KR20020013463A - Methods to enhance and confine expression of genes - Google Patents

Abstract

The present invention provides novel gene therapies for restricted areas such as tumors. The method of the present invention comprises: (a) placing the gene in a plasmid vector driven by a thermal or photoinducible promoter, (b) including a tetracycline reactive fusion protein that acts as a transcriptional activator and thereby leveling the drug Modifying said vector to control gene expression by altering it; (c) modifying said vector by including a DNA sequence that reduces or eliminates the expression of a gene in normal surrounding cells. Also provided are a set of vectors for sustained back control expression. Also provided are novel vectors for gene therapy of local and metastatic breast cancer, ovarian cancer and prostate cancer.

Description

Methods to enhance and confine expression of genes

One of the major obstacles to the success of chemotherapy and radiation therapy in cancer is the difficulty of killing tumor-specific cells. Since radiation or cytotoxic chemotherapeutic agents cannot distinguish between tumor cells and normal cells, they are necessarily limited in their applied doses. As a result, it is often seen that disease recurs due to surviving tumor cells.

The use of gene therapy in treating cancer presents many disadvantages as do chemotherapy and radiation therapy. The problem with state-of-the-art gene therapies is the inability to deliver therapeutic genes specifically to target cells. This problem is toxic to cells that are not intended. For example, manipulation of the p53 gene inhibits the growth of both tumor and normal cells, and intravenous injection of tumor necrosis factor alpha (TNFα) results in systemic toxicity with clinical complications such as high fever and hypertension. cause.

Attempts have been made to solve these problems. For example, the use of tissue-specific receptors to induce genes into desired tissues (Kasahara, N., et al., Science, 266: 1373-1376 (1994)), the expression of genes in specific tissues Use of tissue-specific promoters (e.g., prostate-specific antigen promoters) to limit the expression of heat, heat to enhance expression of therapeutic genes in a space-time controlled manner (Voellmy R., et al., Proc Natl.Acad. Sci. USA, 82: 4949-4953 (1985)) or ionizing radiation inducible enhancers and promoters (Trainman, RH, et al., Cell 46: 567-574 (1986); Prowess, R., et al., Proc. Natl. Acad. Sci. USA 85, 7206-7210 (1988)). Heat-induced heat shock protein (HSP) promoters have been used to induce expression of genes such as cytokine IL-2.

Weichselbaum and colleagues first discovered the radioinduced response of the early growth response (Egr-1) gene promoter. Accordingly, they attempted to induce expression of cytotoxic genes such as TNF-α in tumor cells in order to enhance radiation cell death by the promoter means described above. Previously, systemic administration of cytokine TNF-α as an adjuvant to ionizing radiation has been reported to enhance death in mouse xenograft tumor systems. Since then, systemic administration of cytokine TNF-α has been shown to be partially effective in human tumors. The effect of TNFα appears to be dose-dependent since its tumor-killing effect is correlated with its serum concentration. However, due to the systemic toxicity of TNFα, the dosages that can be applied are limited and thus TNFα has a limited utility of treatment regimens. Attempts have also been made to deliver TNFα genes to tumor cells via adenovirus vectors and / or liposomes. Unfortunately, the expression of the TNF-α gene is not limited to the tumor site due to the 'leakiness' of the promoter.

In an attempt to reduce systemic toxicity by confining the level of TNFα to the overall radiation exposure area, Bachselbaum and colleagues used a radioinducible Egr-1 promoter to activate the TNFα gene in situ. Early studies have shown that expression of certain immediate-early genes such as jun / fos and Egr-1 is activated in cells exposed to ionizing radiation (Sherman, ML, et al., Proc. Natl. Acad. Sci. USA, 87: 5663-5666 (1997); Hallahan, DE, et al., Proc. Natl. Acad. Sci. USA, 88: 2156-2160 (1991)). By placing the TNFα gene under the control of the Egr1 promoter (EGRp), expression of TNFα is enhanced when exposed to ionizing radiation in cells containing the EGRp-TNFα plasmid. In vivo, serum levels of TNFα are significantly enhanced within several hours after irradiation (Weichselbaum R.R., et al., Cancer Res. 54: 4266-4269 (1994)). Combination treatment with this plasmid and radiation induces partial regression of xenografted tumors during the treatment period. The level of TNFα dropped sharply within 24 hours. The decrease in serum levels of TNFα was consistent with tumor regrowth.

There are several possible reasons for the tumor to recur when discontinued. The most obvious reason lies in the same constraints as seen in chemotherapy or radiation therapy, i.e., insufficient dosage levels. The main problem that limits the amount of TNFα produced is the weak and transient nature of the Egr-1 promoter. This promoter is inherently weak and increases expression by up to less than threefold upon induction. In addition, induced expression is necessarily transient. This transient expression, coupled with the weakness of the promoter, causes tumor cells to be exposed only briefly to TNFα.

Another factor limiting the production of sufficient doses of TNFα is that not all tumor cells absorb TNFα plasmids. Although repeated dosing has been suggested to help improve treatment outcomes, it is not clear whether repeated doses of sub-optimal low doses of TNFα are useful with regard to the problem of immune response. It may be conceivable to introduce higher doses of plasmids, but the weakness of the promoter has been an obstacle to such a solution. It is known that substantial baseline levels of activity (20-30%) can be detected with the Egr-1 promoter without ionizing radiation (Weichselbaum, et al., Supra). This is not surprising since the CArG box, which is a radiation response component, is part of the serum response component.

HSP promoters are also prone to leakage. In the absence of fever, this promoter exhibits 25-30% background levels of expression, but this degree is not suitable for most cytotoxic genes. Since this level of expression will be detrimental to unirradiated normal cells that take up genes, administration of this plasmid has therefore been limited to small doses of intratumoral injections to minimize systemic toxicity.

Thus, it may be advantageous to use spatiotemporally regulated promoters such as HSP and Egr-1 promoters to enhance the specificity of gene expression at the site of heat treatment or radiation treatment, but these promoters in their current state constrain their use. Has serious problems. In order to use these promoters for cancer therapy, it is necessary to eliminate or greatly reduce background expression in unheated or unirradiated cells. Ideally, expression of cytotoxic genes should be limited to sites of external stimulation (heat or irradiation). In addition, the weak and transient nature of gene expression driven by these promoters should be improved to ensure sufficient expression levels of therapeutic genes.

Even when an improved inducible vector system has been developed that can limit the expression of therapeutic genes to sites of external stimulation, it is important to recognize that there still remains expression problems in heated or irradiated surrounding normal cells. Therefore, it is important to be able to further limit the expression of the therapeutic genes only to their intended targets (eg tumor cells).

The prior art is incomplete because it suppresses unnecessary toxic side effects of gene therapy on cancer as well as the lack of effective means to enhance and maintain gene expression in a tumor target cell in a modifiable manner. The present invention satisfies the above requirements that have been longing in the field for many years.

Summary of the Invention

The present invention provides compositions and methods for modulating the activation of DNA molecules for gene therapy. Activation of these DNA molecules leads to the production of protein products that can provide opportunities for therapeutic manipulation of cells containing the mentioned DNA molecules. This may be achieved through changes in cell growth and metabolism of the target cells, and may include effects on neighboring cells through the secretion of the therapeutic product. The present invention provides a choice of activation that can be controlled by sustained activation or application of antibiotics. The present invention also provides novel expression vectors for use in gene therapy of local and metastatic breast cancer, ovarian cancer and prostate cancer.

A novel method of confining and enhancing the expression of therapeutic genes to tumors in time and space in the form of expression vectors designed for use in local and metastatic breast cancer, ovarian cancer and prostate cancer is also presented.

In one aspect of the invention, a method is provided for continuously and enhanced expression of a gene through activation of a heat or light inducible promoter. As a variant of this method, heat or light is used to activate the promoter, but the sustained level of gene expression is dependent on the concentration of antibiotics (tetracycline or derivatives thereof) that act on the fusion protein with the tetracycline-reactive element. Adjusted.

In another aspect of the present invention, a method of constructing a vector for an active aspect of gene therapy is provided.

In another aspect of the present invention, improved vectors are provided for reducing background expression in unheated and unirradiated cells.

In another aspect of the invention, improved vectors are provided for reducing expression in normal peripheral cells that have been heat treated and irradiated.

In another aspect of the invention, expression vectors are provided for use in gene therapy of local and metastatic breast and ovarian cancers.

In another aspect of the invention, an expression vector is provided for use in gene therapy of local and metastatic prostate cancer.

Further aspects, features and advantages of the invention will be apparent from the following description of the preferred embodiments of the invention provided for the purpose of publication.

The present invention generally relates to the field of gene therapy of cancer. More specifically, the present invention provides a method of regulating the expression of a therapeutically valuable gene product through an inducible promoter. The present invention provides a method for enhancing and sustaining induced gene expression at an intended cellular target in a time-space-controlled manner by thermal or photo-inducible promoter means. The present invention also provides a method of reducing or eliminating background gene expression in non-target cells.

The specific description of the invention summarized above may refer to specific aspects of the invention illustrated in the accompanying drawings so that the above-described features, advantages, and objects as well as other obvious aspects can be achieved and understood in detail. These drawings form part of the specification. The accompanying drawings, however, illustrate preferred embodiments of the invention and are not to be understood as limiting the scope of the invention.

Figure 1 is a schematic diagram of plasmid pDATH-X (dominant negative, antisense, TET-ON regulating heat shock promoter plasmid) -p53 consisting of four cassettes as follows. (1) TET-ON is amino acid 1-207 of the tetracycline (tet) repressor and the C-terminal last 130 amino acid transcriptional activation domain of VP16 protein of herpes simplex virus (Gossen M., et al. , Science, 268: 1766-1769 (1995)). In cassette 1, the TET-ON sequence is placed under the control of the HSP and tet operator binding sites and pCMV. (2) HSP is a human heat shock 70 gene promoter (Voellmy R., et al.) Linked to a minimal CMV promoter pCMV (Gossen M., et al., Science, 268: 1766-1769 (1995)). , Proc. Natl. Acad. Sci. USA 82: 4949-4953 (1985)) is a heat shock promoter consisting of the heat shock reaction elements (# 260- # 30). In cassette 2, therapeutic gene X is placed under the control of the tetp-pCMV promoter. (3) ptet is the operator O2 of TN10 (Gossen M, and Bujard H., Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)) to which the tet repressor and TET-ON are bound. Is a tet operator consisting of 19 base pairs (bp) reversed sequence of. In cassette 3, antisense TET-ON is placed under the control of the pCMV promoter. (4) Antisense TET-ON is an antisense sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence, including ATG. In cassette 4, the dominant negative TET-ON is placed under the control of the pCMV promoter. The dominant negative TET-ON consists of a tet-repressor excluding the VP16 transcriptional activation domain and is placed under the control of the pCMV promoter. In the absence of heat or light, the TET-ON sequence will be expressed at background level due to leakage of minimal promoter pCMV.

2 is a schematic diagram of a pDATE vector. Plasmid pDATE-X (dominant negative, antisense, TET-ON regulatory EGR promoter expression plasmid) consists of four cassettes as follows: (1) In cassette 1, the TET-ON sequence is EGRp, tetracycline operator binding Placed under control of the site and pCMV. (2) In cassette 2, therapeutic gene X is placed under the control of the tetp-pCMV promoter. (3) In cassette 3, antisense TET-ON is placed under the control of the pCMV promoter. (4) In cassette 4, the dominant negative TET-ON is placed under the control of the pCMV promoter. "TET-ON" is a fusion of the coding sequence for amino acids 1-207 of the tet repressor and the C-terminal 130 amino acid transcriptional activation domain of the VP16 protein of herpes simplex virus. "EGRp" is a radiation induced promoter consisting of fragments -425 to +65 of the EGR-1 promoter containing four copies of the CArG domain. "ptet" is a tet operator consisting of 19 bp reverse sequences of the operator O2 of TN10 to which the tet refresher and TET-ON are bound. "Antisense TET-ON" is a sequence consisting of sequences complementary to the first 80 bases of a TET-ON sequence including ATG. "Dominant negative TET-ON" consists of the coding sequence for amino acids 1-207 of the tet repressor, which is placed under the control of the pCMV promoter. "M" is a chicken lysosomal matrix linkage site that isolates the positional effect of each cassette.

3 is a schematic of a pRIBs-X (radiation-induced breast-specific promoter) expression vector. The pRIBS vector consists of four cassettes. Gene Cassette 1 is the N-terminus (amino acids 1-147) of the yeast GAL4 protein fused to Max's helix-loop-helix-leucine zipper (bHLHLZ) domain (mx, amino acids 8-112) followed by SV40 poly A. ) Is different from the vector described above in that it contains "Gal-DBD-mx", a fusion open reading frame (OFR) encoding a DNA-binding domain (Gal-DBD). Cassette 2 has a minimal CMV promoter (mCMVp), an antisense construct complementary to the Gal-DBD-mx sequence "antisense Gal-DBD-mx", an internal ribosomal introduction site "IRES" and a Gal-DBD- for pGAL binding site. It consists of "Gal-DBD" which competes with mx. Gene Cassette 3 is the nuclear localization signal of the N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by the SV40 giant T antigen, 130 amino acid C-terminal transcriptional activation domain of herpes simplex virus protein VP16, c It consists of "VP16-TA-mc", a fused open reading frame that encodes the bHLHLZ domain of Myc (amino acids 350-439) and SV40 Poly A. The resulting fusion gene VP16-TA-mc is placed under the control of the c-erbB-2 promoter "perbB2" until the first ATG. Gene cassette 4 contains a "GALp" consisting of five copies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via IRES. Gene Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence, including ATG, which is placed under the control of the pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor, which is placed under the control of the pCMV promoter.

4 is a schematic of a pRIPS-GFP (radiation-inducing prostate-specific promoter) expression vector. The pRIPS vector consists of six cassettes. Gene Cassette 1 is the N-terminus (amino acids No. 1 to 147) of the yeast GAL4 protein fused to Max's helix-loop-helix-leucine zipper (bHLHLZ) domain (mx, amino acids 8-112) followed by SV40 poly A. ) Differs from the vector described above only in that it contains "Gal-DBD-mx", a fusion cleavage reading frame encoding a DNA-binding domain (Gal-DBD). Gene Cassette 2 has a minimal CMV promoter (mCMVp), an antisense construct complementary to the Gal-DBD-mx sequence "antisense Gal-DBD-mx", an internal ribosomal introduction site "IRES" and a Gal-DBD for pGAL binding site It consists of "Gal-DBD" which competes with -mx. Gene Cassette 3 is the nuclear localization signal of the N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by the SV40 giant T antigen, 130 amino acid C-terminal transcriptional activation domain of herpes simplex virus protein VP16, c It consists of "VP16-TA-mc", a fused open reading frame that encodes the bHLHLZ domain of Myc (amino acids No. 350-439) and SV40 Poly A. The resulting fusion gene VP16-TA-mc is placed under the control of the provasin gene promoter “pprovacin” up to the first ATG. Gene cassette 4 contains a "GALp" consisting of five copies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via IRES. Gene Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence, including ATG, which is placed under the control of the pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor, which is placed under the control of the pCMV promoter.

5 is a schematic diagram of the mode of action of pRIBS-GFP.

6 illustrates the leakage of the HSP promoter. FIG. 6 summarizes the results of testing a thermally inducible system containing the hsp70 promoter for expression of therapeutic genes p53 and TNFα. 6A shows plasmid constructs for both genes p53 and TNFα. 6B shows p53 transcriptional activity. To analyze the inducibility of the hsp promoter, a person with homozygous deletion of p53 with plasmid pHSP.3p53CD1 or control pHSP.3 vector with Post-2-CAT (containing CAT coding sequence linked to consensus p53 binding site) Co-transfection of ovarian carcinoma cell line SKOV3. After 36 hours of transfection, the cells are not heated or heat treated. CAT activity is measured after 24 hours. Little or no activity was observed in SKOV3 parental transfected cells (lane 1-untreated cells; lane 2-heated cells). Likewise, no activity was observed when transfected with the pHSP.3 vector alone (lane 3 and lane 4). In the case of pHSP.3p53 plasmid, high levels of CAT activity were observed after 24 hours of heat treatment (lane 6). However, even without heat treatment (lane 5), significant levels of p53 expression were observed (about 25%).

7 illustrates the induction of TNFα by heating or photodynamic therapy (PDT). The coding sequence of TNFα is cloned into plasmid pHSP.3 and transfected SKOV3 cells. Selected from G418 to isolate stable colonies. Cells are not heat treated or heat treated at 45 ° C. After 6 hours of treatment, the level of TNFα in the medium is measured with a Genzyme TNFα ELISA kit. TNFα was induced 4-fold by heat treatment and 3-fold induced and secreted by PDT. However, background expression was significant (27%).

FIG. 8 shows the kinetics of expression of p53 in H358 lung carcinoma cell line by feed-forward reaction, where a, b, c, d and e reached p53 reached 10 hours after feedforward reaction. Level). Six hours after heat shock, transfected cells are treated with different doses of doxycycline. After addition of doxycycline, cells are stained with p53 antibody at various time points. Digital images of 50 immunostained cells during each time point are selected with a Nikon microscope. The amount of protein expressed in each cell is proportional to the staining degree expressed as I = 1 / T, where T is the value of transmitted light / unit area. This graph shows the results of experiments with 0.01-0.1 μg / ml doxycycline.

9 is a schematic of the pHIBS-X (heat-induced breast-specific promoter) expression vector. The pHIBS vector consists of six cassettes. Gene Cassette 1 is the N-terminus (amino acids No. 1 to 147) of the yeast GAL4 protein fused to Max's helix-loop-helix-leucine zipper (bHLHLZ) domain (mx, amino acids 8-112) followed by SV40 poly A. ) Differs from the vectors described above only in that they contain "Gal-DBD-mx", a fusion open reading frame that encodes a DNA-binding domain (Gal-DBD). The resulting fusion gene GAL-DBD-mx is regulated by the heat induced HSP promoter. Gene Cassette 2 has a minimum CMV promoter (mCMVp), an antisense construct complementary to the Gal-DBD-mx sequence "antisense Gal-DBD-mx", an internal ribosomal introduction site "IRES" and a Gal-DBD for pGAL binding site It consists of "Gal-DBD" which competes with -mx. Gene Cassette 3 is the nuclear localization signal of the N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by the SV40 giant T antigen, 130 amino acid C-terminal transcriptional activation domain of herpes simplex virus protein VP16, c It consists of "VP16-TA-mc", a fused open reading frame that encodes the bHLHLZ domain of Myc (amino acids No. 350-439) and SV40 Poly A. The resulting fusion gene VP16-TA-mc is placed under the control of the c-erbB-2 promoter "perbB2" until the first ATG. Gene cassette 4 contains a "GALp" consisting of five copies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via IRES. Gene Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence, including ATG, which is placed under the control of the pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor, which is located under the control of the pCMV promoter.

10 is a schematic of the pHIPs-GFP (heat-induced prostate-specific promoter) expression vector. The pHIPS vector consists of six cassettes. Gene Cassette 1 is the N-terminus (amino acids No. 1 to 147) of the yeast GAL4 protein fused to Max's helix-loop-helix-leucine zipper (bHLHLZ) domain (mx, amino acids 8-112) followed by SV40 poly A. ) Differs from the vectors described above only in that they contain "Gal-DBD-mx", a fusion open reading frame that encodes a DNA-binding domain (Gal-DBD). The resulting fusion gene GAL-DBD-mx is regulated by the heat induced HSP promoter. Gene Cassette 2 has a minimal CMV promoter (mCMVp), an antisense construct complementary to the Gal-DBD-mx sequence "antisense Gal-DBD-mx", an internal ribosomal introduction site "IRES" and a Gal-DBD for pGAL binding site It consists of "Gal-DBD" which competes with -mx. Gene Cassette 3 is the nuclear localization signal of the N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by the SV40 giant T antigen, 130 amino acid C-terminal transcriptional activation domain of herpes simplex virus protein VP16, c It consists of "VP16-TA-mc", a fused open reading frame that encodes the bHLHLZ domain of Myc (amino acids No. 350-439) and SV40 Poly A. The resulting fusion gene VP16-TA-mc is placed under the control of the provacin gene promoter (pprovacin) up to the first ATG. Gene cassette 4 contains a "GALp" consisting of five copies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via IRES. Gene Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence, including ATG, which is placed under the control of the pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor, which is located under the control of the pCMV promoter.

As used herein, the term “heat” means thermal energy generated by any means, including microwaves.

As used herein, the term "light" means light energy having a spectral frequency in the visible as well as in the visible region, including ionizing radiation generated by any means. This includes radiation sources such as radionuclides or linear accelerators that can emit gamma and / or beta particles.

According to the present invention, conventional molecular biology, microbiology and recombinant DNA techniques belonging to the art in the art can be used. These techniques are described in detail in Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982);" DNA Cloning: A Practical Approach, "Volumes I and II (DN Glover ed. 1985). "Oligonucleotide Synthesis" (MJ Gait ed. 1984); "Nucleic Acid Hybridization" [BD Hames & SJ Higgins eds. (1985)]; "Transcription and Translation" [BD Hames & SJ Higgins eds. (1984)]; Animal Cell Cuture ”[RI Freshney, ed. (1986)];“ Immobilized Cells and Enzymes ”[IRL Press, (1986)]; B. Perbal,“ A Practical Guide To Molecular Cloning ”(1984)].

The present invention relates to novel gene therapies for defined regions, such as tumors. For the above-mentioned purposes, expressing structurally active and controllable genes through plasmids containing elements that are activated by heat and / or light and react with the presence and concentration of antibiotics (tetracycline and its derivatives) Mechanisms are provided. In regulating the expression of a gene, heat or light initiates expression but the gene is structurally expressed only in the presence of antibiotics (tetracycline and derivatives thereof). Antibiotic concentrations control the level and duration of gene expression.

In order to limit gene expression to tumor cells, two mechanisms are provided for inhibiting gene expression in normal cells as peripheral targets of heat or radiation. If normal cells not exposed to heat or light accidentally absorbed the plasmid, the expression of the therapeutic gene due to the background activity of the promoter may result in antisense and dominant negative for the heat or light-induced antibiotic-dependent transcriptional activator placed in the plasmid. Inhibited by structural expression of the DNA sequence. When normal cells that have absorbed plasmids are exposed to heat or light, there is an additional mechanism that inhibits the expression of therapeutic genes. This is accomplished by the use of modified “two hybrid” systems in which antibiotic dependent transcriptional activators are under the control of both expression of tissue-specific transcriptional activators and exposure to heat or light. Thus, expression of therapeutic genes is found only in cells that are exposed to heat or light and at the same time express tissue-specific transcription factors.

In one aspect of the invention, a recombinant vector pDATH-X (dominant negative, antisense, TET-ON regulating heat shock promoter plasmid) is provided for reducing background levels of expression. This vector consists of the following cassettes: (a) A fusion of coding sequences for amino acids 1-207 of the tetracycline refresher and the C-terminal last 130 amino acid transcriptional activation domain of VP16 protein of the herpes simplex virus ; (b) a heat shock promoter consisting of the heat shock response elements (# -260-30) of the human heat shock 70 gene promoter linked to the minimum cytomegalovirus promoter pCMV; (c) a tet operator consisting of a 19 bp reverse repeat sequence of operator O2 of TN10 to which the tet refresher and TAKON bind; And (d) an antisense sequence consisting of a sequence complementary to the first 80 bases of the TAKON sequence including ATG.

In another aspect of the invention, a method is provided for achieving sustained expression of a gene under the control of a heat or light inducible promoter, comprising introducing a vector containing the gene into a host organism and applying heat or light energy. . In another aspect of the invention, the host organism is a human.

In another aspect of the invention, a recombinant vector pDATE-X (dominant negative, antisense, TET-ON regulatory EGR promoter expression plasmid) is provided. This vector consists of the following cassettes: (a) In cassette 1, the TET-ON sequence is arranged under the control of EGRp, tetracycline operator binding site and pCMV; (2) in cassette 2, therapeutic gene X is placed under the control of the tetp-pCMV promoter; (3) In cassette 3, antisense TET-ON is placed under the control of the pCMV promoter; (4) In cassette 4, the dominant negative TET-ON is placed under the control of the pCMV promoter.

In another aspect, the present invention provides a recombinant expression vector pRIBs-X (radiation-induced breast-specific promoter). This vector consists of the following cassettes: (a) Cassette 1 is a yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper domain (amino acids 8-112) and subsequently to SV40 poly A of Max Contains the "Gal-DBD-mx" fusion open reading frame encoding the N-terminal (amino acids 1-147) DNA-binding domain (Gal-DBD) -generated fusion gene GAL-DBD-mx Regulated by the sex Egr-1 promoter; (b) Cassette 2 is a minimal CMV promoter, an antisense construct complementary to the Gal-DBD-mx sequence "antisense Gal-DBD-mx", an internal ribosomal introduction site "IRES" and a Gal-DBD- for pGAL binding site It consists of "Gal-DBD" which competes with mx; (c) Cassette 3 is the N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by nuclear localization signal of SV40 large T antigen, 130 amino acids C-terminal transcriptional activation domain of herpes simplex virus protein VP16 consisting of the basic helix-loop-helix-leucine zipper domain of amino acids c-Myc (amino acids 350-439) and the fusion open reading frame "VP16-TA-mc" encoding SV40 poly A-generated fusion gene VP16 -TA-mc is placed under the control of the c-erbB-2 promoter "perbB2" until the first ATG; (d) Cassette 4 contains a "GALp" consisting of five copies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via IRES; (e) Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence including ATG, which antisense TET-ON is placed under the control of the pCMV promoter; (f) Cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor.

Also provided are variants of the vector wherein the perbB2 promoter has been replaced with a whey acidic protein promoter or a stromelysin 3 promoter.

In another aspect, the invention provides a method of treating local and metastatic breast and ovarian cancer, comprising administering to a patient a pRIBs-X expression vector (or variant thereof) containing a cytotoxic gene. Representative cytotoxic genes are tumor necrosis factor alpha.

The present invention also provides a recombinant expression vector pRIPs-X (radiation-inducing prostate-specific promoter). This vector consists of the following cassettes: (a) Cassette 1 is a yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper domain (amino acids 8-112) and subsequently to SV40 poly A of Max Contains the "Gal-DBD-mx" fusion open reading frame that encodes the N-terminal (amino acids 1-147) DNA-binding domain-generated fusion gene GAL-DBD-mx is a radiation-induced Egr-1 promoter Controlled by; (b) Cassette 2 competes with Gal-DBD-mx for a minimal CMV promoter, antisense Gal-DBD-mx, an antisense construct complementary to the Gal-DBD-mx sequence, IRES and pGAL binding sites, an internal ribosomal introduction site Consists of Gal-DBD; (c) Cassette 3 is the N-terminal first 11 amino acids of Gal4 followed by nuclear localization signal of SV40 large T antigen, 130 amino acids C-terminal transcriptional activation domain of herpes simplex virus protein VP16, the basic helix of c-Myc Consists of a loop-helix-leucine zipper domain (amino acids 350-439) and a fusion open reading frame “VP16-TA-mc” encoding SV40 Poly A-generated fusion gene VP16-TA-mc Up to ATG is placed under the control of the probasin gene promoter "pprovacin"; (d) Cassette 4 contains a "GALp" consisting of five copies of the 17-mer DNA-binding site for Gal4. TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via IRES; (e) Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence including ATG, which antisense TET-ON is placed under the control of the pCMV promoter; (f) Cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor. Also provided are variants of the vector wherein the provasin promoter has been replaced with a prostate specific antigen promoter.

In another aspect, the present invention provides a method for treating local and metastatic prostate cancer, comprising administering to a patient a pRIPs-X expression vector (or variant thereof) containing a cytotoxic gene. Representative cytotoxic genes are tumor necrosis factor alpha.

In another aspect, the present invention provides a recombinant expression vector pHIBs-X (heat-induced breast-specific promoter). This vector consists of the following cassettes: (a) Cassette 1 is a yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper domain (amino acids 8-112) and subsequently to SV40 poly A of Max Contains the Gal-DBD-mx fusion open reading frame encoding the N-terminal (amino acids No. 1 to 147) DNA-binding domain-generated fusion gene GAL-DBD-mx is generated by a heat induced heat shock protein promoter. Is regulated; (b) Cassette 2 competes with Gal-DBD-mx for the minimal CMV promoter, the antisense Gal-DBD-mx construct that is complementary to the Gal-DBD-mx sequence, the internal ribosomal transduction site, and the pGAL binding site Consists of a DBD; (c) Cassette 3 is the N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by nuclear localization signal of SV40 large T antigen, 130 amino acids C-terminal transcriptional activation domain of herpes simplex virus protein VP16 consisting of the basic helix-loop-helix-leucine zipper domain of amino acids c-Myc (amino acids 350-439) and the fusion open reading frame "VP16-TA-mc" encoding SV40 poly A-generated fusion gene VP16 -TA-mc is placed under the control of the c-erbB2 gene promoter "perbB2" until the first ATG; (d) Cassette 4 contains GALp consisting of five copies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via an internal ribosomal introduction site; (e) Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence including ATG, which antisense TET-ON is placed under the control of the pCMV promoter; (f) Cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor. Also provided are variants of the vector wherein the perbB2 promoter has been replaced with a whey acidic protein promoter or a stromelysin 3 promoter.

In another aspect, the present invention provides a method for treating local and metastatic breast and ovarian cancer, comprising administering to a patient a pHIBs-X expression vector (or variant thereof) containing a therapeutic gene. Representative therapeutic genes are tumor necrosis factor alpha.

The present invention also provides recombinant pHIPs-X (heat-induced prostate-specific promoter) vectors. This vector consists of the following cassettes: (a) Cassette 1 is a yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper domain (amino acids 8-112) and subsequently to SV40 poly A of Max Contains the Gal-DBD-mx fusion open reading frame encoding the N-terminal (amino acids No. 1 to 147) DNA-binding domain-generated fusion gene GAL-DBD-mx is generated by a heat induced heat shock protein promoter. Is regulated; (b) Cassette 2 competes with Gal-DBD-mx for a minimal CMV promoter (mCMVp), an antisense Gal-DBD-mx construct that is complementary to the Gal-DBD-mx sequence, an internal ribosomal transduction site, and a pGAL binding site Consists of Gal-DBD; (c) Cassette 3 is the N-terminal first 11 amino acids of Gal4 followed by nuclear localization signal of SV40 large T antigen, 130 amino acids C-terminal transcriptional activation domain of herpes simplex virus protein VP16, the basic helix of c-Myc Consists of a loop-helix leucine zipper domain (amino acids 350-439) and a fusion open reading frame “VP16-TA-mc” encoding SV40 polyA—generated fusion gene VP16-TA-mc up to the first ATG Is placed under the control of the probasin gene promoter “pprovacin”; (d) Gene Cassette 4 contains GALp consisting of five copies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via IRES; (e) Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence including ATG, which antisense TET-ON is placed under the control of the pCMV promoter; (f) Cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor. Also provided are variants of the vector wherein the provasin promoter has been replaced with a prostate specific antigen promoter.

In another aspect, the present invention provides a method for treating local and metastatic prostate cancer, comprising administering to a patient a pHIPs-X vector (or variant thereof) containing a therapeutic gene. Representative therapeutic genes are tumor necrosis factor alpha.

It is specifically contemplated that the pharmaceutical compositions of the present invention may be prepared for gene therapy. In this case the composition comprises a vector of the invention and a pharmaceutically acceptable carrier. One of ordinary skill in the art of cancer chemotherapy will readily be able to determine the appropriate dose and route of administration without the heavy burden of experimentation. The gene of interest contained in one of the plasmid vectors of the present invention for gene therapy can be delivered to the target cell via a viral vector or liposome.

The level of average technology common in the field of molecular cancer biology has increased significantly in recent years. One of ordinary skill in the art will be able to readily construct and use plasmids for novel therapies according to the taught gene therapy of the present invention.

The following examples illustrate various aspects of the invention. However, these examples should not be understood as limiting the invention in any way.

Example 1

pDATE vector: structure and behavior

2 is a schematic diagram of a pDATE vector. The pDATE-X plasmid acts through a feedforward reaction to amplify the expression of TET-ON and X. Background expression due to leakage of EGRp in the absence of radiation will lead to the synthesis of TET-ON mRNA. Translation of this mRNA is reduced by the simultaneous expression of antisense TET-ON RNA. In addition, TET-ON protein leaked and decoded all the time is inactive in the absence of tetracycline. The leaked (detoxified) TET-ON protein in the presence of tetracycline is active, but the feedforward response is inhibited by the structurally expressed dominant negative TET-ON protein, competing for the same DNA binding site of the ptet promoter. .

Two chicken lysosomal matrix linkage sites (MARs) are inserted to isolate the positional effect of the cassette (McKnight, RA, et al., Mol. Reprod. & Dev., 44: 179-184 (1996) ). MAR may be unnecessary when antisense and dominant negative TET-ON expression is driven by the minimal CMV promoter, but MAR may be necessary if more potent promoters, such as human β actin promoters, drive their expression.

When cells containing pDATE-X are exposed to radiation, an initially explosive TET-ON transcription occurs, inducing two to four times the synthesis of the TET-ON against a background level of TET-ON in a greater amount than the dominant negative TET-ON. . In the presence of tetracycline this excess of TET-ON protein binds to the tetp promoter, to which the coding sequences of both TET-ON and X are linked, and participates in the feedforward reaction. This reaction is controlled by the level of tetracycline. In this case, the expression of X is increased and the period is extended until the tetracycline is removed, at which point the half-life of the TET-ON protein is restarted for a long time using the tetracycline without further radiation exposure of the feedforward response. Will determine if it can be.

This vector uses a feedforward response to achieve and maintain high levels of inducible gene expression. This feedforward feature overcomes the temporal and fragile nature of inducible promoters. When the feedforward reaction is limited within a few hours, there are many differences in the level of TET-ON achieved in heat treated and unheated cells. Thus, by adding and removing tetracycline selectively to enhance the irradiated cells, the level difference of amplified TET-ON in irradiated and non-irradiated cells can be controlled. However, even though the addition and removal of tetracycline can be precisely controlled in cell culture, in vivo due to heterogeneity of tetracycline levels in tissues and variations in the uptake and removal of tetracycline in vivo due to individual differences It is difficult to do so. Therefore, it is important to minimize leakage through the expression of TET-ON with antisense and dominant negative cassettes so that the feedforward response does not significantly amplify expression of TET-ON in unirradiated cells.

If desired, the action of this vector can be further refined by replacing the pCMV minimal promoter with a much more potent promoter such as the human β actin promoter to drive expression of antisense and dominant negative TET-ON. It is also possible to increase the number of copies of antisense and dominant negative coding sequences.

For in vivo induction of TET-ON expression, oxytetracycline is used because of its short in vivo half-life. In humans, the highest plasma concentration of oxytetracycline is reached at 2-4 hours after a single oral administration (Goodman & Gilman's The Pharmaceutical Basis of Therapeutics). Thus, the level of TET-ON expression can be monitored as a function of oxytetracycline concentration. Oxytetracycline acts for a short period of time with an in vivo half-life of just 9 hours (half-life of doxycycline is 18 hours). At 24 hours, oxytetracycline levels decrease to less than 25% of the dose (about 10 to 30% never). Not absorbed and secreted in the active form).

Example 2

pDATH vectors: structures and how they work

1 is a schematic diagram of a pDATH-X vector. This vector works in the same way as the pDATE-X vector except that the Egr-1 promoter is replaced by the HSP promoter and uses heat instead of photo / ionizing radiation.

Example 3

Demonstration of the concept of amplifiable and sustained expression of TET-ON and p53 as a feedforward inducible promoter

Two plasmids are constructed to demonstrate the concept of heat induced tetracycline feedforward amplification of gene expression. The "ptet-splice p53wt" was obtained by acloning the wild type p53 cDNA into the ptet-insertion vector (Gibco BRL) such that p53 is placed under the control of the tetp promoter consisting of a regulatory sequence from the upstream tetracycline-resistant operon of the minimum hCMV promoter. To construct. The plasmid "HSP-tetp-TET-ON" was constructed by replacing the CMV promoter in ptet-on (Clontech) with a 300 bp human heat shock protein promoter and tetp promoter.

Non-small cell lung carcinoma cell line H358 with homozygous deletion of p53 is grown in RPMI + 10% fetal bovine serum. Exponentially grown 10 ⁷ cells were electroporated with 1180 μF and 240 V BRL cell-porers in 0.08 ml RPMI + 6 mM glucose simultaneously to 50 μg of “ptet-splice p53wt” and 10 μg of “HSP-tetp-TET-ON” Transfect. Transfected cells are 25% confluence plated for 36 hours, then half of them are heat treated at 45 ° C. for 20 minutes. Six hours after heat shock, cells are treated with different doses of doxycycline. At several time points after the addition of doxycycline, cells are immunohistochemically stained with the monoclonal p53 antibody DO-1 (Santa Cruz Biologicals) using the immunoperoxidase cell staining kit (Vector) and diaminebenzidine (DAB). At each time point, a Nikon microscope is used to select digital images of 50 immunostained cells. The amount of protein expressed in each cell is proportional to the staining degree expressed as I = 1 / T, where T is the value of transmitted light / unit area. Experimental results using 0.01 to 0.1 μg / ml doxycycline are shown in FIG. 8.

After 6 hours of heat treatment (when the level of induced TET-ON peaked), 0.1 μg / ml of doxycycline was added, and at least 12 times amplification of p53 was achieved within 10 hours (curve a in FIG. 8). And b). During this time doxycycline also initiated a feedforward response in unheated cells as indicated by significant levels of TET-ON. However, since amplification began at lower levels, the amplified levels of TET-ON at 10 hours remained only at low levels (curves c and d in FIG. 8).

The level of p53 induced in the feedforward system can be controlled by adding and removing tetracycline from other regimes. At the point when TET-ON (eg, level [c] in FIG. 8) is removed from the unannealed cell and then drops back to background level [e], the level [a] of TET-ON in the heat treated cells is in similar proportion. (same as [c]-[e]). However, since these levels ([a]-[c]-[e]) are much higher than in untreated cells [e], the addition of tetracycline ([a]-[c]- From [e]) the feedforward reaction will be resumed for the heat treated cells. In this case, the level of background p53 in unannealed cells may be maintained at or below the low level ([c]) reached at 10 hours, while the p53 level in heat treated cells may continue to increase. will be. Thus, TNFα and p53 expression directly driven by HSP is transient, while expression driven by the feedforward system continues as long as tetracycline can be used. It is important to know the half-life of TET-ON in tumor cells because the regimen of in vivo tetracycline addition is determined by the rate of tetracycline extinction in vivo.

In vivo, the pharmacodynamics of tetracycline is heterogeneous for different tissues. Preferred concentrations of tetracycline in certain tissues will lead to higher background expression of TET-ON in some tissues. For example, in humans, 10 to 35% of oxytetracycline is removed through the kidneys and a significant amount thereof is secreted in the active form. Thus, to prevent false amplification in unintended tissues, it is desirable to minimize the level of background expression at initiation. pDATE and pDATH inducible systems use structurally expressed antisense TET-ONs to suppress background levels of TET-ON translation and to inhibit expression using dominant negative TET-ONs that compete with leak-expressed TET-ONs all the time . The timing of tetracycline addition with inhibited background expression is only affected by the desired expression level and duration of the therapeutic gene and not by the desire to suppress the level of background expression in normal unirradiated cells.

Example 4

Decrease in background expression levels

When using a 300bp HSP promoter, the background expression level in the absence of heat or light is about 25% to the observed level of heat or light. To reduce this, connect the HSP to -80 to +30 of the minimum pCMV promoter. The pCMV promoter is preferred because of its low background level expression. In addition, the pCMV promoter significantly amplifies the expression of therapeutic genes regardless of the weaker HSP promoter used to initiate the reaction with heat or light.

To further solve the problem of background expression, two cassettes are introduced into plasmid pDATH. Antisense against TAKON is placed under the control of the pCMV promoter. Structurally produced antisense binds to any TAKON sense mRNA from background transcription and inhibits this mRNA from being translated. A dominant negative TAKON with no transcription activation domain but with a DNA binding site further blocks background transcription in cassette 4, which is placed under the control of pCMV. This induces background transcription that drives the production of TAKON and dominant negative TAKON, allowing them to compete for the ptet binding site.

Example 5

monitoring of p53 expression levels

To assure adequate levels of antisense TAKON RNA and dominant negative TAKON protein production, p53 expression levels are monitored to determine the copy number and intensity of the promoter required to reduce background. First, cell lines containing pDATH are isolated in the absence of tetracycline. The level of p53 or cotransfected ptet-EGFP is then monitored to determine the number of copies of dominant negative TAKON and antisense TAKON that need to be inserted into pDATHso to reduce background expression.

Sealing example 6

Expression vector pRIBs for the treatment of local and metastatic breast and ovarian cancer

As mentioned above, genes placed under the control of a promoter, such as a radiation-inducible promoter of the Egr-1 gene, are often expressed only temporarily and at low levels. This makes them unsuitable for use in cancer therapy. To solve this problem, the expression vector pRIBs-X (radiation-induced breast-specific promoter) is designed.

The gene expression level is optimized using a feedforward reaction with a tetracycline-dependent transcriptional activator Tet-On placed under the control of a tetracycline promoter (tetp) followed by a GAL-4 promoter (pGAL). Transitory transcription initiated in pGAL induces low levels of Tet-On synthesis and binds tetp in the presence of tetracycline. Tet-On then feeds its own expression through a feedforward response and of the therapeutic gene linked to Tet-On. Amplify expression. Expression of therapeutic genes is regulated by six gene cassettes in the pRIBs vector (FIG. 3). In cassette 1, the fusion gene GAL-DBD-mx (the HLH-LZ domain of max fused to the DNA-binding domain of GAL-4) is regulated by EGRp. Background expression of GAL-DBD-mx is inhibited by antisense GAL-DBD-mx and dominant negative GAL-DBD structurally expressed in cassette 2. In cassette 3, the transcriptional activation domain of herpes simplex virus protein VP16 is fused to the HLH-LZ domain of c-Myc. The resulting fusion gene VF16-TA-mc is placed under the control of the c-erbB-2 promoter and is expressed in breast tumor cells overexpressing c-erbB-2. The GAL-DBD-mx fusion protein binds to and activates transcription from the pGAL promoter (cassette 4) by recruiting the VP16-TA-mc protein.

In unirradiated cells, translation of background GAL-DBD-mx mRNA is reduced and dominant negative GAL-DBD (no mx) competitively occupies GALp in cassette 4 to block Tet-On expression. Upon irradiation, GAL-DBD-mx is transiently induced three to four fold and temporarily overcomes inhibition by cassette 2. GAL-DBD-mx recruits VP16-TA-mc (a fusion gene of the VP16 transcriptional activation domain under the control of the c-erbB-2 promoter and the leucine zipper of myc) to GALp and activates low-level Tet-On transcription to feed Initiate a forward reaction.

For example, in therapeutic approaches with pRIBs-TNFα, it can be delivered systemically to tumors and normal cells via liposome complexes or as recombinant viruses. Without radiation and tetracycline no TNFα is expressed. The oxytetracycline is then administered systemically, then X-rays are irradiated to known metastatic tumor sites. As a result, TNFα expression is induced at the tumor site by X-ray and amplified and maintained by oxytetracycline. Although not all tumor cells are able to absorb pRIBs-TNFα, tumor cells around the absorbed cells are locally exposed to very high secreted TNFα. The design of pRIBs-TNFα provides TNFα expression only to breast tumor cells and not to irradiated normal cells that were in the X-ray pathway. As such, systemic toxicity is limited to low levels of TNFα, somehow spreading from tumor cells. Other therapeutic genes X other than or instead of TNFα may be used with the pRIBS vector.

The structure of pRIBs-GFP-1 is shown in FIG. 3 and the mode of action is summarized in FIG. 5. Background GAL-DBD-mx expression and action in unirradiated cells is inhibited in two ways by cassette 2. Antisense against GAL-DBD-mx inhibits the translation of background GAL-DBD-mx mRNA, while GAL-DBD protein acts as a dominant negative inhibitor by competing with GAL-DBD-mx for the pGAL promoter. In unirradiated cells, GAL-DBD-mx expression is transiently induced three to four fold and overcomes inhibition by cassette 2. GAL-DBD-mx recruits VP-16-TA-mc (fusion gene of VP16 transcriptional activation domain under control of the c-erbB-2 promoter and leucine zipper of Myc) to GALp and transient expression of the transcriptional activator TET-ON Activate. In the presence of tetracycline, Tet-ON is activated and combined with the tetp promoter (Gossen, M., et al., Science, 268: 1766-1769 (1995)) to transactivate the tetp promoter and to a feedforward reaction. Amplify its own level and GFP. Background expression of TET-ON and GFP does not occur in the absence of irradiation or tetracycline.

Example 7

Formation of cell lines and xenografts stably expressing pRIBs-GFP

Two pRIBs-GFP plasmids, pRIBs-GFP-1 and pRIBs-GFP-4, with one and four copies of an antisense and dominant negative gene cassette, respectively, were constructed and fibrosarcoma cell line HTB152 and breast tumors for in vitro analysis. Cell lines SK-BR-3 and MDAMB231 are stably transfected. All three human cell lines form incompletely differentiated tumors, but only SK-BR-3 expresses high levels of c-erbB-2. In fact, anti-erbB-2 intracellular single-chain antibodies that down-regulate cell surface erbB-2 induce apoptosis only in SK-BR-3 and not MDA-MB-231 (see Chumakov). AM, et al., Oncogene 8: 3005-3011 (1993)).

Thus, pRIBs-GFP-1 and -4 plasmids are used as models for optimizing test conditions for the treatment of metastatic breast tumor xenografts in nude mice with cytotoxic genes. Cytotoxic genes linked to EGRp are only derived from irradiated cells, thus eliminating toxicity to unirradiated cells. However, it is important to inhibit the expression of cytotoxic genes in normal cells in the X-ray pathway. Three cell lines that differ in c-erbB-2 expression are restricted to living breast tumor cells whose expression is limited to irradiated and living metastatic tumor cells when VP16-TA-mc expression is regulated by tissue- or tumor-specific promoters. It is shown that expression does not occur in the normal cells irradiated of the organ.

The pRIBs-GFP plasmid is constructed as shown in FIG. 3. GAL-DBA-mx and VP16-TA-mc are modified from two mammalian hybrid systems (Fearon, ER, et al., Proc. Natl. Acad. Sci. USA. 89: 7958-7962 (1992)). It is. Two plasmids pRIBs-GFP-1 and pRIBs-GFP-4 with 1 and 4 copies of antisense and dominant negative GAL-DBD driven by a minimum CMV promoter, respectively, are tested.

All three cells are cotransfected with pRIBs-GFP and SVneo plasmids. Cells stably expressing pRIBs-GFP-1 and pRIBs-GFP-4 are selected and isolated from G418. For in vivo analysis, 5 x 10 ⁶ cells for each cell line stably expressing the pRIBs-GFP plasmid are implanted into the flanks of SCID mice (4 per group) and allowed to grow to 0.5 cm in diameter. Expression of GFP in vitro and in xenografts in the absence of radiation or oxytetracycline is analyzed by extracting proteins from comminuted tumors in EBC buffer and quantifying the amount of protein by RIA.

Cells were irradiated at 0-4 Gy with a Varian Clinac 2000 X-ray generator, followed by administration of 0-2 μg / ml of oxytetracycline to determine in vitro inducible levels of GFP by Western analysis. And quantified by RIA. The data using HSPp shows that the feedforward response is very efficient and 0.01 μg / ml is sufficient to increase fold p53 expression 9 times in 10 hours. Tumors are exposed to 0-4 Gy / X-rays for in vivo analysis. After 6 hours of irradiation, 0-15 μg / g of oxytetracycline is injected intraperitoneally. The tumor mass is removed at 3 hour intervals (for 24 hours) after injection and the amount of TET-ON and GFP is measured by comparison with the total amount of actin protein. To obtain higher or lower levels of GFP, the experiment is repeated with modification of TET-ON levels by adjusting the dose of oxytetracycline. The rate of removal of oxytetracycline by secretion is monitored by analyzing plasma concentrations at three hour intervals.

Example 8

Targeting Breast Metastases to WAPp or ST3p

Since human cancers that overexpress c-erbB-2 have an incomplete prognosis, the c-erbB-2 promoter has been selected to confirm the pRIB-X concept. However, it is unlikely that one particular promoter will raise the issue of treating other breast tumors. Thus, the whey acidic protein promoter WAPp (McKnight, RA, et al., Mol. Reprod. & Dev., 44: 179-184 (1996)) or stromelysin 3 promoter ST3p (Ref. Ahmad, A) , et al., Int. J. of Cancer, 73: 290-296 (1997)). It is also important to target GAL-DBD-mx expression for metastatic breast tumors. WAPp targets expression into breast epithelial cells, while ST3p targets expression of matrix-metalloproteinase-secreting stromal cells adjacent to the tumor.

pRIBs are reconstructed by replacing the c-erbB-2 promoter with WAPp or ST3p. Breast and other tumor cell lines are screened for high and low expression of WAP and ST3. Cell lines that differ in the expression of WAP and / or ST3 are used to test the expression of GFP.

The WAP promoter appears to be very specific for lactating breast epithelial cells of transformed animals (Tzeng YJ., Et al., Oncogene 16 (16): 2103-2114 (1998)) and the stromelysin 3 promoter ST3p appears to be expressed only in stromal fibroblasts adjacent to cancer cells. Evidence suggests that production of matrix cells (including ST3) of matrix-metalloproteinases involved in the tumor metastasis process is stimulated by cancer cells. Therefore, targeting VP16-TA-mc to stromal cells allows for expression and release of therapeutic gene products in the vicinity of metastatic tumor cells. It should be appreciated that further treatment specificities are obtained by delivering pRIBs-x to c-erbB2 using liposomes coated with antibodies.

Example 9

Expression vector pRIPs for the treatment of local and metastatic prostate cancer

As mentioned above, genes placed under the control of a promoter, such as the radiation-inducible promoter of the Egr-1 gene, are often transiently expressed only at low levels. This makes these genes unsuitable for use in cancer therapy. To solve this problem, the expression vector pRIPs-X (radiation-inducing prostate-specific promoter) is designed.

The pRIPS vector consists of six cassettes. Gene Cassette 1 is the N-terminus (amino acids No. 1 to 147) of the yeast GAL4 protein fused to Max's helix-loop-helix-leucine zipper (bHLHLZ) domain (mx, amino acids 8-112) followed by SV40 poly A. ) Differs from the aforementioned vectors only in that they contain "Gal-DBD-mx", a fusion OFR encoding a DNA-binding domain. Gene Cassette 2 has a minimal CMV promoter (mCMVp), an antisense construct complementary to the Gal-DBD-mx sequence "antisense Gal-DBD-mx", an internal ribosomal introduction site "IRES" and a Gal-DBD for pGAL binding site It consists of "Gal-DBD" which competes with -mx. Gene Cassette 3 is the nuclear localization signal of the N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by the SV40 giant T antigen, 130 amino acid C-terminal transcriptional activation domain of herpes simplex virus protein VP16, c It consists of the bHLHLZ domain of Myc (amino acids 350-439) and "VP16-TA-mc", a fusion ORF encoding SV40 poly A. The resulting fusion gene VP16-TA-mc is placed under the control of the provasin gene promoter “pprovacin” up to the first ATG. Gene cassette 4 contains a "GALp" consisting of five copies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via IRES. Gene Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence, including ATG, which is placed under the control of the pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor, which is located under the control of the pCMV promoter. In other variants of pRIPs-X pprovacin is replaced with PSA, a promoter region of prostate specific antigen or other prostate-specific genes.

Example 10

Expression Vector pHIBs-X for Treatment of Local and Metastatic Breast Cancer and Ovarian Cancer

The expression vector pHIBS-X is designed to consist of six cassettes. Gene Cassette 1 is the N-terminus (amino acids No. 1 to 147) of the yeast GAL4 protein fused to Max's helix-loop-helix-leucine zipper (bHLHLZ) domain (mx, amino acids 8-112) followed by SV40 poly A. ) Differs from the aforementioned vectors only in that they contain "Gal-DBD-mx", a fusion OFR encoding a DNA-binding domain. The resulting fusion gene GAL-DBD-mx is regulated by the heat induced HSP promoter. Gene Cassette 2 has a minimal CMV promoter (mCMVp), an antisense construct complementary to the Gal-DBD-mx sequence "antisense Gal-DBD-mx", an internal ribosomal introduction site "IRES" and a Gal-DBD for pGAL binding site It consists of "Gal-DBD" which competes with -mx. Gene Cassette 3 is the nuclear localization signal of the N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by the SV40 giant T antigen, 130 amino acid C-terminal transcriptional activation domain of herpes simplex virus protein VP16, c It consists of the bHLHLZ domain of Myc (amino acids 350-439) and "VP16-TA-mc", a fusion ORF encoding SV40 poly A. The resulting fusion gene VP16-TA-mc is placed under the control of the c-erbB-2 promoter "perbB2" until the first ATG. Gene cassette 4 contains a "GALp" consisting of five copies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via IRES. Gene Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence, including ATG, which is placed under the control of the pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor, which is placed under the control of the pCMV promoter.

The pHIBs-X expression vector is identical to the plasmid pRIBs-X plasmid except gene cassette 1 in which the Egr-1 promoter in pRIBs-X was replaced with the HSP 70 promoter. pHIBs-X specifically targets local and metastatic breast tumors and ovarian tumors when exposed to heat.

Example 11

Expression Vector pHIPs-X for Treatment of Local and Metastatic Prostate Cancer

10 illustrates the structure of a pHIPs-GFP (heat-induced prostate-specific promoter) expression vector. This vector consists of six cassettes. Gene Cassette 1 is the N-terminus (amino acids No. 1 to 147) of the yeast GAL4 protein fused to Max's helix-loop-helix-leucine zipper (bHLHLZ) domain (mx, amino acids 8-112) followed by SV40 poly A. ) Differs from the aforementioned vector only in that it contains "Gal-DBD-mx", a fusion ORF that encodes a DNA-binding domain. The resulting fusion gene GAL-DBD-mx is regulated by the heat induced HSP promoter. Gene Cassette 2 has a minimal CMV promoter (mCMVp), an antisense construct complementary to the Gal-DBD-mx sequence "antisense Gal-DBD-mx", an internal ribosomal introduction site "IRES" and a Gal-DBD for pGAL binding site It consists of "Gal-DBD" which competes with -mx. Gene Cassette 3 is the nuclear localization signal of the N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by the SV40 giant T antigen, 130 amino acid C-terminal transcriptional activation domain of herpes simplex virus protein VP16, c It consists of the bHLHLZ domain of Myc (amino acids 350-439) and "VP16-TA-mc", a fusion ORF encoding SV40 poly A. The resulting fusion gene VP16-TA-mc is placed under the control of the provacin gene promoter (pprovacin) up to the first ATG. Gene cassette 4 contains a "GALp" consisting of five copies of the 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter and therapeutic gene X is linked to TET-ON via IRES. Gene Cassette 5 contains antisense TET-ON, a sequence consisting of sequences complementary to the first 80 bases of the TET-ON sequence, including ATG, which is placed under the control of the pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207 of the tet repressor, which is located under the control of the pCMV promoter.

The pHIBs-X expression vector is identical to the plasmid pRIBs-X plasmid except gene cassette 1 in which the Egr-1 promoter in pRIBs-X and pRIPs-X was replaced with the HSP 70 promoter. pHIBs-X specifically targets local and metastatic prostate tumors when exposed to heat.

All patents and publications mentioned in the specification are indicative of the level of skill in the art to which this invention pertains. These patents and publications are hereby incorporated by reference to the same extent when the individual publications are specifically and individually indicated to be incorporated by reference.

Those skilled in the art will readily appreciate that the present invention can be modified to carry out the objects referred to herein and to obtain results and advantages. This example, along with the methods, procedures, treatments, molecules and specific compounds described herein, illustrate preferred embodiments and should not be construed as limiting the scope of the invention. Skilled artisans may modify and otherwise use the invention, but such modifications and other uses are within the scope of the invention as set forth in the claims.

Claims (28)

(a) a fusion cassette of amino acid 1-207 of the tetracycline refresher and the coding sequence for the C-terminal last 130 amino acid transcriptional activation domain of the VP16 protein of herpes simplex virus; (b) a heat shock promoter cassette consisting of a heat shock response element (# -260-30) of the human heat shock 70 gene promoter linked to the promoter of minimal cytomegalovirus pCMV; (c) a tet operator cassette consisting of a 19 bp reverse repeat sequence of operator O2 of TN10 to which the tet refresher and TAKON are bound; And (d) Recombinant vector pDATH-X (dominant negative, antisense, TET-ON regulated heat shock promoter plasmid) comprising a cassette of antisense sequences consisting of sequences complementary to the first 80 bases of the TAKON sequence including ATG. Introducing a vector containing the gene into the host organism; And A method of achieving sustained expression of a gene under control of a heat or light inducible promoter, comprising applying heat or light energy. The method of claim 2, wherein the host organism is human. (a) Cassette 1 comprising an EGRp, tetracycline operator binding site and a TET-ON sequence under control of pCMV; (b) cassette 2 comprising therapeutic gene X under the control of the tetp-pCMV promoter; (c) cassette 3 comprising antisense TET-ON under control of the pCMV promoter; And (d) Recombinant vector pDATE-X (dominant negative, antisense, TET-ON regulatory EGR promoter expression plasmid) comprising cassette 4 comprising dominant negative TET-ON under control of the pCMV promoter. (a) the N-terminal (amino acids 1-147) DNA- of yeast GAL4 protein fused to Max's basic helix-loop-helix-leucine zipper (bHLHLZ) domain (amino acids 8-112) followed by SV40 poly A Cassette 1 comprising “Gal-DBD-mx”, wherein the fusion open reading frame encodes a binding domain (Gal-DBD) and the resulting fusion gene GAL-DBD-mx is regulated by a radiation induced Egr-1 promoter; (b) competes with Gal-DBD-mx for a minimal CMV promoter, an antisense construct complementary to the Gal-DBD-mx sequence, "antisense Gal-DBD-mx", an internal ribosomal transduction site (IRES) and a pGAL binding site Cassette 2 comprising “Gal-DBD”; (c) N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by nuclear localization signal of SV40 large T antigen, 130 amino acids C-terminal transcriptional activation domain of herpes simplex virus protein VP16, c- Myc's primary helix-loop-helix-leucine zipper domain (amino acids 350-439) and the fusion ORF encoding SV40 poly A and the resulting fusion gene VP16-TA-mc are the c-erbB2 promoter "perbB2" until the first ATG. Cassette 3 comprising “VP16-TA-mc”, under the control of; (d) Cassette 4 containing "Galp", five copies of the 17-mer DNA-binding site for Gal4, wherein the TET-ON sequence is under the control of the GALp-ptet promoter and therapeutic gene X is via IRES Connected to TET-ON); (e) cassette 5 comprising an antisense TET-ON sequence consisting of a sequence complementary to the first 80 bases of the TET-ON sequence comprising ATG and under the control of the pCMV promoter; And (f) Recombinant vector pRIBs-X (radiation-induced breast-specific promoter) expression vector comprising cassette 6 comprising a dominant negative TET-ON consisting of coding sequences for amino acids 1-207. The recombinant vector of claim 5, wherein the perbB2 promoter of cassette 3 is replaced with a whey acidic protein promoter. The recombinant vector of claim 5, wherein the perbB2 promoter of cassette 3 is replaced with a stromelysin 3 promoter. The recombinant vector of claim 5, wherein gene X is a gene encoding tumor necrosis factor alpha. A method of treating local and metastatic breast cancer and ovarian cancer, comprising administering the expression vector of claim 5 to an individual in need thereof. A method of treating local and metastatic breast cancer and ovarian cancer, comprising administering the expression vector of claim 6 to an individual in need thereof. A method of treating local and metastatic breast cancer and ovarian cancer, comprising administering the expression vector of claim 7 to an individual in need thereof. (a) the N-terminal (amino acids 1-147) DNA-binding domain of the yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper domain (amino acids 8-112) followed by SV40 poly A of Max; Cassette 1, which encodes a fusion open reading frame and which comprises “Gal-DBD-mx” controlled by a radiation induced Egr-1 promoter; (b) a minimal CMV promoter, antisense Gal-DBD-mx, an antisense construct complementary to the Gal-DBD-mx sequence, Gal-DBD-competition with Gal-DBD-mx for the IRES and pGAL binding sites, an internal ribosomal introduction site Cassette 2 comprising; (c) N-terminal first 11 amino acids of Gal4 followed by nuclear localization signal of SV40 giant T antigen, 130 amino acids C-terminal transcriptional activation domain of herpes simplex virus protein VP16, basic helix-loop- of c-Myc A fusion open reading frame that encodes the helix leucine zipper domain (amino acids 350-439) and SV40 poly A and the resulting fusion gene VP16-TA-mc is under the control of the provasin gene promoter “pprovacin” up to the first ATG. , Cassette 3 comprising “VP16-TA-mc”; (d) Cassette 4 comprising GALp, five copies of the 17-mer DNA-binding site for Gal4, wherein the TET-ON sequence is under the control of the GALp-ptet promoter and the therapeutic gene X is via an internal ribosome introduction site Is connected to TET-ON); (e) cassette 5 comprising an antisense TET-ON sequence consisting of a sequence complementary to the first 80 bases of the TET-ON sequence comprising ATG and under the control of the pCMV promoter; And (f) Recombinant pRIPs-X (radiation-induced prostate-specific promoter) expression vector comprising cassette 6 comprising a dominant negative TET-ON consisting of coding sequences for amino acids 1-207. The recombinant vector of claim 12, wherein the provasin promoter of cassette 3 is replaced with a prostate specific antigen promoter. 13. The recombinant vector of claim 12, wherein gene X is tumor necrosis factor alpha. A method of treating local and metastatic prostate cancer, comprising administering the expression vector of claim 12 to an individual in need thereof. A method of treating local and metastatic prostate cancer, comprising administering the expression vector of claim 13 to an individual in need thereof. (a) the N-terminal (amino acids 1-147) DNA-binding domain of the yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper domain (amino acids 8-112) followed by SV40 poly A of Max; Cassette 1 comprising Gal-DBD-mx, which is a fused open reading frame that encodes and is regulated by a heat induced heat shock protein promoter; (b) a minimal CMV promoter, a construct complementary to the Gal-DBD-mx sequence, an antisense Gal-DBD-mx, a gal-DBD that competes with Gal-DBD-mx for the internal ribosomal introduction site and the pGAL binding site Cassette 2; (c) N-terminal first 11 amino acids of Gal4 (amino acids 1-147) followed by nuclear localization signal of SV40 large T antigen, 130 amino acids C-terminal transcriptional activation domain of herpes simplex virus protein VP16, c- A fusion open reading frame encoding Myc's primary helix-loop-helix leucine zipper domain (amino acids 350-439) and SV40 poly A and the resulting-fusion gene VP16-TA-mc is a c-erbB2 gene promoter up to the first ATG cassette 3 comprising “VP16-TA-mc”, under the control of “perbB2”; (d) Cassette 4 comprising GALp, five copies of the 17-mer DNA-binding site for Gal4, wherein the TET-ON sequence is under the control of the GALp-ptet promoter and the therapeutic gene X is via an internal ribosome introduction site Is connected to TET-ON); (e) cassette 5 comprising an antisense TET-ON sequence consisting of a sequence complementary to the first 80 bases of the TET-ON sequence comprising ATG and under the control of the pCMV promoter; And (f) Recombinant expression vector pHIBs-X (heat-induced breast-specific promoter) comprising cassette 6 comprising a dominant negative TET-ON consisting of coding sequences for amino acids 1-207. 18. The recombinant vector of claim 17, wherein the perbB2 promoter of cassette 3 is replaced with a whey acidic protein promoter. The recombinant vector of claim 17, wherein the perbB2 promoter of cassette 3 is replaced with a stromelysine 3 promoter. The method of claim 17, wherein the therapeutic gene is tumor necrosis factor alpha. A method of treating local and metastatic breast cancer and ovarian cancer, comprising administering the expression vector of claim 17 to an individual in need thereof. A method of treating local and metastatic breast cancer and ovarian cancer, comprising administering the expression vector of claim 18 to an individual in need thereof. A method of treating local and metastatic breast cancer and ovarian cancer, comprising administering the expression vector of claim 19 to an individual in need thereof. (a) the N-terminal (amino acids 1-147) DNA-binding domain of the yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper domain (amino acids 8-112) followed by SV40 poly A of Max; Cassette 1 comprising Gal-DBD-mx, which encodes a fusion open reading frame and the resulting fusion gene GAL-DBD-mx is regulated by a heat induced heat shock protein promoter; (b) Gal-DBD competing with Gal-DBD-mx for the minimal CMV promoter (mCMVp), an antisense Gal-DBD-mx construct that is complementary to the Gal-DBD-mx sequence, an internal ribosomal transduction site, and a pGAL binding site Cassette 2 comprising; (c) N-terminal first 11 amino acids of Gal4 followed by nuclear localization signal of SV40 giant T antigen, 130 amino acids C-terminal transcriptional activation domain of herpes simplex virus protein VP16, basic helix-loop- of c-Myc A fusion open reading frame encoding the helix leucine zipper domain (amino acids 350-439) and SV40 poly A and the resulting fusion gene VP16-TA-mc under the control of the provasin gene promoter “pprovacin” up to the first ATG. , Cassette 3 comprising “VP16-TA-mc”; (d) Cassette 4 comprising GALp, five copies of the 17-mer DNA-binding site for Gal4, wherein the TET-ON sequence is under the control of the GALp-ptet promoter and the therapeutic gene X is via an internal ribosome introduction site Is connected to TET-ON); (e) cassette 5 comprising an antisense TET-ON sequence consisting of a sequence complementary to the first 80 bases of the TET-ON sequence comprising ATG and under the control of the pCMV promoter; And (f) Recombinant expression vector pHIPs-X (heat-induced prostate-specific promoter) comprising cassette 6 comprising a dominant negative TET-ON consisting of coding sequences for amino acids 1-207. The recombinant vector of claim 24, wherein the provasin promoter is replaced with a prostate-specific antigen promoter. The recombinant vector of claim 24, wherein the therapeutic gene is tumor necrosis alpha. A method of treating local and metastatic prostate cancer, comprising administering the expression vector of claim 24 to an individual in need thereof. A method of treating local and metastatic prostate cancer, comprising administering the expression vector of claim 25 to an individual in need thereof.