis against Dulbecco's phosphate buffered saline. The SH group introduction rate quantified from the absorbance was 30 per polylysine molecule, that is, the SH group introduction rate per polylysine residue was about 11%.

[0028] ₍₂₎ α 2 - Introduction of pyridyl disulfide groups into the macroglobulin alpha ₂ - macroglobulin were prepared according to a conventional method from human blood (Arakawa, H., Osada, T , Ika
i, A. Arch. Biochim. Biophy
s. 244: 447-453 (1986)), it was converted to the F form using methylamine (Mitsuda, S.,.
Nakagawa, T .; , Osada, T .; , Shim
amoto, T .; , Nakazato, H .; , Ika
i, A. , Biochem. Biophys. Res.
Commun. , 194: 1155-1160 (199
3)). 11.3 μl of 20 mM SPDP solution was added to 3 ml of a solution containing 13.44 mg of Fα ₂ -macroglobulin, and after stirring for 30 minutes, S was used as an eluent with 50 mM phosphate buffer.
Gel filtration was performed using ephadex G-25. By monitoring the gel filtration process by OD280, 7
Fα ₂ -macroglobulin introduced with a pyridyl disulfide group at a recovery rate of 0% or more was separated and recovered from the unreacted low molecular weight substance. The introduced pyridyl disulfide group is α ₂
-Equivalent to 4.5 per macroglobulin molecule, α
Considering that _2- macroglobulin is a tetramer, it was estimated that approximately one pyridyl disulfide group was introduced per monomer molecule.

(3) Gene DNA β-galactosidase expression gene DNA (pCMVβ, a reporter vector transcribed by cytomegalovirus promoter in mammalian cells and constructed to express β-galactosidase) was obtained from Clontech.

(4) SH group-added polylysine (pL-S
Association of β-galactosidase-expressing gene DNA with H) 8 μl of 10 μM SH-added polylysine was added to 1 μg of gene DNA, and the mixture was allowed to stand at room temperature for 1 hour to electrostatically form an aggregate.

[0031] (5) SH group addition F.alpha to polylysine · DNA aggregate ₂ - F.alpha was introduced macroglobulin binding pyridyl disulfide group ₂ - macroglobulin (Fα ₂ M-DP) was added to 130pmol aggregates, 6- By allowing it to stand for 24 hours, Fα ₂ -macroglobulin was bound to the SH group-added polylysine-DNA aggregate. Under these conditions, the ratio of the number of molecules of polylysine and Fα ₂ -macroglobulin that is involved in complex formation is 8
0: 130, which is a much smaller ratio of the gene DNA present in the complex.

(6) Introduction of Fα ₂ -macroglobulin / polylysine / β-galactosidase gene DNA complex into animal cells Human hepatoma-derived cell line HepG2 was cultured in MEM medium supplemented with 10% fetal calf serum. The Fα ₂ -macroglobulin / polylysine / β-galactosidase gene DNA complex prepared as described above was treated with 10% fetal bovine serum and 10%.
It was added to 1 ml of MEM medium supplemented with 0 μM chloroquine.
The cells were cultured in this medium for 4 hours. Then a new ME
The medium was replaced with M medium and the cells were cultured for 2 days.

(7) Expression of β-galactosidase in introduced cells Chlorophenol red-β-galactopyranoside used as a substrate was obtained from Boehringer Mannheim. The cells were lysed, and the β-galactosidase activity of the supernatant was measured using chlorophenol red-β-galactopyranoside as a substrate.

1) Comparison of transgene expression level between Fα ₂ -macroglobulin / polylysine / β-galactosidase gene DNA complex and SH group-added polylysine / β-galactosidase gene complex as a control

Culturing HepG2 cells in a 35 mm culture dish
Then, gene transfer was performed according to the above method. Addition
The obtained polylysine derivative (pL-SH) per culture dish
It is 2.9 μg. The amount of added gene DNA is 1μ
g. Figure 1 shows β-galactosidase after 2 days of culture.
Measured activity, measured in untreated cells as control
Is expressed as a relative value with 100 as 100. this
As the results show, Fα_Two-Macroglobulin porili
By the gin / β-galactosidase gene DNA complex
Significantly higher than the untreated group (control)
The expression level of β-galactosidase was observed (DNA + pL-S
H + Fα_TwoM-DP). In addition, SH-modified polylysine and DN
The association with A (DNA + pL-SH) and the reactive group
Not introduced Fα _Two-Macroglobulin and SH-modified poly
A mixed solution of gin and DNA (DNA + pL-SH +
Fα_TwoM (free) does not contribute to gene transfer
Was. That is, they do not work unless they are combined.
No. The value entered in the bar graph in Fig. 1 is pL.
-Fα for SH_Two-Macroglobulin derivative (Fα_Two
M-DP) molar ratio is shown in the ratio of 1: 1.6.
In addition, the expression level was 5 times or more that of the control group.

2) Correlation between applied amount of Fα ₂ -macroglobulin / polylysine / β-galactosidase gene DNA complex and transgene expression level The cell culture conditions are the same as above. In addition, the amount of added gene DNA was all 1 μg, and the added Fα ₂ M-
DP is also 260 pmol. In this experiment, maximum gene transfer efficiency was obtained when the amount of pL-SH added was about 80 pmol, and a high β-galactosidase expression amount that was 6 times or more that of the case of no addition was observed. Larger amount of pL
The expression level was decreased by adding -SH (Fig. 2). This result indicates that the gene DNA and pL-SH contribute to the gene DNA transfer by stoichiometric association, and the presence of a large amount of polylysine shows that Fα ₂ -macroglobulin and its receptor are present on the cell surface. It suggests to antagonize the interaction.

[0037]

INDUSTRIAL APPLICABILITY Since the complex of the present invention binds to the cell surface with high affinity, it can be compared with a gene transfer medium based only on electrostatic affinity such as SH group-introduced polylysine. The effect of gene transfer is good. Since the complex of the present invention does not use a heterologous protein, virus, etc., it is highly safe with gene transfer.

In the complex of the present invention, a basic polymer having a molecular weight of about 35,000 and Fα ₂ -macroglobulin having a molecular weight of about 700,000 are the main components constituting the molecular weight of the complex. There is no need to use large and complex molecules of as a medium.

The α ₂ -macroglobulin used in the present invention has been converted into F type, and it has an affinity in comparison with the normal type α ₂ -macroglobulin having no affinity for the α ₂ -macroglobulin receptor. Because of its nature, the normal type α ₂
-Even in an environment in which macroglobulin is present in a large amount, it has an affinity for the receptor, and compared with a gene transfer medium using transferrin, the effect of gene transfer in the presence of a large amount of serum protein can be expected. Unlike transferrin, Fα ₂ -macroglobulin contained in the complex of the present invention has a large amount of the receptor in cell types such as macrophages and hepatic parenchymal cells that normally have low cell proliferation and few transferrin receptors. Therefore, a target cell different from the ligand complex gene transfer method using transferrin can be used as the target cell. In particular, since the receptor is present in cells having antigen-presenting ability such as macrophages, a vaccine by gene transfer, namely DN
It is possible to apply to A vaccine.

[Brief description of the drawings]

FIG. 1 is a graph showing the transgene expression levels of the method of the present invention and the control method.

FIG. 2 is a diagram showing the relationship between the amount of polylysine and the expression amount of transgene.

Claims (7)

[Claims] 1. A complex for gene transfer in which F-type α ₂ -macroglobulin, a molecule having a property of associating with DNA, and DNA are bound. 2. F-type α ₂ -macroglobulin and DNA
The gene-introducing complex according to claim 1, wherein the molecule is associated with a molecule having a property of associating with DNA, or a DNA is associated with a conjugate bound through a crosslinking agent. 3. A molecule having the property of associating with DNA and D
F-type α ₂ − in association with NA, directly or through a cross-linking agent
The complex for gene transfer according to claim 1, which is bound with macroglobulin. 4. A molecule having the property of associating with DNA,
The fusion peptide of (A) a basic polymer, (B) a basic polymer and a positively charged fatty acid or its derivative, or (C) a basic polymer and a virus fusion peptide. Complex for gene transfer. 5. The gene transfer complex according to claim 4, wherein the basic polymer is polylysine. 6. The cross-linking agent is N-succinimidyl-3- (2
The complex for gene transfer according to claim 2 or 3, which is -pyridyldithio) propionate (SPDP). 7. A human or animal cell having the receptor for specifically interacting with F-type α ₂ -macroglobulin in the cell surface of the complex for gene transfer according to any one of claims 1 to 6. A gene transfer method characterized by contacting.