JP2002523032A - Methods for enhancing and limiting gene expression - Google Patents

Abstract

  (57) [Summary] The present invention provides a new method of gene therapy for a limited area such as a tumor. These methods comprise the steps of (a) placing the gene in a plasmid vector that is activated by a heat or light inducible promoter, and (b) including a tetracycline responsive fusion protein that functions as a transcriptional activator. Modifying the vector and (c) modifying the vector by including a DNA sequence that reduces or eliminates expression of the gene in normal peripheral cells. A series of vectors for persistent and controllable expression are also provided. New vectors for gene therapy of localized and metastatic breast, ovarian, and prostate cancers are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to the field of gene therapy for cancer. More specifically,
The present invention describes a method of controlling a therapeutically useful gene product via an inducible promoter. The present invention provides a method wherein the triggered gene expression in the intended cellular target is enhanced and extended in a manner that can be spatially and temporarily regulated by a heat or light inducible promoter. Further, the present invention provides a method for reducing or eliminating background gene expression in non-target cells.

[0002] One of the major obstacles to the success of chemotherapy and radiation therapy for cancer is the difficulty in achieving tumor-specific cell killing. Radiation and chemotherapeutic agents cannot discriminate between tumor cells and normal cells, which limits the applicable dose. as a result,
Disease recurrence due to remaining surviving tumor cells is often observed.

[0003] Performing gene therapy in cancer therapy involves many of the same difficulties as chemotherapy and radiation therapy. Current gene therapy strategies are based on the premise that therapeutic genes cannot be individually delivered to intended target cells. This can lead to toxicity in unintended cells. For example, manipulation of p53 gene silencing arrests the growth of both tumor and normal cells, and intravenous administration of tumor necrosis factor alpha (TNFα) results in systemic toxicity with clinical signs such as fever and hypertension. Is shown.

[0004] Various attempts have been made to overcome these problems. These attempts include the use of tissue-specific receptors to direct genes to desired tissues (Kasahara, N., et al., Science, 266: 1373-1376 (1994)).
), The use of tissue-specific promoters to limit gene expression to particular tissues (eg, the use of prostate-specific antigen promoter), the use of heat (Voelmy et al.
, et al.), Proceeding National Academy Sciences USA (Proc.
Natl. Acad. Sci. USA), 82: 4949-4953 (1995)), or ionizing radiation-induced enhancers and promoters (trains) for enhancing the expression of therapeutic genes in a temporally and spatially controlled manner. (Trainman, RH, et al.), Cells (
Cell) 46: 567-574 (1986); Probes, R, et al., Proceeding National Academy Sciences USA (Proc. Natl. Acad. Sci. USA) 85
, 7206-7210 (1988)). This heat-induced heat shock protein (HSP) promoter has been used to direct the expression of genes such as the cytokine IL-2.

[0005] Weichselbaum and colleagues report an early growth reaction (Egr-
1) Radiation-induced response of the gene promoter was first discovered. Therefore, they attempt to use this promoter to limit the expression of cytotoxic genes such as TNFα to tumor cells to enhance radiation-induced cell killing. Previously, systemic administration of the cytokine TNF-α as an adjunct to ionizing radiation was reported to lead to increased lethality in the mouse xenograft tumor system. Since then, it has been shown to be partially effective in human tumors. The effect of TFNα is
It appears to be dose dependent as its tumor lethal effect is related to its serum concentration. However, the systemic toxicity of TFNα limits the applicable dose, thus limiting the usefulness of this method of treatment. Attempts have also been made to transfer the TFNα gene to tumor cells via adenovirus vectors and / or liposomes. Unfortunately, expression of the TFNα gene is not limited to tumor sites because of the "leakyness" of its promoter.

In an attempt to localize TFNa levels to the entire area of irradiation, thereby reducing systemic toxicity, Beyselselbaum and colleagues in si
The radiation-inducible Egr-1 promoter was used to activate the TFNα gene in tu. Early studies have shown that the expression of certain immediate-early genes, such as jun / fos and Egr-1, are activated in cells exposed to ionizing radiation (Sherman, ML, et al. .) Proceeding National Academy Sciences USA (Proc. Natl. Acad. Sci. USA), 87: 5663-5666 (1997); Hallman (Hallaman, DE et al.), Proceeding National Academy Sciences USA (Proc. Natl.) , Acad Sci. USA), 88: 2156-2160 (1991)). TFN
Exposure to ionizing radiation by placing the α gene under the control of the Egrl promoter (EGRp) enhances the expression of TFNα in those cells harboring the EGRp-TNFα plasmid. In vivo, within hours after irradiation,
Serum levels of TFNα are greatly increased. Combination treatment with this plasmid and radiation results in partial regression of the xenograft tumor during the course of treatment. TFNα levels dropped sharply within 24 hours, and further, a decrease in serum levels of TFNα was consistent with tumor regrowth.

[0007] There are several possible reasons for tumors to recur when treatment is discontinued. The most obvious reasons are probably the same limitations generally found in chemotherapy and radiation therapy,
That is, insufficient dose levels. One significant problem that limits the amount of TFNa produced is the weak and transient nature of the Egr-1 promoter.
This promoter is inherently weak, with up to 3-fold increase in expression upon induction. In addition, the induced expression is necessarily transient. This, combined with the weakness of the promoter, results in short exposure of tumor cells to TFNα.

[0008] Another factor limiting the production of sufficient doses of TFNa is that not all tumor cells take up the TFNa plasmid. Repeated doses have been shown to improve the completeness of treatment, but T
It is not clear whether repeated administration of FNα is beneficial, and problems due to the immune response remain. It is conceivable to provide plasmids at higher doses, but weak promoters have hindered the realization of such a scheme. It is known that significant basal levels of activity (20-30%) can be detected by the Egr-1 promoter in the absence of ionizing radiation (Beiseselbaum et al., Supra). Radioactive reaction elements,
This is not surprising since the GArG box is part of the serum response element.

[0009] The HSP promoter is also easy to leak. In the absence of heat, this promoter expresses 25-30% background levels, which is not suitable for most cytotoxic genes. This level of expression is detrimental to non-irradiated normal cells that take up the gene. Therefore, administration of this plasmid has been limited to small intratumoral injections to reduce systemic toxicity.

[0010] Thus, to enhance the specificity of gene expression at the site of heat or radiation treatment,
While it is advantageous to use partially or temporarily regulated promoters, such as the SP and Egr-1 promoters, such promoters currently available have significant problems that limit their applicability. In order to be able to use these promoters in cancer therapy, it is necessary to completely eliminate or significantly reduce background expression in cells that are not heated or irradiated. Ideally, cytotoxic gene expression should be restricted to the area of the external stimulus (heat or radiation). In addition, there is a need to improve the weak and transient nature of gene expression induced by these promoters to ensure that sufficient levels of the therapeutic gene are expressed.

[0011] Even though improved inducible vector systems have been developed that allow the expression of therapeutic genes to be limited to the area of the external stimulus, the problem of expression in normal, heated or irradiated peripheral cells still exists. It is important, therefore, that the therapeutic gene can be restricted to only the intended target, such as a tumor cell.

[0012] The prior art lacks effective means to reduce the undesirable toxic side effects of gene therapy for cancer and no way to enhance and maintain gene expression in tumor cells targeted in a controllable manner. Technology is inadequate. The present invention addresses the needs and desires that have been long felt in the art.

[0013] The present invention provides compositions and methods for controlled activation of DNA molecules for gene therapy. Activation of these DNA molecules leads to the production of protein products that provide an opportunity for therapeutically manipulating cells containing the DNA molecules. This can be achieved through denaturation in the growth and metabolism of the targeted cells, and may include effects on neighboring cells through the secretion of therapeutic products. The present invention offers the option of either persistent or regulatable activation through the use of antibiotics. The present invention further provides new expression vectors for use in gene therapy of localized and metastatic breast, ovarian and prostate cancers.

[0014] Original strategies for the expression of therapeutic genes and their spatial and temporary containment in tumors are also in the form of expression vectors for use in gene therapy of localized and metastatic breast, ovarian and prostate cancers. Is shown.

In one embodiment of the invention, a method is applied for expressing a gene in a sustained and enhanced manner via activation of a heat or light induced promoter. In a modified version of this method, heat or light is used to activate the promoter, but the level of gene expression is continuously dependent on the concentration of the antibiotic (tetracycline or its derivative) acting on the fusion protein with a tetracycline-responsive element. Is adjusted.

[0016] In another embodiment of the present invention, there is provided a method of constructing a vector for activating gene therapy.

In another embodiment of the present invention, there is provided an improved vector for reducing background expression in unheated or unirradiated cells.

In another embodiment of the present invention, there is provided a reduced vector for reducing expression in heated or irradiated normal surrounding cells.

In another embodiment of the present invention, there is provided an expression vector for use in gene therapy of localized and metastatic breast and ovarian cancer.

In another embodiment of the present invention, there is provided an expression vector for use in gene therapy of localized and metastatic prostate cancer.

[0021] Other and further aspects and advantages of the present invention will become apparent with reference to the following description of a presently preferred embodiment of the invention, provided for disclosure.

As used herein, “heat” refers to thermal energy generated by any means, including ultrashort waves.

As used herein, “light” means light energy having a frequency in the visible and invisible spectrum, and includes ionizing radiation generated by any means. This includes radiation sources, such as radionuclides, that can emit gamma and / or beta particles or are generated by a linear accelerator.

In the present invention, conventional molecular biology and recombinant DNA techniques as can be performed by those skilled in the art can be used. Such techniques are described, for example, in Maniatis, Fritsch & Sambrook, Molecular Cloning: A Laboratory Manual (1982); D
NA Cloning: Procedures 1 and 2 (DNA Cloning: A Practical Approach V
olumes I and II) (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization [Hams and Higgins (BD) Hames & S
J. Higgins eds.) (1985)]; Transcription and Translation
) [BD Hames & SJ Higgins eds. (1984)]; Animal Cell Culture [Freshney (RI Freshney, ed.) (1)
986)]; Immobilized Cells and Enzymes [IRL Press, (1986
Perbal (B. Perbal), A Guide to Molecular Cloning (A Practical Guid)
e To Molecular Cloning) (1984).

The present invention relates to a new method of gene therapy for a limited area such as a tumor. According to the above-mentioned task, constitutive activity can be regulated via a plasmid containing elements activated by heat and / or light and responsive to the presence and concentration of antibiotics (tetracycline and its derivatives). A mechanism is provided for both gene expression. In regulating gene expression, heat or light initiates expression, but genes are constitutively expressed only in the presence of antibiotics (tetracycline and its derivatives). Antibiotic concentration controls the level and duration of gene expression.

To limit gene expression to tumor cells, two mechanisms are provided to suppress gene expression in normal cells, which are peripheral targets of heat or radiation.
In normal cells that have not been exposed to heat or light that accidentally incorporates the plasmid, the expression of the therapeutic gene due to the background activity of its promoter will result in the heat or light-induced, antibiotic-dependent transcriptional activity incorporated into the plasmid. And the constitutive expression of the dominant negative DNA sequence against the antioxidant. In instances where normal cells that take up the plasmid are subsequently exposed to heat or light, another mechanism exists to prevent expression of the therapeutic gene. This is achieved through the use of a modified "2-hybrid" system in which the antibody-dependent transcription activator itself is under control of both the tissue-specific transcription activator and exposure to heat or light. Expression of this therapeutic gene is therefore only exposed to heat or light and is found only in cells that express tissue-specific transcription factors.

[0027] In one embodiment of the present invention, a recombinant vector, pDATA-X (dominant negative, antisense, TET-ON controllable heat shock promoter. Plasmid). This vector contains the following cassettes: (A) a fusion of amino acids 1-207 of the tetracycline repressor with the coding sequence for the last 130 amino acid transcriptional activation region at the C-terminus of the herpes simplex virus V16 protein; (b) binding to the minimal cytomegalovirus promoter, pCVM Human heat shock 70
A heat shock promoter composed of the heat shock response element (-260 to 30) of the gene promoter; (c) a tet operator composed of the 19-base inverted repeat of the TN10 operator O2 to which the tet repressor and TAKON bind. And (d) an antisense sequence consisting of a sequence complementary to the first 80 bases of the TAKON sequence containing ATG.

[0028] In another embodiment of the present invention, a vector comprising said gene is introduced into a host organism and the gene is sustained under the control of a heat or light inducible promoter comprising the step of applying heat or light energy. Methods for achieving high expression are provided. In yet another embodiment, the host organism is a human.

[0029] In still another embodiment of the present invention, a recombinant vector, pDATE-X
(Dominant negative antisense, TET-ON controllable EGR promoter expression plasmid) is provided, which vector contains the following cassettes: (A) cassette 1 contains the TET-ON sequence, the teracycline operator binding site and pCMV placed under the control of EGRp; (b) cassette 2 contains the therapeutic gene X under the control of tetp-pCMV (C) cassette 3 has antisense TET-ON under control of the pCMV promoter, and (d) cassette 4 has a dominant negative TET-ON under control of the pCMV promoter. Has been placed.

Another embodiment of the present invention provides a recombinant vector, pRIBs-X (radiation-inducible, breast-specific promoter) expression vector, which comprises (a) S
Yeast GAL4 fused to the basic helix-loop-helix-leucine zipper region of Max (amino acids 8-122) followed by V40 poly A
Including "Gal-DBD-mix", a fusion open reading frame encoding the N-terminal (amino acid 1-147) DNA binding region of the protein (Gal-DBD), the resulting Gal-DBD-mix is a radiation-induced Egr-1 promoter. , A minimal CMV promoter, an antisense construct complementary to the Gal-DBD-mx sequence, antisense Gal-DBD-mx, an internal ribosome entry site, and Gal for the pGAL binding site. -DBD-m
cassette 2, containing Gal-DBD that competes with ix, (c) the first 11 amino acids of Gal4 (amino acids 1-147) at the N-terminus, followed by the nuclear localization signal of the SV40 large T antigen, herpes simplex virus protein VP16 130 amino acid C-terminal transactivation region of c-MYc (amino acids 350-439) basic helix-loop-helix-leucine zipper region followed by SV40
The fusion gene VP16-TA- created in the fusion reading frame encoding poly A
cassette 3 containing VP16-TA-mc, whose mc is controlled by the c-erbB2 gene "perbB2" to the first ATG; (d) Galp, Gal4
Case 5 containing 5 copies of the 17-mer DNA binding site for TET-
(E) a TET-ON sequence containing an ATG controlled by the pCMV promoter, wherein the ON sequence is under the control of the GAPp-ptet promoter and the therapeutic gene X is linked to TET-IN via the IRES An antisense TET-ON consisting of a sequence complementary to the first 80 bases of
And a cassette 6 composed of a dominant negative TET-ON composed of a coding sequence for

Various variants on the above-described vectors are provided, in which perb
B2 promoter is whey acidic protein promoter or stromelysin 3
Replaced by a promoter.

[0032] Yet another embodiment of the present invention provides a method for treating localized and metastatic breast and ovarian cancer, the method comprising providing a pRIB comprising a cytotoxic gene to a patient.
administering the sX vector (or a variant thereof). A representative cytotoxic gene is tumor necrosis factor alpha.

The present invention also provides a recombinant vector, pRIPs-X (radiation-inducible, prostate-specific native promoter) expression vector, which comprises (a) SV40
Poly A encodes the N-terminal (amino acids 1-147) DNA binding region of yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper region of Max (amino acids 8-112) followed by poly-A. In the fusion open reading frame, the resulting fusion gene GAL-DBD-mx contains radiation-induced Egr-1
Cassette 1 containing "GAL-DBD-mx" controlled by a promoter,
(B) Minimal CMV promoter, antisense GAL-DBD-mx, an antisense construct complementary to the GAL-DBD-mx sequence, IRES, an internal ribosome entry site, and Gal-DBD-, for the pGAL binding site. cassette 2 containing Gal-DBD competing with mx, (c) Gal4 (amino acid
1-147), the first 11 amino acids followed by the nuclear localization signal of the SV40 large T antigen, the 130 amino acid C-terminal transactivation region of the herpes simplex virus protein VP16, the basics of c-MYc (amino acids 350-439). Helix-
In the fusion reading frame encoding the loop-helix-leucine zipper region followed by SV40 polyA, the fusion gene VP16-TA-mc created is controlled by the probasin gene promoter "p-probasin" to the first ATG. It consists of cassette 3 containing VP16-TA-mc and (d) five copies of the 17-mer DNA binding site for GALp and Gal4, in which the TET-ON sequence is placed under the control of the GAPp-ptet promoter. The therapeutic gene X contains cassette 4 linked to TET-ON via an internal ribosome entry site, and (e) TE containing ATG controlled by the pCMV promoter.
Antisense T composed of a sequence complementary to the first 80 bases of the T-ON sequence
It comprises a cassette 5 containing ET-ON and (f) a cassette 6 consisting of dominant negative TET-ON consisting of the coding sequence for amino acids 1-207. Variations of the above-described vectors are also possible, in which the probasin promoter is replaced with a prostate-specific antigen promoter.

[0034] Another embodiment of 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) comprising a cytotoxic gene. provide. A representative cytotoxic gene is tumor necrosis factor alpha.

In still another embodiment of the present invention, a recombinant vector, pHIBs-X (
Heat-inducible, breast-specific promoter), the vector comprising the following cassettes: (A) N-terminal (amino acids 1-174) DNA binding of yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper region of Max (amino acids 8-112) followed by SV40 polyA A cassette 1 composed of a fusion reading frame "GAL-DBD-mx", which encodes a region, wherein the fusion gene GAL-DBD-mx thus produced is controlled by a heat-inducible protein promoter; and (b) a minimal CMV promoter. , An antisense construct complementary to the Gal-DBD-mx sequence, antisense Gal-DBD-mx, an internal ribosome entry site, and Gal-DBD-m for the pGAL binding site.
cassette 2, consisting of a Gal-DBD that competes with ix, (c) the first 11 amino acids at the N-terminus (amino acids 1-147), followed by the SV40 large T antigen nuclear localization signal, herpes simplex virus Created with a 130 amino acid C-terminal transactivation region of protein VP16, a basic helix-loop-helix-leucine zipper region of c-MYc (amino acids 350-439), followed by a fusion reading frame encoding SV40 polyA. Fusion gene VP16-TA-mc
Is cassette 3 containing VP16-TA-mc controlled by the c-erbB2 gene "perbB2" to the first ATG, and (d) 17-me for Gal4
Contains GALp, five copies of the rDNA binding site, where TET-ON
The sequence is under the control of the GAPp-ptet promoter and the therapeutic gene X
Consists of cassette 4 linked to TET-ON via an internal ribosome entry site, and (e) a sequence complementary to the first 80 bases of the TET-ON sequence containing the ATG controlled by the pCMV promoter. The cassette 6 comprises an antisense TET-ON and (f) a dominant negative TET-ON comprising a coding sequence for amino acids 1-207. Variants of the above vectors are also contemplated, in which case the perbB2 promoter is replaced with the whey protein promoter or the stromelysin 3 promoter.

The present invention is further directed to a method for treating local and metastatic breast and ovarian cancer, comprising the step of administering to a patient a pHIBs-X expression vector (or a variant thereof) containing a therapeutic gene. ing. A representative therapeutic gene is tumor necrosis factor alpha.

Another embodiment of the present invention provides a recombinant vector, pHIPs-X (heat-induced, prostate-specific native promoter), which comprises the following cassettes: (a) SV40 polyA Followed by a fusion read encoding the N-terminal (amino acids 1-174) DNA binding region of yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper region of Max (amino acids 8-112) A cassette 1 composed of a frame “Gal-DBD-mx”, in which the fusion gene Gal-DBD-mx thus produced is controlled by a heat-inducible protein promoter; (b) a minimal CMV promoter (mCMVp); DBD-m
Antisense Gal-DBD, an antisense construct complementary to the x sequence
Ga for the mx, internal ribosome entry site, and pGAL binding site
Cassette 2 composed of Gal-DBD competing with 1-DBD-mix, (c)
The first 11 amino acids of Gal4 (amino acids 1-147) at the N-terminus, followed by SV
Nuclear localization signal of 40 large T antigen, herpes simplex virus protein VP16
Fusion gene VP16 created with a 130 amino acid C-terminal alternating region, a basic helix-loop-helix-leucine zipper region of c-MYc (amino acids 350-439) followed by a fusion reading frame encoding SV40 polyA. -TA-m
c) cassette 3, containing VP16-TA-mc, where c is controlled by the probasin gene promoter "p-probasin" to the first ATG, (d) for Gal4
Contains 5 copies of the 17-mer DNA binding site, GALp, where TET
Cassette 4, in which the ON sequence is placed under the control of the GAPp-ptet promoter and the therapeutic gene X is linked to TET-ON via an internal ribosome entry site, (e) ATG controlled by the pCMV promoter TET-ON containing
Antisense TET-O composed of a sequence complementary to the first 80 bases of the sequence
N and (f) cassette 6 consisting of the dominant negative TET-ON consisting of the coding sequence for amino acids 1-207. Variants of the above-described vectors are also contemplated, where the probasin promoter is replaced by a prostate-specific antigen promoter.

In another embodiment of the present invention, there is provided a method for treating local and metastatic prostate cancer comprising administering to a patient a pHISs-X vector containing a therapeutic gene (or a variant thereof). Is done. A representative therapeutic gene is tumor necrosis alpha.

In particular, it is conceivable to prepare a pharmaceutical composition according to the invention for the purpose of gene therapy. In such a case, the composition comprises the vector according to the invention and a pharmaceutically acceptable substrate. Those skilled in the art of cancer treatment will be able to determine appropriate dosages and routes of administration without undue experimentation. In the case of gene therapy, the gene contained in one of the plasmid vectors according to the present invention can be provided to target cells via a viral vector or liposome.

The ordinary skills of the average scientist in the field of molecular cancer biology have increased considerably in recent years. Those skilled in the art, with the teachings herein, will be able to easily construct and utilize plasmids for this new format for gene therapy.

The following examples are intended to illustrate various embodiments of the present invention,
No limitation of the invention is intended in any way.

Example 1 FIG. 2 schematically shows the pDATE vector. The pDATE-X plasmid functions to amplify TET-ON and X expression via a feed forward reaction. In the absence of radiation, background expression due to the ease of ERGp leakage leads to the synthesis of TET-ON mRNA. Translation of this mRNA is reduced by the concurrent expression of antisense TET-ON RNA. Furthermore, the leaked translated TET-ON protein shows no activity without tetracycline. In the presence of tetracycline, leaked (translated) TET-
The ON protein is activated, but the feed forward reaction is a constitutively expressed dominant negative TE that competes for the same DNA binding of the pret promoter.
Blocked by the T-ON protein.

Two chicken lysosomal matrix attachment sites (MAR) were inserted to isolate the cassette (McKnight, RA, et al.,
Mol. Reprod. & Dev., 44: 179-184 (1996)). If antisense and predominant negative TET-ON expression are driven by the minimal CMV promoter, they are unnecessary, but if a strong promoter, such as the human β-actin promoter, promotes its expression, MAR is required. Will be.

When cells carrying pDATA were exposed to radiation, the first burst of TET-ON transcription occurred, and TET compared to dominant negative TET-ON.
Synthesis is performed at 2-4 times the background level of -ON. This excess TE
The T-ON protein binds to the tetp promoter, which in the presence of tetracycline, binds both the TET-ON and X coding sequences and performs a feed-forward reaction. This reaction is controlled at the level of tetracycline. Thus, X expression is enhanced and its expression is maintained until tetracycline is removed, TET
At the half-life of the ON protein, determine how long the feed forward reaction can be restarted with tetracycline without radiation exposure.

This vector utilizes a feed-forward reaction to achieve and sustain high levels of inducible gene expression. This feed-forward property overcomes the transient nature and weakness of the inducible promoter. If the feed-forward reaction is limited to a few hours, there is a significant difference in the level of TET-ON achieved in heated and unheated cells. Thus, by adding or removing tetracycline, it is possible to enhance the former to adjust the level of amplified TET-ON in irradiated and non-irradiated cells. However, while the addition and removal of tetracycline can be precisely controlled in cell culture, the heterogeneity of tetracycline levels in tissues and the variation in tetracycline adsorption and removal in vivo in each individual is different. Hence i
It is difficult to do it in vivo. Therefore, it is important to minimize leakage through expression of TET-ON with antisense and dominant negative cassettes so that the feed forward reaction does not significantly amplify its levels in non-irradiated cells.

If necessary, the pCMV minimal promoter can be replaced with a stronger promoter, such as the human β-actin promoter, to replace the antisense and dominant negative TET-O.
By promoting the expression of N, the action of the vector can be further fine-tuned. In addition, the copy number of this antisense and dominant negative coding sequence can be increased.

Oxytetracycline is used to induce TET-ON expression in vivo because of its short half-life in vivo. In humans, after a single oral dose, peak plasma concentrations of oxytetracycline are achieved in 2-4 hours (eg, Goodman &Gilman's Basic Therapeutic Pharmacy (The Phar
macological Basis of Therapautics). The level of TET-ON expression as a function of oxytetracycline concentration can thus be monitored. Oxytetracycline has a short half-life in vivo of only 9 hours (in contrast to a half-life of 18 hours for deoxycycline).
At the end of 24 hours, oxytetracycline levels are reduced to less than 25% of input (about 10-30% are never absorbed and excreted).

Example 2 pDATH Vector: Structure and Mode of Operation FIG. 1 schematically shows the pDATH-X vector. This vector contains the E
It works on the pDATE-X vector in exactly the same way, except that the gr-1 promoter is replaced by the HSP promoter and heat is used instead of light / ionizing irradiation.

Example 3 Confirmation of the Concept of Amplifiable and Sustainable Expression of TET-ON and p53 with a Feed-Forward Inducible Promoter The concept of heat-induced gene expression, tetracycline feedforward amplification, is further examined. For this, two plasmids were constructed. Plasmid "ptet-splice p53wt" transcribes wild-type p53 cDNA
ptet-splice induces p53 under control of the p promoter (consisting of regulatory sequences from the tetracycline resistant operon upstream from the minimal hCMV promoter)
It was constructed by subcloning into a vector (Gibco BRL). Plasmid "HSP-tep-TET-ON" is ptet-on (Clontec
h) the 300 bp human heat shock protein promoter
And tetp promoter.

The non-small cell lung carcinoma cell line with homozygous deletion of p53, H358, was
Co-transfected with + 10% fetal calf serum. The cells growing exponentially to 10 ⁷ were transferred to a BRL cell porator at 1180 μF in 0.8 ml RPMI + 6 m.
50 V by electroporation using M glucose at 240 V
μg of “ptet-splice p53wt” and 10 μg of “HSP-te
tp-TET-ON ". The transfected cells were place-out at 25% confluence in 36 hours, and then half of them were heat shocked at 45 ° C for 20 minutes. Six hours after shock, cells were treated with different amounts of deoxycycline.At various time points after the addition of deoxycycline, monoclonals were obtained using the immunoperoxidase cell staining kit (Vector) and diaminobenzidine (DAB). p53 antibody DO-1 (Santa Cruz Biol
ogicals). At each time point, digital images of 50 immunostained cells were made using a Nikon microscope. The amount of protein expressed in each cell is proportional to the intensity of staining, indicated as I = 1 / T, as a measure of light transmitted by T / unit area. FIG. 8 shows the results of one such experiment performed at 0.01-0.1 μg / ral.

When 0.1 μg / ml deoxycycline is added 6 hours after heating (at which point the induced TET-ON should have reached its peak), at 10 hours p
53 was amplified by a factor of 10 (FIG. 8, curves a and b). At this time, deoxycycline also initiated a feed-forward reaction in unheated cells as indicated by substantial levels of TET-ON. However, the level of amplification of TET-ON at 10 hours only reached a low level as amplification started at lower levels (FIG. 8, curves c and d).

In feed-forward systems, it is possible to modulate the levels of induced p53 by altering the intake of tetracycline addition and removal. When the level of TET-ON (eg, level c in FIG. 8) was reduced to the background level [e] after removal in non-heated cells, the level of TET-ON in heated cells [a]
] Also decreases at the same ratio (same as [c]-[e]). However, since this level ([a]-[c]-[e]) is much higher than the level [e] in unheated cells, the addition of tetracycline will increase the level ([a]-[c]-[ e]
) To restart the feedforward reaction for the heated cells. Therefore,
Background p53 levels in unheated cells can be kept below the low levels ([c]) reached after 10 hours, while p53 levels in heated cells continue to increase. Thus, TFNα and p53 expression caused directly by HSP is transient, but expression caused by the feed-forward system is continued as long as tetracycline is present. It is important to know the half-life of TET-ON in tumor cells, because the method of adding tetracycline in vivo can be determined by the rate of tetracycline decay in vivo.

In vivo, the pharmacodynamics of tetracycline are heterogeneous from tissue to tissue. Preferred concentrations of tetracycline in certain tissues result in higher background expression of TET-ON in some tissues. For example, in humans, 10-35% of oxyterolacycline is removed via the kidney, which is a significant amount excreted in the active form. Therefore, it is desirable to minimize background expression levels at the start of the reaction to prevent runaway amplification in unintended tissues. The pDATE and pDATH inducible systems are used to suppress the background level of TET-ON translation and to express constitutively expressed antisense TET-ON.
At the same time as using dominant negative TET-ON to address TET-ON expressed via leakage in order to suppress background expression. When background is suppressed, the timing of tetracycline addition depends only on the desired level and duration of therapeutic gene expression, and it is necessary to suppress the level of background expression in normal, non-irradiated cells. Is not affected.

Example 4 Reduction of Background Level of Expression By using the 300 bp HSP promoter, the background level of expression without heat or light is about the level observed with heat or light. It is about 25%. To reduce this, the HSP should have a minimum pC in the range of -80 to +30.
Connected to MV promoter. The pCMV promoter preferably has low background expression. In addition, it allows for further amplification of expression of the therapeutic gene, independent of the constraints of the weaker HSP promoter used to initiate the reaction with heat and light bursts.

To further overcome the problem of background expression, two cassettes of plasmid pDTAH are introduced. Antisense to TAKON is under the control of the pCMV promoter. Constitutively created antisense binds any TAKON sense mRNA from background transcription, making it impossible to translate. Within it, the dominant negative TAKON, which has a DNA binding site but no transcriptional activation region, is further blocked from background transcription by cassette # 4 placed under the control of pCMV. This leads to background transcription, which promotes TAKON and dominant negative TAKON, which compete for the ptet binding site.

Example 5 Monitoring p53 Expression Levels To ensure that appropriate levels of antisense TAKON and dominant negative TAKON proteins are made, monitor p53 expression levels and reduce background to reduce The copy number and strength of the promoter were adjusted. First, cell lines harboring pDATH are isolated in the absence of tetracycline. Subsequently, p53 or co-transfected ptet-
EGFP is monitored to determine the copy number of antisense and dominant negative TAKON that must be incorporated into pDATH to reduce background expression.

Example 6 Expression Vector pRIB for the Treatment of Localized and Metastatic Breast and Ovarian Cancers Often expressed only transiently and at low levels. To overcome these problems, the expression vector pRIBs-X (a radiation-induced breast-specific promoter) was designed.

A tetracycline-dependent transcriptional activator, Tet-O, placed under the control of the tetracycline promoter (tepp) followed by the GAL-4 promoter
The gene expression level was optimized using a feed-forward reaction with n.
Transient transcription initiated by pGAL results in low levels of synthesis of Tet-On, which then binds tetp in the presence of teracycline. Tet-On then amplifies itself and its associated therapeutic gene expression through a feed-forward reaction. The expression of the therapeutic gene is controlled by six gene cassettes in the pRIB vector (FIG. 3). In cassette 1, the fusion gene GAL-
DBD-mx (the HLHLZ region of max fused to the DNA binding region of GAL-4 is regulated by EGRp. Background expression of GAL-DBD-mx is constitutively expressed by antisense GAL- Inhibited by DBD-mx and dominant negative GAL-DBD In cassette 3, the transcriptionally active region of herpes simplex virus protein VP16 is fused to the HLH-LZ region of C-Myc. mc is expressed in breast tumor cells under the control of the c-erb-2 promoter and overexpresses c-erbB-2.
The AL-DBD-mx fusion protein locates the VP16-TA-mc protein and binds to and activates transcription from the pGAL promoter (cassette 4).

In non-irradiated cells, background GAL-DBD-mx mRNA translation was reduced and dominant negative GAL-DBD (no mx)
Competitively occupy p and block Tet-On expression. When irradiated, GAL-D
BD-mx is transiently induced 3-4 fold, temporarily overcoming cassette 2 suppression. GAL-DBD-mx recruits VP-16-TA-mc (a fusion gene of the myc leucine zipper under the control of the VP16 transactivation region and the c-erbB2 promoter) to GALp, and reduces the level of Tet-On.
Activate transcription to initiate the feed-forward reaction.

For treatment with pRIBs-TFNα, for example,
Alternatively, it can be similarly provided systemically to both tumor and normal cells as a recombinant vector. In the absence of irradiation and tetracycline, TFNa is not expressed. Thereafter, X-ray irradiation is performed on metastatic tumor sites known after systemic administration of oxytetracycline. As a result, TFNα expression is induced at the tumor site by X-rays, and is enhanced and maintained by oxytetracycline. Even though all tumor cells do not take up pRIBs-TFNa, tumor cells near the uptake cells are exposed to very high local concentrations of secreted TFNa. pRIBs
-TFNa expresses TFNa only in breast tumor cells, and TFNa is not expressed in irradiated normal cells in the X-ray pathway. Thus, even if there is systemic toxicity, it is limited to only low levels of TFNa spread from the tumor cells. TF
Another therapeutic gene, designated X, in addition to or in place of Nα, is designated pR
It can be used with an IBS vector.

The structure of pRIBs-GFP-1 is shown in FIG. 3, and the mode of action is summarized in FIG. In unirradiated cells, background GAL-DBD-mx expression and function is suppressed by cassette 2 in two ways. Antisense to GAL-DBD-mx suppresses translation of background GAL-DBD-mx mRNA, while GAL-DBD protein binds GAL to the pGAL promoter.
-Functions as a dominant negative inhibitor by competing with DBD-mx.
In irradiated cells, GAL-DBD-mx expression is transiently induced 3-4 fold, overcoming cassette 2 suppression. GAL-DBD-mx is VP-16
-TA-mc (m under control of the VP16 ac region and the c-erbB2 promoter
yc leucine zipper fusion gene) to GALp and low levels of Te
Activates t-On transcription. In the presence of teracycline, Tet-On is activated, which cross-activates the tep promoter (Gossen, M. e.
al.), Science 268: 1766-1769 (1995)), amplifying its own levels and GFP in a feed-forward reaction. In the absence of irradiation or tetracycline, the background expression of TET-ON and GFP is zero.

Example 7 Generation of cell lines and xenografts stably expressing pRIBs-GFP Two pRIBs-GFP plasmids, having one and four antisense and dominant negative gene cassettes, respectively, pRIBs- GFP-1 and pR
To construct and analyze IBs-GFP-4 in vitro, the fibroma cell line H
TB152 and breast tumor cell lines SK-BR-3 and MDAM231 were stably transfected. 5 × 10 ⁶ cells were xenografted into SCID mice. Although all three human cell lines formed poorly differentiated tumors, only SK-BR-3 expresses high levels of c-erbB-2. In fact, an anti-erB-2 intracellular single-chain antibody that down-regulates erbB-2 on the cell surface induces cell necrosis only with SK-BR-3, but not MDA-MB-231 (Chumakov et al. (Chumakov et al.). AM
, et al.), Oncogene 8: 3005-3011 (1993)).

The pRIBs-GFP-1 and -4 plasmids have therefore been used as models for optimizing the conditions for therapeutic testing of metastatic breast tumor xenografts in nude mice with cytotoxic genes. The cytotoxic gene bound to EGRp is induced only in irradiated cells and is not toxic to non-irradiated cells. However, it is important to prevent the expression of cytotoxic genes in normal cells in the X-ray pathway. These three cell lines, which show differences in c-erbB-2 expression, show that when VP16-TA-mc expression is regulated by a tissue or tumor-specific promoter, expression is confined only to irradiated breast tumor cells and metastatic tumor cells are expressed. This indicates that expression is not performed in normal cells of the remaining organ.

The pRIBs-GFP plasmid is constructed as shown in FIG. GA
L-DBD-mx and VP16-TA-mc were modified from two mammalian hybrids (Fearon, ER, et al., Proceeding National Academy Sciences USA (Proc. Natl. Acad. Sci. USA), 89: 79.
58-7652 (1992)). Two plasmids with the predominant negative GAL-DBD derived by the antisense copies of 1 and 4 and the minimal CMV promoter, p
RIBs-GFP-1 and pRIBs-GFP-4 were examined.

All three cell lines were co-transfected with pRIBs-GFP and SVneo plasmids. Cell lines stably expressing pRIBs-GFP-1 and pRIBs-GFP-4 are isolated by selection in G418. For in vivo analysis, 5 × 10 ⁶ cells were removed from each of the cell lines stably expressing the pRIBs-GFP plasmid and SCID mice (4 ×
) And grown to a size of 0.5 cm in diameter. The expression of GFP in vitro and in xenografts without irradiation or oxyterolacycline was determined by extracting the protein from the crushed tumor into EBC buffer and reducing the amount of protein by R
Quantified by IA.

Inducible levels of GFP in vitro were determined by Western analysis and the cells were irradiated with 0-4 Gy on a Varian Clinac X-ray generator, followed by 0-2 μg / ml oxytetracycline. And then quantified by RIA. The data obtained with HSPp showed that the feed-forward reaction was performed very efficiently and was sufficient to enhance p53 expression 9-fold after 10 hours at 0.01 μg / ml. Tumors were exposed to 0-4 Gy / X-rays for in vivo analysis. Six hours after irradiation, 0-15 μg / g oxytetracycline was injected intraperitoneally. Three hours after injection (24 hours), tumor material was removed and the amount of TET-ON and GFP relative to the total amount of actin protein was measured. To achieve higher or lower GFP levels, the experiment was repeated with modified TET-ON levels by adjusting the dose of oxytetracycline. The amount of oxytetracycline removed by secretory excretion was monitored by analyzing plasma concentrations at 3 hour intervals.

Example 8 Targeting of Metastatic Breast Tumors by WAPp or ST3p Human cancers overexpressing c-erbB-2 have poor predictors, so to first validate the pRIB-X concept , C-erbB-2 promoter had been selected. However, it is unlikely that one particular promoter will address the challenge of treating various breast tumors. Thus, using the whey acidic promoter, WAPp (McKnight, RA, et al.
, Mol. Reprod. & Dev., 44: 179-184 (1996)) or using the stromelysin 3 promoter, ST3p (Ahmad, A., et al.), (International Journal of Cancer (Int. J. of Cancer), 73: 290-296 (1997)
)) It is also important to limit GAL-DBD-mx expression to metastatic breast tumors. WAPp restricted expression to breast epithelial cells, whereas ST3p restricted expression to stromal cells secreting the substrate metalloproteinase adjacent to the tumor.

[0068] pRIB was reconstituted by replacing the c-erbB-2 promoter with either WAPp or ST3p. Breast and other tumor cell lines were selected based on high or low expression levels of WAP and ST3. GFP expression was tested using sub-strains with different levels of WAP and / or ST3 expression.

The WAP promoter has been shown to be highly unique to lactating mammary epithelial cells of genetically modified animals (Tzeng YI, et al. Oncogene 16 (16) : 2013-2114 (1998)), and the stomerisin 3 promoter, ST3p, has been shown to be expressed only in stromal fibroblasts adjacent to cancer cells. Various evidence suggests that the production of stromal cells, a substrate involved in the process of tumor metastasis, including metallo-p-rotasease (including ST3), is stimulated by cancer cells. Thus, directing VP16-TA-mc to stromal cells results in the expression and release of the therapeutic gene product in adjacent regions of metastatic tumor cells. c-
Providing pRIBs-X in liposomes coated with an antibody to erbB32 provides additional therapeutic effects.

Example 9 Expression Vector pRIPs for Treating Local and Metastatic Prostate Cancer As mentioned above, many genes placed under the control of a promoter, such as the radiation-induced promoter of the Egr-1 gene, Is expressed transiently and at low levels. Therefore, these genes are unsuitable for use in cancer therapy. To overcome these problems, the expression vector pRIPs-X (radiation-induced prostate-specific promoter) was designed.

The pRIPS vector is composed of six cassettes. Gene cassette 1 contains yeast GA fused to the basic helix-loop-helix-leucine zipper region of Max (amino acids 8-112) followed by SV40 polyA.
Fusion O encoding N-terminal (amino acids 1-147) DNA binding region of L4 protein
It differs from the vector described above only in that it contains the RF (open reading frame) "Gal-DBD-mx". Cassette 2 contains a minimal CMV promoter (mCMVp), an antisense construct complementary to the Gal-DBD-mx sequence, antisense Gal-DBD-mx, an internal ribosome entry site "IRES", and a pGAL binding site. It is composed of Gal-DBD that competes with Gal-DBD-mx. Cassette 3 contains a fusion open reading frame encoding the first 11 amino acids of Gal4 at the N-terminus followed by SV40.
VP16-TA-mc followed by large T antigen nuclear localization signal, 130 amino acid C-terminal alternating region of herpes simplex virus protein VP16, bHLHLZ region of c-MYc (amino acids 350-439) followed by SV40 polyA. It is configured. The resulting fusion gene VP16-TA-mc is controlled by the probasin gene promoter, "p-probasin" to the first ATG.

Gene cassette 4 contains GALp, which consists of five copies of the 17-mer DNA binding site for Gal4. Inside, the TET-ON sequence is GAPp-
Under the control of the ptet promoter, the therapeutic gene X is linked to TET-ON via the IRES. Gene cassette 5 contains antisense TET-ON, a sequence composed of a sequence complementary to the first 80 bases of the TET-ON sequence containing ATG controlled by the pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequence for amino acids 1-207 of the tet repressor placed under the control of the pCMV promoter. pR
In other variants of IPs-X, pProbasin is replaced by PSA, the promoter region of the prostate-specific antigen, or other prostate-specific genes.

Example 10 Expression Vector pHIBs-X for the Treatment of Localized and Metastatic Breast and Ovarian Cancer The expression vector pHIBs-X was designed and consists of six cassettes. Gene cassette 1 contains M40 followed by SV40 polyA.
yeast GAL4 protein (G) fused to a basic helix-loop-helix-leucine zipper (bHLHLZ) of ax (mx, amino acids 8-112)
al-DBD) fusion O encoding the N-terminal (amino acids 1-147) DNA binding region
It differs from the vector described above only in that it contains the RF "Gal-DBD-mx". The resulting fusion gene GAL-DBD-mx is heat-inducible HS
Controlled by the P promoter. Cassette 2 has the minimum CMV promoter (mCM
Vp), antisense Gal-DBD-mx, an antisense construct complementary to the Gal-DBD-mx sequence, and "I", an internal ribosome entry site.
RES ", and G that compete with Gal-DBD-mx for the pGAL binding site.
It is composed of al-DBD. Cassette 3 is a fusion ORF encoding the first 11 amino acids of Gal4 at the N-terminus, followed by the SV40 large T antigen nuclear localization signal, VP16-TA-mc, herpes simplex virus protein VP16.
Of the c-MYc (amino acids 350-439) followed by SV40 polyA. Fusion gene VP to be created
16-TA-mc is the first AT from the c-erbB2 promoter "perbB2".
G control. Gene cassette 4 is a 17-mer for Gal4
It contains GALp composed of five copies of the DNA binding site. TET-
The ON sequence is under the control of the GAPp-ptet promoter and the therapeutic gene X is linked to TET-ON via the IRES. Gene cassette 5 contains p
Antisense TET-ON, which is a sequence composed of a sequence complementary to the first 80 bases of the TET-ON sequence containing ATG controlled by the CMV promoter. Gene cassette 6 contains a dominant negative TET consisting of the coding sequence for amino acids 1-207 of the tet lip resor placed under the control of the pCMV promoter.
-ON is included.

The pHIBs-X expression vector was constructed by using the Egr-1 promoter of pRIBs-X as HS.
Except for gene cassette 1 which has been replaced by the P70 promoter, pRIB
Same as the sX plasmid. pHISs-X shows that when the tumor is exposed to heat,
Local and metastatic breast and ovarian tumors are individually targeted.

Example 11 Expression Vector pHIPs-X for Treatment of Localized and Metastatic Prostate Cancer FIG. 10 shows the structure of pHISs-GFP (a heat-induced, prostate-specific promoter). This vector is composed of six cassettes. Gene cassette 1
Shows the N-terminus of yeast GAL4 protein (Gal-DBD) fused to the basic helix-loop-helix-leucine zipper (bHLHLZ) of Max (mx, amino acids 8-112) followed by SV40 polyA. Amino acid 1
-147) "Gal-DBD-mx" which is a fusion ORF encoding a DNA binding region
The resulting fusion gene GAL-DBD-mx is controlled by a heat-inducible HSP promoter.
Cassette 2 contains the minimal CMV promoter (mCMVp), an antisense construct complementary to the Gal-DBD-mx sequence, antisense Gal-DBD-mx.
, An internal ribosome entry site “IRES”, and a Gal-DBD that competes with Gal-DBD-mx for the pGAL binding site. Cassette 3 is a fusion ORF encoding the first 11 amino acids of Gal4 at the N-terminus, followed by the nuclear localization signal of the SV40 large T antigen, VP16-TA-mc.
C-MYc (amino acids 350-439) followed by SV40 polyA, a 130 amino acid C-terminal transactivation region of herpes simplex virus protein VP16
BHLHLZ region. The produced fusion gene VP16-TA-mc is controlled by the promoter of the probasin gene (p-probasin) to the first ATG. Gene cassette 4 contains GALp, which consists of five copies of the 17-mer DNA binding site for Gal4. The TET-ON sequence is GAPp
Under control of the -ptet promoter, the therapeutic gene X is linked to TET-ON via the IRES. Gene cassette 5 contains antisense TET-ON, a sequence composed of a sequence complementary to the first 80 bases of the TET-ON sequence containing ATG controlled by the pCMV promoter. Gene cassette 6 contains amino acids 1-207 of the tet repressor placed under the control of the pCMV promoter.
And a dominant negative TET-ON composed of a coding sequence.

The pHIBs-X expression vector contains the Egr-1 promoter of pRIBs-X and the pR
Identical to the pRIBs-X plasmid, except for gene cassette 1 in which IPs-X has been replaced by the HSP70 promoter. pHISs-X individually targets localized and metastatic breast and ovarian tumors when the tumors are exposed to heat.

All patents or publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually incorporated.

It will be readily apparent to one skilled in the art that the present invention is well adapted to carry out the tasks set forth above and pertinent thereto, and to achieve the objects and advantages. Would. The examples, methods, procedures, procedures, molecules, and specific compounds described therein are illustrative of the presently preferred embodiments, are specific examples, and are not intended to limit the scope of the present invention. I haven't. Those skilled in the art will readily envision changes and other uses within the spirit of the invention as defined in the appended claims.

For a better understanding of the features, advantages, objects, and other ways in which the invention as described above is achieved, the embodiments of the invention summarized above are set forth in the accompanying claims. The following description is made with reference to several embodiments thereof, which are illustrated in the drawings. These drawings constitute a part of the specification. However, it should be noted that the attached drawings are for describing preferred embodiments of the present invention, and are not intended to limit the scope thereof.

[Brief description of the drawings]

FIG. 1 shows a plasmid consisting of the following four cassettes, pDATH-X.
(Dominant negative, antisense, TET-ON controllable heat shock promoter plasmid) -p53 is shown schematically. (1) TET-ON is a fusion of the coding sequence for amino acids 1-207 of the tetracycline (tet) repressor with the C-terminal last 130 amino acid exchange region of the herpes simplex virus. In cassette 1, the TET-ON sequence is under the control of the HSP and tet operator binding sites and pCMV. (2) HSP is the minimal CMV promoter, pCMV (Gossen M, et al., Science, 268).
: 1766-1769 (1995)), a heat shock promoter composed of a heat shock response element (−260 to 30) of the human heat shock 70 gene promoter (Voellmy R., et al., Procedural National Academy USA (Proc. Natl. Acad. Sci. USA) 82, 4949-4953 (1985)). In cassette 2, the therapeutic gene X is under the control of the tetp-pCMV promoter. (3) ptet is a tet operator composed of an inverted repeat of 19 base pairs (bp) of operator O2 of TN10 to which the above suppressor and TET-ON bind (Gossen and Bujad) M, and Bujard H
Proc. Natl. Acad. Sci. USA
89: 5547-555 (1992)). Cassette 3, antisense TET-ON
Is under the control of the pCMV promoter. (4) Antisense TET-ON is an antisense sequence composed of a sequence complementary to the first 80 bases of the TET-ON sequence including ATG. In cassette 4, the dominant negative TET-ON is p
It is under the control of the CMV promoter. The dominant negative TET-ON is composed of the tet repressor but has no VP16 transactivation region and is under the control of the pCMV promoter. In the absence of heat or light, there is a background level of TET-ON sequence expression due to the minimal leakiness of the promoter pCMV.

FIG. 2 shows the pDATE vector. The plasmid, pDATE-X (dominant negative, antisense, TET-ON controllable EGR promoter expression plasmid) is composed of the following four cassettes. 1) In cassette 1, TET
The -ON sequence is under the control of EGRp, the tetracycline operator binding site and pCMV, and 2) in cassette 2, the therapeutic gene X
3) In cassette 3, TET-ON is pC
4) In cassette 4, the dominant negative TET-ON is under control of the pCMV promoter. TET-ON
Is a fusion of the coding sequence for amino acids 1-207 of the tet repressor with the C-terminal 130 amino acid alternating activation region of the herpes simplex virus VP16 protein.
EGRp is a radiation-induced promoter composed of fragment -425- + 65 containing four copies of the CArG region. ptet is a tet operator composed of the 19 bp inverted repeat of operator O2 of TN10, to which the tet repressor and TET-ON bind, and is associated with the minimal CMV promoter. Antisense Tet-On is a sequence composed of a sequence complementary to the first 80 bases of the TET-ON sequence including ATG. Dominant negative TET-ON is pCM
Consists of the coding sequence for amino acids 1-207 of the tet repressor placed under the control of the V promoter. M is a chicken lysosomal substrate attachment site for separating the position effect of the above-mentioned cassette.

FIG. 3 shows the structure of the pRIBs-X (radiation-induced, breast-specific promoter) expression vector. The pRIB vector is composed of four cassettes. Cassette 1 contains the N-terminus of the yeast GAL4 protein (Gal-DBD) fused to the basic helix-loop-helix-leucine zipper (bHL.HLZ) region of Max (mx, amino acids 8-122). -147) differs from the previously described vectors in that it contains the fusion reading frame Gal-DBD-mx encoding the DNA binding region, followed by SV40 polyA. Gene cassette 2 contains the minimum CMV promoter (mCMVp), Gal-DBD
Antisense Gal-D, an antisense construct complementary to the mx sequence
It is composed of BD-mix, IRES which is an internal ribosome entry site, and Gal-DBD which competes with Gal-DBD-mix for pGAL binding site. Gene cassette 3 encodes the first 11 amino acids of Gal4 (amino acids 1-147) at the N-terminus, followed by the fusion reading frame VP16-TA-, followed by the SV40 large T antigen nuclear localization signal. mc, a 130 amino acid C-terminal alternating region of herpes simplex virus protein VP16, followed by the bHLHLZ region of c-Myc (amino acids 350-439) followed by SV40 polyA.
The resulting fusion gene, VP-16TA-mc, is under the control of the C-erb-2 promoter, the region from "perbB2" to the first ATG. Gene cassette 4 is composed of five copies of the 17-mer binding site for Gal4. The TET-ON sequence is placed under the control of the GALp-ptet promoter, the therapeutic gene X is linked to TET-ON via the IRES, and the gene cassette 5 contains the TET- containing ATG placed under the control of the pCMV promoter. The first of the ON array
It is composed of a sequence complementary to 80 bases. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequence for amino acids 1-207 of the tet repressor placed under the control of pCMV.

FIG. 4 shows the structure of pRIPS-GFP (radiation-induced prostate-specific promoter). The pRIPS vector is composed of six cassettes. Gene cassette 1 contains Max (mx, amino acids 8-1), followed by SV40 polyA.
12) Basic helix-loop-helix-leucine zipper (bHLHL)
Z) contains the fusion open reading frame "Gal-DBD-mix" which encodes the N-terminal (amino acids 1-147) DNA binding region of the yeast GAL4 protein (Gal-DBD) fused to the region. It is different from the vector mentioned earlier. Gene cassette 2 contains the minimum CMV promoter (mCMVp), Gal-
“Antisense Gal-DBD-mix”, an antisense construct complementary to the DBD-mix sequence, “IRES”, an internal ribosome entry site
And "Gal" which competes with Gal-DBD-mix for pGAL binding site
Gene cassette 3 comprises the first 11 of Gal4 (amino acids 1-147) at the N-terminus followed by the SV40 large T antigen nuclear localization signal.
"VP16-TA-" which is a fusion open reading frame encoding
mc ", a 130 amino acid C-terminal alternating region of herpes simplex virus protein VP16, followed by C-Myc (amino acids 350-4) followed by SV40 polyA.
39) bHLHLZ region. The resulting fusion gene VP16-TA
-Mc is placed under the control of the initial ATG from the probasin gene promoter "p-probasin". Gene cassette 4 contains "GALp" composed of five copies of the 17-mer DNA binding site for Gal4. The TET-ON sequence is G
Under the control of the ALp-ptet promoter, the therapeutic gene X is linked to TET-ON via the IRES; gene 5 is the first 80 of the TET-ON sequence containing the ATG placed under the control of the pCMV promoter. And antisense TET-ON, which is a sequence composed of a sequence complementary to the bases described above. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequence for amino acids 1-207 of the tet repressor placed under the control of the pCMV promoter.

FIG. 5 is a diagram schematically showing the mode of action of pRIBS-GFP.

FIG. 6 shows the ease of HSP promoter leakage. This figure summarizes the test results of a heat-inducible system containing the therapeutic gene p53 and the hsp70 promoter in TFNα. FIG. 6 (A) shows two genes, p53 and TFNα. FIG. 6 (B) shows p53 transcription activity. To analyze the inducibility of the hsp promoter, the plasmid pHSP. 3p53CD1 or pHSP. Only three vectors were co-transfected into a human ovarian carcinoma cell line SKOV3 containing a homozygous deletion of p53 with Post-2-CAT (containing the CAT coding sequence linked to the consensus p53 binding site). Was. 36 hours after transfection, some of the cells were heated and some were not. After 24 hours, CAT activity was measured. Little or no activity is seen in parental SKOV untransfected cells (lane 2: heated, lane 1: unheated). Similarly, in the case of the pHSP vector alone, no activity was observed with or without heating (lanes 3 and 4). pHSP. In the case of the 3p53 plasmid, a high level of CAT activity is observed 24 hours after heating (lane 6).
However, without heating (lane 5), there is a significant level of p53 expression (about 25%).

FIG. 7 shows induction of TFNα by thermal or photodynamic therapy (PDT).
The coding sequence of TFNα is the plasmid pHSP. 3 subcloned into SKO
Transfected into V3 cells. Stable colonies were isolated by selection of G418. The cells were partially heated at 45 ° C. and partially unheated. Six hours after the treatment, the TFNα in the medium was measured using a Genzyme TFNα ELISA kit. TFNa was induced and secreted 4-fold by heat and 3-fold by PDT. However, there was also considerable background expression (27%).

FIG. 8 shows p53 in H358 carcinoma cell line by feed forward reaction.
In this figure, a, b, c, d and e show the levels of p53 10 hours after the feedforward reaction. Six hours after heat shock, transfected cells were treated with different amounts of deoxycis lines. At various times after addition of the deoxycis line, cells were stained with the p53 antibody. At each time point, a digital image of 50 immunostained cells was made using a Nikon microscope. The amount of protein expressed in each cell is proportional to the stained area expressed as I = 1 / T (T is a measure of transmitted light / unit area). This figure is 0.01-
One of such experiments performed with 0.1 μg / ml deoxycycline is shown.

FIG. 9 shows the pHIBS-X (heat-inducible breast-specific promoter) expression vector. The pHIBS vector is composed of six cassettes. Gene cassette 1 contains Max (mx, amino acids 8-112), followed by SV40 polyA.
Basic Helix-Loop-Helix-Leucine Zipper (bHLHLZ)
Previously, it contained "Gal-DBD-mix," a fusion open reading frame encoding the N-terminal (amino acids 1-147) DNA binding region of the yeast GAL4 protein (Gal-DBD) fused to the region. It differs from the vector described. The resulting fusion gene Gal-DBD-mix is placed under the control of the HSP promoter. Gene cassette 2 contains the minimum CMV promoter (mCMVp), Gal
"Antisense Gal-DBD-mix" which is an antisense construct complementary to the DBD-mix sequence, "IRES" which is an internal ribosome entry site
And "Ga which competes with Gal-DBD-mix for the pGAL binding site.
1-DBD ". The gene cassette 3 is followed by SV40 large T
The first of Gal4 (amino acids 1-147) at the N-terminus followed by the nuclear localization signal of the antigen
"VP16-TA", a fusion open reading frame encoding 11 amino acids
-Mc ", a 130 amino acid C-terminal alternating region of herpes simplex virus protein VP16, followed by C-Myc (amino acid 350) followed by SV40 polyA.
-439) bHLHLZ region. The resulting fusion gene VP16-
TA-mc is placed under the control of the first ATG from the C-erbB-2 promoter "perbB2". Gene cassette 4 contains "GALp" composed of five copies of the 17-mer DNA binding site for Gal4. The TET-ON sequence was placed under the control of the GALp-ptet promoter, and the therapeutic gene X was IRES
Gene 5 is an antisequence composed of a sequence complementary to the first 80 bases of the TET-ON sequence containing the ATG placed under the control of the pCMV promoter. Sense TET-ON is included. Gene cassette 6 contains amino acids 1-2 of the tet repressor placed under the control of the pCMV promoter.
It contains a dominant negative TET-ON consisting of the coding sequence for 07.

FIG. 10 shows the composition of a pHIPs-GFP (heat-induced prostate-specific promoter) expression vector. The pHIPS vector is composed of six cassettes.
Gene cassette 1 contains a basic helix-loop-helix-leucine zipper (bH) of Max (mx, amino acids 8-112) followed by SV40 polyA.
N-terminal (amino acid) of yeast GAL4 protein (Gal-DBD) fused to LHLZ) region
1-147) "Gal-DBD" which is a fusion reading frame encoding a DNA binding region
-Mix ". The resulting fusion gene Gal-DBD-mix is controlled by a heat-inducible HSP promoter. Gene cassette 2 contains a minimal CMV promoter (mCMVp). ), Gal-DBD-mi
"Antisense Gal-DB, an antisense construct complementary to the x sequence
D-mix ", an internal ribosome entry site" IRES ", and pGA
It is composed of "Gal-DBD" which competes with Gal-DBD-mix for the L-binding site. Gene cassette 3 is a fusion open reading frame encoding the first 11 amino acids of Gal4 (amino acids 1-147) at the N-terminus followed by the SV40 large T antigen nuclear localization signal, "VP16-TA-mc "The 130 amino acid C-terminal alternating region of herpes simplex virus protein VP16, followed by S
Consists of the bHLHLZ region of C-Myc (amino acids 350-439) followed by V40 polyA. The resulting fusion gene VP16-TA-mc is placed under the control of the initial ATG from the probasin gene promoter "pProbasinn". Gene cassette 4 contains "GALp" composed 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, the therapeutic gene X is linked to TET-ON via the IRES; gene 5 is the TET-O containing the ATG placed under the control of the pCMV promoter.
Antisense TET-ON, which is a sequence composed of a sequence complementary to the first 80 bases of the N sequence, is included. Gene cassette 6 contains a dominant negative TET-ON consisting of the coding sequence for amino acids 1-207 of the tet repressor placed under the control of the pCMV promoter.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Gormer, Charles United States 91741 California, Glendora, Fountain Springs Lane 1059 (72) Inventor Tang, Anne United States 90039 California, Los Angeles, Ivanhoe Drive 2455 F-term (reference) 4B024 AA01 BA01 CA01 CA07 DA03 EA04 FA02 GA11 HA17 4B065 AA93X AB01 AC01 AC09 BA02 CA24 CA44 4C08A AA13 CA53 NA05 NA06 ZB26 4C087 AA01 AA03 CA12 NA05 NA06 ZB26

Claims (28)

[Claims] 1. pDATH-X (dominant negative antisense, TET-ON controllable heat shock promoter plasmid) comprising the following cassettes:
Genetically modified vector. (A) fusion of the coding sequence for amino acids 1-207 of the tetracycline repressor with the transcriptional activation domain of the last 130 amino acids at the C-terminus of the VP16 protein of herpes simplex virus (b) pCMV of the minimal cytomegalovirus promoter Heat shock promoter composed of the heat shock response element (-260 to 30) of the human heat shock 70 gene promoter linked to (c) 19 bp reverse of the operator O2 of TN10 where tet repressor and TAKON are linked (T) an antisense sequence consisting of a complementary sequence of the first 80 bases of the TAKON sequence containing ATG. 2. introducing a vector containing the gene into a host organism;
And a method for achieving sustained expression of a gene under the control of a heat or light inducible promoter, comprising the step of applying heat or light energy. 3. The method according to claim 2, wherein the host organism is a human. 4. pDATE-X (predominant negative,
Antisense, TET-ON controllable EGR promoter expression plasmid) Genetic recombination vector. (A) cassette 1 containing the TET-ON sequence under the control of EGRp, the tetracycline operator binding site, and pCMV; (b) cassette 2 containing the therapeutic gene X under the control of the tetp-pCMV promoter; (C) Cassette 3 containing antisense TET-ON under control of pCMV promoter, and (d) Cassette 4 containing dominant negative TET-ON under control of pCMV promoter. 5. pRI which is a recombinant vector containing the following cassettes:
Bs-X (radiation-induced breast-specific promoter) expression vector. (A) Yeast GAL fused to the basic helix-loop-helix-leucine zipper region of Max (amino acids 8-112) followed by SV40 polyA.
A fusion open reading frame encoding the N-terminal DNA binding region (amino acids 1-147) of the four proteins (Gal-DBD), and the resulting fusion gene Gal-DBD-mx
Is the cassette 1 containing "Gal-DBD-mx" controlled by the radiation-inducible Egr-1 promoter, (b) the minimal CMV promoter, "anti-antibodies," an antisense construct complementary to the Gal-DBD-mx sequence. "Sense Gal-DBD-mx", internal ribosome entry site (IRES), and "Gal-DBD-mx" for pGAL binding site.
Cassette 2 containing "Gal-DBD" which competes with "DBD-mx"; (c) the first 11 amino acids of Gal4 (amino acids 1-147) at the N-terminus, followed by the nuclear localization signal of SV40 large T antigen; Herpes simplex virus protein V
C-M, the 130 amino acid C-terminal alternating region of P16 followed by SV40 polyA
Basic helix-loop-helix-leucine of Yc (amino acids 350-439)
The fusion gene VP16-TA generated with the fusion ORF encoding the zipper region
Cassette 3 containing "VP16-TA-mc" controlled by c-erbB2 promoter "perB2" to the first ATG; (d) "G" which is five copies of the 17-mer DNA binding site for Gal4.
cassette 4, wherein the TET-ON sequence is under the control of the GAPp-ptet promoter and the therapeutic gene X is linked to TET-IN via the IRES, (e) the pCMV promoter A cassette 5 containing an antisense TET-ON composed of a sequence complementary to the first 80 bases of the TET-ON sequence containing the ATG controlled by: and (f) a coding sequence for amino acids 1-207 Dominant negative TET
-Cassette 6 configured with ON. 6. The recombinant vector according to claim 5, wherein the perbB2 promoter of the cassette 3 is replaced with a whey acidic protein promoter. 7. The genetic recombination vector according to claim 5, wherein the perbB2 promoter of the cassette 3 is replaced with a stromelysin 3 promoter. 8. The recombinant vector according to claim 5, wherein the gene X is a gene encoding tumor necrosis factor alpha. 9. A method for treating local and metastatic breast and ovarian cancer, comprising the step of administering the expression vector according to claim 5 to an individual in need thereof. 10. A method for treating local and metastatic breast cancer and ovarian cancer, comprising administering the expression vector according to claim 6 to an individual in need of treatment. 11. A method for treating local and metastatic breast and ovarian cancer comprising administering the expression vector of claim 7 to an individual in need of treatment. 12. A recombinant pRIPs-X (radiation-induced prostate-specific promoter) expression vector comprising the following cassette: (A) N-terminal (amino acids 1-147) DNA of yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper region of Max (amino acids 8-112) followed by SV40 polyA Cassette 1, containing "Gal-DBD-mx" in which the resulting fusion gene GAL-DBD-mx is controlled by a radiation-inducible Egr-1 promoter, in the fusion reading frame encoding the binding region, (b) the minimal CMV promoter , An antisense construct complementary to the Gal-DBD-mx sequence, antisense Gal-DBD-mx, an internal ribosome entry site, IRES, and a Gal-DBD for the pGAL binding site.
Cassette 2 containing Gal-DBD that competes with -mx, (c) simple followed by the first 11 amino acids of Gal4, SV40 polyA at the N-terminus followed by the nuclear localization signal of SV40 large T antigen. The 130 amino acid C-terminal alternating region of herpes virus protein VP16, c-MYc (amino acid 350
-439) a fusion open reading frame encoding a basic helix-loop-helix-leucine zipper region wherein the fusion gene VP16-TA-mc created is controlled by the probasin gene promoter, "p-probasin" to the first ATG Cassette 3 containing "VP16-TA-mc", (d) GALp of 5 copies of 17-mer DNA binding site for Gal4
Where the TET-ON sequence is placed under the control of the GALp-ptet promoter and the therapeutic gene X is linked to the TET-ON via the IRES, (e) the pCMV promoter A cassette 5 containing an antisense TET-ON consisting of a sequence complementary to the first 80 bases of the TET-ON sequence containing the controlled ATG, and (f) a dominant consisting of the coding sequence for amino acids 1-207 Negative TET
Cassette 6 containing -ON. 13. The recombinant vector according to claim 12, wherein the probasin promoter of cassette 3 is replaced with a prostate-specific antigen promoter. 14. The recombinant vector according to claim 12, wherein the gene X is tumor necrosis factor alpha. 15. A method for treating local and metastatic prostate cancer comprising administering the expression vector of claim 12 to an individual in need of treatment. 16. A method for treating local and metastatic prostate cancer comprising administering the expression vector of claim 13 to an individual in need of treatment. 17. A recombinant expression vector for pHIBs-X (heat-inducible breast-specific promoter) comprising the following cassette: (A) N-terminal (amino acids 1-147) DNA binding of yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper region of Max (amino acids 8-112) followed by SV40 polyA. "Gal-DBD-mx" in which the resulting fusion gene GAL-DBD-mx is controlled by a heat-inducible heat shock protein promoter in the fusion reading frame encoding the region
(B) Minimal CMV promoter, antisense Gal-DBD-mx, an antisense construct complementary to the Gal-DBD-mx sequence, an internal ribosome entry site, and Gal-DBD for the pGAL binding site. Cassette 2 containing Gal-DBD competing with -mx, (c) the first 11 amino acids of amino acids (amino acids 1-147) at the N-terminus, followed by the nuclear localization signal of SV40 large T antigen, herpes simplex virus protein V
Created with a 130 amino acid C-terminal transactivation region of P16, a basic helix-loop-helix-leucine zipper region of c-MYc (amino acids 350-439), followed by a fusion open reading frame encoding SV40 polyA. Cassette 3 containing "VP16-TA-mc" in which the fusion gene VP16-TA-mc is controlled by the c-erbB2 gene "perbB2" to the first ATG; (d) five of the 17-mer DNA binding sites for Gal4 GA that is a copy
Lp, where the TET-ON sequence is under the control of the GALp-ptet promoter, and the therapeutic gene X is inserted into the TE via an internal ribosome entry site.
Cassette 4 linked to T-ON, (e) Antisense TET-ON consisting of a sequence complementary to the first 80 bases of TET-ON sequence containing ATG controlled by pCMV promoter Cassette 5, and (f) a dominant negative TET consisting of the coding sequence for amino acids 1-207
-Cassette 6 configured with ON. 18. The genetic recombination vector according to claim 17, wherein the perbB2 promoter of cassette 3 has been replaced with a whey acidic protein promoter. 19. The perbB2 promoter of cassette 3 is stromelysin 3
18. The genetic recombination vector according to claim 17, wherein the vector is replaced with a promoter. 20. The method according to claim 17, wherein the therapeutic gene is tumor necrosis factor alpha. 21. A method for treating local and metastatic breast and ovarian cancer comprising the step of administering the expression vector of claim 17 to an individual in need of treatment. 22. A method for treating local and metastatic breast and ovarian cancer comprising administering the expression vector of claim 18 to an individual in need of such treatment. 23. A method for treating local and metastatic breast and ovarian cancer comprising administering the expression vector of claim 19 to an individual in need of treatment. 24. A recombinant vector of pHIPs-X (heat-inducible prostate-specific promoter), comprising the following cassette: (A) N-terminal (amino acids 1-147) DNA binding of yeast GAL4 protein fused to the basic helix-loop-helix-leucine zipper region of Max (amino acids 8-112) followed by SV40 polyA Cassette 1 containing the fusion open reading frame Gal-DBD-mx encoding the region, wherein the resulting fusion gene GAL-DBD-mx is controlled by a heat-inducible heat shock protein promoter; and (b) a minimal CMV promoter. mCMVp, an antisense construct complementary to the Gal-DBD-mx sequence, antisense Gal-DBD-mx, an internal ribosome entry site, and Gal-DBD-m for the pGAL binding site.
cassette 2 containing Gal-DBD competing with x, (c) the first 11 amino acids of Gal4 at the N-terminus, followed by the nuclear localization signal of the SV40 large T antigen, the 130 amino acid C-terminal exchange of the herpes simplex virus protein VP16 Region, basic helix of c-MYc (amino acids 350-439)-loop-
A fusion open reading frame encoding a helix-leucine zipper region followed by SV40 polyA, where the resulting fusion gene VP16-TA-mc is regulated by the probasin gene promoter "p-probasin" to the first ATG.
Cassette 3, containing 16-TA-mc ", (d) GA, five copies of the 17-mer DNA binding site for Gal4
Lp, where the TET-ON sequence is placed under the control of the GAPp-ptet promoter and the therapeutic gene X is inserted into the TE via an internal ribosome entry site.
Cassette 4 linked to T-ON, (e) Antisense TET-ON consisting of a sequence complementary to the first 80 bases of TET-ON sequence containing ATG controlled by pCMV promoter Cassette 5, and (f) a predominant negative TET comprising the coding sequence for amino acids 1-207
-Cassette 6 configured with ON. 25. The recombinant vector according to claim 24, wherein the probasin promoter has been replaced by a prostate-specific antigen promoter. 26. The recombinant vector according to claim 24, wherein the therapeutic gene is tumor necrosis factor. 27. A method for treating local and metastatic prostate cancer comprising administering the expression vector of claim 24 to an individual in need of treatment. 28. A method of treating local and metastatic prostate cancer comprising administering the expression vector of claim 25 to an individual in need of treatment.