Title:  Genetically modified fibroblast cells

United States Patent:  6,846,674

Issued:  January 25, 2005

Inventors:  Angel; Peter (Leimeu, DE); Fusenig; Norbert (Heidelberg, DE); Kolbus; Andrea (Rastatt, DE); Schorpp-Kistner; Marina (Durmersheim, DE); Szabowski; Axel (Heldelberg, DE); Maas-Szabowski; Nicole (Heidelberg, DE); Andrecht; Sven (Darmstadt, DE)

Assignee:  Deutsche Krebsforschungszentrum Stiftung des Offentlichen rechts (Heidelberg, DE)

Appl. No.:  196000

Filed:  July 15, 2002

Abstract

The invention relates to genetically modified fibroblast cells which comprise the following properties: (a) a gene, encoding a subunit of the transcription factor AP-1, of its genome is inactivated and/or a component, acting on the AP-1 transcription factor of the signal transduction pathway is modified resulting in a modified AP-1 activity; (b) functionally linked with a promoter, it contains in an expression vector the gene from (a) in a form such that the active subunit is only expressed after induction of the promoter or the subunit encoded by the gene is present in inactive form and a form that can be activated. Furthermore, the invention relates to a method which uses the fibroblast cell according to the invention to identify a compound which can support, inhibit or modify the AP-1-dependent differentiation and/or proliferation of keratinocytes or a gene which is involved in the AP-1-dependent differentiation and/or proliferation of keratinocytes as well as to the use of the test compound identified according to the method of the invention and the gene for treating or diagnosing diseases associated with a disturbed proliferation and/or differentiation of keratinocytes.

Description of the Invention

The present invention relates to genetically modified fibroblast cells in which a gene, coding for a subunit of the AP-1 transcription factor, of the genome thereof is inactivated and/or a component, acting on the AP-1 transcription factor, of the signal transduction path is modified resulting in a modified AP-1 activity and which functionally linked with the promoter contain the above-mentioned gene in an expression vector in a form such that the active subunit is only expressed after induction of the promoter or in a form such that the subunit encoded by the gene is present in inactive form which can, however, be activated. The present invention also relates to a method of identifying compounds which can support, inhibit or modify the AP-1-dependent differentiation and/or proliferation of keratinocytes and enable the identification of genes involved in the AP-1-dependent differentiation and/or proliferation of keratinocytes.

The proliferation and differentiation of keratinocytes and thus the structure and function of the epidermis are dependent on the interaction with fibroblasts, these processes at least in part depending on the controlled expression of genes which are dependent on the AP-1 transcription factor (e.g. c-Jun, JunB). It may be assumed that abnormal or lacking regulation of these genes results in a disturbed proliferation and/or differentiation of the keratinocytes, which in turn may lead to a number of diseases, e.g. a disturbed metabolism of the skin (disturbed wound healing, psoriasis, chronic inflammatory reactions, etc.) or the formation of tumors of the skin. The involved genes and/or factors are hardly known thus far and the former search for such factors, which is substantially based on pharmacological studies made in connection with cell cultures or animals and partially also on clinical studies, has the drawback that the analyses to be carried out are very complex and time-consuming and above all not very selective either.

It is thus the object of the present invention to provide products for identifying compounds directly or indirectly correlated with an expression of the AP-1 transcription factor and/or directly or indirectly regulated by this transcription factor and involved in the proliferation and/or differentiation of keratinocytes of the skin. On the one hand, these are compounds which directly control genes via the AP-1 transcription factor, are responsible for growth regulation and epidermis differentiation and are defective or regulated imperfectly in pathological states. These compounds comprise a large number of known signal substances which are not yet sufficiently known, such as interleukins and other cytokines and growth factors as well as synthetic agonists and/or antagonists for their receptors. The genes controlled by them via AP-1 and/or their products comprise a similarly large number of known still insufficiently known growth and differentiation factors, enzymes, their inhibitors and structural proteins of the epidermal cells. In addition to these components directly controlling AP-1 or dependent thereon, the test system is to detect regulating factors or proteins which are released indirectly by pathological or therapeutic influences in keratinocytes and then induce reaction mechanisms secondarily controlled by AP-1 within the genetically modified fibroblasts.

This object is achieved by providing the embodiments characterized in the claims.

It was found surprisingly in the present invention that by means of genetically modified fibroblast cells a system for identifying the above substances can be established. These fibroblast cells have the following properties: (a) a gene coding for a subunit of the AP-1 transcription factor, of its geneome is inactivated and/or a component, acting on the AP-1 transcription factor, of the signal transduction pathways is modified resulting in a modified AP-1 activity and (b) functionally linked with a promoter they contain the gene from (a) in an expression vector in a form such that the active subunit is only expressed after induction of the promoter or in a form such that the subunit encoded by the gene is present in inactive form which can, however, be activated. This genetically modified fibroblast cell line enables a more specific and simpler search for new active substances, e.g. new agonistic or antagonistic pharmacons, or for corresponding genes which can be of use for treating diseases associated with the proliferation and/or differentiation of keratinocytes. In addition, substances already identified in other systems can also be analyzed more specifically in test systems based on the fibroblast according to the invention.

An embodiment of the present invention thus relates to a genetically modified fibroblast cell having the following properties: (a) a gene coding for a subunit of the AP-1 transcription factor, of the genome thereof is inactivated and/or a component, acting on the AP-1 transcription factor, of the signal transduction pathway is modified, resulting in a modified AP-1 activity; and (b) functionally linked with the promoter it contains the gene from (a) in an expression vector in a form such that the active subunit is only expressed after induction of the promoter or in a form such that the subunit encoded by the gene is present in inactive form which can, however, be activated.

Examples of genes coding for subunits of the AP-1 transcription factor are jun-c, JunB, JunD, c-Fos, FosB, Fra-1 and members of the ATF-protein family (Angel and Karin, 1991).

The gene coding for a subunit of the AP-1 transcription factor can be inactivated according to methods known to a person skilled in the art. Here, the use of the antisense technique (Riabowol et al., 1992, PNAS 89, pp. 157-161) or the injection of neutralizing antibodies (Kovary et al., 1991, Mol. Cell. Biol. 11, pp. 4466-4472) has to be mentioned by way of example. The preferred approach of inactivating a gene is, however, the production of "knockout" (mammalian) animals and the isolation of cells derived therefrom. This comprises the production of a suitable gene-targeting vector, the isolation or correctly genetically modified embryonic non-human stem cells, the provision of (mammalian) animal blastocysts (preferably: murine blastocysts) with these cells by way of injection, the establishment of chimeras and the pairing of these (mammalian) animals to generate (mammalian) animals (preferably: mice) having the desired genotype (A. L. Joyner: Gene targeting: A practical approach, Oxford University Press, Oxford, 1993, p. 1-234). Thereafter, mammals are paired with a suitable genotype, the embryos are isolated during pregnancy (see Example 1), primary embryonic fibroblasts are isolated therefrom according to standard protocols and immortal lines are spontaneously established therefrom (Todaro and Green, 1995; Schreiber et al., 1995). Thus, a preferred embodiment of the fibroblast cell according to the invention is a fibroblast cell in which the gene coding for a subunit of the AP-1 transcription factor was inactivated by means of "knockout". The gene coding for a subunit of the AP-1 transcription factor can also be inactivated by interfering with (e.g. inhibiting) AP-1 higher regulatory proteins. In place of or in addition to the inactivation of the gene coding for a subunit of the AP-1 transcription factor the activity of AP-1 can also be modified so as to modify a component, acting on AP-1, of the signal transduction pathway. This modification can be a modification of the influencing component on the gene or protein level, e.g. an upward-regulated or downward-regulated expression or a conformational or structural change. The component thus no longer adopts its role in the signal transduction pathway as usual and effects a change in the activity of AP-1.

It is preferred to have the genes to be expressed in a suitable expression vector controlled by a constitutive promoter. Regulatory units of viruses (SV40, RSV, CMV) or of cellular genes expressed constitutively (e.g. the ubiquitin C protein) are suited for this purpose. It is preferred to design the expression vector such that a subunit of the AP-1 transcription factor is expressed constitutively in inactive form which can, however, be activated. Here, an inactive fusion protein is produced which only changes its conformation after addition of an inductor such that the protein becomes active.

In order to produce the expression vector of step (b), the person skilled in the art can use common in vitro recombination methods, as described e.g. in Sambrook et al., 1989.

In a preferred embodiment, the genetically modified fibroblast cell according to the invention is characterized in that the gene which codes for a subunit of the AP-1 transcription factor is c-jun (Angel et al., 1988; Hilberg et al., 1993), or junB (Schorpp-Kistner et al., 1999), i.e. the fibroblast cells are c-jun-deficient or JunB-deficient.

In a particularly preferred embodiment, the genetically modified fibroblast cell according to the invention is a human fibroblast cell or murine fibroblast cell, and a genetically modified fibroblast cell which contains c-JunER.TM. or JunB-ER.TM. is most preferred. c-JunER.TM. contains the N-terminal half of c-Jun and the tamoxifen-binding site (ER) (Bossy-Wetzel et al., 1997). In order to optimize the expression, the c-Jun-ER.TM. sequences are controlled by the human ubiquitin-C promoter/enhancer sequence which represents an efficient regulatory unit (Schorpp et al., 1996). On the basis of JunB a JunB-ER.TM. construct is prepared analogously (see Example 9).

Another embodiment of the present invention relates to a method of identifying a compound which can support, inhibit or modify the AP-1-dependent differentiation and/or proliferation of keratinocytes, the method comprising the steps of: A) contacting the test compound with the genetically modified fibroblast cell according to the invention in coculture with primary keratinocytes in a test system I and/or test system II, test system I being characterized in that the protein encoded by the gene from (b) is not available or only available in inactive form, and test system II being characterized in that the protein encoded by the gene from (b) is present in active form, and B) determining the expression of marker genes associated for the differentiation state or proliferation state of keratinocytes and/or the epithelial structures, a change in the expression of the marker genes and/or the epithelial structure in test system II only indicating incompletely in test system I that the test compound can support, inhibit or modify the AP-1-dependent differentiation and/or proliferation of keratinocytes. Although proliferation but not differentiation of the keratinocytes can be measured in test system I, test system I distinguishes itself in that keratinocytes and fibroblasts are cocultured in a two-dimensional system on (plastic) culture carriers, whereas in test system II both cell types together with a matrix form a three-dimensional tissue structure, similar to the skin.

Test system I represents a two-dimensional feeder-layer coculture in which keratinocytes together with irradiated fibroblasts (feeder cells) are cultured in submersed fashion (Rheinland and Green, 1975). Primary human keratinocytes are isolated from skin biopsies of plastic surgery, expanded in feeder layer coculture (passage 1) and introduced into the experiments in passage 2. Both irradiated, post-mitotic murine 3T3 fibroblasts and human fibroblasts or fibroblast lines can be used as a feeder layer, and the differentiation process of the epithelial cells remains unchanged, i.e. the specific markers are expressed adequately. The proliferation of the keratinocytes is induced significantly by the mesenchymal cells together with which they are cultured. Here, it is a close vicinity of epithelial and mesenchymal cells that is decisive but not the direct cell contact. This indicates a paracrine effect of the mediators produced by the mesenchymal cells (Smola et al., 1993). For example, mediators produced by keratinocytes, such as IL-1, can stimulate the feeder cells to synthesize keratinocte-specific growth factors, such as KGF (Maas-Szabowski et al., 1999), which refers to a regulatory circuit between fibroblasts and keratinocytes. Both cell types can be cocultured according to known methods, e.g. by means of the methods described in the below examples, preferably by means of the organotypical culture described in below Examples 3 and 11 to 13. However, if keratinocytes are cultured under conventional conditions in the feeder-layer system while submersed in medium, they will proliferate and, as soon as they have reached confluence, will start growing in several layers. Depending on the culturing conditions, they form one or more cell layers resembling squamous epithelia but differing considerably in vivo from the epidermis as regards structure and function.

In test system II, i.e. the much more complex, three-dimensional organotypical culture, the keratinocytes are cultured on a collagen gel admixed with fibroblasts while exposed to air (type I collagen, from rat or cow) so as to achieve an improved epithelial tissue architecture more easily comparable with the structure of the skin (Fusenig et al., 1994). Medium components diffusing through the collagen gel immersed in the medium feed the fibroblasts and keratinocytes. In organotypical cultures, the keratinocytes already form a multi-layered, orderly epithelium after only 7 days, consisting of basal layer, spinous layer, regeneratirve layer horny layer. From a morphological point of view, the epithelium corresponds to the epidermis, since it has the specific epidermal differentiation markers, such as keratin 1/10, transglutaminase, filaggrin, loricrin (Stark et al., 1999).

The test compounds may be widely differing compounds, both naturally occurring compounds and synthetic, organic and inorganic ones as well as polymers (e.g. oligopeptides, polypeptides, oligonucleotides and polynucleotides) as well as small molecules, antibodies, sugar, fatty acids, nucleotides and nucleotide analogs, analogs of naturally occurring structures (e.g. peptide imitators, nucleic acid analogs, etc.) and a number of further compounds as well as mixtures of different compounds. One the one hand, cytokines and growth factors and, on the other hand, low-molecular lipophilic substances imitating cytokine or growth factors (agonists) or inhibiting cytokine-induced or growth factor-induced signal paths (antagonists) are of special interest. Furthermore, testing of proteins is of interest, whose expression is regulated by c-Jun or JunB, e.g. the matrix metalloproteinases MMP-9 and MMP-13 (Schorpp-Kistner, 1999). Uptake into the cells is effected in the test compounds within short distances in the culture system. The direct passage of lipophilic substances takes place through the cell membrane. In the case of cytokines and growth factors, binding to corresponding surface receptors and triggering of intracellular signal paths to the cell nucleus take place. In addition, a large number of possibly useful compounds can be screened in extracts of natural products as a starting material. Such extracts may be derived from a large number of sources, e.g. the following species: fungi, actinomycetes, algae, insects, protozoa, plants and bacteria. The extracts showing activity can then be analyzed to isolate the active molecule; see in this connection e.g. Turner, 1996, and Suh, 1995.

Basically skin-specific genes indicating a certain differentiation type are in consideration as marker genes. The genes used in FIGS. 4-6 represent usable markers. The expression is determined by in situ hybridization or preferably immunohistochemistry (IHC) or immunofluorescence. The criteria for proliferation and differentiation are both histological criteria (e.g. thickness of the epithelium) and the expression strength -and localization of the marker genes ki-67 (proliferation) and of the genes used in FIGS. 4-6 (differentiation).

Changes in the expression level of the marker gene can be studied using methods well known to a person skilled in the art. They comprise the monitoring of the mRNA concentration (e.g. using suitable probes or primers), immunoassays as regards the protein concentration (e.g. by means of corresponding antibodies), RNAse protection assays, amplification assays or any other means suitable for detection and known in the art.

New compounds whose expression is regulated directly or indirectly by AP-1 or which regulate the proliferation and/or differentiation of skin keratinocytes and are thus of therapeutic value, can also be searched for on a large scale, e.g. by screening a very large number of candidate compounds in substance libraries, the substance libraries being able to contain synthetic or natural molecules.

The above method according to the invention can be modified by means of protocols described in the scientific literature and patent literature and known in the art. For example, a large number of possibly useful molecules can be screened in a single test. For example, when a field of 1000 compounds shall be screened, in principle all of the 1000 compounds can be placed in a microtitration plate well and tested at the same time. When the presence of a compound having the desired activity is discovered, the pool of 1000 can be divided into 10 pools of 100 and the process can be repeated until an individual compound is identified. In any case, the production and simultaneous screening of large libraries from synthetic molecules can be carried out by means of well known methods of combinatorial chemistry, see e.g. van Breemen, 1997, and Lam, 1997.

The method according to the invention can also be accelerated greatly as high throughput screening. The assays described herein can be modified correspondingly so that they can be used in such a method. It is obvious to the person skilled in the art that a number of methods are available for this purpose.

By means of the method according to the invention the molecular modes of action of cytokines, for example, can be studied as to the growth and differentiation processes of the skin.

The present invention also relates to a method of identifying a gene involved in the AP-1-dependent differentiation and/or proliferation of keratinozyctes. Here, the expression pattern of genes in standard fibroblasts and genetically modified fibroblasts, such as c-Jun-/- and JunB-/- cells, is compared. Genes regulated differently are potential candidates for genes regulating the proliferation and differentiation in keratinozcytes. The c-Jun-/- cell line which carries the Ubi-cJun-ER.TM. expression vector and/or the JunB-/- cell line which carries the Ubi-JunB-ER.TM. expression vector is preferably used for comparative gene expression analysis. Here, the expression in untreated and tamoxifen-treated cells shall be compared. In all of the cases, RNA is prepared from the corresponding cells and transcribed into cDNA. The cDNA can in this connection be labeled either radioactively or by a fluorescent dye. The cDNAs serve as probes for the hybridization of filters or glass carriers on which a plurality of indicative sequences of individual genes are immobilized. Genes whose expression changes under the above described-conditions can be identified by means of the differences in the hybridization strength. This comparative gene expression analysis by means of "high density filter screening" and "DNA chip technology" are described in Iyer et al. (1999) and Fambrough et al. (1999). In known genes, the corresponding substance may either be added directly to the culture medium as recombinant protein (e.g. in the case of a cytokine or growth factor) or be inserted by means of gene transfer in the modified fibroblasts and expressed. The same applies correspondingly for DNA sequences unknown thus far.

In a particularly preferred embodiment of the method according to the invention, the marker gene is a gene coding for K1/K10, transglutaminase, filaggrin or loricrin (Stark et al., 1999). The expression of these genes can be determined in accordance with the above statements, e.g. also in accordance with the methods described in below Examples 11 to 13, using suitable antibodies.

A compound identified by the method according to the invention or an identified gene can then be used for treating diseases, the expression of the target gene being reduced or eliminated depending on the kind of disease (e.g. by knocking out the gene in the target cell or by blocking the translation via antisense RNAs or ribozymes or via vectors containing polynucleotides as insertions, which code for antisense RNAs or ribozymes) or being increased, e.g. by administering a vector containing the target gene under the control of a suitable promoter, e.g. an inducible promoter or a promoter resulting in an intensive expression. This vector may be derived from a virus, e.g. from an adeno-associated virus (e.g. AAV type 2), vaccinia virus or adenovirus which is of use for a gene therapy. Retroviruses are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV. Methods of producing suitable vectors based on the above viruses are known to the person skilled in the art. The genes or the vectors containing them can also be transported to the target cells in the form of colloidal dispersions for the purpose of gene therapy. They comprise liposomes or lipoplexes, for example. Antibodies directed against proteins encoded by the above genes and preferably having a neutralizing effect are also suited for a therapy. These antibodies may be monoclonal, polyclonal or synthetic antibodies or fragments thereof. In this connection, the term "fragment" refers to all parts of the monoclonal antibody (e.g. Fab, Fv or single chain Fv fragments) which have an epitope specificity the same as that of the complete antibody. The production of such fragments is known to the person skilled in the art. The antibodies according to the invention are preferably monoclonal antibodies. The antibodies according to the invention can be produced according to standard methods, the protein encoded by the genes to be studied or a synthetic fragment thereof preferably serving as an immunogen. Methods of obtaining monoclonal antibodies are also known to the person skilled in the art. When the substances, genes or antibodies identified according to the method of the invention are administered, they are preferably available in combination with a pharmaceutically compatible carrier. Suitable carriers are e.g. phosphate-buffered common salt solutions, water, emulsions, e.g. oil/water emulsions, wetting agents, sterile solutions, etc.

The medicaments containing these substances can be administered orally or preferably parenterally. The methods of parenteral administration comprise the topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathekal, intraventricular, intravenous, intraperitoneal or intranasal administration. The suitable dose is determined by the attending physician and depends on various factors, e.g. on the patient's age, sex and weight, the stage of the disease, e.g. the skin tumor, the kind of administration, etc.

Thus, the present invention also relates to the use of the compound identified according to the method of the invention, the gene or antibody directed against a protein encoded by this gene for treating diseases associated with a disturbed proliferation and/or differentiation of keratinocytes, preferably skin diseases, such as psoriasis, chronic inflammations of the skin, delayed wound healing or skin tumors. Furthermore, acne, neurodermatitis, eczemas and scar treatment should be mentioned. Two examples of antibodies whose desired properties can be identified in test systems I and II (FIG. 7) are those directed against interleukin 1 and GM-CSF and neutralize the activity of these cytokines. Likewise compounds can be identified by means of the present invention, which relate to the proliferation and differentiation of various epithelia or disturbances which are accompanied by corresponding diseases and are likewise controlled via interactions with the associated connective tissue cells. The identified compounds can then be tested in corresponding coculture models as to their relevancy. Corresponding models are available or under development for oral mucosa, liver, mammary gland, prostate/urinary bladder, intestine.

Finally, the present invention relates to the use of the gene identified according to the method of the invention or a fragment thereof or an antibody against the protein encoded by this gene for the diagnosis of diseases associated with a disturbed proliferation and/or differentiation of keratinocytes. Here, a suitable tissue sample is withdrawn according to methods known to the person skilled in the art and the diagnostic detection is carried out in accordance with the probe used, e.g. as a Southern blot, Northern blot, PCR, sequencing or as immunohistochemical or immunological detection, e.g. as RIA or ELISA. As to the expression "antibody" reference is made to the above definition.

The above described preferred embodiments of the system according to the invention have the advantage that, on the one hand, the actual direct target genes, e.g. of c-Jun or Jun-B, can be detected by this, since there is a defined starting point of activity (e.g. addition of hormone). On the other hand, a large amount of active protein is immediately available, which represents an advantage over vector systems based on an inducible promoter (in this case the protein amount can only be raised by neosynthesis in the course of several hours). Clonal differences between the cell lines can also be ruled out in the system according to the invention.

Claim 1 of 14 Claims

What is claimed is:

1. A genetically modified fibroblast cell comprising:

(a) a gene of the genome of said fibroblast cell encoding a subunit of the transcription factor AP-1, wherein the gene is inactivated and/or

a modified component of the signal transduction pathway which is acting on the AP-1 transcription factor, resulting in a modified AP-1 activity; and

(b) the gene of (a) operably linked to a promoter in an expression vector, wherein the gene encodes the subunit of the transcription factor AP-1 in an inactive form which can, be activated.

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