WO2000023582A1

WO2000023582A1 - Gene transfer cell sheet and process for preparing the same

Info

Publication number: WO2000023582A1
Application number: PCT/JP1999/005794
Authority: WO
Inventors: Minoru Ueda; Nobuhiko Emi; Hirokazu Mizuno
Original assignee: Japan Tissue Engineering Co., Ltd.
Priority date: 1998-10-21
Filing date: 1999-10-20
Publication date: 2000-04-27
Also published as: AU6227599A; JP2000125863A

Abstract

A gene transfer cell sheet consisting of a plural number of layers of mucosal epithelial cells having a gene transferred thereinto. By selecting the subject gene, this sheet can elevate the taking efficiency upon a living body and the product of the thus transferred gene can be formed over a long time after the transfer. Owing to these characteristics, this sheet is usable as a graft having a high taking efficiency or as a carrier in gene therapy. Moreover, the mucosal epithelial cells constituting the sheet are undifferentiated cells into which a subject gene can be easily transferred. Thus, the sheet can be readily prepared.

Description

TECHNICAL FIELD The present invention relates to a transgenic cell sheet and a method for producing the same, and more particularly, to a transgenic cell sheet useful for gene therapy and for transplantation, and a method for producing the same. Landscape technology

Living cells and tissues may be transplanted or introduced into living organisms, including humans.

For example, tissue may be implanted as a wound dressing. In this case, the part where the tissue of the living body has been partially removed by surgery or accident is replaced with another tissue (graft) to supplement the removed part or improve the appearance. Various implants have been used for such purposes.

In particular, sheets of epidermal cells can be made from autologous epidermal cells. The epidermal cell sheet produced in this way is highly evaluated as a rejection-free graft useful for burns and the like.

In addition, epithelial cells isolated from oral mucosal cells have shorter metabolic cycles than epidermal cells because keratinocytes of the skin, that is, epidermal cells are also undifferentiated cells. This has the advantage that long-term maintenance is possible without keratinization. From these properties, it is considered that oral mucosal cells are more useful as tissue for transplantation than epidermal cells.

In the field of gene therapy, on the other hand, cells are sometimes used as carriers for therapeutic genes, and the therapeutic genes are introduced into cells extracted from living organisms and returned to living organisms.

Cells used for the use of such genes as carriers include cells such as hematopoietic stem cells, vascular endothelial cells, and intestinal epithelial cells. After being returned to the living body, these cells express the target gene in the living body.

However, when transplanting a tissue into a living body, it is necessary to prepare a graft in a short time after the necessity of transplantation. In addition, engraft tissues that are not originally at the transplant site In some cases, the graft may not survive successfully. In particular, since the original site of the transplant site has been removed, its resistance to bacteria and the like is weak, and if bacterial infection occurs, the transplant cannot survive.

In addition, when performing gene transfer, it is necessary to collect cells as carriers for the target gene from the individual to be treated, but cells that can be easily used as carriers may be difficult to collect from living organisms. For example, hematopoietic stem cells are undifferentiated cells and can be easily transduced and expressed, but must be collected from bone marrow. In addition, even if the target gene is introduced, its gene product cannot always be produced in a living body for a long time.

For this reason, for example, when trying to introduce a gene via epidermal cells, the growth rate is slow, and the gene is difficult to be introduced. Even if the gene can be introduced, the gene product cannot be produced for a long period of time.

Therefore, an object of the present invention is to provide a transgenic cell sheet that can easily introduce a target gene and can be prepared in a short time.

In particular, an object of the present invention is to provide a gene-introduced cell sheet useful for transplantation, which has an enhanced engraftment efficiency to a living body.

Another object of the present invention is to provide a transgenic cell sheet useful for gene therapy that can produce a transgene product for a long time after transfection. It is an object of the present invention to provide a method for producing a transgenic cell sheet which can easily produce any useful transgenic cell sheet. Disclosure of the invention

The transgenic cell sheet of the present invention is characterized by comprising a plurality of layers of mucosal epithelial cells into which a target gene has been introduced. In addition, among the above mucosal epithelial cells, preferably, the target gene is introduced into 40% to 60% or 90% or more of the cells. Further, the gene-introduced cell sheet of the present invention is characterized in that the target gene is selected from the group consisting of a blood coagulation factor, insulin, a growth factor, a tumor antigen gene and a tumor suppressor gene.

The gene transfer cell sheet of the present invention is for gene therapy used as a gene carrier in gene therapy. The transgenic cell sheet of the present invention is used as a transplant. The method for producing a transgenic cell sheet of the present invention is a method for producing a transgenic cell sheet comprising a plurality of layers of mucosal epithelial cells into which a target gene has been introduced, comprising: a mucosal epithelial cell for producing a cell sheet; Obtaining a supporting cell compatible with the mucosal epithelial cell, introducing the expression system containing the target gene and the marker gene into the mucosal epithelial cell, and culturing in a serum-free selective culture medium containing no serum; A mucosal epithelial cell into which the expression system has been introduced is selected, and the mucosal epithelial cell into which the expression system has been introduced is cultured in a serum-containing growth medium together with the feeder cells to form a mucosal epithelium having a plurality of layers. Obtaining cells. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the construction of a plasmid containing human blood coagulation factor IX used for gene transfer.

FIG. 2 is a graph showing the infection efficiency for oral mucosal epithelial cells.

Figure 3A is a diagram of transduced mucosal epithelial cells selectively cultured in a serum-containing selective medium, Figure 3B is a 100-fold enlarged view of Figure 3A, and Figure 3C is a selective culture in a serum-free selective medium FIG. 3 is a diagram of the obtained transfected mucosal epithelial cells.

Fig. 4A shows a 10-fold enlarged view showing the staining state of the transgenic mucosal epithelial sheet one week after transplantation, and Fig. 4B shows the staining state of the transgenic mucosal epithelial sheet three weeks after transplantation. FIG. 4C is a 10-fold enlarged view showing the stained state of the transduced mucosal epithelial sheet 5 weeks after transplantation.

FIG. 5A is a 250-times enlarged view showing the staining state of the transgenic mucosal epithelial sheet one week after transplantation, and FIG. 5B is the staining state of the transgenic mucosal epithelial sheet three weeks after transplantation. FIG. 5C is a 250 × magnification showing the stained state of the transduced mucosal epithelial sheet 5 weeks after transplantation.

FIG. 6 is a graph showing changes in the concentration of human blood coagulation factor IX in the blood of a mouse transplanted with a transgenic mucosal epithelial cell sheet. Detailed description of the invention

The transgenic cell sheet of the present invention comprises a plurality of layers of mucosal epithelial cells into which a target gene has been introduced. As the mucosal epithelial cells in the present invention, oral mucosal cells, nasal mucosal cells, pharyngeal mucosal cells, vaginal mucosal cells, gastrointestinal mucosal cells, etc. can be used, but oral mucosal cells are particularly preferable from the viewpoint of easy collection. . By being composed of such mucosal epithelial cells, it can be directly used as a graft-gene carrier. Moreover, these mucosal epithelial cells are unlikely to be keratinized, unlike epidermal cells, and thus can be transplanted to a wide variety of sites in a living body. -These mucosal epithelial cells can be collected from a suitable organism.

For example, oral mucosal cells can be collected from the oral cavity of a healthy subject together with submucosal tissues. Since the collected mucosal tissue contains connective tissue in addition to mucosal cells, it is necessary to isolate mucosal cells. In the isolation of mucosal cells, an appropriate enzyme, such as dispase or tribusin, is used to remove connective tissue and dermis from the collected mucosal tissue. Debris is removed using a nylon mesh.

The target gene is introduced into the mucosal cells isolated as described above together with a commonly used expression system. Expression systems used in such applications may be those well known in the art, and include, for example, expression plasmids.

The expression system that can be used here includes a marker gene, a gene introduction site into which the target gene is inserted, and a control site such as a promoter that controls the expression of the inserted target gene.

The control part is provided with a promoter. Those commonly used can be applied as they are, for example, retroviruses such as Rous sarcoma virus (RSV), human immunodeficiency virus (HIV), Sendai virus (HVJ), adenovirus, adeno-associated virus, etc. Things can be used.

The marker gene is a gene for confirming whether or not the target gene has been introduced into the expression plasmid, and a marker gene known in the art can be applied. Examples of such a marker gene include an antibiotic resistance gene such as a neomycin resistance gene and a hygromycin resistance gene, a chromogenic gene that develops a specific color such as -galactosidase, and luciferase.

The target gene is inserted upstream or downstream of the promotion. The target gene used here is selected according to the use of the transgenic mucosal cell sheet of the present invention. Can be

When the gene-introduced mucosal cell sheet of the present invention is used as a graft, a factor for facilitating graft engraftment, for example, a factor for preventing bacterial infection at the time of graft engraftment, for example, antibiotics Substances, antimicrobial peptides (such as defensins), and factors that promote graft engraftment, such as growth factor genes such as platelet growth factor (PDGF), fibroblast growth factor (FGF), and hepatocyte growth factor (HFG) Can be the target gene. These genes are known in the literature and can be easily obtained from public institutions.

When the gene-introduced mucosal cell sheet of the present invention is used as a carrier for gene therapy, a therapeutic gene can be used as a target gene. Target genes that can be targeted for such gene therapy include congenital and acquired deficiency factors, such as blood coagulation factors, insulin, various growth factors, tumor suppressor genes, and tumor antigen genes. The sequences of these target genes are already known in the literature and the like, and are available from various public institutions.

Two or more types of expression systems as described above may be used simultaneously, and if possible, two or more types of target genes may be arranged in one expression system.

The above-mentioned expression plasmid is transfected into virus-producing cells by a conventional method such as calcium phosphate, ribosome, or electroporation. Various types of virus producing cells are known, and any of them can be used. For gene transfer into mucosal epithelial cells, the virus-producing cells may be used as they are, for example, by co-culture, or the culture supernatant of the virus-producing cells may be used. It is preferable to use a culture supernatant of virus-producing cells from the viewpoint of easily removing the possibility that virus-producing cells are mixed into the cell sheet and from the viewpoint of easy operation.

Gene transfer into mucosal epithelial cells using a viral supernatant can be performed by adding the viral supernatant to a culture system of mucosal epithelial cells seeded on a plate. At this time, it is preferable to add 3 to 8 μg / ml of polypropylene together with the virus supernatant. The mucosal epithelial cells are infected with the virus in the virus supernatant within a few hours, and the target gene is introduced into the mucosal epithelial cells. After infection, the infected cells are cultured in a selective medium described below, whereby mucosal epithelial cells into which the target gene has been introduced (hereinafter, introduced cells) are selected. The selection medium used in the present invention contains a selection agent but does not contain serum.

The selection drug corresponds to the primary gene introduced into the cell together with the target gene, and the target drug is introduced by killing cells other than the cell into which the marker gene has been introduced, that is, the cell into which the target gene has been introduced. A drug that can be used to select only cells. Combinations of such marker genes and selection agents are well known in the art; for example, if a neomycin resistance gene is selected as the marker gene, an agent such as G418 is used. In the case of the transfected cell sheet of the present invention, the amount of G418 used may be smaller than that of epidermal cells and the like, and may be 100 to 200 / g / ml, preferably 100 to 150 g / ml. is there.

In the present invention, it is essential that the selection medium does not contain serum. By not containing serum in the selection medium, the differentiation potential of the cells can be suppressed and the undifferentiated state can be maintained.

Such selection media include epidermal cell selection media (KGM). This GM is commercially available from Kurabo Industries as a serum-free liquid medium for growing normal human epidermal keratinocytes (Hu Media-KG2).

By culturing in such a selective medium, the ratio of transfected cells in the transgenic cell sheet can be adjusted. The percentage of transfected cells in the transgenic cell sheet affects the amount of transgene product produced from the sheet and the preparation period.

In the gene-introduced epithelial cell sheet of the present invention, the ratio of transduced cells in the obtained cell sheet can be 40 to 60%. Such a cell sheet allows the transgene product to be produced properly and has a short culture period and can be prepared early. A transgenic cell sheet into which a gene has been transfected at a rate of 40% to 60% can be easily obtained by culturing it for about 5 days in a selective medium.

Further, in the transgenic epithelial cell sheet of the present invention, the ratio of introduced cells in the obtained cell sheet can be 90% or more. Such a cell sheet can sufficiently produce the product of the transgene. The transfected cell sheet into which the gene has been introduced at a rate of 90% or more can be obtained by culturing in a selective medium for 10 to 14 days. The 100% ratio can be obtained by further extending the culture period in the selective medium. The ratio of transfected cells in the transgenic cell sheet may be changed depending on the type of transgene. For example, when a gene such as an antimicrobial peptide is used as a transgene to increase engraftment efficiency, the ratio is as high as 90% or more, and when a gene of a defective factor is a transgene, about 40 to 60% May be effective. In the present invention, a supporting cell is used in order to grow a mucosal epithelial cell, which is an introduced cell, in a layered manner with high efficiency. The supporting cells include, for example, fibroblasts, for example, mouse NI H3T3 cells, 3T3J2, and Swiss 3T3.From the viewpoint of the thickness of the obtained transfected cell layer and the growth rate of the transfected cells on the supporting cells, 3T3J2 cells are preferred. The proliferative capacity of the feeder cells is eliminated so as not to interfere with the growth of the transfected cells. The disappearance of the proliferative ability can be performed by a method known in the art, for example, treatment with mitomycin C / irradiation. Culture is performed using a culture vessel such as a normal plastic dish.

The transfected cells may be co-cultured with the supporting cells depending on the type of the supporting cells, or may be seeded on the supporting cells that have been seeded and grown in a layered manner. The transfected cells can then be seeded at a cell density of 1 × 10 ⁵ / cm ² , where the supporting cells are 1 × 10 5

It can be seeded at l x 10 ³ / cm ³ or more. If the number of the supporting cells is less than this, it cannot sufficiently serve as a supporting cell, and if it is more than this, the growth of the introduced cells is hindered, which is not preferable. In addition, if the number of introduced cells is smaller or larger than this range, it cannot be efficiently propagated, which is not preferable.

In the present invention, a growth medium is used to grow the transfected cells on feeder cells. The growth medium used for this purpose can be selected from various media known in the art. By culturing in a growth medium, the transfected cells easily form multiple layers. On the other hand, the supporting cells die only by supporting the growth of the mucosal epithelial cells, and when the mucosal epithelial cells form multiple layers, they no longer exist in the culture system.

It is preferable to use an epithelial sheet forming medium (EFM) capable of forming a sheet as the growth medium. EFM is a medium usually used to grow epidermal cells in layers, and consists of a 3: 1 mixture of DMEM and Ham F-12 medium, 5% FBS, 5 g / ml insulin. , transferrin 5〃 / 1111, 2x 10- ⁹ M of Toriyo one Dosaironin, 1 xlO- ⁹ M cholera toxin, 0.4 g / ml of Hyde port cortisone, 10 OU / ml penicillin, of 0.1 / g / ml Kanama It is supplemented with isin, 0.25 μg / ml amphotericin B and 1 ng / ml human recombinant epithelial cell growth factor (EGF).

The multi-layered mucosal epithelial cells are separated from the culture vessel while maintaining the layer structure. This separation can be performed by a method known in the art, and can be easily performed, for example, by using a suitable enzyme such as dispase to inhibit the adhesion of the basal layer. -The obtained mucosal epithelial cell sheet is transplanted into a living body by a method known in the art. Since the gene-introduced cell sheet of the present invention is composed of multiple layers of mucosal epithelial cells, it can be directly transplanted to a transplant site, for example, as a graft. Further, since the gene-introduced mucosal epithelial cell sheet of the present invention is composed of mucosal epithelial cells, it can be transplanted onto the epidermis in contact with the outside world. Also suitable for transplantation. In the case of gene therapy, the gene product of the target gene can be produced at a specific fixed site.For example, if a transplant site where the gene product is released into the bloodstream is selected, the gene product can be produced. Can be delivered to the whole body by blood flow.

The method of transplantation can be changed depending on the type of the transfected cell sheet and the expression site of the transfected gene, and can be carried out by applying a suitable known method as it is. For example, the transfected cell sheet can be transplanted under the skin, in the oral cavity, in the small intestine epithelium, or in the gastrointestinal epithelium with or without a suitable transplant carrier, and a flap is created on the back and placed underneath. You may.

The transduced mucosal epithelial cell sheet taken into the living body produces a substance corresponding to the introduced gene by the action of the expression plasmid. The gene product-producing ability of this transduced mucosal epithelial cell sheet is expected to last for at least 5 weeks, preferably for as long as 8 weeks. The produced substance can be confirmed for its production by various known methods.

Various methods are known in the art for confirming the product. However, when an antibody against the product is known, it is preferable to carry out the ELISA method from the viewpoint of simplicity and sensitivity.

Thus, the transgenic cell sheet of the present invention can be easily prepared because it is composed of mucosal epithelial cells having high proliferative properties and easy transfection at the undifferentiated stage. Also, because they are mucosal epithelial cells, they can be converted into mucosal cells in a humid environment. And can be used as a substitute for epidermal cells under dry conditions such as skin.

In addition, when a gene that promotes graft survival, such as a growth factor or a gene for an antimicrobial peptide to prevent infection, is selected as the target gene, the transplant can be used to obtain a more efficient biological treatment. Can survive.

Further, when a gene for treatment is selected as a target gene for the purpose of gene therapy, it can be easily prepared and used as a useful gene carrier having a long-term gene product producing ability. Example

Hereinafter, examples representing the present invention will be described.

I. Cell preparation

Mucosal tissue was obtained from healthy oral mucosa with patient's consent. Submucosal tissue was removed with scissors and chopped into small pieces. A small piece of mucosal tissue was buffered in phosphate buffer containing 100 OU / ml penicillin (Sigma, St. Louis, M0), 1 mg / ml kanamycin and 2.5 g amphotericin B (Gibco, Gland Island, NY). The cells were immersed twice in PBS (PBS) at 37 ° C for 30 minutes.

The tissue is then immersed in Dulbecco's Modified Minimum Essential Medium (DMEM) containing 100 OPU / ml Dispase® (manufactured by Godo Shusei Co., Ltd.) for 16 hours at 4 ° C., followed by 0.25 minutes at room temperature for 30 minutes. % Trypsin (Gibco, Gland

(Island, NY) to separate cells. Enzyme activity was stopped by washing with DMEM containing 1 %% fetal calf serum (FBS) (Hyclone, UT). Thereafter, the mixture was stirred in DMEM containing 10% FBS for 30 minutes, and then the debris was filtered with a 50 μm nylon gauze to purify the mucosal cells.

The purified mucosal cells were centrifuged at 1500 rpm for 5 minutes, and the obtained cell pellet was resuspended in KGM (Kurabo). Purified mucosal cells were cultured at 37 ° C. in a 10% CO ₂ incubator. The culture was replaced with fresh culture every 2-3 days.

II. Plasmid construction

The Moroni murine leukemia virus (MoMLV) -based retroviral vector pLRNL is a neoma under the control of the Rous sarcoma virus (RSV) promoter. Isin resistance (contains the Neo gene (Li et al., Virology, (1989) 171: 33, 341) There is a Pstl site immediately upstream of this RSV promoter, where a galactosidase is located. The entire code of the enzyme: a Pstl fragment (Matsushita et al., Thromb. Res., (1993) 69: 387-393) containing the cDNA for the region or blood coagulation factor IX was inserted, thereby resulting in pLBZ or pL IXRNL, respectively. (Fig. 1) (Matsushita et al., Thromb. Res., 69: 387-393 (1993)) _0-

III. Virus generation and transfection

PA317, a virus-producing cell, was grown in DMEM containing 10% fetal calf serum (FCS) supplemented with high concentration of glucose.

Plasmid pLBZ or pLIXRNL was transfected into the amphotropic packaging cell line PA317 by the calcium phosphate method as described previously (Emi et al., J. Virol., (1991) 65: 1202-1207). Over the following two weeks, these cells were selected for expression of the neomycin resistance gene by G418 (400 xg / ml). This resulted in a virus producing cell, PA317 / LAZ or PA317 / LI XRNL. The virus titer of each virus-producing cell relative to 208 F cells was approximately 4 × 10 ⁴ / ml.

IV. Efficiency of gene transfer into mucosal epithelial cells

To examine the efficiency of gene transfer into mucosal epithelial cells, mucosal epithelial cells were infected by different infection methods.

Source of infection, were prepared as seeded and virus supernatant of the virus-producing cells, the virus-producing cells as feeder cells in 1 x 10 ^6/35 mm Didzushu. When a virus-producing cell was used as a feeder cell, the virus-producing cell was treated with mitomycin C to lose its growth ability.

Mucosal epithelial cells were seeded at each of these sources. Mucosal epithelial cells were 1 × 10 ^{6 in} 35 mm dishes. Transfected cells were identified 24 hours later by 5-bromo-4-chloro-3-indolyl BD D-galactopyranoside (X-gal) staining. The evaluation was performed by comparing the number of positive cells in the 2 × 2 mm area.

As shown in FIG. 2, the gene could be similarly introduced into the mucosal epithelial cells by the virus-producing cells as supporting cells or by the virus supernatant of the virus-producing cells. Therefore, the operability is good and the virus-producing cells are not used directly. It was decided to carry out gene transfer using a virus supernatant that could transfer the gene into the cell.

V. G418 concentration assay-The optimal amount of G418 used to select for transfected cells was examined.

Mucosal epithelial cells without neomycin resistance gene and other epithelial cell lines (PA317) were prepared as described above, cultured for 10 days, treated with trypsin, and then treated with 3-6 x 35 mm dishes. Seeded at a density of 10 ⁶ cells. When the cells became confluent, various amounts of G418 were added to the culture. Table 1 shows the results. "One" indicates that the cells are killed by the action of G418 (appropriately selectable), and "10" indicates that the cells are not killed by the action of G418 (not properly selectable). For mucosal epithelial cells, two rounds were performed. Table 1 Mucosal epithelial cells and epithelial cell lines

Effects of neomycin

G418 concentration

Mucosal epithelial cells Epithelial cell line

600

500

400 +

300 +

200 +

150 +

100 +++ As shown in Table 1, mucosal epithelial cells were selectable by G418 at lower concentrations than normal epithelial cell lines. Therefore, it was decided to use 0418 at a concentration of 150/1111 when introducing genes into mucosal epithelial cells.

VI. Formation of transgenic mucosal epithelial cell sheet

In the same manner as above, the prepared mucosal epithelial cells were cultured for 10 days, then treated with tribcine, and then seeded on a 35 mm dish at a density of 3-6 × 10 ⁴ cells. After overnight culture, a virus containing 5 μg / ml of polypropylene (Sigma, St. Louis, MO) The mucosal epithelial cells were infected with 2 ml of the supernatant for 3 hours.

Twenty-four hours after infection, transduced mucosal epithelial cells were selected by culturing for 10-14 days in KGM medium containing G418 (150 g / ml; (Gibco, Gland Island, NY)). The mucosal epithelial cell population was treated with trypsin-EDTA to prepare a cell suspension of 1 × 10 ⁵ cells / ml.

3T3-J2 cells as feeder cells were treated with 4 g / ml mitomycin C (Kyowa Hakko, Tokyo) in serum-free DMEM. Two hours later, mitomycin C was removed by rinsing several times with PBS (-), and the cells were treated with trypsin to prepare a cell suspension of 110 ⁴ cells / ml.

1 and 10 ⁵ cells / ml of mucosal cell suspension, and a support cell suspension 1 X 10 ⁴ cells / ml, was added in lml each of 6 © El plates Ueru, in KGM selected culture ground Cultured. Every two or three days, half of the medium was replaced with fresh EFM selective medium and maintained to form a layered epithelial cell sheet.

The mucosal epithelial cells seeded and cultured on the supporting cells thus proliferated on the supporting cells. In addition, the mucosal cells at the undifferentiated stage could be maintained by the selective culture in the KGM selection medium for 10 to 14 days, and the ability to form a layer and to form an epithelial cell sheet was maintained (Fig. 3A and Fig. 3). B) By culturing on feeder cells for about 2 weeks, it became thick enough as a transplant (3 to 5 layers, about 100 m). Since the epithelial cell sheet can be formed by layering in this manner, the transgenic cell sheet can be used as a graft or a gene carrier for therapy.

In contrast, cells cultured in serum-containing EFM selective media alone without KGM selective media formed an irregular layer and were enucleated (Figure 3C). This means that terminal differentiation has already taken place and the ability to form sheets to be uniformly layered has been lost.

VII. Transplantation of transgenic mucosal epithelial sheet

The transfected mucosal epithelial cells became confluent 10 to 14 days after the start of culture and became stratified. This cell sheet was used as a graft.

The epithelial cell sheet was detached with dispase (400 PU / ml) and washed twice with PBS. The cell sheet was transferred to a nude mouse (5-6 weeks old, male, BALB / c nu / nu) (Nippon SLC, Hamamatsu) under general anesthesia by intraperitoneal administration of Bentobarbi 0.04 mg / g. Transplanted. The transplantation method was performed by the method of Barrandon et al. (Barrandon et al., J. Invest. Dermatol., (1998) 91, 315-318) and the method of Sugimura et al. (Sugimura et al., J. cranio-maxillofac)

Surg., (1997) 24, 352-359). -That is, after the back of the mouse was thoroughly disinfected with 10% Hibiten alcohol, a 30 x 30 mm rectangular flap was made to expose the muscle layer. A sterilized silicone membrane was placed over the entire exposed mouse muscle layer to isolate the flap from the muscle layer. Next, the prepared cultured mucosal epithelial cell sheet was placed on a sterilized silicone membrane as a carrier for transplantation, and transplanted onto the silicone membrane so that the cultured mucosal epithelial cells were in contact with the inside of the flap. Then, the flap was returned so as to cover the transplanted mucosal epithelial cell sheet and silicone membrane, and sutured with 5-nylon thread to complete the transplantation. Thus, the mucosal epithelial cells were fixed such that the surface (the basal layer) that had been in contact with the plate during the culture of the mucosal epithelial cells was in contact with the inside of the mouse flap.

VIII. Histological analysis

Histological analysis was performed to confirm the engraftment of the transplanted transgenic mucosal epithelial cells. Engraftment of the transplanted transduced mucosal epithelial cell sheet was confirmed by staining with X-Ga1 for ガ -galactosidase expressed by pLBZ (including the gene for —-galactosidase).

At 1, 3 and 5 weeks after transplantation, flaps containing the transduced mucosal epithelial cells were collected and frozen in cryoprotection medium (O.T., Tissue Tek, Miles). Frozen sections (5 μm) of the above flaps embedded in cryoprotective medium were fixed in a 2.5% glutaraldehyde solution in PBS for 15 minutes at 4 ° C. The sections were then washed twice in PBS and stained

(Dimethylformamide solution containing ImM MgCl ₂ , 5 mM K ₃ Fe (CN) ₆ , 5 mM K ₄ (CN) ₆ and 400/1111 -0 & 1) C soaked and stained. The results are shown in FIGS.

Transplants of the transduced mucosal epithelial cell sheet were stained almost blue one week after transplantation (Fig. 4A, X5). This means that one week after the transplantation, the transplanted transfected mucosal epithelial cells have produced a sufficient amount of the galactosidase protein and have been fully engrafted. One week after transplantation, the graft was completely adhered, and many epidermal cells had infiltrated along the graft (Fig. 5A, X250).

The staining of such grafts weakens after 3 weeks (Figure 4B) and after 5 weeks Almost no staining (Fig. 4C). Three weeks after transplantation, expression of? -Galactosidase was reduced, and the grafts became progressively thicker and showed signs of cell degradation, such as a lack of basement membrane (Figure 5B). However, 5 weeks after the transplantation, expression of -galactosidase was detectable (Fig. 5C).

IX. Blood coagulation Factor IX production

To confirm the production of the gene product introduced into mucosal epithelial cells, the gene for blood coagulation I-X factor was introduced, and the blood concentration of this factor was measured. It has been already proved that the used blood coagulation factor IX expression product is active. The production of blood coagulation factor IX by the transduced mucosal epithelial cells is a model of gene therapy for hemophilia.

The transduced mucosal epithelial cells transgenic and transfected using pLIXRNL were selectively cultured with G418 for 2 weeks as described above, seeded on a 35 mm culture dish, and co-cultured with the support cells. After culturing, human blood coagulation factor IX released into the growth medium over time was measured for each dish. Measurement of blood coagulation factor IX was performed by the ELISA method described previously (Takahashi et al., J. Lab. Clin. Med., (1991), 118: 317-325). Table 2 shows the results. Table 2 Transgenic oral mucosal epithelial cells

In vitro expression of blood coagulation factor X

As shown in Table 2, the maximum value of blood coagulation factor IX detected in the growth medium was 30 days after the start of the culture. Under culture conditions, the transgene was shown to be expressed for at least 7 weeks.

Next, the in vivo expression of the transgene was analyzed.

Transfection of human blood coagulation factor IX into mucosal epithelial cells and selection After the co-culture, the transgenic mucosal epithelial sheet formed as described above was placed on a 3 × 3 cm silicon membrane and transplanted to the back of two nude mice. Each mouse's blood was collected by infraorbital puncture and collected in a 1/10 volume of 3.8% sodium citrate solution. Remove cells by centrifugation and store plasma sample at -80 ° C. Blood coagulation factor IX levels were measured by ELISA as described above. Two mice transplanted with the transfected mucosal epithelial cells were found to produce 1.5-1.8 ng / ml of human blood coagulation factor IX early after transplantation (Fig. 6). Human blood coagulation factor IX in this blood was detected for more than 3 weeks. This means that the transduced mucosal epithelial cells stably produce gene products for a long time after transplantation, as under the culture conditions.

As described above, the transgenic cell sheet of the present invention can be easily prepared, and even when transplanted to the back in a sheet form, human blood coagulation factor IX can be produced in blood for a long time. Industrial applicability

According to the gene-introduced mucosal epithelial cell sheet of the present invention, since it is composed of more undifferentiated mucosal epithelial cells, the target gene can be easily introduced and can be prepared in a short time. This can provide a useful carrier for gene therapy that produces the gene product of the transgene for a long period of time, and provides a graft with high engraftment efficiency in which graft engraftment is promoted. be able to.

Further, according to the method for producing a gene-introduced mucosal epithelial cell sheet of the present invention, it is possible to easily produce a useful carrier for gene therapy as described above and a graft with high engraftment efficiency.

The description in this specification is merely for the purpose of describing the present invention, and should not be treated as limiting the present invention, and various modifications are possible without departing from the spirit and scope of the present invention.

Claims

The scope of the claims

1. A transgenic cell sheet composed of multiple layers of mucosal epithelial cells into which the target gene has been introduced.

2. The transgenic cell sheet according to claim 1, wherein the target gene is introduced into 40% to 60% of the mucosal epithelial cells. -

3. The transfected cell sheet according to claim 1, wherein the target gene is introduced into 90% or more of the mucosal epithelial cells.

4. The transgenic cell sheet according to claim 1, wherein the target gene is selected from the group consisting of a blood coagulation factor, insulin, a growth factor, a tumor antigen gene, and a tumor suppressor gene.

5. The gene-transferred cell sheet according to any one of claims 1 to 4, for gene therapy used as a gene carrier in gene therapy.

6. The transgenic cell sheet according to any one of claims 1 to 4, which is used as a graft.

7. A method for producing a transgenic cell sheet comprising a plurality of layers of mucosal epithelial cells into which a target gene has been introduced,

Obtaining mucosal epithelial cells for preparing a cell sheet, and supporting cells compatible with the mucosal epithelial cells,

An expression system containing the target gene and the marker gene is introduced into the mucosal epithelial cells,

The cells were cultured in a serum-free selective culture medium containing no serum, and the mucosal epithelial cells into which the expression system was introduced were selected.

The mucosal epithelial cells into which the expression system has been introduced are cultured together with the feeder cells in a growth medium containing serum,

Obtain mucosal epithelial cells with multiple layers,

A method for producing a transgenic cell sheet comprising: