WO2003074692A2

WO2003074692A2 - Specific gene expression in colonic cancer cell lines

Info

Publication number: WO2003074692A2
Application number: PCT/DE2003/000669
Authority: WO
Inventors: Andreas Block
Original assignee: Universitätsklinikum Hamburg-Eppendorf Körperschaft des Öffentlichen Rechts
Priority date: 2002-03-05
Filing date: 2003-03-04
Publication date: 2003-09-12
Also published as: AU2003229263A8; WO2003074692A3; DE10209451A1; AU2003229263A1

Abstract

Das Verfahren dient zur Durchführung einer spezifischen Genexpression in Kolonkarzinom-Zell-Linien. Es werden zur Genexpression rekombinante adenovirale Vektoren verwendet. Die Genexpression wird durch einen Mucin-1/DF3-Promoter vermittelt. Zur Amplifikation der Genexpression wird ein Gal4/VP16 Fusionsprotein erzeugt.

Description

Specific gene expression in colon cancer cell lines

The invention relates to a method for carrying out a specific gene expression in colon carcinoma cell lines, in which recombinant adenoviral vectors are used for gene expression.

At 20%, colorectal cancer is one of the main causes of death for malignant diseases in the western world. The development of colorectal cancer from adenomas is now considered to be certain and the timely endoscopic removal of these precancerous diseases can prevent the development of colon cancer. However, if the tumor is already advanced or metastatic, the therapeutic options are limited and 5-year survival is only 5%. Therefore, metastatic tumor disease poses one of the greatest challenges in the Oncology. By introducing therapeutic genes into tumor cells, gene therapy approaches open up new perspectives in the treatment of these diseases.

Gene therapy approaches were only made possible by the development of suitable vector systems. A variety of different vector systems of both viral and non-viral origin have been developed in the past 10 years. The direct injection of DNA into tissue resulted in no significant gene expression. Packaged in liposomes, DNA can be taken up by cells using endocytosis. Complexation with suitable proteins enables receptor-mediated uptake. As a rule, however, only a slight and temporary transgene expression is possible with these non-viral systems. With the help of viral vectors, foreign genetic material can be smuggled into the tumor cells much more efficiently via the highly developed viral infection mechanism. In particular, adenoviruses and retroviruses, as well as adeno-associated viruses, herpes simplex viruses and hepatitis B viruses, were used for the viral transfer of genes. Retroviruses integrate their genome into the genome of the infected cell.

With gene therapy of metabolic diseases for permanent gene expression, which may be desirable, permanent gene expression is not required for gene therapy of malignant diseases. Preparations with higher virus titers and significantly higher transduction efficiency even at the time of infection of non-dividing cells make recombinant adenoviruses appear more suitable for experimental therapy of colorectal and other carcinomas. The adenoviral genome is not integrated into the host genome and the expression of adenoviral genes is only temporary. Long-term expression is not or is not required in the treatment of malignant diseases desirable because the eradication of the tumor cells is in the foreground and the expression of cytotoxic genes or cytokines genes should be limited to the duration of the tumor eradication. The adenoviral expression of suicide genes in pancreatic and colon carcinomas leads to significant tumor regression in the animal model after administration of the corresponding prodrugs. A prolongation of survival through antitumor immunostimulation could not be achieved by suicide therapeutic approaches alone. The adenoviral-mediated expression of cytokines opened up new possibilities for the induction of cytotoxic T lymphocytes (in particular interleukin-2 and interleukin-12).

With the adenoviral expression of interleukin-18 and interleukin-12, an efficient activation of NK cells has also become possible, which will be of particular importance in the gene therapy of MHC-I negative tumors. Not only suicide therapeutic approaches but especially approaches to adenoviral expression of cytokines are limited by the transgene-dependent toxicity. This is caused not only by infected tumor cells but in particular also by the transduction of surrounding parenchyma cells. The systemic and portal venous administration of recombinant adenoviruses in the tumor-free animal model leads to the predominant transduction of hepatocytes. Even with direct intratumoral injection of hepatic metastases with predominant transduction of the tumor cells, there is significant infection of neighboring hepatocytes with dose limitation due to the resulting hepatotoxicity.

The use of tumor- and tissue-specific promoters enables the reduction of transgene expression in surrounding non-malignant cells with the aim of lower toxicity of these gene therapy approaches. Tissue-specific and even tumor-specific gene expression can be achieved by tumor and tissue-specific promoters. Such tumor-specific approaches in retroviral and adenoviral gene therapy have been developed for a large number of different malignancies. Table 1 gives an overview of tumor-specific or tissue-specific promoters.

An ideal tumor-specific promoter combines the following properties: (1) high gene expression in the tumor tissue with (2) comparatively low gene expression in the surrounding tissue, and (3) the wide range of uses within a tumor entity.

The carcinoembryonic antigen (CEA) was detected in 70% of all colorectal carcinomas and in 60% of all patients with colon carcinoma the CEA serum levels were increased. In the development of a specific adenoviral gene therapy approach to the treatment of hepatic metastases of colorectal cancer, the use of the CEA promoter enables selective gene expression in CEA-positive colon cancer with negligible expression in hepatocytes. However, transgene expression is only limited to differentiated colon carcinoma cells expressing CEA and gene expression is significantly lower than when using the cytomegalovirus promoter which is widespread in gene therapy.

Mucins were first described in the endothelium of the gastrointestinal tract and the expression of these mucins was already discussed in 1994 as a potential immunological target in the treatment of gastrointestinal carcinomas. Mucin-1 mRNA expression was regularly detected in colorectal carcinomas, but not in hepatocytes and in contrast to CEA expression, mucin expression correlates positively with hepatic metastasis and tumor development. The publication "SCHWARTZ, B., BRESALIER, RS and KIM, YS (1992). The role of mucin in coloncancer metastasis. Int J Cancer 52, 60-65" explains the corresponding background.

The object of the present invention is to develop ade-viral vectors for the gene therapy of colorectal carcinomas which enable a specific but nevertheless efficient transgene expression and thus increase the therapeutic range of such an experimental approach.

The expression of adenoviral genes is said to be kept low by the use of a singular adenoviral vector system and the recombinant, replication-deficient adenoviruses should be able to be synthesized in high titers with good transduction efficiency in relation to a large number of different colon carcinoma cell lines. This results in the following targets:

1. Recombinant, replication-deficient adenoviruses of the Ad5 serotype

2. Specific gene expression in mucin-positive cells

3. Efficient gene expression comparable to the cytomegalovirus promoter

4. Construction of singular adenoviral vectors

The object is achieved according to the invention in that the gene expression is mediated by a mucin-1 / DF3 promoter and in that a GAL4 / VP16 fusion protein is generated for the amplification of the gene expression.

During the development of these specific and at the same time potent adenoviral vectors for the gene therapy of colorectal carcinomas, a system for the amplification of the transgenic Expression integrated. For this purpose, a gene coding for Gal4 / VP16 fusion protein was cloned into the El region of recombinant adenoviruses under the control of the specific DF3 / Muc-1 promoter. Downstream Gal4 binding sites for the induction of transgene expression were then integrated in the same adenovirus.

Gal4 was already described in 1974 in the regulation of galactose metabolism in Saccharomyces cerevisiae. The corresponding binding site was not identified until 11 years later. The fusion of this Gal4 with VP16, a Herpes Simplex Virus transcription activator of the "Immediate early viral genes", resulted in a transcription activator that was unusually potent in eukaryotic cells. The combination of 5 Gal4 binding sites also resulted in a 36-fold amplification of expression compared to a Gal binding site. This system was described, for example, by "Tantin, D., CHI, T., HORI, R., PYO, S., and CAREY, M (1996). Biochemical mechanism of transcriptional activation by GAL4-VP16. Methods Enzymol 274, 133- 149 "and used in adenoviral vectors to express the BAX gene.

The approach described here represents the first-time use of the Gal4VPl6 transactivator system in an adenoviral vector for mucin-specific gene expression. The quantification of the gene expression was carried out using the luciferase system.

With widespread mucin expression in a variety of different gastrointestinal tumors, the use of the DF3 / Muc-1 promoter described for the first time in breast cancer enabled a new, more universal approach to gene therapy of colorectal cancer and its hepatic metastases. The previous gene therapy Breast cancer studies using the DF3 / Muc-1 promoter in "CHEN, L., CHEN, D., MANOME, Y., DONG, Y., FINE, HA, and KUFE, DW (1995) Breast cancer selective gene expression and therapy mediated by recombinant adenoviruses containing the DF3 / MUC1 promoter. J Clin Invest 96, 2775-2782 "showed a specific but significantly weaker gene expression than the CMV promoter.

Immunohistochemical detection of Muc-1 in tumor tissue and cell culture

Tissues from primary tumors or hepatic metastases were fixed in 10% formalin (pH 7.3) and embedded in paraffin. The paraffin was extracted in 5 μm thin sections with xylene and the samples after unmasking of antigenic structures by citrate pretreatment with a 1: 100 dilution of a murine, monoclonal anti-mucin-1 antibody (clone VU 4-H-5, mouse igGl, # sc-7313, Santa Cruz, USA) incubated for 1 hour at room temperature. Alternatively, slides with cultured tumor cells were acetone-fixed, air-dried and preincubated with a 1:50 dilution of sheep serum. Bound mucin-specific antibodies were detected by alkaline phosphatase-anti-alkaline phosphatase antibodies (APAAP). The alkaline phosphatase activity was finally detected with the help of Neufuchsin and the tissue sections were stained with hematoxylin, in the cell culture preparations for the inhibition of endogenous alkaline phosphatase activity preincubation with 0.8 mg / ml levamisole.

Cell-lines

The 293 renal carcinoma cell line was cultivated in HGDMEM cell culture medium (Gibco, Rockville, MD). The human HepG2 HCC cell line and the human colon carcinoma cell lines Colo320, Colo205, HT29, Lovo, SkCol, SW620 were propagated in the recommended media (ATCC). All Cell culture media were supplemented with 10% fetal calf serum, 1% glutamine and 1% penicillin-streptomycin. The cultivation was carried out according to standard cell culture procedures.

Cloning adenoviral expression plasmids

Cloning of the pAd.CMV-luc: The luciferase gene was excised from pGL3 using Kpnl / Sall digestion and ligated into the adenoviral expression vector pAd.CMV-pA. Cloning of pAd.Mucl / DF3-luc: The mucin promoter was isolated by Xbal / Kpnl digestion of pDF3-CAT + 3-7 and replaced the CMV promoter in pAd.CMV-pA. The luciferase gene was cloned into the pGreenLantern vector after Kpnl / Sall digestion and was inserted into the multiple cloning site of the pAd.Mucl / DF3pA after Kpnl / Not1 digestion.

Cloning of the pAd.Mucl / DF3 _bιn -luc: The Hindlll fragment of the pSGVP containing Gal4VP16 was cloned into the Hindlll region of the pGal4B5-CAT 5 "terminal of the Gal4 binding site. The SV40pA from pBK-CMV was subsequently digested by Kpnl / Mlul and blunt-end ligation integrated into the Psti region between Gal4VPl6 and Gal4B5, partial digestion with Hindlll and Sacl led to the isolation of the cassette containing the binary system, which was cloned into the multiple cloning site of pBK-CMV, followed by digestion with Clal and Sacl and blunt-end ligation into the Kpnl region 3'-terminal of the mucin promoter of the pAd.Mucl / DF3-luc resulted in the adenoviral expression plasmid pAd.Mucl / DF3bin-luc.

Plasmid transfection of 293 cells

2 * 10 ⁵ 293 cells were incubated for 12 hours in 6-well cell culture plates and subsequently co-precipitated with 0.3 μg of purified DNA and 0.3 μg of a pSV-ß-galactosidase control plasmid. 4.5 hours later Medium was added and after 24 hours the cells were lysed for luciferase and galactosidase activity in 150 μl cell culture lysis reagent. Galactosidase and luciferase were determined in accordance with the manufacturer's protocols (Tropix, Foster City, CA).

Generation of recombinant, replication-deficient adenoviruses

After reducing the LPS contamination from plasmid DNA by

With Triton-X extraction, recombinant adenoviruses were generated by coprecipitation and homologous recombination of the corresponding adenoviral expression plasmids with pBHGIO in 293 cells. Recombinant adenoviruses were purified after replication in 293 cells by CsCl density gradient centrifugation. Plaque-forming units were determined by infecting 293 cells with serial dilutions and agarose overlay. The resulting titers were 7.5 * 10 ⁹ pfu / ml (Ad.CMV-luc), 8.4 * 10 ⁹ pfu / ml (Ad.Muc-luc) and 2.5 * 10 ⁹ pfu / ml (Ad. Muc _bin -luc). Viral DNA was isolated using a Qiagen DNA Blood Kit and partially sequenced to confirm the integration of the promoter and transactivator elements.

Adenoviral infection of human tumor cells

The hepatocellular carcinoma cell line HepG2 and the human colon carcinoma cell lines HT29, Colo205 and SkCol were incubated with 10 ^ε cells per well 6 hours before planned infection with recombinant adenoviruses in 6 well cell culture plates. purified

Virus preparations were diluted with McCoy cell culture medium and the tumor cells were incubated for 1 hour in 500 μl each. The cultivation then took place over 24 hours in the appropriate growth medium. The luciferase activity was finally determined from the cell lysates. Quantification of luciferase gene expression

The transgene expression was determined by quantifying the luciferase reporter gene in the cell lysate. Standard curves using recombinant luciferase in concentrations from 1 pg / ml to 300 ng / ml were used to determine a biphasic exponential approximation. The function was calculated using the PRISM software (GraphPad Software, Ine, SanDiego, CA). Luciferase expression was correlated with the total cell protein, which was determined using a DC protein assay kit.

SDS-Page and Immunobiotting

Cell lysates of HepG2, HT29, Colo205 and SkCol after infection with Ad.Muc-luc or Ad.Muc _bin -luc were separated after boiling in Laemmli sample buffer under reducing conditions using SDS-acrylamide gel electrophoresis and the proteins on 0.45 μm Immobilon -P transferred. After blocking, actin and Gal4VP16 were detected by anti-actin antibodies and polyclonal anti-VP16 antibodies. After washing the blots with 0.1% Tween-20 in TBS, pH 7.5, they were incubated with a peroxidase-labeled second antibody and visualized by chemiluminescence detection according to the manufacturer's instructions.

test results

Mucin expression in colon carcinoma

To test this mucin-specific adenoviral system with Gal4VP16 amplified gene expression, colon carcinoma tissues and various Zeil lines were examined for their mucin gene expression. Mucin detection in human colon carcinoma tissues was carried out in accordance with FIG. 1. The mucin-1 expression in human colon carcinoma (A) and hepatic metastasis (B) is illustrated here. In paraffin-fixed sections, mucin-1 was detected using a monoclonal antibody and an alkaline phosphatase-mediated neufuchsin color reaction. In the colon carcinoma cells there are intracytoplasmic mucin deposits both in the primary tumor and in the hepatic metastasis. Surrounding hepatocytes (B, lower section of the image) show no mucin-1 staining.

Colonic carcinoma cells were stained after fixation with acetone as shown in FIG. 2. The detection of mucin-1 in human colon carcinoma cell lines is illustrated here. After fixation and incubation with a monoclonal antibody against mucin-1, mucin could be detected in various human colon carcinoma cell lines (HT29, SW620, Lovo, Colo205, SkCol). Colo320 and the human HCC cell line HepG2 showed no mucin-1 expression. Clusters of colon carcinoma cell groups can be seen in the primary tumor, surrounded by mucin-negative fibroblasts. The hepatic metastases of this colon carcinoma express significant amounts of mucin without detection of Muc-1 in the surrounding hepatic tissue. In the human HCC cell line HepG2, too, no Muc-1 could be detected, which corresponds to preliminary examinations at the muc-1 mRNA level. In the different human colon carcinoma cell lines, a differently developed mucin

Expression can be demonstrated. Apart from Colo320, in which no Muc-1 expression was detectable, all other Zeil lines tested expressed mucin with maximum expression in SkCol. The HCC cell line HepG2 and HT29 with low, Colo205 with medium and SkCol with high cin-1 expression was used for the subsequent studies on adenoviral-mediated, mucin-1-dependent aplified transgene expression.

Adenoviral vectors for amplified mucin-specific

gene expression

Recombinant adenoviral vectors were constructed according to FIG. 3 with the following promoter elements: cytomegalovirus (CMV) -controlled luciferase gene expression (A), mucin-1 specific gene expression using the DF3 / Muc-1 promoter (B) and a mucin-1 specific gene expression system for luciferase gene expression using an integrated cassette for Gal4VPl6 transactivator-fusion protein-mediated amplification of gene expression (C). The luciferase gene expression takes place secondarily after mucin-1 dependent Gal4VP16 expression and binding to the further downstream Gal4 binding sites with VP16 mediated transactivation of this secondary gene expression. These elements were cloned into the EI region of adenoviral expression plasmids and the expression plasmids were homologously recombined with pBHGIO as described for the generation of recombinant adenoviruses in 293 cells.

Partial sequencing verified promoter insertion, transactivator elements and the luciferase gene. After pacification of the adenoviral expression plasmids in 293 cells and infection of HT29 cells with the corresponding adenoviruses, the luciferase gene expression could be detected. 3 graphically illustrates the system for amplified gene expression. The mucin-1 specific promoter (DF3 / Mucl) is not used directly to control transgene expression (B) but first to express a Gal4VPl6 fusion protein (C). This very potent one After binding to a Gal4 binding site further downstream, the transactivator leads to activation of the downstream transgene expression via the VP16 domain. These constructs were compared to transgene expression by a CMV promoter (A). Gal4B5: 5-fold copy of the Gal4 binding site.

Figure 4 illustrates the cloning of adenoviral vectors for specific, amplified gene expression. The constructs described in FIG. 3 were inserted into the El region of corresponding recombinant, replication-deficient adenoviruses. These adenoviruses were constructed by subsequent coprecipitation with pBHGIO for homologous recombination in 293 cells.

Amplification of gene expression by Gal4VPl6-

transactivation

In order to quantify the amplification of the gene expression by integrating a Gal4VP16 fusion protein gene and the corresponding Gal4 binding sites, various mucin-negative (HepG2) and mucin-positive (HT29, Colo205, SkCol) cell lines with Ad. Muc-luc or Ad.Muc _bin -luc infected, and after 24 hours compared the luciferase gene expression per total cellular protein, the results are illustrated in Fig. 5.

5 shows in particular the amplification of gene expression by the binary system. HepG2 (HCC) and various human colon carcinoma cell lines (HT29, Colo205 and SkCol) with different viral doses (multiplicity of infection = moi) of the adenoviruses Ad.Muc _b: LI.-Luc and Ad.Muc -luc infected and the luciferase activity quantified as a measure of transgene expression.

The recombinant adenoviruses Ad.Muc-luc and Ad.Muc _bin -luc both contain the mucin-1 specific promoter, but differ in the presence of a binary system for amplifying gene expression. In HepG2 it was possible to achieve a 50-200 fold amplification of the gene expression, regardless of the moi of the recombinant adenoviruses used. In the mucin-1 positive cell lines, the amplification was dependent on the moi used and was up to 250-fold. With a moi of 100, the amplification of the gene expression in the mucin-negative system corresponded to the amplification in the mucin-positive Zeil lines. Comparison of amplified, specific expression with that

CMV promoter

The constitutive viral cytomegalovirus (CMV) promoter enables transgene expression, which is only temporary but high, in a large number of adenoviral gene therapy preclinical and clinical studies. The question was therefore asked whether the adenoviral vectors for mucin-specific and Gal4VP16-amplified gene expression have a gene expression comparable to that of the CMV promoter and thus enable effective gene therapy approaches for specific gene therapy.

For this purpose, different mucin-positive and mucin-negative cell lines were infected with different moi of either Ad.CMV-luc, Ad.Muc-luc or Ad.Muc _bin -luc and after 24 hours the luciferase transgene expression based on the total Cell protein determined. 6 shows a comparison of the amplified specific gene expression with the CMV-mediated gene expression. Different mucin-1 positive and negative cell lines were again infected with different moi of the adenoviruses Ad.Muc _bin -luc, Ad.Muc-luc and Ad.CMV-luc and the luciferase gene expression was quantified.

Compared to Ad.CMV-luc, the use of the Ad.Muc _bin -luc resulted in a 7 to 1.3-fold (moi = 100) higher luciferase gene expression in the mucin-negative cell line HepG2, in mucin-positive cell line The Ad.Muc _bin -luc mediated gene expression was clearly superior to Ad.CMV-luc with up to 590-fold lines. Particularly in the case of the higher and thus more side effect-relevant mois (10 and 100), a more specific gene expression by the DF3 / Muc-1 promoter in mucin-positive cells resulted in a clear superiority of this system in the equal to Ad.CMV-luc. These data illustrate the potential of the Ad.Muc _bin -luc for a specific and highly efficient gene therapy of mucin-1 positive tumor cells.

In addition, the transgene expression of Ad.Mucluc, that is to say the adenoviral vector for specific but not amplified transgene expression, was compared with the Ad.CMV-luc; the results are also illustrated in FIG. 6. Despite specific gene expression, higher m.o.i. underlie this transgene expression even in mucin-positive cell lines without amplification of the use of the CMV promoter.

Table 2 compares various mucin-positive colon carcinoma cell lines with the mucin-negative HepG2 cell line for transgene expression after infection with either Ad.Muc-luc (left) or Ad.Muc _bin -luc (right). One can see a lower specificity of the mucin-dependent gene expression compared to the non-amplified DF3 / Muc-1, but in particular obtained with higher moi, due to the binary system.

Dose-corrected transgene expression and Western blot analysis

The Zeil lines HepG2, HT29, Colo205 and SkCol differ in their different adenoviral transduction efficiency. In order to be able to determine a specific and amplified transgene expression independently of the transduction efficiency of the cell line examined, the adenoviral titers were first determined which, using the Ad.CMV-luc, resulted in a transgene which was comparable with respect to the total cell protein -Expression lead. With a moi of 6 (HepG2), 80 (HT29), 240 (Colo205) and 100 (SkCol), infection of these Zeil lines with Ad.CMV-luc with regard to luciferase resulted in Gene expression no significant difference, this is shown in Fig. 7. In particular, the dose-adapted transgene expression is illustrated here. To compensate for a different transduction efficiency of the different carcinoma cell lines, different moi were determined, which led to a comparable transgene expression using the Ad.CMV-luc (HepG2: 6 mOi, HT29: 80 moi, Cθlθ205: 240 moi , SkCol: 100 moi). If the Ad.CMV-luc vector is exchanged for the recombinant adenovirus for specific, amplified gene expression, Ad.Muc _bin -luc, there is an increase in transgene expression which is significant for HT29 and Colo205 and SkCol is highly significant compared to HepG2. Otherwise, the transgene expression rates are significantly higher than with the Ad.CMV-luc under the corresponding moi

The above-mentioned Zeil lines were now infected with the corresponding moi Ad.Muc _bin -luc and the luciferase gene expression was quantified based on the total cell protein after 24 hours. In comparison to HepG2, there was now a significant - (p <0.05) for the mucin-1 positive Zeil lines HT29 and for Colo205 (p <0.01) and SkCol (p <0.005) a highly significantly superior transgene expression , Cell lysates of uninfected cells, cells infected with Ad.CMV-luc and Ad.Muc _bin -luc were subsequently examined for the expression of the Gal4VP16 fusion protein. The expression is illustrated in FIG. 8, in particular here the Western blot analysis of the Gal4VPl6- Transactivator expression shown. Cell lysates from the experiment presented in FIG. 7 were examined for the presence of the Gal4VP16 fusion protein in a Western blot. The fusion protein could be detected in mucin-1 positive cells after infection with Ad.Muc _bin -luc, but not in HepG2, uninfected or Ad.CMV-luc infected cells. These data confirm the adenoviral mediated mucin-1 dependent expression of the Gal4VP16 fusion protein and resulting transactivation of the luciferase gene expression.

The fusion protein could be detected in mucin-1 positive tumor cells infected with Ad.Muc _bin -luc, but not in HepG2 cells, in non-infected or Ad.CMV-luc infected tumor cells. As expected, actin gene expression showed no difference in the cell lysates examined.

Evaluation of the test results

The experiments explained above describe for the first time a singular recombinant adenoviral vector system with integrated Gal4VP16 and corresponding Gal4 binding sites for the amplification of specific gene expression.

A DF3 / Muc-1 promoter was used for the specific gene expression in mucin-1 positive colon carcinoma cells and the transgene expression was quantified by means of luciferase gene expression. The high transgene expression in mucin-positive colon carcinoma cells and the comparatively low transgene expression in mucin-negative cells enable tissue- and tumor-specific adenoviral gene therapy. It was shown that the amplified, tumor-specific gene expression in this system was significantly higher than in comparable adenoviral vectors using the cytomegalovirus promoter.

The amplification of the transgene expression by integration of the Gal4VP16 system resulted in an up to 250-fold increased transgene expression. Remarkably, a dependence of the amplification on the moi used could be shown in mucin-1 positive cells. These results may be The effect of transgene expression after multiple infections of the same target cell can be attributed to the fact that Gal4VP16 fusion proteins formed can lead to transactivation of the luciferase gene expression at all available Gal4 binding sites.

A lack of amplification due to higher m.o.i. in the mucin-negative HepG2 cell line, the higher transduction efficiency and saturation compared to the other Zeil lines may already be due to the lower m.o.i. due. Further investigations are necessary to adequately explain this phenomenon, with high and therefore toxicity-relevant m.o.i. however, there are no differences between mucin-positive and mucin-negative cell lines with regard to the amplification of the specific gene expression.

The amplification of the specific gene expression by the binary system (Gal4VP16 - Gal4 binding sites) did not lead to a significant loss of the specificity of this therapeutic approach and the comparison with the cytomegalovirus promoter showed up to 590 times higher transgene expression in mucin 1 positive line. In the mucin-negative HepG2 cell line, the amplified transgene expression was 7-1.3 times higher than with comparable CMV promoter constructs. These data illustrate the potency of adenoviral vectors with integrated Gal4VPl6 - Gal4B5 in tumor-specific gene therapy; compared to conventional promoters, this high transgene expression efficiency will even reduce the adenoviral dose (m.o.i.) and thus adenoviral-related toxic side effects.

The superiority of the adenoviral vectors for specific and amplified transgene expression over Ad.CMV-luc in mucin-positive tumor cells was also demonstrated Compensation of the different transduction efficiencies in the different Zeil lines can be illustrated by appropriately adapted moi and the expression of the Gal4VP16 fusion protein in the esters blot correlated with the luciferase gene expression.

These results support further experiments on portal venous and even intravenous systemic application in mucin-1 positive hepatic micrometastasis animal models. Organ distribution of transgene expression after appropriate application of recombinant adenoviruses for the specific and amplified expression of a red fluorescent protein (RFP2) is currently being investigated in vivo. This distribution of gene expression is compared by the simultaneous administration of recombinant adenoviruses for CMV-controlled gene expression of green fluorescent protein (GFP) with the distribution pattern of conventional adenoviral gene therapy approaches. The expression is both in mucin-1 positive intrahepatic colon carcinoma metastases and in mucin-1 positive non-malignant cells e.g. of the gastrointestinal tract of particular interest.

The use of the Muc-1 / DF3 promoter system has several advantages over the CEA promoter: Gastrointestinal tumors and in particular colorectal carcinomas express mucin-1 more often than CEA, and in contrast to CEA expression there is an increase in metastasis and tumor progression Mucin-1 gene expression. Before starting such an approach, an immunohistological test for mucin-1, as illustrated in this work, could provide information about the possible efficiency of this experimental therapy.

So far, 12 different mucins have been characterized. With some mucins restricted to certain organs the membrane-associated mucin-1 is found on almost all glandular epithelial cells, but not in hepatocytes. This favors mucin-1 specific therapeutic approaches for the treatment of intrahepatic metastases of a variety of different carcinomas of glandular origin. In the systemic administration of these vectors, however, the high potential of amplified transgene expression in glandular, epithelial cells must also be considered. The transduction of these cells, which has so far not been noteworthy for systemic application of recombinant adenoviruses, is advantageous.

The above-mentioned studies on the in vivo expression of fluorescence proteins will help to estimate the potential of a possibly even systemic application. In intratumoral intrahepatic and portal venous application, these vectors will significantly increase the safety of adenoviral gene therapy approaches. The integration of tumor-specific or tissue-specific Gal4VP16 expression and corresponding Gal4 binding sites with the desired transgene in an adenoviral construct led to an amplification of transgene expression which is superior to that of two vector systems, since this approach does not involve simultaneous infection of a target cell with two different adenoviruses for amplification presupposes.

6. Technical use and economic prospects

This system for specific and amplified adenoviral transgene expression represents an innovative concept in adenoviral gene therapy for gastrointestinal and other tumors. These constructs can increase the safety of future gene therapy approaches through specific expression. By amplifying the specific gene expression, a significant transgene expression can be achieved even with systemic administration is comparable to the use of the constitutive CMV promoter.

Depending on a possible transduction of other, mucin-1 positive, non-malignant cells, these vectors will be suitable for systemic administration. Irrespective of such a transduction, the portal venous administration of these vectors already opens up new possibilities in the gene therapy of intrahepatic metastases of colorectal cancer. The results to date show that corresponding vectors for cytokine expression (interleukin-12 and interleukin-18) have a higher therapeutic breadth and efficiency in the animal model compared to the use of constitutive promoters (CMV). If a heterogeneous tumor population is infected, it is possible to induce antitumoral T lymphocytes and NK cells, which is not directed against the cytokine-producing, mucin-1 positive carcinoma cells in isolation, but against common cells present on mucin-1 positive and negative cells immunogenic determinants of the corresponding carcinoma.

The system presented for amplified gene expression using adenoviral vectors can be transferred to the therapy of other tumor entities (therapy of hepatocellular carcinomas by AFP-specific, amplified gene expression) by integrating other tumor or tissue-specific promoters.

In summary, these adenoviral vectors for the first time enable adenovirally mediated, proven tissue-specific and high transgene expression, which makes this innovative therapeutic approach more efficient and safer. A reduction in possible, therapy-associated side effects by reducing the adenoviral dose with high intratumoral transgene expression is likely. Summary of the test results

Mucin expression has been demonstrated in a variety of colon cancers. The systemic toxicity in gene therapy approaches could be reduced by the specific gene expression mediated by mucin-1 / DF3 promoter. This specific, but in comparison to constitutive viral promoters (cytomegalovirus promoters) too weak gene expression in mucin-positive tissues has hitherto prevented corresponding effective gene therapy approaches. New recombinant adenoviral vectors for amplified, specific gene expression have therefore been developed. The mucin-specific promoter Muc-1 / DF3 was used to express a Gal4VP16 fusion protein.

The same adenoviral vector also contains Gal4 binding sites immediately before the transgene region. In mucin-positive cells, the Gal4VP16 fusion protein is thus expressed as an intermediate product, which leads to transcription of the therapeutic gene through its VPl6 transactivator domain after binding via the Gal4 domain at the Gal4 binding sites. To validate this concept of amplified, specific gene expression, adenoviruses were constructed in which the El region was replaced on the one hand by the mucin promoter and the luciferase gene (Ad.Muc-luc), and on the other hand between the mucin-specific promoter and the luciferase gene the genes just mentioned for the Gal4VP16 fusion protein and the five-fold Gal4 binding sites were inserted (Ad.Muc _bin -luc). These innovative adenoviral vectors were compared to adenoviruses for luciferase gene expression under the control of the constitutive viral CMV promoter (Ad.CMV-luc). Mucin-1 gene expression was demonstrated in a variety of colorectal cancer cell lines using a monoclonal anti-Mucl / DF3 antibody. These and mucin-negative Zeil lines were infected with the various recombinant adenoviruses and characterized with regard to the luciferase gene expression.

The integration of the binary system, consisting of the Gal4VP16 fusion protein and a corresponding Gal4 binding site, resulted compared to adenoviral vectors only with the mucin promoter Muc-1 / DF3 in up to 250-fold amplification of the gene expression in the various carcinoma cell lines. lines. The transgene expression in these mucin-1-specific and Gal4VP16-amplified adenoviral vectors was up to 590 times higher in mucin-1 positive colon carcinoma cell lines than after infection with recombinant adenoviruses for luciferase gene expression under the control of the CMV- Promoters. In contrast, the gene expression by these binary vectors in muzin-1 negative Zeil lines was only 1.3 to 7 times higher than with the CMV promoter.

Various colon carcinoma cell lines and an HCC cell line were subsequently infected with Ad.CMV-luc in different adenoviral titers, which compensate for the transduction efficiencies and resulted in a comparable gene expression. If these carcinoma cell lines were infected instead with adenoviruses for mucin-1-dependent luciferase gene expression with amplification by a binary system, a significantly to significantly higher transgene expression could be detected in the mucin-1 positive colon carcinoma cells. In the Western blot, the Gal4VP16 fusion protein was infected with the corresponding adenovi after infection of mucin-1 positive Zeil lines. be proven. These novel adenoviral vectors combine the tissue-specific gene expression by the Muc-1 / DF3 promoter with the highly efficient gene expression by transactivation by means of the Gal4VP16 fusion protein while maintaining specificity. The experimental results show that these vectors will enable tumor-specific and efficient adenoviral gene therapy and will significantly increase the therapeutic breadth of this experimental therapeutic approach. In addition, these adenoviral vectors in mucin-1 positive cell lines are superior to recombinant adenoviruses using the CMV promoter in terms of their transgene expression.

Tumor-specific promoters in gene therapy

Tumor promoter / enhancer traπsgeπ vector author year

Glioma myelin basic protein ß-galactosidase retrovirus Miyao 1993 glial fibrillary acidic protein HSV-Thymidiπ-Kinase lipofection Vaπdier 1998 glial fibrillary acidic protein HSV-Thymidiπ-KJnase Adenoviπjs Vaπdier 2000

Prostate Prostate Specific Aπtigen Ludferase Transfection Pang 1995

Prostate-specific aπtigen polyglutamine transfection Sega a 1998

Spec. Membrane Antigen Enh. Cytosine deaminase transfection O'Keefe 2000

Prostate-specific antigen itroreductase adenovirus Latham 2000

Prostate specific antigen diptheria toxin lipofection Pang 2000

Kallikrein 2 Green fluorescent protein Adenoviπjs Xie 2001

Bladder L-plastin cytosine deaminase adenovirus Peπg 2001

Hepatom Albumin ß-Galactosidase Retrovirus Kuriyama 1991 α-Fetoproteiπ HSV-Thymidiπ-KJnase Retrovirus Ido 1995 α-Fetoprotein HSV-Thymidine Kinase Adenovirus Kaneko 1995 α-Fetoprσtein ß-Galactosidase Adenovirus Arbuthnotein 1996-F-Albuminovin-1996

Albumin HSV thymidine kinase retrovirus Kuriyama 1997

AFP & phosphoglycerol kiase HSV thymidine kiase retrovirus Cao 2001

Melanoma Tyrasiπase ß-galactosidase retrovirus Vile 1993

Tyrosinase HSV-Thymidine Kinase & IL-2 Retrovirus Vile 1994

Melanocyte-specific Enh. CAT transfection Artuc 1995

Tyrosinase PNP transfection Hughes 1995

Tyrosinase β-galactosidase adenovirus siders 1996

Tyrosinase HSV Thymidiπ Kinase Adenovirus Siders 1998

Tyrosinase cytosine deaminase retrovirus Cao 1999

Thyroglbulin thyroid HSV thymidine kinase Retrovirus Braideπ 1998

Kalzitoπiπ CALC-I HSV thymidine kinase Adenoviπjs Minemu ra 2000

Cervical secretion. Leukoprotease inhibitor HSV thymidine kinase Adenoviπjs Robertson 1998

Ovarian DF3 / Muc1 BAX Adenoviπjs Tai 1999

L-plastin cytosine deaminase Adenoviπjs Peng 2001

Ovar spec. Promoter-1 HSV thymidine kinase transfection Sel akumaran 2001

Breast DF3 / Muc1 HSV thymidine kinase retrovirus Mano e 1994

DF3 / Muc1 HSV thymidine kinase Adenoviπjs Chen 1995

DF3 / Muc1 Eariy 1 region adenovirus Kurihara 2000

Estrogen-responsive elements E1-α & E4 adenovirus Hemandez 2001

Lung CEA HSV thymidine KJπase transfection Osaki 1994

SCLC gastric releasiπg peptide HSV thymidine kinase transfection Inase 2000

Gastrin-releasing peptide HSV thymidine kinase adenovirus Morimoto 2001

Neuron-specific enolase HSV thymidine kinase transfection Taπaka 2001

Osteosarcoma Osteokalziπ HSV thymidine kinase adenovirus Shirakawa 1998

MM leukemia CD11b glucocerebrosidase retrovirus Malik 1995

Stomach CEA cytosine deaminase Adenoviπjs Lan 1996

CEA HSV thymidine kinase adenovirus Tanaka 1996

CEA cytosine deaminase adenovirus Lan 1997

CEA HSV Thymidiπ Kinase Adenovirus Tanaka 1997

Colon CEA HSV thymidine kinase adenovirus fire 1998

Table 1: Tumor-specific promoters in gene therapy. List of all gene therapy approaches using tumor or gcwebspecific promoters. Relative adenoviral gene expression

Ad.Muc-luc Ad.Muc _b iπ-luc - fold p - fold P

Colo205 / HepG2 m.o.i .: 1 54 <0.005 2.0 n.s.

10 129 <0.0005 5.8 <0.005

100 20.3 0.0005 17.6 <0.0001

HT29 / HepG2 m.o.i .: 1 165 <0.0005 4.3 <0.005

10 135 <0.0001 7.2 <0.0001

100 6.6 n.s. 15.4 <0.001

SkCol / HepG2 m.o.i .: 1 545 <0.0005 23.8 <0.0005

10 1343 <0.0001 116 <0.0005

100 337 <0.05 310 <0.05

m.o.i .: multiplicity of infection.n.s .: not significant, Muc: mucin promoter, luc: luciferase, bin: binary system.

Table 2: Relative adenoviral gene expression. The mucin-1 specific gene expression was compared by comparison of the transgene expression in Ad.Muc-luc (left) and Ad.Muc _b , .- luc (right) infected mucLn-1 cells (Colo205, HT29 and SkCo l) of the mucin-1 negative HepG2 cell line.

Claims

Patent claims

1. A method for carrying out a specific gene expression in colon carcinoma cell lines, in which recombinant adenoviral vectors are used for gene expression, characterized in that the gene expression is mediated by a mucin-1 / DF3 promoter and that a Gal4 for amplification of the gene expression / VP16 fusion protein is generated.

2. The method according to claim 1, characterized in that the gene coding for Gal4 / VP16 fusion protein is cloned into the El region of recombinant adenoviruses.

3. The method according to claim 1 or 2, characterized in that further downstream in the same adenovirus at least one further Gal4 binding site is integrated for the induction of transgene expression.

4. The method according to any one of claims 1 to 3, characterized in that the El region is replaced by the mucin promoter and the luciferase gene.

5. The method according to any one of claims 1 to 4, characterized in that between the mucin-specific promoter and the luciferase gene genes for the Gal4 / VP16 fusion protein Gal4 binding sites are inserted.

6. The method according to claim 5, characterized in that five times Gal4 binding sites are inserted for the Gal4 / VP16 fusion protein.

7. The method according to any one of claims 1 to 6, characterized in that adenoviruses are used as viral vectors.

8. The method according to any one of claims 1 to 6, characterized in that retroviruses are used as viral vectors.

9. The method according to any one of claims 1 to 6, characterized in that herpes simplex viruses are used as viral vectors.

10. The method according to any one of claims 1 to 6, characterized in that hepatitis B viruses are used as viral vectors.

11. The method according to any one of claims 1 to 10, characterized in that the luciferase gene expression is carried out under the control of a viral CMV promoter.

12. The method according to claim 11, characterized in that the luciferase gene is replaced by a gene for inducing an anti-tumor immune response.

13. The method according to claim 12, characterized in that the luciferase gene is replaced by an IL-2 gene.

14. The method according to claim 12, characterized in that the luciferase gene is replaced by an IL-12 gene.

15. The method according to claim 12, characterized in that the luciferase gene is replaced by an IL-18 gene.

16. The method according to claim 12, characterized in that the luciferase gene is replaced by a TNF-α gene.

17. The method according to claim 12, characterized in that the luciferase gene is replaced by an iFN-γ gene.

18. The method according to any one of claims 1 to 17, characterized in that the luciferase gene expression is replaced by genes for apaptosis induction.

19. The method according to claim 18, characterized in that the luciferase gene expression is replaced by BAX genes.

20. The method according to claim 18, characterized in that the luciferase gene expression is replaced by FAS-L genes.

21. The method according to any one of claims 1 to 20, characterized in that the luciferase gene expression is replaced by a suicide gene.

22. The method according to claim 21, characterized in that the luciferase gene expression is replaced by a thymidine kinase gene.

23. The method according to claim 21, characterized in that the luciferase gene expression is replaced by a cytosine deaminase gene.

24. The method according to any one of claims 1 to 20, characterized in that the luciferase gene expression is replaced by a marker gene.

25. The method according to claim 24, characterized in that the luciferase gene expression is replaced by a β-galactosidase gene.

26. The method according to claim 24, characterized in that the luciferase gene expression is replaced by a fluorescent protein.

27. The method according to any one of claims 1 to 26, characterized in that the mucin promoter Mucin-1 / DF3 is replaced by another tissue-specific promoter.

28. The method according to claim 27, characterized in that a myelin basic protein is used as a promoter.

29. The method according to claim 27, characterized in that a glial fibrillary acidic protein is used as a promoter.

30. The method according to claim 27, characterized in that a prostate-specific antigen is used as the promoter.

31. The method according to claim 27, characterized in that Kallikrein 2 is used as promoter.

32. The method according to claim 27, characterized in that L-plastin is used as promoter.

33. The method according to claim 27, characterized in that an α-fetoprotein is used as a promoter.

34. The method according to claim 27, characterized in that tyrosinase is used as a promoter.

35. The method according to claim 27, characterized in that thyroglobulin is used as a promoter.

36. The method according to claim 27, characterized in that calcitonin CALC-I is used as a promoter.

37. The method according to claim 27, characterized in that an ovary spec. Promoter-1 is used.

38. The method according to claim 27, characterized in that CEA is used as a promoter.

39. The method according to claim 27, characterized in that gastrin-releasing peptide is used as a promoter.

40. The method according to claim 27, characterized in that neuron-specific enolase is used as a promoter.

41. The method according to claim 27, characterized in that osteocalcin is used as a promoter.