WO2003006657A2

WO2003006657A2 - Gene-regulatory elements for gene therapy, for the prevention and diagnosis of metastases and for the gene therapy of tumours

Info

Publication number: WO2003006657A2
Application number: PCT/EP2002/007857
Authority: WO
Inventors: Karsten Brand
Original assignee: Custos Biotechnologie Gmbh
Priority date: 2001-07-13
Filing date: 2002-07-15
Publication date: 2003-01-23
Also published as: AU2002328897A1; WO2003006657A3

Abstract

The invention relates to a method for producing promoters or enhancers, which can be induced by tumorous tissue and/or metastases. The promoters or enhancers are suitable for use in gene-therapy vector systems, in particular for treating cancer and metastases.

Description

Gene regulatory elements for gene therapy, for the prevention and diagnosis of metastases and for gene therapy of tumors

The present invention relates to a method for producing promoters / enhancers which can be induced by tumor tissue and / or metastases. Furthermore, the present invention relates to promoters / enhancers, the activity of which is regulated by at least one factor from tumor tissue or metastases, and to the use of the promoters / enhancers in diagnostics and cancer therapy.

By far the most common cause of death in malignant tumors is organ metastasis. While the primary tumor can be surgically resected at least in the early and middle stages, this is rarely possible in the case of metastasis. With a few exceptions, the conventional methods of chemotherapy and radiation can, if at all, only achieve temporary improvements in the clinical picture of metastatic tumors.

The most common tumors include colorectal cancer. Liver metastases of colorectal origin are the leading cause of death for patients with colorectal cancer. Since they are often the only manifestation of the disease over a long period of time after surgical removal of the primary tumor, they are a possible target for curative therapeutic approaches (Dreben, JA and Niederhuber, JE. (1993) Cancer of the lower gastrointestinal tract - colon cancer. In : Niederhuber, JE ed. Current Therapy in Oncology. St. Louis, MO: Decker, 426-431). The potentially curative surgical removal of liver metastases is only possible for a small percentage of the patients and temporary remissions are shown for chemotherapy but no life extension. There is therefore an urgent need for alternative forms of therapy. Due to their complexity, the gene therapy approaches that have been in development for around 10 years have a dramatically increased level of controllability compared to conventional forms of therapy. In the area of cancer gene therapy, this can be used, among other things, for tumor-specific targeting of the vectors or restriction of gene expression to tumor tissue and for the specific adaptation of the transferred transgenes to points of attack of the respective tumor type.

Apart from immunological approaches, the gene therapy approaches pursued so far, like the conventional methods, almost exclusively focus on the infiltrating tumor tissue. This harbors the problem of inadequate reaching of the tumor, which presents itself with increased intratumoral pressure and limited blood supply and is insufficiently met even with the direct intratumoral injection of gene therapy transfer vehicles which is usually carried out. In the case of multiple metastases, it has been shown that the systemic or regional administration of the vectors then required makes it much easier to infect the surrounding normal tissue than the tumor cells. This fact can be exploited by circumventing the problem of the difficult access to the tumor tissue, by declaring the easily accessible normal tissue as the primary gene therapy goal. The method is characterized in that the normal organ tissue is equipped with defense functions directly at the site of a potential or existing metastasis, which prevent the establishment or further growth of the metastases. The further spread of an inoperable primary tumor can also be prevented. This strategy of impregnating healthy tissue differs fundamentally from all gene therapy and non-gene therapy approaches carried out to date.

A problem with this approach, however, is that when ubiquitously active or organ-specific gene regulatory elements are used, constitutive expression of antitumorous transgenes takes place which is independent of the extent of the actual invasive event and is potentially too significant Side effects. It is therefore necessary to limit the activation of antitumorous transgenic products to the normal tissue in the vicinity of actually affected tumor areas.

The invasion of tumor cells into normal tissue is a very complex, fine-tuned process. The example of liver metastasis shows that the cells of normal tissue react to the invasion in a variety of ways. For example, the hepatocytes react with vacuole formation, fat storage or massive deformation of the cell shape. Such changes, which are to be interpreted as defense reactions or at least reactions to the invasion process, depend on the transcription of the corresponding genes involved in these processes (invasion-induced genes). In addition to the reactions of the normal tissue that is directly adjacent to the tumor, genes in distant normal tissue also react to tumor activity (genes that can be induced to spread).

The invention was based on the problem of providing gene regulatory elements which can be activated by spread or invasion and which make gene therapy of metastases and primary tumors more effective and safer or enable early diagnosis of activated micrometastases. Furthermore, the invention was based on the problem of specifying a method for producing the elements mentioned.

The invention is implemented according to the claims.

An essential point of the invention are the enhancers / promoters. These gene regulatory elements of spread and invasion-induced genes are used to place antitumor genes such as tumor suppressor genes, suicide genes, angiostatic and antiproteolytic genes under their control. This then enables the activation of these genes in normal tissue at exactly the desired time of active tumor invasion.

The promoters / enhancers in question can also be used diagnostically. In this case, reporter genes are used as transgenes, their gene products be secreted into the blood when e.g. B. Micrometastases become active and activate the promoters / enhancers in the surrounding normal tissue. In this way, active micrometastases can be identified, which at least would not yet be recognizable with imaging methods, and which accordingly react therapeutically.

Furthermore, combinations of diagnostic genes under the control of spread-activated or invasion-activated enhancers / promoters and antitumor genes under the control of externally regulated enhancers / promoters are used. This provides the opportunity to intervene in a controllable, externally controllable manner after detection of metastasis activation.

Viral, liposomal and other non-viral vectors are used as the gene transfer vehicle.

The approach according to the invention can be inserted into the following clinical scenarios:

1. The preoperative or intraoperative vector application in target organs in order to prevent the extravasion of any metastatic tumor cells detached during operation of the primary tumor.

2. The prevention of the outgrowth or killing of occult micrometastases, especially in high-risk groups such as operated breast cancer with lymph node involvement or colorectal tumors.

3. Limiting the growth of already established metastases or inoperable primary tumors.

4. The early diagnosis of active micrometastases.

The technical problems mentioned are solved according to the invention by a promoter / enhancer which is more active in the invasion front by at least factor 2, preferably by at least factor 3, than in normal tissue further away from the tumor, and by a method for producing a promoter / enhancer which at least one factor is regulated from tumor tissue and / or metastases, and / or the influence of the tumor tissue or metastases on the surrounding normal tissue is regulated either directly or indirectly, comprising the following steps:

a) Provision of tissue containing tumor tissue and healthy tissue

b) isolating healthy tissue I which is adjacent to the tumor tissue and / or metastatic tissue (invasion front) and healthy tissue II which is not adjacent to the tumor tissue and / or metastatic tissue;

c) determining the gene expression pattern in tissue I and II;

d) comparison of the expression patterns obtained from c); and

e) isolating the promoter / enhancer belonging to a gene whose expression in tissue I and tissue II is different.

In a preferred embodiment, the tissue for identifying suitable promoters is selected from liver tissue, lung tissue, nerve tissue, bone tissue, lymph nodes and skin.

In a further preferred embodiment, the tissue is a material that has been obtained by means of biopsy.

In a further preferred embodiment, the desired material is obtained by means of microdissection techniques, with laser microdissection being particularly preferred. This technology is described, for example, in M.R. Emmert-Buck et al., Laser capture microdissection. Science 274 (1996), 998-1001.

In a further preferred embodiment, at least two cell layers are isolated for the tissue of type I, the material being removed from the contact area with the tumor in the direction of the normal tissue. Preferably at least three cell layers of material are isolated. In a further preferred embodiment, at least three cell layers are removed for the type II tissue, but these are located further from the contact area with the tumor than the type I tissue, so that the type II tissue is removed from healthy tissue located further away. At least five cell layers of this type are preferably removed.

In a further preferred embodiment, the removed tissue of type I or type II each comprises approximately 10,000 cells.

In a further preferred embodiment, RNA, preferably mRNA, is isolated both from the type I tissue and from the type II tissue. This is then analyzed using microarrays. The respective RNA sample is placed on a microarray with known probes and after hybridization under suitable conditions it is determined whether and if so which species from the RNAs of the respective tissue types hybridize with probes of the microarray and thus give a signal. This so-called gene expression analysis is, for example, described in detail in M. Noordewier et al., Gene expression microarrays and the Integration of biological knowledge, Trends in Biotechnology 19 (2001), 412-415.

In a further preferred embodiment, the RNA is first amplified again after its isolation, before it is then fed to the analysis described above by means of microarrays. Several methods are possible for amplification, including in vitro transcription and the known PCR techniques. Care must be taken to ensure that the RNA of both tissue types is amplified to the exact same degree, in order to avoid artificial results in the gene expression analysis. A different degree of amplification of RNA of tissue type I compared to the RNA of tissue type II would lead to incorrect conclusions regarding a possible activation of the respective DNA by the tumor tissue or the metastases.

In a further preferred embodiment, the isolated RNA is provided with a label which delivers the measurement signal in the later analysis by means of microarrays. This marker can, for example, coincide with the amplification of the RNA done. Suitable markers for this purpose are well known to the person skilled in the art, as described, for example, in: http://www.affymetrix.com/products/reagents/specific/enzotranscript.affx.

In a further preferred embodiment, the expression patterns of the genes in tissue type I and tissue type II are compared with one another, for which purpose in particular the comparison of the gene expression patterns obtained by means of microarrays is used. Since these gene expression patterns provide not only qualitative, but also at least semi-quantitative information about individual RNA species, a comparison of the patterns obtained with tissue type I and tissue type II enables conclusions to be drawn as to which RNA species is present in tissue of type I in a different concentration than in type II tissue.

In a further preferred embodiment, the difference in the amount of RNA of a particular species should be at least a factor of 3 when comparing tissue type I and tissue type II. Such a difference is highly significant and shows the influence of the tumor tissue, or one or more factors thereof, on the activity of the corresponding gene.

It is preferred to look for promoters / enhancers in which the proximity of the tumor tissue leads to an increase in promoter activity. According to this, genes and their promoters of particular interest are those which are expressed more strongly by a factor of 3 in type I tissue than in type II tissue.

In a further preferred embodiment, the promoter is then identified, characterized and possibly isolated, which promoter is responsible for the changed RNA expression of a particular RNA species. The promoter can be isolated, for example, from genomic banks, the sequence serving as a probe from the microarray, for example, also being used to isolate the complete gene, including the regulatory sequences from a gene bank. Alternatively, the corresponding promoter can also be identified using the bioinformatics in corresponding databases. Once the gene on which the changed signal is based in the gene expression analysis has been identified, it is possible to isolate and provide the associated promoter using routine methods. The present invention also relates to a promoter / enhancer which can be obtained by the process described above and which is regulated by at least one factor from tumor tissue and / or metastases. "Regulated" in this context means that the promoter / enhancer activity changes under the influence of tumor tissue or metastases. The activity of a particular promoter / enhancer can be reduced under the influence of the factor mentioned, while in the case of another promoter / Enhancers the activity is increased. Preferred are promoters / enhancers in which the activity is increased by at least a factor of 2 when the promoters come to lie in the invasion front compared to their activity in normal tissue which is further away from the tumor tissue or the This activity preferably differs by at least a factor of 3, the activity preferably being increased in the invasion front compared to normal tissue located further away.

In a further preferred embodiment, the promoter / enhancer controls a heat shock gene, an apoptosis inducer gene or a tissue transglutaminase gene, these genes representing the natural environment of the promoter / enhancer.

In a further preferred embodiment, the promoter / enhancer according to the invention can be induced by metabolic stress and / or hypoxia. Metabolic stress includes e.g. following metabolic states: lack of nutrients (especially glucose), lack of removal of toxic metabolites. Hypoxia is characterized, for example, by the presence of reactive oxygen molecules, such as oxyl, peroxyl or hydroxyl radicals or non-radical oxygen molecules such as oxygen, ozone, peroxyl nitrite, by nitrogen oxide, lipid peroxides and others, as well as by the enzymes and other proteins which are responsible for their formation or their degradation and as a defense reaction, such as superoxide dismutase, catalase, NAD (P) H oxidase or glutathione.

In a further preferred embodiment, the promoter / enhancer is induced in hepatocytes or copper star cells or sinus endothelial cells when these come into contact with a tumor and / or metastases or when these cells are located in the invasion front. The invasion front encompasses at least 2 Cell positions calculated from the point of contact with tumor tissue into healthy normal tissue.

In a further preferred embodiment, the promoter / enhancer is induced in lung tissue or nerve tissue or bone tissue or lymph nodes or skin when these cell types come into contact with a tumor and / or metastases.

The present invention also relates to a vector containing the promoter / enhancer according to the invention. Common plasmids, viruses and non-viral vectors are suitable as vectors. The person skilled in the art is familiar with inserting the promoter / enhancer into a vector, cf. e.g. Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989).

In a particularly preferred embodiment, the vector is constructed in such a way that it is used in the context of gene therapy. Suitable gene therapy vector systems are described, for example, in Brand, K., Strauss, M. (1998) Molecular basis of gene transfer and application for gene therapy, in: Handbook of Molecular Medicine, Vol. 2: Tumor Diseases (Ganten, D., Ruckpaul, K., ed.), Springer, Berlin, Heidelberg, New York, pp. 110-145.

In a further preferred embodiment, the vector contains a gene which is under the control of the promoter / enhancer according to the invention. Suitable arrangements are familiar to the person skilled in the art.

In a further preferred embodiment, the gene encodes a reporter molecule and / or an active pharmaceutical ingredient. The use of a reporter molecule is of particular interest in diagnostics. Switching on the promoter / enhancer under the influence of a tumor or metastases leads to the production of a reporter molecule, for example a protein, which either directly or indirectly generates a detectable signal. Such well-known reporter molecules include, for example, the well-known Lac-Z system. More suitable Reporter molecules / detection systems include, for example, the luciferase system, the green fluorescent protein system or the alpha 1 antitrypsin system.

In the second alternative, in which the gene brings about a pharmaceutical active ingredient, a variety of possible genes can be selected depending on the desired therapeutic effect.

In a particularly preferred embodiment, the gene encodes a factor that can limit tumor growth, destroy the tumor or protect normal tissue from tumor invasions.

In a further preferred embodiment, the gene codes for a metalloprotease inhibitor, TIMP-1, TIMP-2, TIMP-3, TIMP-4, PAI-1, PAI-2 or a C-terminally shortened TIMP-2, an extracellular matrix protein or a cell adhesion - molecule. The respective genes for insertion into the vector according to the invention are available to the person skilled in the art from the prior art.

In a further preferred embodiment, the factor is a protein which is selected from the polypeptide chains, collagen, fibronectin and laminins, claudins, occludins, cadherins and an integrin. Several polypeptide chains of the proteins mentioned can also be encoded by the vector according to the invention. In a further preferred embodiment, the gene codes for a protein which comprises a membrane anchor sequence or a secretory leader sequence.

In a further preferred embodiment, the gene is a suicide gene. The protein is particularly preferably a cytosine deaminase or nitroreductase.

In a further preferred embodiment, the gene product of the reporter gene is a product which is secreted into the blood or which can be detected in tissue sections. In the first case, the activation of the promoter, and thus the spread of the tumor or the metastases, can be recognized by the fact that the reporter gene product increases in the blood. In the second alternative, the protein can be detected, for example, by means of antibodies in tissue sections. Alternatively, the protein can also be an enzyme, the activity of which can then be detected in tissue sections.

In a further preferred embodiment, the vector according to the invention encodes both a reporter molecule and an active pharmaceutical ingredient.

In a further preferred embodiment, the vector is a vector suitable for gene therapy, which is selected in particular from an adenovirus, an adeno-associated virus, a minimal adenovirus, an HSV and lentivirus-based vector. Other possible vectors are described, for example, in Brand, K., Strauss, M. (1998) Molecular Foundations of Gene Transfer and Application for Gene Therapy, in: Handbook of Molecular Medicine, Vol. 2: Tumor Diseases (Ganten, D., Ruckpaul, K., ed.), Springer, Berlin, Heidelberg, New York, pp. 110-145.

The present invention also relates to a cell, preferably a mammalian cell, which contains a promoter / enhancer according to the invention and / or a vector according to the invention, the promoter or the vector being integrated into the cell's genome at a position which is not its own corresponds to natural position. This is usually the result of the transformation of the cell with a corresponding construct, the construct being randomly integrated into the genome.

The present invention also relates to a kit for diagnosing tumors and / or metastases, the kit containing the vector according to the invention together with further reagents, such as, for example, buffer solutions, reagents for the detection of a reporter molecule, and corresponding instructions for use.

The present invention also relates to a pharmaceutical composition containing the promoter or vector according to the invention or the cell genetically modified with the products mentioned. Such a composition usually contains other auxiliaries which are however familiar to the person skilled in the field of galenics. The promoter according to the invention is particularly suitable for producing a medicament for the treatment of cancer. In this connection, the promoter according to the invention is administered, for example, together with a gene which produces a tumor-toxic substance. After administration of the medicament to the patient, the spreading of a tumor or metastases leads to an activation of the promoter according to the invention, which in turn produces tumor-toxic product which is suitable for inhibiting tumor growth or eliminating the tumor. In a further preferred embodiment, the promoter according to the invention or the vector containing the promoter is used to diagnose cancer or to monitor the course of cancer therapy. With the means mentioned, it can be tracked, for example, whether tumor spread or metastatic spread can be reduced when cancer therapy is started.

Further preferred embodiments relate to the following subjects

1. Comprehensive means for the prophylaxis, therapy and diagnosis of tumor diseases

- a vector in the sense of a gene transfer vehicle

- an enhancer / promoter

- one or more transgenes,

an enhancer / promoter is selected which is activated in the normal tissue when the tumor spreads into the surrounding normal tissue.

2. Means in which the enhancer / promoter is a heat shock gene enhancer / promoter.

3. Means in which the enhancer / promoter is an apoptosis inducer gene enhancer / promoter.

4. Means in which the enhancer / promoter is the enhancer / promoter of the tissue transglutaminase. 5. Means in which the enhancer / promoter can be induced by metabolic stress.

6. Means in which the enhancer / promoter can be induced by hypoxia.

7. Means in which the enhancer / promoter can be activated in hepatocytes or copper star cells or sinus endothelial cells by spreading the tumor.

8. Means in which the enhancer / promoter can be activated in the lung tissue or nerve tissue or bone tissue or lymph nodes or skin by spreading the tumor.

9. agents containing transgenes for substances,

- which limit the growth of the tumor

- destroy the tumor

- protect normal tissue from tumor invasion.

10. Agent containing genes from metalloprotease inhibitors

11. Agent containing an antitumor transgene coding for: TIMP-1 or TIMP-2

12. Agent containing a protease inhibitory transgene coding for: TIMP-3 or TIMP-4 or PAI-1 or PAI-2.

13. Agent containing a modified transgene, the antitumor effect of which was enhanced by this modification.

14. Agent which contains C-terminally truncated TIMP-2 as the relevant transgene.

15. Agent containing a transgene of the extracellular matrix. 16. Agents containing at least two polypeptide chains of collagen or fibronectin or laminin or genes whose products are responsible for the synthesis of non-protein components of the ECM.

17. Agent containing a modified transgene of the extracellular matrix that it is difficult or not degradable.

18. Agent containing a transgene coding for an adhesion molecule.

19. Means in which the adhesion molecule in question is claudin or occludin or a cadherin or an integrin or a gene from the immunoglobulin superfamily is a selectin or a mucin.

20. Agent for the prophylaxis and treatment of tumor diseases containing an antitumor transgene or sequences thereof, which is provided with a membrane anchor sequence.

21. Use of a gene transfer vector for the production of an agent for the prophylaxis and treatment of tumor diseases containing an antitumor transgene or sequences thereof, which is provided with a membrane anchor sequence or secretion sequence.

22. A method for the prophylaxis and treatment of tumor diseases in which an antitumor transgene or sequences thereof, which is provided with a membrane anchor sequence or secretion sequence, is transferred.

23. Agent which contains a suicide gene or another chemotherapeutically active gene as the relevant transgene

24. Means in which the transgene in question

Cytosine deaminase or active partial sequences thereof or nitroreductase or active partial sequences thereof. 25. Agent containing at least 1 transgene, which can be used to detect tumor activity.

26. Agent containing at least 1 transgene, the gene product of which is secreted into the blood, such as alpha fetoprotein or alpha 1 antitrypsin.

27. Agent containing at least 1 transgene whose gene product can be made visible by isotope diagnosis.

28. Agent containing both a therapeutic and a diagnostic transgene.

29. Agent in which the therapeutic gene is not by a spread-inducible promoter / enhancer but by an externally regulable promoter / enhancer, e.g. the tet promoter is regulated.

30. Means in which an enhancer / promoter is selected which is activated when the tumor spreads into normal tissue in normal tissue which is directly adjacent to the invasion front of the tumor.

31. Means in which the vector is a virus.

32. means in which the virus is a first generation adenovirus or an adeno-associated virus or a minimal adenovirus or an HSV or a lentivirus.

33. Agent in which the virus is a lentivirus / minimal adenovirus hybrid.

34. Agent in which the vector is a non-human mammalian adenovirus.

35. Means in which the vector is not a virus. 36. Agent in which the vector is a liposomal formulation or carrier proteins are used.

37. Agent in which the surface is modified so that a specific gene transfer into normal tissue is achieved.

The following example illustrates the invention

Determination of promoters with activity in the liver part of the tumor / liver invasion front

Principle: non-tumorous organ tissue, which is adjacent to tumors, is isolated by laser microdissection (LCM), mRNA isolated, amplified by in vitro transcription or unamplified on genomic microarrays (Affimetrix, Santa Clara, California, US). Differential gene expression is determined in comparison to organ tissue not adjacent to the tumor. Promoters of genes that are differentially upregulated in the invasion front are candidate promoters for invasion-regulated gene expression (see example of use).

1. Obtaining tissue

a) Animal model: 5x106 LS 174 colon tumor cells are inserted into the liver

Injected to nude mice. After 14 days, intrahepatic tumors have grown. The animals are sacrificed, the livers are removed and immediately in pieces of approx. 1 cm 2 in OCT Compound (Sakura Tissue

Tek, Zoeterwoude, Netherlands). The embedded pieces of liver are immediately pre-cooled in nitrogen-cooled 2-methylbutane, then frozen in liquid nitrogen and stored at -80 ° C. b) Alternatively, human liver metastases are surgically removed,

Regions containing the invasion front were cut into 1 cm pieces and frozen as described above. 2. Preparation of tissue sections

Cryopreserved tissue is placed in a cryotome at an object temperature of -18 ° C in a section thickness of 5 μm on a slide. The sections are dried for 3-5 minutes at approx. 50 ° C on a hot plate or 1-2 minutes at 80 ° C in the hybridization oven.

This is followed by a brief fixation in 100% ethanol. The sections are subjected to hematoxylin-eosin staining for tissue differentiation. A short washing step in 70% ethanol is followed by a 3 minute incubation in Mayer's haematoxylin solution. The sections are blued in autoclaved tap water for 2 min. Eosin staining (0.25% in 70% ethanol and addition of glacial acetic acid) takes place for 5 seconds and excess color is washed out in DEPC-treated water. Then the cuts go through an ascending ethanol row (2x70%, 2x95%, 100%). An additional washing step in 70% ethanol at the end prevents drying artifacts. The slides are again dried for approx. 5 min at 50 ° C on the heating plate or ^Λ A up to 1 min at 80 ° C in the hybridization oven.

3. Laser microdissection

With the help of the laser capture microdissection method, 3 cell layers of liver tissue (= liver part of the invasion front, L1) are isolated from the tumor / liver contact area. The number of cut cells is approximately 10,000. An equal number of cells which are at least 5 cell layers and further away from the invasion front are isolated as comparative tissue.

4. Total RNA isolation

The total RNA of the microdissected tissue is isolated using the standard Chomczynski method (Chomczinski and Sacchi, 1987, AnalBiochem, 162, 156).

5. Amplification of mRNA by two rounds of reverse transcription followed by in vitro transcription

With the help of a poly-dT primer, which carries the promoter sequence of the T7 RNA polymerase, the mRNA from the total RNA of the microdissected Tissue rewritten into cDNA. Superscript II is used as the reverse transcriptase

(Invitrogen, Karlruhe, Germany).

The synthesized cDNA is transcribed in vitro using T7 RNA polymerase, for which a commercially available kit is used (Ambion Megascript T7, Austin,

Texas).

In the second round of amplification, random hexamers initiate the cDNA

Synthesis. Then proceed as in the first round.

6. Biotinylation of the amplified mRNA or the isolated total RNA

The biotinylation reaction corresponds to another round of amplification. There is again a cDNA synthesis with a defined starting amount of RNA (Affymetrix protocol, Affimetrix, Santa Clara, CA, USA). In the subsequent in vitro transcription, biotinylated UTP nucleotides are used for the labeling (Enzo-Kit, Farmingdale, New York, USA).

7. Purification and fragmentation of the antisense RNA (aRNA)

The amplified and labeled aRNA is purified using RNA columns (RNeasy kit from Qiagen, Hilden, Germany) and split into 35-200 bp fragments to avoid steric interactions due to their size (Affymetrix protocol, http: //www.affvmetrix .com / products / reagents / specific / enzotranscript.affx .. Affimetrix, Santa Clara, CA, USA).

8. Array hybridization

The biotinylated aRNA is applied to microarrays (MG U74Av2, Affimetrix) for hybridization. These are glass supports onto which 25 nucleotide long oligos have been synthesized, which represent a total of over 12,000 mouse genes and ESTs. The hybridization takes place at 45 ° C. in the buffer described by Affimetrix

(http://www.affymetrix.com/products/reagents/specific/enzotranscript.affx.) for 14 hours. The chips are then washed and scanned. 9. Evaluation

In order to be able to make a statistical statement about genes upregulated in the invasion front, at least three times two arrays must be hybridized and evaluated.

Using the Affimetrix software MAS 5.0, batch analyzes are carried out to determine the extent of potential upregulation

Describe candidate genes (signal log ratio).

Genes that are consistently up to 3 times upregulated on all three chips are confirmed by quantitative PCR.

10. Quantitative PCR

To confirm the upregulation of invasion front genes, non-amplified mRNA is isolated from the invasion front and the non-invasion front liver. This mRNA is first reverse transcribed and then subjected to a SYBRgreen-based quantitative PCR (Röche, Mannheim, Germany) according to the manufacturer's instructions. Genes whose differential regulation is confirmed in qPCR are candidate genes for invasive gene expression.

11. Determination of invasion-inducible promoters

The promoters of those differentially regulated genes of the invasion front that are found in steps 1-10 are determined in the following manner. a) Promoters that have already been known and characterized are requested from the responsible laboratories and can be integrated into a suitable gene therapy vector without modification. b) Known promoters which are not yet sufficiently characterized for gene therapy use (size limitation, for example viral vectors) are initially characterized using promoter restriction digests and reporter gene assays. Then proceed as described under a). 3) The genomic fragment of promoters that have not yet been cloned is isolated from a genomic library using the corresponding cDNA probe, the transcription start is determined by a primary extension assay, and a sequence up to 8 kB in size, 5 'of the transcription start is analyzed as described under b).

Reporter gene assays are carried out for cases b) and c) under a kind of stimulation that simulates the in vivo situation. Such stimuli can e.g. be pro-apoptotic (e.g. transfection of a caspase gene or p53), hypoxic (oxygen deprivation) or anti-metabolic (serum deprivation). If such a stimulus does not lead to promoter activation, a 4 kB fragment is cloned into an adenoviral vector without further in vitro testing and tested according to the application example in vivo.

Assay for the validation of identified promoters for their inducibility by tumor / metastases

1. Vector construction:

Gene transfer vectors are constructed which contain the HSP 70 promoter or the TG promoter as a promoter / enhancer, the TIMP-2 gene (inhibits tumor spread) as a transgene or either carry the reporter gene β-galactosidase or α antitrypsin and a first-generation adenovirus as a vector component to have.

2. Tumor model

As a tumor model, an injection of colon carcinoma cells (eg LS174 T) into the portal vein circuit of the liver of immunodeficient nude mice is used to induce liver metastases, or the tumor cells are injected directly into the liver to induce a liver tumor. 3. Gene transfer

10 days after tumor induction, when small tumors have formed, recombinant adenoviruses according to item 1 are administered via the tail vein of the animals. In a dose of 3x10 ¹⁰ infectious units, about 50% of the liver cells are infected.

4. Detection of tumor spread

From day 3 after the gene transfer, blood is drawn from the animals and the concentration of the αi-antitrypsin is determined. The concentration of the protein in the serum of animals with tumor induction and transfer of the αrAntitrypsin adenovirus is several times higher than in the serum of animals which either had only tumor induction or only gene transfer. The concentration will increase with increasing size of the tumor invasion front, i.e. with increasing spread of the tumor.

Similar results are obtained when ß-galactosidase adenovirus is used, except that it is not the serum concentration that is measured here, but the intracellular ß-galactosidase concentration, which is visualized by a histochemical reaction (blue color). Liver cells in the immediate vicinity of spreading tumors are stained more blue than more distant cells. No blue staining is seen in tumor animals without gene transfer. Little or no blue coloration can be seen in gene transfer without tumor induction.

5. Efficiency

The efficacy of the approach in terms of inhibiting tumor spread is demonstrated with the TIMP-2 gene as a transgene. Tumor-bearing animals that received a TIMP-2 adenovirus are killed at different times, the tumor volumes are determined and compared with those of animals that received reporter gene viruses. The TIMP-2 animals have significantly smaller tumors.

Claims

claims

1. A method for producing a promoter / enhancer which is regulated by at least one factor from tumor tissue and / or metastases, comprising the following steps:

a) providing tissue containing tumor tissue and healthy tissue b) isolating healthy tissue I which is adjacent to the tumor tissue and / or metastatic tissue (invasion front) and healthy tissue II which is not adjacent to the tumor tissue and / or metastatic tissue; c) determining the gene expression pattern in tissue I and II; d) comparison of the expression patterns obtained from c); and e) isolating the promoter / enhancer belonging to a gene whose expression in tissue I and tissue II is different.

2. The method according to claim 1, characterized in that the tissue in step a) is selected from liver tissue, lymphatic tissue, brain tissue, bone, skin, kidney and adrenal gland.

3. The method according to any one of claims 1 or 2, characterized in that the tissue in step a) is a biopsy tissue.

4. The method according to any one of claims 1 to 3, characterized in that the isolation after step b) is carried out by means of microdissection.

5. The method according to claim 4, characterized in that the microdissection is carried out by means of a laser.

6. The method according to any one of claims 1 to 5, characterized in that in step b) the tissue I comprises at least two cell layers, preferably at least three cell layers, starting from the contact area with the tumor.

7. The method according to any one of claims 1 to 5, characterized in that in step b) the tissue II comprises at least three cell layers, preferably at least five cell layers, which are further away from the contact area with the tumor than the tissue I.

8. The method according to any one of claims 1 to 7, characterized in that in step b) approximately 10,000 cells of tissue I or tissue II are removed.

9. The method according to any one of claims 1 to 8, characterized in that in step c) RNA is isolated from tissues I and II and each is analyzed separately by means of microarrays.

10. The method according to claim 9, characterized in that the RNA is amplified after isolation and before analysis.

11. The method according to claim 9 or 10, characterized in that the isolated RNA is provided with a label.

12. The method according to any one of claims 1 to 11, characterized in that the expression patterns in tissue I and tissue II are compared.

13. The method according to any one of claims 1 to 12, characterized in that a difference of factor 2 or more in the amount of a particular RNA species between tissue I and tissue II indicates a tumor or metastasis-regulated promoter / enhancer.

14. The method according to claim 13, characterized in that an increased by a factor of 3 of an RNA species in tissue I compared to tissue II indicates a tumor or metastasis-inducible promoter / enhancer.

15. The method according to any one of claims 1 to 14, characterized in that the promoter / enhancer associated with an altered RNA expression between tissue I and tissue II is isolated.

16. The method according to claim 15, characterized in that the promoter is isolated from a genomic bank.

17. promoter / enhancer which is regulated by at least one factor from tumor tissue and / or metastases and which is obtainable by one of the methods according to any one of claims 1 to 16.

18. Promoter / enhancer according to claim 17, characterized in that it is more active in the invasion front by at least factor 2, preferably by at least factor 3, than in normal tissue further away from the tumor.

19. Promoter / enhancer according to one of claims 17 or 18, characterized in that it regulates a heat shock gene, apoptosis inducer gene or a tissue transglutaminase gene in its natural environment.

20. Promoter / enhancer according to one of claims 17 to 19, characterized in that it is induced by metabolic stress and / or hypoxia.

21. Promoter / enhancer according to one of claims 17 to 20, characterized in that it is induced in hepatocytes or copper star cells or sinus endothelial cells when they come into contact with a tumor and / or metastases.

22. Promoter / enhancer according to one of claims 17 to 21, characterized in that it is induced in the lung tissue or nerve tissue or bone tissue or lymph nodes or skin when they come into contact with a tumor and / or metastases.

23. Vector containing a promoter / enhancer according to one of claims 17 to 22.

24. Vector according to claim 23, characterized in that it is suitable for use in gene therapy.

25. Vector according to one of claims 23 or 24, characterized in that it further contains a gene under the control of the promoter according to one of claims 17 to 22.

26. Vector according to claim 25, characterized in that the gene encodes a reporter molecule and / or a pharmaceutical agent.

27. Vector according to one of claims 17 to 26, characterized in that the gene encodes a factor which can limit tumor growth, destroy the tumor and / or protect normal tissue against tumor invasions.

28. Vector according to one of claims 22 to 27, characterized in that the gene encodes a protein selected from metalloprotease inhibitors, TIMP-1, TIMP-2, TIMP-3, TIMP-4, PAI-1, PAI-2, C terminally truncated TIMP-2, an extracellular matrix protein and a cell adhesion molecule.

29. Vector according to claim 28, characterized in that the protein is selected from polypeptide chains of collagen, fibronectin, laminins, claudins, occludins, cadherins and an integrin.

30. Vector according to one of claims 28 or 29, characterized in that the protein comprises a membrane anchor sequence and / or a secretory leader sequence.

31. Vector according to one of claims 26 to 30, characterized in that the gene represents a suicide gene or encodes a protein selected from cytosine deaminase and nitroreductase.

32. Vector according to one of claims 23 to 31, characterized in that the reporter gene encodes a gene product which is secreted into the blood or which is a protein which can be detected in tissue sections.

33. Vector according to one of claims 23 to 32, characterized in that it contains a gene which encodes a reporter molecule and a gene which encodes a pharmaceutical active substance.

34. Vector according to one of claims 23 to 33, characterized in that it is selected from a vector suitable for gene therapy, preferably based on an adenovirus, an adeno-associated virus, a minimal adenovirus, an HSV and lentivirus.

35. Cell containing a promoter according to one of claims 17 to 22 and / or a vector according to one of claims 23 to 34, preferably in a position which does not correspond to the position of the naturally occurring promoter.

36. Kit for diagnosing tumors and / or metastases, containing a vector according to one of claims 23 to 34 and further reagents together with an instruction manual.

37. Pharmaceutical composition containing a promoter according to one of claims 17 to 23 and / or a vector according to one of claims 23 to 34 and / or a cell according to claim 35.

38. Use of a promoter according to one of claims 17 to 23 and / or a vector according to one of claims 23 to 34 for the manufacture of a medicament for the treatment of cancer, in particular micrometastases.

39. Use of a promoter according to any one of claims 17 to 23 and / or a vector according to any one of claims 23 to 34 for the diagnosis of cancer or for follow-up in cancer therapy.