WO1997017844A1

WO1997017844A1 - Composition for gene introduction and method for gene introduction by using the composition

Info

Publication number: WO1997017844A1
Application number: PCT/JP1996/003360
Authority: WO
Inventors: Ryuichi Morishita; Yasushi Kaneda; Toshio Ogiwara; Tsuneaki Sakata; Mamoru Hasegawa
Original assignee: Dnavec Research Inc.
Priority date: 1995-11-16
Filing date: 1996-11-15
Publication date: 1997-05-22
Also published as: AU1131297A

Abstract

A method for efficiently introducing a gene specifically into the epidermis which comprises bringing a composition containing a compound having affinities for both of DNA and the cells and a vector for gene introduction into direct contact with the epidermis of a mammal involving vital epidermic cells.

Description

TECHNICAL FIELD The present invention relates to a composition for gene transfer and a method for gene transfer using the composition.

The present invention relates to a gene transfer technique or a gene therapy technique. The present invention particularly relates to a composition for gene transfer including a vector used for gene transfer or gene therapy.

Technology background

Conventionally, when a gene is specifically introduced into the skin, cells such as fibroblasts, epithelial cells, and keratinocytes present in the skin are taken out of the living body, and the following methods are used: ① Electrical transfer method, (Steinberg, RM et al., J Virol., 63, 957 (1989)) ② Microinjection method (Roth, P., et al., Nucl. Acids. Res., 18, 5009 (1990) / Schlegel, R. et al., EMBO J., 7, 3181 (1988 ) / / Munger, K. et al., J. Virol., 63, 4417 (1989) Bruno Pecoraro, G., P roc. Natl . Acad. Sci. USA, 86, 563 (1989)), ③ Retroviral vector (Morgan, JR et al., Science, 237, 1476 (1987), Teumer, J. et al., FASEB J., 4, 3245 (1990) / Gerrad, AJ et. al., Nature Genet, 3, 180 (1993)), アデノ Adenovirus vector (Aneskievich, BA, J. Invest. Dermatol., 91, 309 (1988)), リ Liposomes including cation liposomes Gene transfer in vitro using vectors (Jiang, K. et al., J. Invest. Dermatol., 97, 969 (1991)). And returned to the living body. However, in this method, the operation of removing skin cells from a living body and returning them to the living body requires skill, and the cells returned to the living body have a life span of several months. Long-term survival was difficult. In addition, this method has the disadvantage that several months are required for the previous step, and it is particularly difficult to grow cells in culture. The envelope protein of the Moroni mouse retrovirus vector and the hematopoietic factor Erythropoietin, one of the offspring, is made into a fusion protein and appears on the virus surface

Although vectors have been developed to introduce genes specifically for erythroblasts (N. Kashiharae et al., Science, 266, 1373-1376 (1994)), there have been few successful cases and technically immature I have to say. In particular, there has been no success in tissue-specific gene transfer in vivo. Disclosure of the invention

An object of the present invention is to provide a gene transfer composition that allows simple and efficient gene transfer. Another object of the present invention is to introduce a gene into the epidermis of a mammal in vivo using the composition.

The present inventors have made it possible to directly contact a composition containing a compound having affinity for both DNA and cells and a gene transfer vector with the epidermis of a mammal including living epidermal cells. The inventors have found that gene transfer can be performed efficiently, and have completed the present invention. More specifically, the present inventors have introduced a “gene introduction vector containing a compound having an affinity for both DNA and cells and a gene introduction vector into a fetal amniotic fluid”. As a result, they found that the gene was introduced into the entire body surface specifically in the body surface, and completed the present invention.

That is, the present invention

(1) a compound having affinity for both DNA and cells and a gene transfer vector for specifically introducing a foreign gene into the epidermis of a mammal including living epidermal cells, A composition for gene transfer,

(2) The composition according to (1), wherein the compound having affinity for both DNA and cells forms a liposome.

(3) The composition according to (1) or (2), which comprises an inactivated Sendai virus (HVJ).

(4) The composition according to any one of (1) to (3) or a lysate thereof, A method of introducing a gene into the epidermis of a mammal other than a human, comprising directly contacting the epidermis of a mammal containing epidermal cells.

About.

The "compound having an affinity for both DNA and cells" in the "composition for gene transfer" of the present invention includes ribosome, which is an artificially produced membrane structure composed of phospholipid / glycose glycolipid And cationic ribosomes and complexes composed of cationic phospholipids (PL Feigner et al., Proc. Natl. Acad. Sci. USA, 84, 7413 (1987), CM Corma et al., Mol. Cell. Biol., 2, 1044 (1982), N. Haga, K. Yagi, J. Clin. Biochem. Nutr., 7, 175 (1989), JR Neuman et al., Biotechniques 12, 643 (1987). ), And ribosomes containing HVJ-liposome (“The Manual of Ribosome Z Experiments in Life Sciences” Springer's Falarak Tokyo (1992) p. 282-287) and the like. As the “gene transfer vector” in the “gene transfer composition” of the present invention, basically all gene transfer vectors are used. For example, retroviral vectors, adenovirus vectors, adenoviruses, A virus vector for gene transfer such as an associated virus vector is used. For administration, a “gene transfer composition” dissolved in water or various buffers can be injected as a water-soluble substance into a desired site using a syringe or the like.

The term “surviving epidermis cells” in the present invention includes any living epidermis that is not keratinized, and includes, for example, skin epithelial cells and skin fibroblasts.

The method of administering the “composition for gene transfer” of the present invention includes a method of administering the composition ex vivo to cells and a method of administering the composition into fetal amniotic fluid. When administered in fetal amniotic fluid, gene transfer into the entire epidermis becomes possible. By selecting the stage of embryo development to be injected into amniotic fluid, fertilized eggs, which are pre-implantation embryos during embryo development, are of course, 2-cell stage, 4-cell stage, 8-cell stage, and moromi Embryos are inherited in all stages, including embryos, blastocysts, late blastocysts, early tubule embryos, tubule embryos, and late tubule embryos It is possible to introduce children. It is also possible to transfer genes to embryos after implantation.

By introducing a gene into the “whole epidermis”, gene therapy for skin hereditary disorders such as fish scales and scalp abnormalities can be suitably performed. In addition, by introducing a gene in a “stage-specific” manner, a gene can be placed at a specific site in the embryo that forms the epidermis at that stage, and the gene expression can be controlled regardless of the vector and the motor used. It can be controlled in a tissue-specific manner and can be applied to the treatment of genetic diseases.

Further, examples of the gene introduced by the “gene transfer vector” of the present invention include all types of genes such as development stage-specific genes, organ-specific genes, particularly skin-specific genes, and genes for treating various diseases. Can be

Furthermore, the range of animals to which the composition of the present invention can be administered is applicable to all mammals such as humans, mice, rabbits, rabbits, and monkeys.

The administration of the “composition for gene transfer” of the present invention can be applied to the treatment of genetic diseases in general, but it can be inferred that it is particularly effective for skin disorders such as fish and abnormalities of the skin such as abnormalities of the scalp. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (A) -FIG. 1 (B) are photomicrographs showing the distribution of FITC-labeled 0DN introduced into fetal rat amniotic fluid by direct injection at 2 hours. (A) was observed at 40x and (B) at 100x.

Figure 2 is a micrograph showing the distribution of FITC-labeled 0DN introduced into fetal rat amniotic fluid by direct injection 5 days later.

FIG. 3 is a diagram showing the structure of a plasmid pSV40Gal that expresses a / 3-galactosidase gene.

FIG. 4 (A) -FIG. 4 (C) are photographs of rat fetal skin 5 days after transfection. (A) and (B) are the three-galactosidase vector and control vector, respectively. (C) relates to the untreated rat.

Figure 5 (A)-Figure 5 (C) are photographs showing histological analysis of rats on day 5 after transfusion. (A) and (B) each relate to a rat transfected with a 5-galactosidase vector and a control vector, and (C) relates to an untreated rat.

Figure 6 (A)-Figure 6 (C) are photographs showing histological analysis of rats 10 days after transfusion. (A) and (B) relate to a rat transfected with a 3-galactosidase vector and a control vector, respectively, and (C) relates to an untreated rat. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[Example 1] Preparation of HVJ-liposome

Phosphatidylserine, phosphatidylcholine, and cholesterol were mixed at a weight ratio of 1: 4.8: 2. The dried lipid was suspended in a 200 1 BSS solution (137 mM NaCl, 5.4 mM KC1, 10 mM Tris-HCl, pH 7.6) containing plasmid MA or FITC-labeled oligonucleotide (ODN). Ribosomes were prepared by shaking and ultrasound (see "The Manual of Ribosome Z Experiments in Life Sciences", Springer-Fairark Tokyo (1992), p. 282-287).

On the other hand, HVJ (Z strain) was purified and inactivated by ultraviolet irradiation (110 erg / imn ² / sec) for 3 minutes immediately before use. The ribosome solution described above (10 mg ribosome / 0.5 ml solution) was mixed with 10,000 hemagglutinating units of HVJ and diluted with BSS solution to a final volume of 4 ml. The mixture was kept at 4 for 5 minutes and then gently shaken at 37 for 30 minutes. Unreacted HVJ and ribosome were removed from HVJ-ribosome by a sucrose density gradient.

[Example 2] In vivo introduction of FITC-labeled oligonucleotide (0DN)

The oligonucleotide (0DN) was labeled with FITC at the 5 'end and 3' end (Antisense Res. Develop. Vol. 2, 27-39 (1992)). The HVJ ribosome containing the labeled ODN was prepared according to Example 1. Prepared by the method. The final concentration of FITC-labeled ODN to be injected into amniotic fluid (0DN relative to the total ribosome solution) was used. HVJ-ribosome 101 containing labeled 0DN prepared by the method of Example 1 was injected into the amniotic fluid of a Sprague-Dawley rat on the day of pregnancy through a 30-gauge injection needle. At this time, the injector was shot directly into the amniotic fluid from the uterine wall. As a control, HVJ-ribosome containing no FITC-labeled 0DN prepared by the method of Example 1 was injected. All rats were sacrificed after 2 hours or 5 days, and the extirpated tissues were fixed in 4% paraformaldehyde solution according to the standard method, and the sections were stained with erythrochrome black and observed with a fluorescence microscope. did.

As a result, fluorescence was observed in several layers of epidermis and epithelium throughout the whole body 2 hours after the introduction (FIGS. 1 (A) and 1 (B)). No fluorescence was observed in rats injected with 101 HVJ ribosomes without FITC-labeled 0DN or uninjected (data not shown). Epidermal cell fluorescence was mainly observed in the nucleus and was observed for at least 5 days after transfection (Fig. 2). Although the fluorescence was low, it was observed in the liver, but not in the kidney, heart, lung, and brain.

[Example 3] Introduction of galactosidase into neonatal rat

PSV40Gal, a plasmid that expresses the / S-galactosidase gene, was constructed as follows. Plasmid DNApAct-c-inyb containing a 370 base pair 5'-promoter region and a 900 base pair first exon region of the chicken ^ actin promoter was restricted to remove the C-myb region from the restriction enzymes Ncol and Xbal. Then, the Sail linker was ligated. 3. The 1-kilobase pair E. coli 3-galactosidase gene was excised from plasmid DN ApMC1871 using the restriction enzyme Sail and ligated to the Sail site of the above plasmid DNA (Figure 3). As a control, Escherichia coli; a plasmid DMpAct-CV containing no S-galactosidase gene was simultaneously prepared.

HVJ-ribosomes containing the β-ii root gene plasmid pSV40Gal or the control plasmid DNA pAct-CV were prepared. To increase transfection efficiency, HMG-1 protein was simultaneously encapsulated in HVJ-ribosomes. Hit the syringe directly into the amniotic fluid from the uterine wall Then, 10 / il of HVJ-ribosome prepared by the method of Example 2 was injected. Rats were sacrificed 5 days and 10 days after injection, and the removed tissue was fixed in a 1% paraformaldehyde solution according to a standard method, and the tissue was removed. Staining solution. Then, X-gal chromadin buffer solution (49 mg / ml 5-promo-4-chloro-3-indolinole-yS-D-galactoside: 1 ramol Mg ₂ and 3 mmol K ₃ Fe ( CN) ₆ : l: 99) was added. After staining, the cells were observed with a microscope. As a control, the same operation was performed on rat hearts into which HVJ-liposome containing plasmid DNA containing no E. coli galactosidase gene was injected.

As a result, expression of 9-galactosidase was observed in fetal skin after 5 days by directly injecting HVJ-liposome containing the galactosidase gene plasmid DNA vector into amniotic fluid (Fig. 4 (A)). On the other hand, expression of -galactosidase was not found in HVJ-ribosomes or untreated fetal skin that did not contain the lactosidase gene plasmid DNA vector (Fig. 4 (B), Fig. 4 (C)). Cells showing expression of S-galactosidase were present in the epidermis and several layers of epithelium (Fig. 5 (A)). These cells were mainly fibroblasts. The expression of yS-galactosidase was observed over 10 days, although the expression level was weak (Fig. 6 (A)). On the other hand, the expression of -galactosidase was not observed in the tissues of mice treated with HVJ-ribosome not containing β-ii lactosidase gene plasmid DNA vector and untreated mice (Fig. (Fig. 5 (B), Fig. 5 (C), Fig. 6 (Β), Fig. 6 (C)) The difference in survival rate between rats with and without HVJ-ribosomes was observed. I couldn't. Industrial applicability

INDUSTRIAL APPLICABILITY According to the present invention, a foreign gene can be specifically introduced into the epidermis of a mammal, and a gene can be introduced into a living body much more easily than in the past. In addition, epidermal-specific gene transfer became possible. This enables skin-specific gene therapy and genetically controls factors that cause rejection during skin transplantation. By controlling it, it is possible to improve the skin engraftment rate. Furthermore, the present invention has opened the way to simple gene therapy and expanded the repertoire of genes targeted for gene therapy.

Claims

The scope of the claims

1. Including a compound having affinity for both DNA and cells and a gene transfer vector for specifically introducing a foreign gene into the epidermis of a mammal including living epidermal cells, A composition for gene transfer.

2. The composition according to claim 1, wherein the compound having an affinity for both DNA and cells forms a liposome.

3. The composition according to claim 1 or 2, comprising inactivated Sendai virus (HVJ).

4. A non-human mammal comprising directly contacting the composition according to any one of claims 1 to 3 or a lysate thereof with the epidermis of a mammal including living epidermal cells. A method of introducing a gene into the epidermis of an animal.