ES2358323T3 - PRIMARY CULTURE ADIPOCITS FOR GENE THERAPY. - Google Patents

Abstract

Pharmaceutical composition comprising a primary culture preadipocyte, in which the preadipocyte stably maintains a foreign gene that codes for a protein that is secreted outside the cell and in which the gene has been inserted into a retroviral vector and has been transferred to the cell by the retroviral vector, in which the protein is insulin or glucagon-like peptide 1 (GLP-1).

Description

Technical field

The present invention relates to primary culture adipocytes for gene therapy, to which a foreign gene (s) has been transferred.

Prior art

Current gene therapies (Toyooka et al., Folia Pharmacol. Jpn. 116: 158-162, 2000) can be classified into two groups: (1) methods of therapeutic gene transfer in patients directly administering viral vectors, naked plasmids, or so that they code for the gene (in vivo), and (2) methods of temporary extraction of patient cells, transfer of a gene to these cells and then return of these cells to the patient (ex vivo).

In in vivo methods, important problems, such as transfer efficiency, continuous expression and selective gene transfer to target cells, remain unresolved. In contrast, ex vivo methods can potentially overcome these problems. Most examples of ex vivo methods have been performed using blood system cells (peripheral lymphocytes and bone marrow cells), since their collection and transplantation is relatively easy and the burden is reduced for patients (Tani et al., Saishin Igaku, 56: 258-267, 2001). With regard to cells other than blood system cells, methods have been carried out that transfer genes to hepatocytes and then return these cells to the patient (Raper, SE et al., Cell Transplant 2 (5): 381-400, 1993) , but most of these methods focus on the recovery, maintenance and enhancement of the function of the transfected cells themselves.

Description of the invention

While looking for suitable cells for ex vivo gene therapy, the present inventors developed the idea of using primary culture adipocytes. The use of adipocytes has the following advantages:

(one)

 There are many reports of humoral factors secreted from adipocytes, and adipocytes comprise the functions of hormone production and can act as secretory organs (Bradley RD, et al., Recent Prog. Horm. Res., 2001, 56, 329-358) ;

(2)

 adipocytes can be easily collected since they also exist subcutaneously, and techniques related to their removal in the fields of plastic and cosmetic surgery are being developed; in addition, even when adipocytes are grafted into subcutaneous tissue, which allows for easy implantation, these cells are not heterotropic since they originally belonged to this region;

(3)

 since isolated primary culture adipocytes actively proliferate, even in vitro, they are suitable for procedures such as gene transfer;

(4)

 Since adipocytes are likely to remain in a limited area after implantation, grafted cells can be removed after implantation if desired (specifically, when gene expression is to be eliminated);

(5)

 since adipocytes themselves produce angiogenic factors (Mick, G.J., et al., Endocrinology 2002, 143 (3): 94853), a high level of graft acceptance can be expected after implantation;

(6)

 the removal or implantation of adipocytes has a small impact on the human organism because the weight of this organ changes greatly in adults; Y

(7)

 Adipocytes are widely recognized as superfluous and obstructive, and consent to their collection can be easily obtained.

Although investigations with similar objectives are currently under way using keratinocytes (J. Gene. Med. January-February 2001, 3 (1): 21-31; Histochem. Cell Biol. 2001 Jan, 115 (1): 73-82) , the removal of the biological barrier of the skin in the isolation procedure of the primary culture is problematic considering the risk of infection. It is predicted that the patient's pain during excision and implantation is strong, and it is not easy to re-remove (4, mentioned above) to eliminate expression. In addition, when keratinocytes or skin are used, which can only be grafted two-dimensionally, the amount of the graft can only be increased by increasing the surface area of the graft. Therefore, adipocytes, which allow three-dimensional transplantation, are considered more useful.

The present inventors designed methods to transfer genes effectively into primary culture adipocytes. They also confirmed that the transferred genes work after implantation, and found that adipocytes can be used effectively in gene therapy. In addition, adipocytes that stably express the foreign gene transferred in vivo for a long period of time can be obtained by the methods of this invention. Mature implanted adipocytes can continue to express foreign genes for a year or more. In addition, if the expression of the foreign gene becomes unnecessary after the implantation of the adipocytes, the expression can be stopped by removing the graft.

The present invention relates to the following points:

one.

A pharmaceutical composition comprising a primary culture preadipocyte, in which the preadipocyte stably maintains a foreign gene that encodes a protein that is secreted outside the cell and in which the gene has been inserted into a retroviral vector and is has been transferred to the cell by the retroviral vector, in which the protein is insulin or glucagon-like peptide 1 (GLP-1).

2.

A primary culture preadipocyte, the preadipocyte stably maintaining a foreign gene that codes for a protein that is secreted outside the cell, and in which the gene has been inserted into a retroviral vector and transferred to the cell by retroviral vector, for use in gene therapy, in which the protein is insulin or GLP-1.

3.

The pharmaceutical composition of item 1, or the preadipocyte for use according to item 2, in which the preadipocyte has the ability to significantly express the protein in vivo for at least 20 days.

Four.

The pharmaceutical composition of item 1 or 3, or the preadipocyte for use according to item 2 or 3, in which the preadipocyte is used to release the protein into the bloodstream.

5.

A method of in vitro production of a preadipocyte for use in gene therapy, the method comprising the steps of:

(one)

Carry out a primary culture of a preadipocyte; Y

(2)

transfer, and then stably maintain a foreign gene that codes for a protein that is secreted out of the cell,

in which the protein is insulin or GLP-1.

6.

The method of item 5, in which the foreign gene is transferred by a retroviral vector.

7.

The preadipocyte for use according to point 2 that has been produced by the method of point 5 or 6.

8.

An implant composition, the composition comprising a primary culture preadipocyte, which stably maintains a foreign gene that codes for a protein that is secreted outside the cell, and in which the gene has been inserted into a retroviral vector and is has been transferred to the cell by the retroviral vector, and a pharmaceutically acceptable carrier, for use in gene therapy, in which the protein is insulin or LPG

one.

9.

The implant composition for use according to item 8, or the pharmaceutical composition according to any one of claims 1, 3 and 4, further comprising an extracellular matrix component.

10.

The implant composition for use according to item 8 or 9, or the pharmaceutical composition according to any one of item 1, 3, 4 and 9, further comprising an angiogenesis factor.

eleven.

A primary culture preadipocyte that stably maintains a gene coding for insulin or glucagon-like peptide 1 (GLP-1) for use in blood glucose lowering, in which the gene has been inserted into a retroviral vector and It has been transferred to the cell by the retroviral vector.

12.

A non-human animal, in whose organism the preadipocyte of primary culture of point 2 has been implanted.

Hereinafter, the way to carry out this invention will be described.

First, the present invention provides primary culture adipocytes for gene therapy, in which the adipocytes stably maintain a foreign gene (s) encoding a protein (s). ) that is secreted (n) outside the cell.

Here, a foreign gene refers to an insulin or GLP-1 gene transferred to the primary culture adipocytes from outside. In addition, primary culture cells refers to non-established cells that are grown from tissues extracted from a living organism. Adipocytes refers to mature adipocytes and cells that comprise the ability to differentiate into adipose tissue, such as preadipocytes. More specifically, unless it is particularly said that adipocytes are "mature" adipocytes, they also include preadipocytes. Mature adipocytes are spherical cells that store fat, and contain droplets of lipids. Fat stored in mature adipocytes can be identified using O oil red staining. Mature adipocytes generally secrete leptin in response to insulin. Preadipocytes normally exist as stromal cells that have not yet differentiated into mature adipocytes. The preadipocytes can be isolated by treating the adipose tissue with collagenase, or they can be isolated as a result of the division of mature adipocytes, using the roof culture method described below (Sugihara, et al. Nippon Rinsho 1995, 53, 115-120; Sugihara , H., et al. J. Lipid Res. 1987, 28, 1038-1045; Zhang HH, et al. J. Endcriniol. 2000, 164, 119-128). Although the existence of adipocyte-specific surface antigens has not been confirmed, high levels of CD36 and similar expression have been found in mature adipocytes (Abumrad NA, et al. J. Biol. Chem. August 25, 1993, 268 ( 24): 17665-8). Therefore, extremely pure adipocytes can be collected using such molecules as markers. By inducing differentiation as described below, preadipocytes can be differentiated into mature adipocytes within a few days to a few weeks (Hauner H., et al., J. Clin. Invest. 84, 1663-1670, 1989 ; Marko, et al., Endocrinology 136, 45824588, 1994). Primary culture adipocytes can be isolated from a desired tissue, for example, subcutaneous adipose tissue or visceral adipose tissue such as tissue surrounding the epididymis or mesenteric tissue.

The term "for gene therapy" refers to using the in vivo expression of a protein (s) encoded by a foreign gene (s) providing a therapeutic effect. In addition, cells for gene therapy refers to cells that carry a foreign gene (s), the cells being used to administer the foreign gene to an organism by ex vivo administration, and the cells comprise the ability to express the protein in that organism. Ex vivo administration refers to extracting adipose tissues or adipocytes from an individual, performing the gene transfer in vitro and then implanting the cells in the same or a different individual.

Cells for gene therapy preferably refers to cells used to treat disorders, which are cells that are implanted so that a specific protein is produced. Preferably, treatment by a specific protein includes replacement therapy, which uses a protein whose absence or physical or functional deficiency causes a disorder. The specific protein is insulin or GLP-1 that shows activity in the bloodstream, or is supplied to a target tissue through the bloodstream, and works on the cell surface of that tissue. A continuous supply of the specific protein is also preferably required for a certain period of time (for example, for a few days to a few weeks or more). The factors and disorders for which protein replacement therapy is already being carried out, or is predicted to be effective, can all become targets.

Hereinafter, representative targets are listed according to their classification, but it is not to be understood that their use is limited to these examples, and the use of similar factors for similar purposes is included within the scope of this invention.

Substitution therapy includes complementation against disorders that develop or are exacerbated by a lack or reduced function of a hormone, complementation against disorders due to a congenital genetic defect, and complementation of a factor for pathological improvement:

insulin / diabetes; Glucagon-like peptide-1 (GLP-1) / diabetes, obesity, eating disorders.

In addition, the adipocytes of the present invention are not limited to those used for the so-called "therapy", but include cells used for in vivo expression of a desired secretory protein. For example, the methods of this invention allow the production of model animals by subsequent expression of a particular protein. Using these methods, disease model animals can be produced with a posteriori expression of pathogenesis or aggravating factors, and these animals can be used to select drugs. In addition, expressing pathological improvement factors, these methods can be used as a working hypothesis test for discoveries of novel drugs in which a given factor improves a pathological state. Animals that are used include desired non-human animals, and preferably non-human mammals (including rodents and primates).

The primary culture adipocytes for the gene therapies of this invention stably maintain an insulin gene (s) or GLP-1 encoding insulin or GLP-1 that is secreted out of the cell. The term "stably maintained" means that the foreign gene passes to the daughter cells during cell division, and more specifically, this expression refers to the incorporation of the foreign gene into a cell chromosome. Adipocytes for gene therapy of this invention comprise an insulin gene (s) or GLP-1, stably transferred (s) by a viral chromosome incorporation vector. The insulin or GLP-1 gene is transferred by a retroviral vector.

The retroviral vector is stably integrated into a cell chromosome and comprises the ability to express a gene transferred over a long period. The transfer efficiency of the vector and the continuation of the expression of the transferred gene depends on the type of cell. For example, a gene transferred by a retroviral vector can show continuous expression while cells grow, but expression can stop when cell growth stops (Lund, AH, et al., J. Biomed. Sci. 1996, 3: 365-378; Niwa, O. et al., 1983, Cell, 32: 1105-1113). It is often observed that the expression of the foreign gene is suppressed, particularly after introducing the gene into an organism by in vivo or ex vivo methods. It is said that such suppression of the expression involves de novo methylation of the promoter or the coding sequence of the transferred gene (Jahner, D. and Jaenisch, R., Nature 315: 594-597, 1985; Challita, P.-M. and Kohn, DB, Proc. Natl. Acad. Sci. USA 91: 2567-2571, 1994; Hoeben, RC et al., J. Virol. 65: 904-912, 1991). In addition, histone deacetylation is implicated in the silencing of the transferred gene (Chen, WY et al., Proc. Natl. Acad. Sci. USA 97: 377-382, 2000; Chen, WY et al., Proc. Natl Acad. Sci. USA 94: 5798-5803, 1997). However, when the present inventors transferred a foreign gene to primary culture adipocytes using a retroviral vector, surprisingly, it was found that the expression of the transferred gene persisted extremely stable, both in vitro and in vivo. The expression of the transferred genes is stable in adipocytes before their differentiation and also in mature adipocytes. It was confirmed that the expression of the transferred gene persisted throughout the duration of the experiment for in vitro cultures (80 days or more), and for the entire duration of the experiment when it was implanted in the organism (360 days or more). Therefore, primary culture adipocytes can be used, to which an insulin gene (s) or GLP-1 has been stably transferred, as implants that stably express a gene (os) ( is) for a long period.

Adipocytes for gene therapy of this invention comprise the ability to significantly express insulin or GLP-1 encoded by a foreign insulin gene (s) or GLP-1 (s) for at least 20 days or more in vitro, or more preferably in vivo. The term "express significantly" means, for example, that expression is detected at a statistically significant level compared to when the foreign gene is not transferred (for example, with a significance level of 5% or a higher significance). More preferably, the adipocytes of the present invention, when transplanted into an organism, comprise the ability to significantly express insulin or GLP-1 encoded by an insulin gene (s) or GLP-1 in the body for at least 30 days or more, preferably 40 days or more, more preferably 50 days or more, even more preferably 60 days or more, still more preferably 80 days or more, even more preferably 100 days or more, even more preferably 150 days or more, even more preferably 200 days or more, even more preferably 250 days or more, even more preferably 300 days or more, and even more preferably 350 days or more.

The adipocytes for gene therapy of this invention are particularly useful as cells for releasing insulin or GLP-1, which are encoded by foreign genes carried by the cells, in the bloodstream. The proteins released into the bloodstream are insulin and / or glucagon-like peptide-1 (GLP-1) to treat diabetes and the like. For example, for insulin, the cleavage sites (site 1 and site 2) can be replaced by the cleavage sequence of a protease expressed in adipocytes, so that mature insulin can be produced efficiently (e.g., Groskreutz, DJ, et al. JBC, 1994, 269 (8), 6241). A modified insulin analog with a single chain can also be used (Lee, H.C., et al., Nature. November 23, 2000, 408 (6811): 483-8). For GLP-1, a desired peptide that acts as a ligand for the GLP-1 receptor can be used (NP_002053; Thorens, B. et al., Diabetes 42, 1678-1682 (1993); Dillon, JS et al., Endocrinology 133, 1907-1910 (1993); Graziano, MP et al., Biochem. Biophys. Res. Commun. 196, 141-146 (1993); Stoffel, M. et al., Diabetes 42, 1215-1218 (1993) ). An example is GLP-1 (7-37) (Diabetes, 1998, 47: 159-69; Endocrinology, 2001, 142: 521-7; Curr. Pharm. Des., 2001, 7: 1399-412; Gastroenterology, 2002 , 122: 531-44).

The present invention also relates to adipocyte production methods for use in gene therapy, the methods comprising the steps of:

(one)

 carry out a primary preadipocyte culture, and

(2)

 transfer cells with a foreign gene (s) encoding (n) for a protein (s) that is secreted outside the cell, preferably using a retroviral vector so that the gene is stably maintained, said protein being insulin or GLP-1.

The present invention also relates to adipocytes for gene therapy produced by this method. "Stably maintained" means the transfer of a foreign gene (s) so that it passes to the daughter cells when the cell divides, and more specifically, refers to the integration of the gene foreign in the chromosome of the cells. Southern blotting or PCR using genomic DNA can demonstrate from the point of view of molecular biology that the foreign gene has achieved stable expression by integrating it into a chromosome. In addition, to concentrate stably transfected cells, for example, a method using fluorescence activated cell sorting (FACS) can be used, which concentrates cells recognizing the GFP expressed jointly by the cells together with the target gene.

1. Methods for collecting primary culture adipocytes

Adipocytes of primary culture can be collected by the methods described in the report of Sugihara et al. (Sugihara, H. et al., Differentiation, 31: 42-49, 1986). More specifically, adipose tissue is removed, and preferably the subcutaneous adipose tissue itself or visceral adipose tissue of the implant recipient, such as the tissue surrounding the epididymis or mesenteric tissue, under sterile conditions, and, for example, after washing with PBS, It is chopped using scissors or a scalpel. This chopped tissue is digested by stirring at 37 ° C in a medium comprising an appropriate amount of collagenase, preferably 1 to 3 mg / ml, for an appropriate period of time, preferably for 20 to 60 minutes, and then separated into a residue. precipitate and a floating layer by centrifugation.

The floating layer is preferably further washed once or twice by centrifugation, and then added to a culture flask filled with medium. The bubbles are removed, and the flask is allowed to stand in a CO2 incubator for cultivation, so that the conventional culture surface is a roof (roof culture). After culturing for an appropriate period, preferably ten to 14 days, cells adhered to the roof surface are collected by trypsin treatment. Subsequently, these cells are subcultured in a conventional culture system.

Adipocytes of primary culture can be stored by freezing before or after gene transfer. This procedure allows multiple use of adipocytes after a single collection.

2. Gene transfer to adipocytes

Gene transfer can be performed using gene transfer reagents (Fugene 6, Roche; Lipofectamin, Invitrogen; Cellphect transfection kit (calcium phosphate method), Amersham; etc.), electroporation methods (Chen, H. et al., J. Biol. Chem. 1997, 272 (12), 8026-31), or viral vectors (Kay, MA, et al., Nat. Med. 2001, 7, 33-40). Preferably the transfer is performed using viral vectors, and more preferably using retroviral vectors (eg, Arai, T. et al., J. Virol., 1998, 72, pp. 1115-21).

When gene transfer is performed using a plasmid, the plasmid is transfected into adipocytes, and adipocytes that stably maintain the transferred foreign gene can be selected. Such adipocytes can be selected, for example, by providing the plasmid that codes for the foreign gene of a drug resistance gene, or by performing transfection together with a plasmid carrying a drug resistance gene, and then selecting the transfected cells using this drug. On the other hand, the cells can be obtained by cloning the cells by limiting dilution techniques. In addition, when gene transfer is performed using a plasmid, a method of transient expression of a phage derived integrase can be used to increase the efficiency of chromosomal insertion (Mol. Cell Biol. 2001 Jun, 21 (12): 3926-34 ).

In the present invention, the foreign insulin or GLP-1 gene is transferred to adipocytes using a retroviral vector. Retrovirus refers to viruses that belong to the Retroviridae family, and include oncovirus, foamvirus (Russell, DW and Miller, AD, J. Virol. 1996, 70: 217-222; Wu, M. et al., J. Virol. 1999, 73: 4498-4501) and lentivirus (eg, HIV-1 (Naldini, L. et al., Science 1996, 272: 263-267; Poeschla, E. et al., Proc. Natl. Acad. Sci USA 1996, 93: 11395-11399; Srinivasakumar, N. et al., J. Virol. 1997, 71: 5841-5848; Zufferey, R., et al. Nat. Biotechnol. 1997, 15: 871-875; Kim, VN, et al., J. Virol. 1998,72: 811-816) and feline immunodeficiency virus (Johnston, JC et al., J. Virol. 1999, 73: 4991-5000; Johnston, J. and Power, C., J. Virol. 1999, 73: 2491-2498; Poeschla, EM et al., Nat. Med. 1998, 4: 354-357)). A preferable retroviral vector for use in this invention is a Moloney murine leukemia virus (VLMM) vector (T. M. Shinnick, R. A. Lerner and J. G. Sutcliffe, Nature 293, 543-548, 1981).

Retroviruses can be self-activating vectors (SIN). A SIN vector can be prepared by deleting a part of the 3 ′ LTR during viral packaging (Yu S.F. et al. (1986) Proc. Natl. Acad. Sci. USA 83: 3194; Yee,

J. K. et al., 1987, Proc. Natl Acad. Sci. USA 84: 5197-5201; Zufferey, R. et al., 1998, J. Virology, 72, 9873-9880). The foreign gene in the retrovirus can be transcribed by the LTR, or it can be expressed from another promoter within the vector. For example, a constitutive expression promoter such as CMV promoter, EF-1 promoter or CAG promoter, or a desired inducible promoter can be used. In addition, a chimeric promoter can be used, in which a part of the LTR is replaced by another promoter.

To transfer genes using retroviruses, specifically, a plasmid carrying a gene to be transferred, such as pBabe CL-SEAP-IRES-GFP, is introduced by gene transfer into the packaging cells, such as 293-EBNA cells (Invitrogen) , using a reagent for gene transfer and the like. This is then cultured for an appropriate period of time, preferably one to three days, and the recombinant viruses produced in the supernatant are collected. Then, these viruses are infected in the adipocytes that are going to be transfected.

Retroviral vectors preferably comprise a envelope protein with broad tropism, so that they can infect a wide range of mammalian adipocytes, including those of humans. For example, amphotropic envelope protein (for example 4070A) (record K02729; Sorge, J. et al., Mol. Cell. Biol. 4 (9), 1730-1737 (1984)) can be used. In the present invention, the retrovirus is preferably pseudotyped (Emi, T. Friedmann and JK Yee, J. Virol., 65 (3), 1202-1207 (1991); Yee, J.-K. et al. (1994) Methods Cell Biol. 43 43: 99-112; Burns, JC et al. (1993) Proc. Natl. Acad. Sci. USA 90 90: 8033-8037) by protein G of vesicular stomatitis virus (VSV-G ) (Rose, JK and Gallione, CJ, J. Virol. 39 (2), 519-528 (1981)). Pseudotyping by VSV-G allows highly efficient transfer of genes to adipocytes. The pseudotyped vector can be produced by VSV-G expressing VSV-G in the packaging cells. More specifically, for example, packaging cells that can induce VSV-G can be favorably used (for example, Arai T. et al., J. Virol., 1998: 72, pp. 1115-21).

The titer of the viruses produced can be determined by infecting cells with virus solutions that have been gradually diluted, and counting the number of colonies of infected cells (for details, see Ausubel et al). (Ausubel, F.M. et al. Eds. (1995) Current Protocols in Molecular Biology. (John Wiley & Sons, NY)). Alternatively, the titer can be determined by the method of Byun et al. (Byun, J. et al. (1996) Gene Ther. 33333: 1018-1020), Tafuro et al. (Tafuro, S. et al. (1996) Gene Ther. 33333: 679-684), Miyao et al. (Miyao, Y. et al. (1995) Cell Struct. Funct. 2020: 177-183), Claudio et al. (Claudio, P. P. et al. (2001) Anal. Biochem. 291: 96-101), or Cashion et al. (Cashion, L. M. et al. (1999) Biotechniques 26: 924-930).

Viral vectors can be introduced into primary culture adipocytes by contacting the vectors with the cells. For example, primary culture adipocytes are incubated in a culture solution comprising viral vectors. Preferably, the adipocytes are infected in the form of preadipocytes. The effectiveness of the infection can be increased by adding more or less from 0.5 to 8 mg / ml of polybrene. The multiplicity of infection (MOI) is not particularly limited, but can be appropriately adjusted within the range of 0.1 to 100. Cells subjected to gene transfer can be selected using a marker gene, for example. However, if the infection is carried out at an MOI of about 2 or more, or preferably about 3, 4, 5 or more, the gene can be transferred to most cells, even without selection. Adipocytes subjected to gene transfer can be used for implantation without further treatment, or in certain cases, they can be converted into mature adipocytes by culture in a medium comprising 3-isobutyl-1-methylxanthine (IBMX), dexamethasone and insulin. In such cases, since mainly IBMX and dexamethasone are used to activate the adipocyte peroxisome proliferator-activated receptor  (PPAR-), drugs that directly activate this receptor (for example thiazolidine derivatives) can be added at the same time. pioglitazone / Takeda Pharmaceutical Company Limited and rosiglitazone / GlaxoSmithKline).

The primary culture adipocytes of this invention, which carry a desired therapeutic insulin or GLP-1 gene, can be implanted in the body of an immunologically compatible receptor, thus allowing gene therapy by in vivo expression of the secretory protein encoded by the therapeutic gene The primary culture adipocytes to be implanted are preferably cells of the same host as the recipient. The gene therapy methods in which the primary culture adipocytes of this invention are implanted can be applied by expressing a desired insulin secreting protein or GLP-1 in an organism, anticipating the effects of that protein. For example, a disorder can be treated or prevented by implanting the adipocytes of this invention, which maintain a foreign insulin gene (s) or GLP-1 (s) encoding an insulin or a GLP-1 comprising a therapeutic or preventive effect against the disorder. In addition, the present invention relates to methods of releasing proteins in the bloodstream, the methods comprising the step of administering the primary culture adipocytes of this invention to an organism. Using these methods, insulin or GLP-1 encoded by a foreign insulin or GLP-1 gene can be significantly secreted into the bloodstream for at least 20 days or more, preferably 30 days or more, more preferably 40 days or more, even more preferably 50 days or more, still more preferably 60 days or more, even more preferably 80 days or more, even more preferably 100 days or more, even more preferably 150 days or more, even more preferably 200 days or more, even more preferably 250 days or more, even more preferably 300 days or more, and even more preferably 350 days or more. The foreign insulin or GLP-1 gene expressed in an organism can be detected and / or quantified, for example by immunoassays such as EIA. Removal of the transplanted cells can stop the expression of the foreign insulin or GLP-1 gene administered at any time. In certain cases, by transferring an inducible suicide gene (for example, HSV-tk) to the graft cells, the graft cells can be removed by administering ganciclovir, for example.

The present invention also provides implant compositions for use in gene therapy, comprising primary culture adipocyte compositions that stably maintain an insulin gene (s) or GLP-1 encoding an insulin or a GLP-1 secreted outside the cell, and pharmaceutically acceptable carriers. Examples of the vehicles are physiological saline, phosphate buffer, culture solutions, sera and body fluids. These can also be combined with a solid or gel support that becomes a framework for cells.

The implant compositions of the present invention preferably comprise an extracellular matrix component (CME). An extracellular matrix component refers to a component such as a protein or a mucopolysaccharide comprised in an insoluble network or fibrous structure accumulated between the cells. They can be isolated from organisms or reconstructed artificially. CME components preferably used in this invention are collagen, fibronectin, vitronectin, laminin, heparan sulfate, proteoglycan, glucosaminoglycan, chondroitin sulfate, hyaluronate, dermatan sulfate, keratin sulfate, elastin or combinations of two or more of the foregoing. Preferably, these CME components are formed on a gel and then mixed with the adipocytes. The CME gels used in this invention are not particularly limited, provided that they are comprised of at least one or more of the aforementioned components, but preferably comprise at least type IV collagen, laminin and heparan sulfate. Such CMEs include a substrate extracted from Engelbreth-Holm-Swarm mouse tumor (Matrigel®) (Becton Dickinson Labware) (US Patent No. 4,829,000). The structure of the compositions comprising the CME component and the adipocytes used in the present invention is not particularly limited, and may be, for example, a gel or paste network structure, a fibrous structure, flat structure (disk), structure in honeycomb and sponge-like structure. The CME components can be gelled according to conventional methods. For example, gelation can be carried out by incubating an aqueous solution comprising approximately collagen at 0.3 to 0.5% at 37 ° C for ten to 30 minutes. On the other hand, CME components can be gelled using a gelling agent.

In addition, the implant compositions of the present invention preferably comprise an angiogenesis factor. The implant compositions of this invention comprising an angiogenesis factor cause blood vessels to form around them after implantation, and can secrete a foreign protein in the bloodstream with superior efficacy. Angiogenesis factors are not particularly limited, provided they are factors that can induce angiogenesis in vivo, and examples are vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), fibroblast growth factor acid (aFGF), platelet-derived growth factor, transforming growth factor- (TGF-), osteonectin, angiopoietin and hepatocyte growth factor (HGF). The most preferred example is bFGF. The bFGFs, which are also called FGF2, are not only fibroblast growth factors, but also comprise the growth promoting activity of various cells such as vascular endothelial cells, cartilage, osteoblasts and epidermal cells (Abraham et al., EMBO J., 5, 2523-2528, 1986; Prats et al., Proc. Natl. Acad. Sci. USA, 86, 18361840, 1989). The bFGFs used in the present invention are not only natural proteins, but can also be produced by genetic engineering using recombinant DNA technology, and modified forms thereof. Examples of the bFGFs are those described in WO87 / 01728, WO89 / 04832, WO86 / 07595, WO87 / 03885, European Patent Application Publications Nos. 237966, 281822, 326907, 394951 and 493737. Alternatively, another expression vector that transiently expresses an angiogenesis factor can be introduced into the adipocytes (see WO97 / 49827). The main objective of the angiogenesis factors used in this way is to form blood vessels around the transplant cells, so that the foreign protein can be effectively secreted in the blood flow from the adipocytes of this invention. Therefore, when a vector encoding a vascular induction factor is used to express that vascular induction factor from adipocytes, the use of a transient expression vector (more specifically, a vector that is not incorporated into the chromosome) is preferred. ). When adipocytes express a vascular induction factor over a long period, excess amounts of blood vessels form around the implanted adipocytes, which can cause systemic side effects. Therefore, it is preferable that the foreign gene encoding an angiogenesis factor is not stably transferred to the primary culture adipocytes of this invention.

3. Adipocyte implantation

Adipocytes subjected to gene transfer were prepared at an appropriate cell concentration, preferably 0.2 x 107 to 2 x 107 cells / ml, or 0.2 x 106 to 5 x 106 cells / ml when transfected with a retrovirus. They are infused as such into subcutaneous tissue or adipose tissue, preferably subcutaneous tissue, or by mixing with an effective medium, preferably a solution comprising an extracellular matrix such as collagen. The injection into the adipose tissue can be done by making an incision and exposing the adipose tissue. Cells that have differentiated terminally into mature adipocytes will not proliferate after transplantation, and will express the foreign gene for a long period at a constant level. The level of expression of a foreign gene in an organism that receives an implant is proportional to the number of implanted cells. Therefore, when an implant is performed, a desired level of expression can be maintained for a long period in an organism that receives an implant by adjusting the amount of adipocytes that are implanted to match the level of expression of the foreign gene in vitro previously measured.

Brief description of the drawings

Figure 1 is a set of photomicrographs of primary culture adipocytes isolated from the subcutaneous fat of ICR mice three weeks old. (A) shows adipocytes that adhere to the culture surface on the roof side after 14 days of roof culture, (B) shows primary culture adipocytes grown in a normal culture, (C) shows mature adipocytes that have stored droplets of lipids due to induction of differentiation, and (D) shows an image stained with oil red O of cells with induced differentiation.

Figure 2 shows the activity of plasma alkaline phosphatase (FA) obtained by implanting primary culture adipocytes in ICR nude mice (derived from subcutaneous fat from ICR mice) that are transiently transfected with the plasmid pcDNA3.1-SEAPmh expressing FA .

Figure 3 shows a comparison of the efficiency of gene transfer when the retroviral vector MLV (VSV) / pBabeCL (PLAP) IP is transduced into primary culture adipocytes derived from various adipose tissues.

Figure 4 is a set of photomicrographs showing images of the induction of differentiation of primary culture adipocytes transduced with MLV (VSV) / pBabeCL (GFP) IP. (A) and (B), respectively, show an optical photomicrograph, and a GPF fluorescence photograph of the same visual field.

Figure 5 shows the duration of FA expression in subcultures of primary culture adipocytes transduced with a viral vector expressing FA. (A) shows the result of the transfer of the SEAP (MLV (VSV) / pBabeCL (SEAPmh) I2G) gene or the PLAP (MLV (VSV) / pBabeCL (PLAP) IP) gene to subcutaneous fat derived cells of C57BL / mice 6. (B) shows the result of the transfer of the PLAP (MLV (VSV) / pBabeCL (PLAP) IP) or GFP (MLV (VSV) / pBabeCL (GFP) IP) gene to adipocytes derived from ICR mice.

Figure 6 is a set of photographs and a graph showing the change in expression in adipocytes subjected to gene transfer with induced differentiation. (A) shows a microscopic light image of GFP of primary culture adipocytes under conditions that do not induce differentiation, in which adipocytes transfected with MLV (VSV) / pBabeCL (GFP) IP are derived from subcutaneous ICR fat. (B) shows a microscopic image of similar GFP taken under conditions that induce cell differentiation. (C) shows the production of AF by primary culture adipocytes transfected with MLV (VSV) / pBabeCL (FLAP) IP (subcutaneous fat derivatives of ICR) under conditions that do not induce differentiation (without differentiation) and conditions that induce differentiation (differentiation).

Figure 7 shows the production of (pro) insulin by transfection of plasmids into primary culture adipocytes.

Figure 8 shows the stable expression of FA in primary culture adipocytes (subcutaneous fat derivatives of C57BL / 6 mice) transfected with VAA expressing FA.

Figure 9 shows insulin expression at the time of induction of differentiation in primary culture adipocytes transfected with the retroviral vector expressing insulin s1s2B10. (A) shows the results using an EIA produced by Morinaga and (B) shows the results using an EIA produced by IBL.

Figure 10 shows the expression of GLP-1 (7-37) in primary culture adipocytes transfected with retroviral vector expressing GLP-1 (7-37). Triplicate measurements were made, and their average values and standard deviations are shown.

Figure 11 shows the effect of the presence or absence of stimulation before the implantation of the induction of differentiation on the expression of AF in vivo in the implantation of primary culture adipocytes expressing FA.

Figure 12 is a set of graphics and a photograph. (A) shows the change in plasma AF activity when primary culture adipocytes expressing FA are implanted in the presence of differentiation stimulation using Matrigel supplemented with basic FGF. (B) shows the loss of plasma AF activity by removing the implanted Matrigel (individual A). (C) shows a GFP optical microscope image of the Matrigel removed from the control group that received cells transfected with GFP. For the group that was implanted with PLAP shown in (A), the values shown are the group average and the standard deviation of measured values for each individual until the 32nd day. The remaining values are average values.

Figure 13 shows the results of the long-term examination of AF activity in the blood of mice that received an implant by the method of Figure 12 (A), and by a variety of other methods.

Figure 14 shows the results of performing an excision test similar to that of Figure 12 (B) in the late phase of the transplant.

Figure 15 shows the dependence of blood FA activity on the number of cells implanted when adipocytes expressing FA were implanted. The indicated values are the group average and the standard deviation of the measurements of each individual.

Figure 16 shows the effect of implantation of adipocytes expressing insulin s1s2B10 in STZ-induced diabetic mice. (A) shows the effect on fasting plasma glucose level, and (B) shows the effect on body weight. The indicated values are the group average and the standard deviation of the measurements of each individual.

Best way to carry out the invention

The present invention will now be described in detail with reference to the examples, but it should not be understood to be limited thereto. All references cited in this document are incorporated in this description.

[Example 1] Primary culture of murine adipocytes

[Methods]

Three week old male ICR mice or four to five week old male C57BL / 6 mice (both from Charles River) were anesthetized with diethyl ether, and sacrificed by collecting whole blood from the heart. Subsequently, the inguinal subcutaneous fat, or fat surrounding the epididymis, and the mesenteric adipose tissue were sterilely removed. The excised tissues were washed with PBS, and then chopped using scissors or a scalpel. This chopped tissue was digested with shaking at 37 ° C for 20 to 60 minutes in normal medium (DMEM-high glucose / SIGMA, 10% FCS) comprising 1 mg / ml collagenase (S1 fraction / Nitta gelatin), and then it was separated into precipitate and suspended layer by centrifugation (300 g, five minutes).

The floating layer was further centrifuged once or twice to remove the collagenase by dilution, and then added to a T-25 flask (IWAKI) filled with medium. The bubbles were removed, and this was grown under a 5% CO2 atmosphere in a CO2 incubator at 37 ° C so that the conventional culture surface was upside down (roof culture). Ten to 14 days after culturing, the cells that adhered to the roof surface were collected by trypsin treatment and transferred to a normal culture system. Then the subculture was performed at a ratio of 1: 3 to 1:10.

To induce differentiation, the medium from cultured cells was transferred to the confluence in a 6-well plate to an induction medium (normal medium supplemented with 0.5 IBMM IBMX, 0.25 M dexamethasone and 10 g insulin / ml) This stimulation was continued for 48 hours. Next, the cells were differentiated in a maturation medium (normal medium supplemented with 10 µg / ml insulin). The ripening medium was exchanged every three days.

The oil red staining solution O was prepared by mixing a stock solution, prepared by mixing 0.3 g of oil red O in 100 ml of isopropanol (99%), with distilled water in a ratio of 3: 2 at the time of use . The cells were washed with PBS and then fixed with 10% neutral formalin solution (WAKO). After washing again with PBS, the cells were stained with oil red staining solution O at room temperature for ten minutes. The cells were washed with PBS again, and then examined under a microscope.

[Results]

Figure 1 is a set of photomicrographs of primary culture adipocytes isolated from the subcutaneous fat of ICR mice three weeks old. After 14 days of roof culture, adhesion of adipocytes carrying lipid droplets was observed on the culture surface of the roof side (A). When these cells were transferred to a normal culture system, they showed fibroblast-like growth, as shown in (B). However, when differentiation was induced by IBMX, dexamethasone and insulin, the cells differentiated again into mature adipocytes that carry lipid droplets (C). The stored fat was stained with oil red staining O (D). The cells isolated by this method were shown to be primary culture adipocytes that comprised the ability to differentiate.

[Example 2] Transient transfer of the alkaline phosphatase gene (AF) secretory thermostable in primary culture adipocytes, and implantation of transfected adipocytes in mice

As a model system for gene expression, the FA gene was transferred, more specifically, the SEAP gene (Clontech) or the PLAP gene (Goto, M. et al. Mol. Pharmacol. Vol.49 860-873 (1996)) to primary culture adipocytes, and changes in AF activity were examined. (Both FA gene products are thermostable and can easily be distinguished from endogenous alkaline phosphatases by heat treatment.)

[Methods]

(1) Production of primary culture adipocytes transiently transfected with the SEAP gene

A plasmid expressing FA (pcDNA3.1-SEAPmh) was constructed by inserting the SEAP sequence, obtained by double digestion of the pSEAP2-basic vector (Clontech) with HindIII-XbaI restriction enzymes, at the HindIII-XbaI site of pcDNA3.1Myc -HisA (Invitrogen), which is a vector for expression in mammalian cells.

For each gene transfer to a 10 cm plate, 500 µl of DMEM-free DMEM medium and 15 µl of Fugene 6 reagent (Roche) were mixed, then 5 µg of pcDNA3.1-SEAPmh was added. This mixture was allowed to stand at room temperature for 15 minutes. This mixture was added to primary culture cells (derived from subcutaneous fat from ICR) grown to a confluence of 70 to 80% in a 10 cm plate. Then this was grown for 24 hours in a CO2 incubator.

(2) Implantation in mice of primary culture adipocytes subjected to alkaline phosphatase gene transfer

Cells subjected to gene transfer were collected by trypsin treatment, and washed twice with PBS by centrifugation. The cells were then suspended in PBS at 1 x 10 7 cells / ml. Animals were anesthetized (ICR nude mice, five weeks of age at the time of operation) by intraperitoneal administration of 50 mg / kg of sodium pentobarbital (Nembutal; Dainippon Pharmaceutical). After disinfecting the area to be operated with the diluted Hibitane solution (Sumitomo Pharmaceuticals), an incision of more or less than 3 mm to 5 mm was made in the skin near the base of the right hind leg, and the subcutaneous inguinal fat. 0.55 ml of the prepared cell suspension solution (5.5 x 106 cells / head) was loaded into a 1 ml syringe, and injected into the subcutaneous fat using a 22 G injection needle. As a control, injected PBS in the same place. To compare this with the protein supplementation method, 1 mg of purified FA (Roche) was dissolved in PBS under sterile conditions, and injected in a similar manner. The skin was sutured with the incision and the site operated with surgical Isodine (Meiji Seika) was disinfected.

Blood was collected using a heparin-coated capillary (Dramond) from the postorbital venous plexus before implantation (day 0) and after implantation over time. Plasma was obtained from whole blood by centrifugation at 2000 g for 15 minutes. FA activity in this plasma was measured using an assay kit (SEAP indicator gene test kit, Roche) following the instructions attached.

[Results]

Figure 2 shows the plasma AF activity achieved by implanting primary culture cells in mice, which have been transiently transfected with pcDNA3.1-SEAPmh plasmid expressing alkaline phosphatase (FA). For comparison purposes, 1 µg of purified FA protein (Roche) was administered to the mice by injection. Seven days after administration, blood FA activity in these mice decreased to the level of control. On the other hand, it was confirmed that blood AF activity in mice that received a cell implant that maintain transiently transferred genes reaches a maximum on the fourth day after implantation, and the duration of expression was 14 days. The duration of expression in vivo by implanting cells that carry the gene transferred transiently was short, and it was found that the concentration in the blood varied greatly, although it remained longer than injecting the protein.

[Example 3] Production, using a viral vector, of adipocytes that express AF stably

[Methods]

(1) Construction of control GFP and FA expression vectors

The PLAP gene of pTK-PLAP was cut using HindIII and BglII, as described in the literature (Goto, M. et al. Mol. Pharmacol. Vol. 49, 860-873 (1996)). The SEAP gene was obtained by double digestion of pcDNA 3.1-SEAPmh with HindIII / PmeI. The GFP gene of pEGFP-N2 was cut using NotI-NcoI.

The plasmid, pBabeCLXI2G, was used for the production of viral vectors, based on pBabePuro (Morgenstern, JP et al. Nucleic Acids Res. Vol. 18, 3587-3596 (1990)), cutting its SV40 promoter and the genes of neomycin resistance using SalI-ClaI, and blunt those ends with Klenow fragments, then replacing these with the internal ribosome reentry site (IRES) of the encephalomyocarditis virus (VEMC), which was cut from pIRES2-EGFP by HincII- HincII, and the green fluorescent protein (GFP); then replacing the part of the long terminal repeat (LTR) at the insertion site of the foreign gene (multiple cloning site) (SspI-BamHI) with a corresponding sequence (SspI-BamHI) of pCLXSN (IMGENEX). In addition, pBabeCLXIP was also used, in which the IRES-GFP part of pBabeCLXI2G had been replaced by the puromycin-IRES resistance gene.

The ends of each of the aforementioned PLAP, SEAP and GFP DNA fragments were blunt, with Klenow fragments, then inserted into the vector pBabeCLXIP or pBabeCLXI2G cleaved with Hpa I, producing pBabeCL (PLAP) IP, pBabeCL ( SEAPmh) I2G and pBabeCL (GFP) IP, respectively.

(2) Production of viral vectors

Each gene transfer was made to a 10 cm plate as follows: 30 µl of TransIT plasmid transfection reagent (MIRUS) was mixed in 500 µl of DMEM medium free of FCS, and allowed to stand at room temperature for five minutes (DMEM / TransIT solution mixed). In a separate tube, 3.3 µg of a vector encoding VSV-G (pCALG, modified according to Arai, T. et al., J. Virol., 1998, 72, pp. 1115-21), were mixed, 3.3 g of a vector encoding Gag-Pol (pCLAmpho / RetroMax system (IMGENEX)) and 3.3 g of a vector comprising a packaging signal and the transferred gene (pBabeCL (PLAP) IP, pBabeCL (SEAPmh) I2G or pBabeCL (GFP) IP), totaling 9.9 g (plasmid dissolution). The plasmid solution was added to the mixed DMEM / TransIT solution, mixed vigorously and then allowed to stand at room temperature for 15 minutes. This was then added to 293-EBNA cells (Invitrogen), grown overnight from 2 x 10 6 cells / 10 cm plate on the previous day.

The medium was exchanged eight hours after the addition, and the culture supernatant was collected for another two days. The collected culture supernatant was centrifuged (300 g, five minutes) or filtered through a 0.45 µm syringe filter (Millipore) to remove contaminants, and this supernatant was used as a virus solution (MLV ( VSV) / pBabeCL (PLAP) IP, MLV (VSV) / pBabeCL (SEAPmh) I2G and MLV (VSV) / pBabeCL (GFP) IP, respectively). Some of the virus solution was concentrated by ultracentrifugation (19,500 rpm, 100 minutes) and then used.

(3) Gene transfer to and culture of primary culture adipocytes

Adipocytes were prepared to be used for gene transfer (derivatives of subcutaneous fat, fat surrounding the epididymis and mesenteric fat of ICR mice, and subcutaneous fat of C57BL / 6 mice) in 6-well or 96-well plates so that had a confluence of 50 to 80% the day before the transfection. The medium was discarded, and equal amounts of 4 mg / ml polybrene solution (SIGMA) and virus solution were added to the cells to transduce the viral vector. Eight hours after transduction, the medium was changed to a normal medium, and additional cultures and subcultures were performed. FA activity of a part of the cells was measured by collecting the 24-hour culture supernatant on day four after transfection (Figure 3).

Subculture was performed according to the method of Example 1 on a 10 cm plate scale. The cells were cultured for four to seven days, and the medium was exchanged upon reaching confluence. FA activity in the culture supernatant was measured 17 hours later. These cells were subcultured continuously and appropriately performing similar manipulations, expression maintenance was examined (Figures 5 and 6). FA activity was not measured each time a subculture was performed.

Differentiation in 6-well plates was induced according to the method of Example 1. However, treatment was carried out for three days with induction medium, which was replaced by maturation every three days thereafter. The FA activity of the culture supernatant was measured using the culture supernatant obtained every three days, and the x-axes in the figures show the day on which the supernatant was collected. For cells transfected with GFP, photomicrographs were taken under appropriate GFP light (Figures 4 and 6). Conditions that do not induce differentiation refers to conditions in which the culture is continued in a normal medium instead of an induction medium or mature medium.

[Results]

Figure 3 is a comparison of gene transfer efficiency for each class of tissue-derived cell when retroviral vectors are used. FA activity was confirmed in the culture supernatant of all cells when gene transfer was performed in the primary culture adipocytes isolated from each of the adipose tissues that exist in the inguinal subcutaneous tissue, the area around the epididymis and the ICR mouse mesentery. This showed that retroviral vectors can transfer genes regardless of the site of cellular origin.

Figure 4 is a set of photomicrographs showing images of the induction of differentiation of transduced cells with a retroviral vector expressing GFP. The induction of differentiation began 13 days after gene transfer, and photographs were taken three weeks later. GFP fluorescence was observed in cells containing lipid droplets, which showed that the viral vector can transfer genes to preadipocytes that have the ability to differentiate, and that gene transfer via the vector does not affect their ability to differentiate.

Figure 5 shows the continuity of expression in subcultures of primary culture adipocytes transfected with a viral vector expressing FA. AF activity was measured in culture supernatant taken 17 hours after the cells reached confluence on a 10 cm plate. Continuous AF production was confirmed throughout the 87 days during which primary culture adipocytes derived from subcutaneous fat from C57BL / 6 (A) mice were examined, and throughout the 63 days during which adipocytes were examined. Primary culture derived from subcutaneous tissue of ICR mice (B). These results showed that transduction of the viral vector into primary culture adipocytes can produce stable expression cells that maintain foreign genes in the daughter cells produced after division.

Figure 6 is a set of photographs and a graph showing changes in expression in adipocytes undergoing gene transfer with induced differentiation. Adipocytes expressing GFP derived from subcutaneous fat from ICR showed strong expression of GFP both under normal culture conditions (A), and conditions that induce differentiation (B). In addition, adipocytes expressing FA derived from subcutaneous fat from ICR showed continuous expression of AF both in conditions that do not induce differentiation (without differentiation) and in conditions that induce differentiation (differentiation) (C). It was found that the primary culture adipocytes that underwent gene transfer stably expressed genes at any stage, not only under the proliferation conditions described in Figure 5, but also under conditions that do not induce differentiation, or more specifically in non-proliferative conditions or mature conditions.

[Example 4] Production, using a plasmid vector, of adipocytes that express insulin stably

Gene transfer methods include methods that use plasmid vectors.

[Methods]

(1) Isolation and modification of the human insulin gene

PCR was performed in a cDNA library derived from human pancreas (Stratagene), using the primers shown in Table 1 (insulin dir. And inv.). A fragment of the human insulin gene was obtained. The nucleotide sequence of this 354 bp fragment obtained was determined, and the fragment was subcloned into the vector pCR2.1TOPO (Invitrogen) as native insulin.

5

10

fifteen

twenty

25

30

Table 1 Sequences of primers used for PCR

Primer nucleotide sequence (5’-)

image 1

Fw insulin

image2

sPL-GLP-1Fw

image3

(Bold letters indicate the initiation codon in Fw, and the antisense sequence of the termination codon in Rv. The underlined indicates mutated parts.)

Next, in order to express mature insulin in adiposites, a genetic modification was made based on the literature (JBC, 1994, 269 (8), 6241-). More specifically, primers from both directions were synthesized individually to contain mutations at each of the binding sites between the human insulin B chain and the C-peptide (site1), between the same C-peptide and the A-chain (site2), and the 10th histidine residue of chain B (B10) (table 1). Mutants were obtained using a Quickchange (Stratagene) mutagenesis kit. Performing this reaction at site1 and site2 produced the s1s2 mutant. Performing the reaction at site1, site2 and B10 produced the mutant insulin s1s2B10. After confirming the nucleotide sequence of the modified human insulin gene obtained, the gene was incorporated into the pcDNA3.1 vector, and then used for gene transfer.

(2) Gene transfer to primary culture adipocytes

After mixing 500 µl of DMEM-free DMEM medium and 15 µl of Fugene 6 reagent (Roche), 5 µg of transfection plasmid was added, and then this was allowed to stand at room temperature for 15 minutes. The mixed solution was added to primary culture adipocytes (derived from adipose tissue around the epididymis of C57BL / 6 mice), which had been grown to a confluence of 70 to 80% in a 10 cm plate. This was grown for 24 hours in a CO2 incubator. Four days after gene transfer, the cells were subcultured in a T225 flask, and grown overnight. The medium was then exchanged for a medium comprising 0.2 mgU / ml of G418 (SIGMA), and the culture was continued for three weeks, after which cells subjected to gene transfer were selected. G418 resistant cells were plated on a 10 cm plate, and the amount of insulin in the culture supernatant was measured using an EIA kit for ultrasensitive insulin (Morinaga). This EIA kit detects both proinsulin, which has not yet been processed, and mature insulin.

[Results]

Figure 7 shows the production of (pro) insulin by transfection of plasmids into primary culture adipocytes. Each of the pcDNA3.1Myc-His vectors that individually incorporated the native (native) human insulin gene and the site 1 / site 2 / B10-modified form (s1s2B10), or an empty (simulated) vector, was transfected into adipocytes derived from adipose tissue surrounding the epididymis of C57BL / 6 mice. Human (pro) insulin was detected in the culture supernatant of resistant cells obtained by selection with G418. This demonstrated that stable gene transfer to primary culture adipocytes is also possible using a plasmid vector.

[Example 5] Production, using an adeno-associated virus, of adipocytes stably expressing AF

Gene transfer methods include methods that use adeno-associated viruses (VAA).

[Methods]

The study was carried out using the Helper-Free AAV system (Stratagene). The PLAP fragment of Example 2 (a fragment cut using HindIII and BglII) was inserted into the same restriction enzyme site of the pAAV-MCS vector, producing pAAV-PLAP.

Production of VAA vector was carried out as follows: 1.75 ml of OPTI-MEM (Invitrogen) was mixed with 220 µl of Fugene plasmid transfection reagent, then mixed in 25 µg of each of pAAV-PLAP, pAAV-RC and pHelper, and allowed to stand at room temperature for 15 minutes (Fugene / plasmid solutions). Meanwhile, 293-EBNA cells grown to a confluence of 60 to 70% on a 15 cm plate were prepared. The culture solution was changed to FCEM free DMEM, then the Fugene / plasmid solution was uniformly instilled, and this was cultured for two to three hours. Then FCS was added to a final concentration of 10%, and this was cultivated for two more days. The cells were collected by trypsin treatment and centrifugation, and then suspended in 50 mM Tris-HCl and 150 mM NaCl solution so that the final volume was 3 ml. The cells were broken by performing three cycles of freezing with carbonic ethanol / thawing at 37 ° C on this suspension solution. In addition, after degrading the genomic DNA of the host using Benzonase (SIGMA), virus dissolution occurred by centrifugation at 9,000 rpm for 30 minutes, followed by filtration of the supernatant.

Primary culture adipocytes (subcutaneous fat derivatives of C57BL / 6 mice) were plated on a 12-well plate at 1 x 104 cells / well the day before gene transfer, and cultured. They were then treated for six hours in a medium containing 40 mM hydroxyurea and 1 mM buriteic acid (both from SIGMA). After removing this medium, 0.5 ml / well of the virus solution, diluted to 1/100 with FCEM-free DMEM, was added. After culturing for one hour, medium containing FCS was added to a final concentration of 10%, and this was grown overnight. After that, exchanges were made with normal medium, and subculture was performed on the 24th day.

The medium was exchanged on the first, seventh and 25th day of transfer, and the culture supernatant collected two days after each exchange was used for AF tests. 10 µl of the supernatant, which was diluted as necessary, was heated at 65 ° C for 20 minutes, then 50 µl of assay buffer (16 mM NaHCO3, 12 mM Na2CO3, 0.8 mM MgSO4) was mixed and mixed 50 The luminescent staining reagent (CDP-Star ready for use with Sapphire II, TROPIX), was reacted in the dark for 30 minutes, and then measured with a luminometer.

[Results]

Figure 8 shows the stable expression of FA in primary culture adipocytes (subcutaneous fat derivatives of C57BL / 6 mice) transfected with VAA expressing FA. AF activity was detected in the culture supernatant throughout the entire examination period. This demonstrated that stable gene transfer to primary culture adipocytes can be carried out using a VAA vector.

[Example 6] Construction of a retroviral vector that expresses human insulin and its transduction into adipocytes

[Methods]

The modified human insulin gene constructed in example 4 (s1s2B10Ins) was inserted into the vector pBabeCLXI2G following the method of example 3 (pBabeCL (s1s2B10Ins) I2G). This plasmid was introduced together with a vector encoding VSV-G (pVPack-VSV-G / Stratagene) and a vector encoding Gag-Pol (modified from pVPack-gp / Stratagene) in 293-EBNA cells according to the method of Example 3, thus producing the retroviral vector expressing modified insulin (MLV (VSV) / pBabeCL (s1s2B10Ins) I2G). The culture supernatant (approximately 200 ml) of 293-EBNA cells from twenty-two 10 cm plates was collected, the insoluble material was removed by centrifugation / filtration treatment, and then the concentrated virus was dissolved by ultracentrifugation (19,500 rpm, 100 minutes) This was transferred to primary culture adipocytes (subcutaneous fat derivatives of C57BL / 6), which had been plated on a 6-well plate on the previous day.

The cells subjected to gene transfer were re-plated on a 6-well plate, and differentiation was induced according to the method of Example 1. Each culture supernatant was collected for three days, from three days before induction to day of the initiation of induction (before induction), and for three days from day 14 to day 17 of induction (after induction). The amount of insulin was tested by the same method as in Example 4. In addition, to confirm that processing occurred at the desired sites, and that mature insulin was produced, measurements were made using the EIA kit for insulin ( IBL), which only recognizes mature insulin. The culture supernatant of cells not subjected to gene transfer, which were simultaneously subjected to induction of differentiation, was used as a control.

[Results]

Figure 9 shows insulin expression at the time of induction of differentiation in primary culture adipocytes transduced with retroviral vector expressing insulin s1s2B10. (A) shows the results of the use of the EIA produced by Morinaga, and (B) shows the results of the use of the EIA produced by IBL. These results demonstrate that insulin is secreted stably both before and after induction of differentiation, and that the transfer of the mutant insulin gene can cause the production of mature insulin from adipocytes.

[Example 7] Construction of a retroviral vector expressing peptide-1 similar to human glucagon (GLP-1), and transduction thereof into adipocytes

GLP-1 is a peptide that is produced from small intestine L cells during food intake, and comprises the effect of stimulating insulin secretion by acting on  pancreatic cells. It is also known that GLP-1 has a variety of other antidiabetic and anti-obesity effects such as a regeneration effect on  pancreatic cells, an appetite suppressing effector and an inhibitory effect on gastric emptying (Meier, JJ et al. Eur. J Pharmacol. 2002, 12; 440 (2-3): 269-79; Drucker, DJ Gastroenterology 2002; 122 (2): 531-544). A peptide comprising positions 7 to 37 of the amino acid sequence of GLP-1 (or up to position 36 in the amide form) is formed, by specific tissue processing of the polypeptide produced from the preproglucagon gene, and it is known comprising the main pharmacological activity (Drucker, DJ et al. Proc. Natl. Acad. Sci. USA. 1987 May; 84 (10): 3434-3438; Kreymann, B. et al. Lancet. 1987, 5; 2 ( 8571): 1300-1304; Mojsov, S. et al. J. Clin. Invest. 1987 Feb; 79 (2): 616-619). The following test was carried out in order to produce this factor from adipocytes.

[Methods]

A nucleotide sequence with a total of 156 base pairs was designed, comprising a sequence (SEQ ID NO: 10 shows the coding sequence) in which human GLP-1 (7-37) and a termination codon bind to the signal peptide (17 amino acids) of the PLAP gene used in example 3. Nucleotides were synthesized so that a superimposition of 22 groupers in the center (sPL-GLP-1Fw and sPL-GLP-1Rv in Table 1) was included. These were paired and a double chain was formed using Pfu polymerase (Stratagene). The target fragment was then amplified by PCR using 5 ′ and 3 ′ end primers (GLP-5 ’and GLP-3’ in Table 1). This fragment was subcloned into the vector pCR2.1, then cut using restriction enzymes and subsequently inserted into the vector pBabeCLXI2G, as in example 3 (pBabeC-L (sPL-GLP1) I2G). This was transfected into 293-EBNA cells by a method similar to that of Example 6, producing a retroviral vector expressing GLP-1 (MLV (VSV) / pBabeCL (sPL-GLP-1) I2G). Approximately 90 ml of the 293-EBNA cell culture supernatant was collected from nine 10 cm plates. The insoluble material was removed by centrifugation / filtration treatment, and then the supernatant was ultracentrifuged (19,500 rpm, 100 minutes) to produce a concentrated virus solution. This was transduced into primary culture adipocytes (C57BL / 6 subcutaneous fat derivatives) that had been plated on a 6-well plate the day before. The transfected adipocytes were then plated again on a 12-well plate, and the induction of differentiation was carried out according to the method of Example 1. "Uninduced" refers to a condition in which the culture was continued. in a normal medium instead of an induction medium or mature medium. Seven days later, the medium was exchanged to FCEM-free DMEM medium comprising 1 mM valine-pyrrolidine (enzymatic inhibitor of GLP-1 degradation; synthesized in Eisai). The culture supernatant was collected 18 hours later, and the amount of active GLP-1 (7-37) was measured using ELISA (LINCO).

[Results]

Figure 10 shows the level of expression in primary culture adipocytes transfected with retroviral vector expressing GLP-1 (7-37). GLP-1 expression (7-37) was actively observed in the culture supernatant of both adipocytes without induced differentiation and induced differentiation. This showed that even when a factor such as the preprotype is produced and then cut by processing, this method allows the production of only that factor from adipocytes.

[Example 8] Implantation in mice of cells that express AF stably (Test 1)

[Methods]

After culturing the adipocytes expressing FA (transduced with MLV (VSV) / pBabeCL (PLAP) IP; subcutaneous fat derivatives of C57BL / 6) produced by the method of Example 3 until confluence, the cells were collected by trypsin treatment, washed with PBS and suspended at 5 x 107 cells / ml in ice-cold Matrigel (Becton Dickinson). Implantation was performed by injecting this into the dorsal subcutaneous (Sc) area of C57BL / 6 mice (eight weeks of age at the time of the operation; Charles River) at a dose of 0.2 ml per mouse (1 x 106 cells / head) (without induction of differentiation). On the other hand, the same cells were grown to confluence, then they were cultured for three days in the induction medium of Example 1, and then implanted by similar methods (with induction of differentiation). Blood was collected over time by the method indicated in Example 2, and the activity of FA in the plasma was measured.

[Results]

Figure 11 shows the change in plasma AF activity in mice that were implanted with primary culture adipocytes expressing FA. Individuals who received an implant of cells undergoing stimulation that induced differentiation for three days before implantation ("with induction of differentiation") showed less fluctuation in continued expression than individuals who received an implant of cells that were not induced. However, both implantation methods showed continued expression throughout all 50 days

or almost exam. This demonstrates that the survival rate after cell transplantation can be improved by providing stimulation that induces differentiation.

[Example 9] Implantation in mice of cells that express AF stably (Test 2)

[Methods]

(1) Implementation

Adipocytes expressing FA (transduced with MLV (VSV) / pBabeCL (PLAP) IP; subcutaneous fat derivatives of ICR) produced in Example 3 were cultured to confluence. Cells were collected by trypsin treatment, washed with PBS and suspended at 5 x 107 cells / ml in an ice-cold Matrigel (Becton Dickinson) to which 1 mg / ml of bFGF (Genzyme Techne) was added. Implantation was performed by injecting this at a dose of 0.2 ml per mouse (1 x 106 cells / head) at each site (dorsal subcutaneous area (Sc), inguinal subcutaneous fat (fat) and intraperitoneal region (ip)), of ICR nude mice (six weeks old at the time of the operation, Charles River). As a control, adipocytes expressing GFP were treated similarly and implanted in the subcutaneous tissue.

Some of the cells expressing FA were cultured for three days by the induction medium of Example 1, and then collected and implanted in the same manner (Dif). After using the induction medium, some of these cells were cultured for four days in a maturation medium, and then collected and implanted in the same manner (Mat).

In addition, some of the cells expressing FA were plated on an eight-well Labteck chamber (Nunc) under the same conditions used for implantation (Matrigel with added 1 x 106 / 0.2 ml bFGF), and solidified. cells heating at 37 ° C. Implantation was carried out by inserting this solidified gel in the subcutaneous area of the mouse. Here, the cells grown in a normal medium after solidification were called (pf) / gr preset, and the cells grown in a medium that induces differentiation were called pf / dif. Implantation was carried out after seven days of culture.

Plasma AF activity was measured over time, before implantation (day 0) and after implantation, according to the method of Example 2.

(2) Removal

In the group implanted after induction of differentiation (Dif / Sc), the implanted cell masses were removed, along with the Matrigel, from individuals A and B, five and 43 weeks after implantation, respectively. The control sample was removed in the fifth week after implantation. Each individual was administered intraperitoneally 50 mg / kg of Nembutal as anesthesia. An incision was then made in your skin and a section of implanted Matrigel was visually confirmed. The surgery site was sutured and disinfected with Isodine (Meiji). The animals were then raised in the same way and blood was collected over time.

[Results]

Figure 12 (A) shows the result of the examination of the change in plasma AF activity over 50 days, when primary culture adipocytes expressing AF were implanted using Matrigel with added basic FGF, in the presence of differentiation stimulation. (Dif / Sc group). The change in blood AF activity was stable for 49 days over a 5-fold interval. This showed that the addition of bFGF at the time of implantation can further improve the graft acceptance rate after implantation. (B) shows the disappearance of plasma AF activity due to the removal of the implanted Matrigel (individual A) over the same period of time. AF activity in excised individuals decreased significantly compared to the average value for the PLAP transduced group. This showed that blood AF is derived from implanted cells, and that removal of the graft can quickly eliminate gene expression. At this time, the excision was also performed in a part of the control group, to which cells transfected with GFP were implanted. GFP positive cells were found in the removed Matrigel, and many of them showed an image of vacuoles (C) similar to that shown in Figure 6 (B). This showed that primary culture adipocytes implanted by this method can be grafted as mature adipose tissue in vivo.

Figure 13 shows the result of a long-term examination of blood FA activity in implanted mice of Figure 12 (A), and in mice that received an implant by a variety of other methods. In the group to which cells transfected with PLAP were implanted, an evident increase in blood AF activity was confirmed for all implantation sites and implantation methods. Blood AF activity was maintained for a long period and, in particular, stable expression of AF was observed for one year during the test period of the Dif / Sc group (the group described in Figure 12 (A)). Continuous AF production was also confirmed for the other implantation methods, during the entire examination period (316 days for the ip group, 54 days for the fat group, 225 days for the Sc group, 317 days for the Mat group / Sc and 314 days for the two preset groups). The peak activity observed within one week of implantation was the highest in the ip group. The highest values were then in the order of Sc> fat> Dif / Sc ≅ pf-dif> pf-gr ≅ Mat / Sc. The variation interval after implantation was observed as a ratio between activity after 13 weeks and peak activity, which can be compared in all groups. The variance was the smallest, approximately three times, in the two preset groups, approximately five times in the ip, Dif / Sc and Mat / Sc groups, and approximately ten times in the Sc and fat groups. The peak value immediately after implantation, and the variation interval after implantation differed for each implantation method. Therefore, any of these methods can be used according to the characteristics of the gene product used, the pathological characteristics and the simplicity of the technique. This demonstrated that the implantation of primary culture adipocytes can be performed, to which genes were introduced stably ex vivo, by a variety of methods, and that stable long-term in vivo gene expression is possible after implantation.

Figure 14 shows the result of performing an excision experiment, similar to that described in Figure 12 (B), in the late stage of implantation. It was confirmed that blood AF activity after removal disappeared rapidly, not only in individuals in whom the excision was performed in the early stages of implantation (individual A), but also in individuals in whom the excision was performed in the late stages of implantation (individual B). This showed that adipocytes implanted by this method are located in the implanted site for a long period after implantation, and their removal, when appropriate, can eliminate gene expression regardless of the moment.

[Example 10] Transplantation in mice of cells stably expressing AF

(Test 3)

The following tests were carried out to confirm the dependence of the "dose" on the number of implanted cells.

[Methods]

Adipocytes expressing FA produced in Example 3 (transfected with MLV (VSV) / pBabeCL (PLAP) IP; subcutaneous fat derivatives of ICR) were grown to confluence. The cells were cultured for three days in the induction medium indicated in example 1, and then collected by trypsin treatment. After washing with PBS, the cells were suspended at 5 x 10 7 cells / ml in Matrigel. A 5-fold gradual dilution was carried out in the suspension solution of cells with AF using Matrigel, and solutions of 1 x 10 7 cells / ml and 2 x 10 6 cells / ml, respectively, were prepared. BFGF was added to these solutions at a final concentration of 1 mg / ml, and then they were implanted in the dorsal subcutaneous zone of ICR nude mice at a dose of 0.2 ml per mouse (high dose: 1 x 10 6 cells / head; medium dose: 2 x 105 cells / head; low dose: 4 x 104 cells / head). As a control, adipocytes expressing GFP were similarly treated, and implanted in the subcutaneous tissue under the same conditions as for high dose conditions (1 x 106 cells / head).

[Results]

Figure 15 shows the dependence of blood FA activity on the number of cells implanted when adipocytes expressing FA were implanted. Dose-dependent blood AF activity was observed when changing the number of implanted cells, and this was not influenced by duration. More specifically, the low or medium dose groups did not show a peak in the early stage of implantation, which was observed in the high dose group, and the fluctuation interval was narrower. This showed that the level of expression in vivo can be easily adjusted using the number of implanted cells, and that by adjusting the optimal number of cells, blood concentration can be stabilized after implantation.

[Example 11] Hypoglycemic effect in diabetes model mice due to implantation of insulin-expressing adipocytes

[Methods]

Diabetic mice were produced by administering intravenously to male C57BL / 6 mice of 8 weeks of age 10 ml / kg of streptozotocin 170 mg / kg (STZ, SIGMA). Fasting blood glucose (GSA) levels were measured individually one and two weeks after STZ administration, and individuals with GSA of 300 mg / dl or more were determined to have diabetes. It was measured in blood sugar level by performing a treatment with perchlorate immediately after the collection of whole blood, and then using the glucose-II test (WAKO).

Adipocytes transfected with MLV (VSV) / pBabeCL (s1s2B10Ins) I2G produced in example 6 were subjected to stimulation of induction of differentiation using the same method as in example 10, and then suspended at 5 x 107 cells / ml in Matrigel to which 1 µg / ml of bFGF had been added. This solution was implanted in suspension in the dorsal subcutaneous area of each diabetic mouse, at 0.2 ml per site, to a total of four sites (four x 106 / head). For the control group, adipocytes not subjected to gene transfer were implanted by the same method. Implantation was performed 19 days after STZ treatment, and after that, the level of

5 GSA over time. Statistical analysis was carried out by comparison with the control group (t test for independent data).

[Results]

Figure 16 shows the effect of implantation of adipocytes expressing insulin s1s2B10 in mice with STZ-induced diabetes. Cells not subjected to gene transfer were implanted as a control. The blood glucose level of the group to which cells expressing insulin were implanted tended to decrease from the seventh day of implantation, and a significant hypoglycemic effect was indicated on day 13 and 21 of implantation (A). The body weight 20 days after implantation was significantly higher in the group to which cells expressing insulin were implanted than in the control group, and therefore the weight loss due to diabetes was improved (B). Exam results using FA suggest that this hypoglycemic effect will be maintained for a long period. By

Therefore, it was shown that the foreign gene product produced from the implanted primary culture adipocytes can contribute to the modification of the recipient's pathology, indicating that this method can treat diabetes.

Industrial applicability

The present invention established methods of ex vivo transfer of a foreign gene into primary culture adipocytes suitable for gene therapy, and established primary culture adipocytes that stably maintain a foreign gene.

SEQUENCE LIST

<110> EISAI CO., LTD.

<120> PRIMARY CULTURE ADIPOCITS FOR GENE THERAPY

<130> E1-A0305Y1P

<150> JP 2002-177648

<151> 06-18-2002

<150> JP 2002-237974

<151> 08-19-2002

<160> 11

<170> PatentIn version 3.1

<210> 1

<211> 30

<212> DNA

<213> Artificial

<220>

<223> an artificially synthesized oligonucleotide to amplify the human insulin gene

<400> 1

image 1

<210> 2

<211> 30

<212> DNA

<213> Artificial

<220>

<223> an artificially synthesized oligonucleotide to amplify the human insulin gene

<400> 2

image 1

<210> 3

<211> 37

<212> DNA

<213> Artificial

<220>

<223> an artificially synthesized oligonucleotide for mutagenesis

<400> 3

image 1

<210> 4

<211> 28

<212> DNA

<213> Artificial

<220>

<223> an artificially synthesized oligonucleotide for mutagenesis

<400> 4

image 1

<210> 5

<211> 29

<212> DNA

<213> Artificial

<220>

<223> an artificially synthesized oligonucleotide for mutagenesis

<400> 5 <210> 6

image 1

<211> 83

<212> DNA

<213> Artificial

<220>

<223> an artificially synthesized oligonucleotide to construct a gene that encodes a human secretory GLP-1

<400> 6

image 1

<210> 7

<211> 95

<212> DNA

<213> Artificial

<220>

<223> an artificially synthesized oligonucleotide to construct a gene that encodes a human secretory GLP-1

<400> 7

image 1

<210> 8

<211> 20

<212> DNA

<213> Artificial <220>

<223> an artificially synthesized oligonucleotide for PCR

<400> 8

image 1

<210> 9

<211> 23

<212> DNA

<213> Artificial

<220>

<223> an artificially synthesized oligonucleotide for PCR

<400> 9

image 1

<210> 10

<211> 144

<212> DNA

<213> Artificial

<220>

<223> a gene that codes for a human secretory GLP-1

<220>

<221> CDS

<222> (1) .. (144)

<223>

<400> 10 <210> 11

image 1

<211> 48

<212> PRT

<213> Artificial

<220>

<223> A GLP-1 human secretor

<400> 11

image 1

Claims (13)

one.

Pharmaceutical composition comprising a primary culture preadipocyte, in which the preadipocyte stably maintains a foreign gene that codes for a protein that is secreted outside the cell and in which the gene has been inserted into a retroviral vector and has been transferred to the cell by the retroviral vector, in which the protein is insulin or glucagon-like peptide 1 (GLP-1).

2.

Primary culture preadipocyte, in which the preadipocyte stably maintains a foreign gene that codes for a protein that is secreted outside the cell, and in which the gene has been inserted into a retroviral vector and transferred to the cell by the retroviral vector, for use in gene therapy, in which the protein is insulin or GLP-1.

3.

Pharmaceutical composition of claim 1, or preadipocyte for use according to claim 2, wherein the preadipocyte has the ability to significantly express the protein in vivo for at least 20 days.

Four.

Pharmaceutical composition of claim 1 or 3, or preadipocyte for use according to claim 2 or 3, wherein the preadipocyte is used to release the protein into the bloodstream.

5.

In vitro production method of a preadipocyte for use in gene therapy, where the method comprises the steps of:

(one)

carry out a primary culture of a preadipocyte; Y

(2)

transfer, and then stably maintain a foreign gene that codes for a protein that is secreted out of the cell,

in which the protein is insulin or GLP-1.

6.

Method of claim 5, wherein the foreign gene is transferred by a retroviral vector.

7.

Preadipocyte for use according to claim 2 which has been produced by the method of claim 5 or 6.

8.

Implant composition, where the composition comprises a primary culture preadipocyte, which stably maintains a foreign gene that codes for a protein that is secreted outside the cell, and in which the gene has been inserted into a retroviral vector and is has been transferred to the cell by the retroviral vector, and a pharmaceutically acceptable carrier, for use in gene therapy, in which the protein is insulin or LPG

one.

9.

Implant composition for use according to claim 8, or pharmaceutical composition of any one of claims 1, 3 and 4, further comprising an extracellular matrix component.

10.

Implant composition for use according to claim 8 or 9, or pharmaceutical composition of any one of claims 1, 3, 4 and 9, further comprising an angiogenesis factor.

eleven.

Primary culture preadipocyte that stably maintains a gene that encodes insulin or glucagon-like peptide 1 (GLP-1) for use in blood glucose reduction, in which the gene has been inserted into a retroviral vector and It has transferred to the cell using the retroviral vector.

12.

Non-human animal, in whose organism the primary culture preadipocyte of the claim has been implanted

2.