JP2003235560A - Complexes and methods for gene transfer - Google Patents

Abstract

(57) An object of the present invention is to provide means and methods capable of introducing a target gene into the nucleus of a target cell at the individual level. A ligand for a receptor on a target cell membrane;
A gene introduction complex comprising a histone and a genetic material, and a method for introducing a gene into a target cell nucleus by endocytosis via a receptor on a target cell membrane.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a complex and a composition capable of introducing a gene of interest into the nucleus of a target cell at the individual level, and a target by a receptor-mediated endocytosis on a target cell membrane. The present invention relates to a method for introducing a gene into the cell nucleus.

[0002]

2. Description of the Related Art Conventionally, as a method for introducing a gene into a cell nucleus, a method using a viral vector or electroporatio
n method is used. For example, plasmids in cultured cells
There are many reports that DNA is introduced and the gene is expressed. Injecting plasmid DNA into seminiferous tubules and liver and applying electroporation to germ cells (Huang, Z. et al. In vivo transfection of test
icular germ cells and transgenesis by using the mi
tochondrially localized jellyfish fluorescentprote
in gene. FEBS Lett. 487, 248-251 (2000); Yamazaki,
Y., Yagi, T., Ozaki, T & Imoto, K. In vivo gene
transfer to mouse spermatogenic cells using green
fluorescent protein as a marker. J. Exp. Zool. 28
6, 212-218 (2000)), and liver (Suzuki, T., Shin,
BC, Fujikura, K., Matsuzaki, T. & Takata, K. Dir
ect gene transfer into rat liver cells by in vivo
Gene expression is observed in electroporation. FEBS Lett. 425, 436-440 (1998).

[0003]

However, the method using a viral vector has a problem of safety for living body,
In addition, the Electroporation method has the drawback that it cannot be applied to the human body. Therefore, a simple method for introducing a plasmid DNA into a target cell at the individual level has not been reported so far.

[0004]

[Means for Solving the Problems] To date, the present inventors have confirmed that gold-labeled testosterone-BSA is taken up by sperm cells by endocytosis and enters the nucleoplasm through a route similar to that of SV40. Suggested (Nishimura,
T. & Nakano, T. Nuclear translocation of gold lab
eled-testosterone-bovine serum albumin conjugate t
hrough the nuclear double membranes in rat spermat
ids. Cell Struct. Funct. 22, 621-629 (1997)). In addition, we have found that hydrocor infused via rat tail vein
We reported that tisone and bovine immunoglobulin G (IgG) conjugate IgG could be detected in the target cell nucleus of the liver by the fluorescent antibody method, suggesting that the protein bound to the steroid hormone enters the target cell nucleus (Nishimura, T. & Nakano, T.
Nuclear localization of bovine immunoglobulin G (b
IgG) in the liver of rats injected with hydrocorti
sone-bIgGconjugate intravenously. Okajimas Folia A
nat. Jpn. 78, 107-114 (2001)).

On the other hand, it is known that cultured cells introduced with a histone / DNA complex express the gene (Fritz, JD, Herweijer, H., Zhang,
G & Wolff, JA Gene transfer into mammalian cell
s using histone-condensed plasmaid DNA.Hum.Gene The
r.7, 1395-1404 (1996)).

[0006] Therefore, the present inventor has noticed that a ligand such as a steroid hormone binds to a receptor (receptor) for the ligand on the target cell membrane and is taken up into the cell by endocytosis, The present invention has been completed as a result of earnest research, considering that the genetic material can be incorporated into target cells by binding the genetic material such as DNA to such a ligand.

That is, the present invention firstly relates to a complex for gene introduction, which comprises a ligand for a receptor on a target cell membrane, histone, and genetic material. This complex can be used, inter alia, to introduce genetic material, such as a gene of interest, into target cells in vivo at the individual level.
Secondly, the present invention relates to a method for introducing a gene into a target cell nucleus in vivo by endocytosis via a receptor on the target cell membrane. Here, endocytosis (phagocytosis) generally refers to a mechanism by which cells take up substances from the external environment via vesicles caused by invagination of cell membranes. In particular, as a preferred example of the introduction method of the present invention, a method of introducing the already described gene introduction complex of the present invention or a composition containing the same into a living body can be mentioned. The present invention further relates to a method for introducing a gene into a target cell nucleus in vivo to express the gene in the target cell by endocytosis via a receptor on the target cell membrane.
As a preferred example of the expression method of the present invention, a method of expressing the gene in a target cell, which comprises administering the complex for gene introduction of the present invention described above in vivo, can be mentioned.

[0008]

More specifically, in the complex of the present invention, the ligand for the receptor on the target cell membrane and histone are, for example, Chang, ES &O'Connor, JD in
Methods of hormone radioimmunoassay 797-814 (Acade
mic Press, New York, 1979). In such a case, the complex of the present invention is prepared by mixing the conjugate of the thus obtained ligand for the receptor on the target cell membrane with histone with the genetic material in a suitable buffer. It can be prepared as a composition containing the body. The concentration of each component in such a composition can be appropriately selected according to the type of each component, administration form, type of target cells, etc., but is usually in the range of several μg to several hundreds μg / ml. Further, the mixing ratio of each component is also arbitrary and can be appropriately selected according to the type of each component, the administration form, the type of target cells, etc. Usually, the ligand for the receptor on the target cell membrane and histone are The mixing ratio of the bound substance and the genetic material is 1 to 2. The buffer used when preparing the composition is, for example, H 2
It can be appropriately selected from any type known to those skilled in the art, such as EPES and physiological saline. Further, the composition may contain other suitable auxiliary agents and additives such as glycerol.

As the genetic material, any material having genetic information can be used. For example, DNA or R
It is a vector composed of NA, and more specifically, an expression vector such as a plasmid containing an exogenous gene to be inserted. Such an expression vector is known to those skilled in the art, and can be appropriately selected depending on the type of target cells and the like. For example, when it is introduced into a mammal, for example, SV40-derived plasmid DNA is suitable.

Those skilled in the art can appropriately select one kind or a plurality of kinds of genes to be introduced into target cells, depending on the purpose of introduction and the like. For example, when the green fluorescent protein (GFP) gene is used, whether or not the gene has been introduced into the nucleus and expressed can be simply measured by observing the fluorescence emitted by the protein encoded by the gene. I can. In addition, various genes that can be the target of gene therapy can be used. In addition, various operations such as insertion and ligation of a gene into the above various vectors can be easily performed by various methods well known in the art.

The complex of the present invention is characterized in that the ligand for the receptor on the target cell membrane and the genetic material constitute the complex via histone. Histone is a protein widely present in eukaryotic cells from single-celled organisms to higher organisms, and is a protein contained in the nucleus of cells in almost the same amount as DNA. There are also commercially available products, and those skilled in the art can easily obtain them. Can be done. H1, H2A, H2B, H
There are 5 types, 3 and H4, all of which are basic proteins,
It forms an ionic bond with DNA. In the present invention, any type of histone can be selected. For example, bovine-derived histone H1 can be used.

[0012] The ligand for the target cell receptor can be appropriately selected by those skilled in the art depending on the type of target cell, the purpose of its introduction and the like. In order to introduce a gene into germ cells, for example, testosterone ( Testosteron
Various steroid hormones such as e) and hydrocortisone are suitable.

The means and method for administration into the living body are arbitrary,
A person skilled in the art can appropriately select, for example, it can be administered in vivo by injecting into a blood vessel such as tail vein or subcutaneously. The type of target cells can be appropriately selected by those skilled in the art depending on the purpose of use, the type of ligand, etc., for example, mammalian cells, in particular, when testosterone is used as a ligand, cells of seminiferous tubules, Germ cells such as intertesticular cells and epithelial cells of seminal vesicles,
Mention may be made of cardiomyocytes, as well as skeletal muscle cells.

[0014]

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

The reagents and the like used in the examples are as follows.・ Testosterone 3- (o-carboxymetyl) oxime (Sigma, St.
Louis, MO, USA, testosterone-CMO) ・ Tributylamine (Nacalai Tesque, Kyoto, Japan) ・ Isobutylchloroformate (Aldorich, Milwaukee, WI, USA) ・ Dioxane (Aldorich, Milwaukee, WI, USA) ・ Bovine Histone H1 (Upstate Biotechnology, Lake Pla
cid, NY, USA) concentration is 5 mg / ml (in H ₂ O) ・ plasmid DNA is pTracer-SV40 (Invitrogen, Carlsbad,
CA, USA).

Example 1 A Testosterone-histone H1 conjugate was prepared according to the following procedure. 1. Add 20 mg of testosterone-CMO to a mixed solution of 400 μl methanol and 50 μl tributylamine. 2. Add 4.4 μl of isobutylchloroformate,
Let stand for 30 minutes at room temperature. 3. In a mixture of dioxane 6 ml and 0.5 N NaOH 600 [mu] l in a separate container, 5mg / mlH ₂ O concentration of histone H1 (calf t
hymus) Add 6 ml. Mix solutions 4.2 and 3 and stir at 4 ° C. for 5 hours. The solution of 5.4 is dialyzed against 4 ° C. H ₂ O overnight. 6. The dialyzed solution is freeze-dried.

Further, according to the following procedure, Testosoteron
The e-hisotone H1-plasmid DNA complex was prepared. 1. Testosterone-histone H1 conjugate was added to HEPES (N-2-hydr
oxyethylpipeazine−N'-2-ethanesulfonic acid) buffe
Dissolve it in r saline (containing 10% glycerol) and adjust the concentration to 30μ.
Adjust to g / ml. 2. Add 1 ml of HEPES buffer saline to 20 μg of plasmid DNA (pTracer-SV40: Invitrogen). Add 1 ml of the solution of 1 to 3.2 and react at room temperature for 30 minutes. 4. A solution having a weight ratio of Testosterone-histone H1 / plasmid DNA of 3/2 is obtained. Final product (Testosterone-histone H1-DNA) is 5% glyce
15 μg Testoste in 1 ml of HEPES buffersaline containing rol
It contains rone-histone H1 and 10 μg plasmid DNA.

Example 2 Testosterone-histone H1- of the present invention prepared in Example 1
The DNA complex was administered to rats according to the following procedure to confirm gene introduction and expression. 1. 1 ml of a testosterone-histone H1-DNA solution having a concentration of II-4 was injected from the tail vein of anesthetized ether rats. After 2 or 6 days, the rats were anesthetized with ether and 3% paraformaldehyde / 0.1M phosphate buffe was added.
phosphate buffered fixative containing r was perfused from the left ventricle,
Remove seminal vesicles, skeletal muscles, heart, thymus, liver, spleen,
After cutting into 5-mm blocks, soaking in the same fixative solution for 2 hours, washing with 0.1 M phosphate buffer, embedding the specimen in OCT compound (Miles INC, Elkhart, IN), and freezing in liquid nitrogen After that, store at -80 ° C until use. Frozen section (thickness 10
(μm) is sliced with a cryostat, fixed on a glass slide and dried. 3. Frozen sections were stained with 0.01% Evans blue for 5 minutes,
After washing with H ₂ O, a small amount of mounting medium (Perma fluor) is used for mounting, and evaluation is performed with a confocal laser scanning microscope (LSM GB200, Olympus, Japan) using a wavelength of 488 nm. The results thus obtained are shown in FIGS.

As is clear from the figure, testosterone-
Three rats sacrificed 144 hours (6 days) after injection of histone H1-pDNA proved to express GFP in seminiferous tubule cells, intertesticular cells, epithelial cells of seminal vesicles, and cardiomyocytes . These cells are reported to have androgen receptors in the nucleus. The inventors have reported that the colloidal gold-labeled testosterone-bovine serum albumin conjugate intravenously injected into rats translocates to these cell nuclei (Nishimura, T. and Ichikawa, I. 1997 Nuclear c
oncentration of gold labeled-testosterone-bovine s
erum albumin conjugate injected intravenously in t
he hormone-target cells of rat. Cell Struct. Funct.
22: 433-442.). Furthermore, it was demonstrated by the present invention that GFP was expressed at a high level in skeletal muscle cells, and it is reported that the androgen receptor is located in the nucleus in these cells as well. On the other hand, it was proved that GFP was not expressed in the liver, spleen and thymus.

[0020]

From the above results, it was revealed that the target gene can be introduced into a target cell and expressed in vivo by using the gene transfer complex of the present invention.

[0021] So far, when a mouse into which the GFP-encoding pDNA is injected into the vas deferens mates with a normal estrus female,
It has been confirmed that transgenic mice carrying GFP are born (Huguet, E. & Esponda, P. Generati
on of genetically modified mice by spermatozoa tran
sfection in vivo: preliminary results. Mol. Repro
d. Dev. 56, 243-247 (2000)). Furthermore, pTracer-SV40
(Invitrogen), when introduced into cultured mammalian cells,
It is known that the gene is expressed and the gene expression is observed in the cells after several generations, that is, the pTracer-SV40 gene is inherited by the offspring (Bennett, RP, Cox, CA & Ho.
effler, JP Fusion of green fluorescent protein
with the Zeocin -resistance marker allows visual s
creening and drug selection of transfected eukaryo
tic cells. Biotechniques. 24, 478-482 (1998)). Therefore, since the target gene can be introduced into the germ cell nucleus by utilizing the complex of the present invention, the transgene may be inherited to the offspring by mating with a normal female thereafter. Be expected. Therefore,
INDUSTRIAL APPLICABILITY The present invention is useful for developing a method for preventing the transmission of a genetic disease affecting the next generation such as hemophilia to the offspring.

[Brief description of drawings]

FIG. 1 is a photograph showing the results of observing cells during testis with a confocal laser scanning microscope in rats sacrificed 48 hours (2 days) after injection of testosterone-histone H1-pDNA.

FIG. 2 shows intertesticular cells of rats sacrificed after 48 hours (2 days) (left) and 144 hours (6 days) (right) after injection of testosterone-histone H1-pDNA. 2 is a photograph showing the result of observation with a confocal laser microscope.

FIG. 3 is a photograph showing the results of observing epithelial cells of seminal vesicles with a confocal laser scanning microscope for rats sacrificed 48 hours (2 days) after injecting testosterone-histone H1-pDNA.

FIG. 4 shows skeletal muscle cells of rats sacrificed 48 hours (2 days) (left) and 144 hours (6 days) (right) after injection of testosterone-histone H1-pDNA. It is a photograph which shows the result observed with the confocal laser scanning microscope.

FIG. 5 shows that cardiomyocytes were co-cultured in rats sacrificed 48 h (2 days) (top) and 144 h (6 days) (bottom) after injection of testosterone-histone H1-pDNA. It is a photograph which shows the result observed with the focus laser microscope.

FIG. 6 shows the results of observing the spleen (left) and thymus (right) with a confocal laser scanning microscope for rats sacrificed 48 hours (2 days) after injecting testosterone-histone H1-pDNA. It is a photograph.

Front page continuation (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 19/00 C12N 15/00 AF term (reference) 4B024 AA20 CA02 CA09 DA02 EA04 FA10 GA11 HA17 4C084 AA01 AA02 AA07 AA13 BA01 BA08 BA22 BA36 CA17 DB10 MA65 NA13 ZA361 ZA96 ZC10 4C087 AA01 BB65 BC83 MA02 MA65 NA13 ZA36 ZA96 ZC10

Claims (24)

[Claims] 1. A gene transfer complex comprising a ligand for a receptor on a target cell membrane, histone, and genetic material. 2. The complex according to claim 1, wherein a ligand for a receptor on a target cell membrane and a histone are bound to each other. 3. The complex according to claim 1, which is a mixture of a binding substance of a ligand for a receptor on a target cell membrane and histone, and a genetic material. 4. The mixture ratio of the binding substance of the ligand for the receptor on the target cell membrane and histone and the genetic material is 1 to 2.
The composite according to claim 2 or 3, which is 5. The complex according to any one of claims 1 to 4, wherein the genetic material is a DNA vector. 6. A plasmid DN whose genetic material contains a gene.
The complex of claim 5, which is A. 7. The complex according to claim 6, wherein the plasmid DNA is derived from SV40. 8. The complex according to any one of claims 1 to 7, wherein the gene is green fluorescent protein. 9. The complex according to any one of claims 1 to 8, wherein the histone is histone H1. 10. The complex according to any one of claims 1 to 9, wherein the ligand for the target cell receptor is a steroid hormone. 11. The complex according to claim 10, wherein the steroid hormone is testosterone. 12. The complex according to claim 10, wherein the steroid hormone is hydrocortisone. 13. A method for introducing a gene into a target cell nucleus in vivo by endocytosis via a receptor on a target cell membrane. 14. A method for introducing a gene into a target cell nucleus, which comprises administering the complex for gene introduction according to any one of claims 1 to 12 into a living body. 15. The method according to claim 14, wherein a composition containing the gene transfer complex and a buffer solution is administered in vivo. 16. The method according to claim 13, wherein the gene transfer complex is injected into a living body by injecting it into a blood vessel.
5. The method according to any one of 5 above. 17. The method according to any one of claims 13 to 16, wherein the target cells are mammalian cells. 18. The method according to claim 17, wherein the target cells are seminiferous tubule cells, intertesticular cells, epithelial cells of seminal vesicles, cardiomyocytes, or skeletal muscle cells. 19. A method for introducing a gene into a target cell nucleus in vivo to express the gene in the target cell by endocytosis via a receptor on the target cell membrane. 20. A method for expressing a gene in a target cell, which comprises administering the complex for gene introduction according to any one of claims 1 to 12 to a living body. 21. The method according to claim 20, wherein a composition containing the gene transfer complex and a buffer solution is administered in vivo. 22. The method of claim 20 or 21, wherein the gene transfer complex is injected into a living body by injecting into the blood vessel.
The method described. 23. The method according to any one of claims 19 to 22, wherein the target cells are mammalian cells. 24. The method of claim 23, wherein the target cells are seminiferous tubule cells, intertesticular cells, seminal vesicle epithelial cells, cardiomyocytes, or skeletal muscle cells.