CN1321093A

CN1321093A - Method to enhance and confine expression of genes

Info

Publication number: CN1321093A
Application number: CN99811269A
Authority: CN
Inventors: 恽凯峰; C·戈梅; A·唐
Original assignee: RESEARCH DEVELOPMENT FOUDATION
Current assignee: RESEARCH DEVELOPMENT FOUDATION; Research Development Foundation
Priority date: 1998-08-18
Filing date: 1999-08-18
Publication date: 2001-11-07
Also published as: AU763183B2; CA2340929A1; IL141473A0; NZ509966A; ZA200101207B; KR20020013463A; WO2000010612A1; RU2226108C2; EP1109582A4; AU5684599A; EP1109582A1; JP2002523032A

Abstract

The present invention provides a novel approach to gene therapy of restricted areas such as tumors. The methods introduced here comprise: (a) placing a gene of interest in a plasmid vector driven by a heat or light inducible promoter; (b) modifying this vector by including a tetracycline responsive fusion protein which acts as a transcriptional activator, thus permitting regulation of gene expression by varying the levels of drug and; (c) modifying this vector by including DNA sequences that reduce or eliminate expression of genes in normal bystander cells. Also provided are a set of vectors for both sustained and regulable expression. There is also presented novel vectors for the gene therapy treatment of local and metastatic breast, ovarian and prostate cancer.

Description

The method of enhancing and confine expression of genes

Background of invention

Invention field

The present invention relates generally to the field of gene of cancer.Say that more specifically the invention provides a kind of inducible promoters that passes through, the method for valuable gene product expression is gone up in the control treatment.Method provided by the invention is to strengthen and prolong derivative gene expression in the cellular targets with the space-time regulative mode by heat or light inducible promoter.In addition, the invention provides a kind of method weakens or eliminates background expression of gene in the non-target cell.

Description of related art

One of successful cancer chemotherapy and radiocurable major obstacle are to be difficult to realize killing and wounding of tumor cell specific.Lonizing radiation or cytotoxicity chemotherapeutics can not be screened tumor cell and normal cell, and the used dosage of essential restriction.As a result, usually observe because the palindromia that the tumor cell of residue survival causes.

In treatment of cancer, use gene therapy, equally with chemotherapy and radiation have many same disadvantages.The problem of strategies in gene therapy comprises and can not pass to target cell with therapeutic gene is specific in current this area.This has caused non-target cell is caused toxicity.For example, handle the p53 gene and suppressed tumor cell and Normocellular growth simultaneously, and intravenous is used tumor necrosis factor (INF α) and caused systemic poisoning, has clinical symptoms such as heating and hypertension.

These problems have been attempted overcoming, comprise following strategy: gene is pointed to required tissue (Kasahara with the tissue specificity receptor, N., Deng, science, 266:1373-1376 (1994)), with tissue-specific promoter with gene expression be limited to specific tissue (as, use the prostate specific antigen promoter) and with heat (Voellmy R etc., Proc.Natl.Acad.Sci.USA 82:4949-4953 (1985)) or derivable enhancer of ion ionizing irradiation and promoter (Trainman, R.H. etc., cell, 46:567-574 (1986); Prowess, R. etc., Proc.Natl.Acad.Sci.USA 85,7206-7210 (1988)) strengthen the expression of therapeutic gene in the mode of space-time control.Instruct gene with heating derivable heat shock protein (HSP) promoter, as the expression of cytokine IL-2.

Weichselbaum and colleague have found the derivable reaction of irradiation of early growth reactivity (Egr-1) gene promoter at first.Therefore, they have attempted with this promoter with cytotoxic gene, as TNF-α guiding tumor cell, strengthen irradiating cell and kill and wound.Before, Preliminary report whole body give the cytokine TNF alpha of ionizing irradiation adjuvant, cause the enhancing that mice xenotransplantation tumor system is killed and wounded.Effective to people's tumor also display part afterwards.The effect of TNF α is dose dependent seemingly, because its tumor-killing effect is relevant with its serum-concentration.Yet the general toxicity of TNF α has limited its consumption, thereby has limited the effect of therapeutic scheme.Attempted TNF α gene being passed to tumor cell by adenovirus vector and/or liposome.Unfortunately, because this promoter very " weak ", TNF α expression of gene can not be confined to tumor locus.

Be confined to shine exposed region in the level with TNF α, thereby weaken in the systemic-toxic trial, Weichselbaum and colleague have used the derivable Egr-1 promoter of irradiation to come in-situ activation TNF α gene.Studies show that more early in being exposed to the cell of ionizing irradiation, activated early gene in some, as expression (Sherman, M.L. etc., Proc.Natl.Acad.Sci.USA, the 87:5663-5666 (1997) of jun/fos and Egr-1; Hallahan, D.E. etc., Proc.Natl.Acad.Sci.USA, 88:2156-2160 (1991)).By TNF α gene is placed under the control of Egrl promoter (EGRp), strengthened the expression of TNF α in the cell that carries EGRp-TNF α plasmid when being exposed to ionizing irradiation.The inherent irradiation of body back is in a few hours, and the serum levels of TNF α improves (Weichselbaum, R.R. is etc., cancer research 54:4266-4269 (1994)) greatly.With this plasmid and irradiation therapeutic alliance, xenotransplantation tumor part in therapeutic process is degenerated.In 24 hours, the level of TNF α sharply descends; The further decline of TNF α serum levels is consistent with regrowing of tumor.

After treatment stopped, tumor recurrence had several possible reasons.The most tangible reason may be the same limitation that sees chemotherapy and radiation usually, that is, and and the dosage level deficiency.The subject matter of the TNF α amount that restriction produces is that Egr-1 promoter character is weak and of short duration.This promoter is innately very weak, induce the back to express to increase at most 3 times less than.And inductive expression is inevitable of short duration, and this is weak relevant with this promoter, only makes the of short duration contact of tumor cell TNF α.

The factor that another restriction produces enough TNF α dosage is or not that each tumor cell has all absorbed TNF α plasmid.Though propose, repeat dispenser and may help to improve therapeutic outcome, do not know whether repeating delivery Asia the suitableeest low dosage TNF α can be useful, though the problem that also exists immunne response to cause.Though send more heavy dose of plasmid may be useful, and the weak problem of promoter has hindered this method.Even known when not having ionizing irradiation, can detect roughly the active baseline values of 20-30% (Weichselbaum, etc., on seeing) with the Egr-1 promoter.This is not wondrous, because irradiation response element CArG box is the part of serum response element.

The HSP promoter is also a bit leaked.When not heating, this promoter shows the background expression of 25-30%, is not suitable for the most cells virulent gene.Because this expression will be harmful to the non-irradiated normal cell that absorbs this gene, therefore, this plasmid be limited in a small amount and have injected in the tumor, so that general toxicity minimizes.

Therefore, though but the promoter of using space-time to regulate, the specificity that strengthens heat or the gene expression of treatment with irradiation position as HSP and Egr-1 promoter may be favourable, present this class promoter has serious problems, has limited their practicality.For these promoteres are used for treatment of cancer, must eliminate or significantly reduce not heating and not in the irradiating cell background express.It is desirable to, the expression of cytotoxic gene should be limited to the zone that is subjected to outside stimulus (heat or irradiation).In addition, express to adequate level, must improve the weak and transience of gene expression that these promoteres drive in order to ensure therapeutic gene.

Be important to note that,, in the time of the expression of therapeutic gene can being confined to be subjected to the outside stimulus zone, still have the problem of in the irrelevant cell of heating and irradiation, expressing normally even when having developed a kind of improved carrier system of inducing.Therefore, the target cell that crucial is should be able to be further only limits to the expression of therapeutic gene to be scheduled to is in tumor cell.

The defective of prior art is, lacks the method that effectively suppresses unwanted toxic side effects in the gene therapy for cancer, and can not be provided in the target tumor cell and to strengthen with controllable way and the method for continuous expression gene.The present invention has filled up this secular needs and hope in this area.

The invention summary

The invention provides activatory compositions of dna molecular and method in the controlling gene treatment.The activation of these dna moleculars causes producing protein product, thereby can provide therapeutic to handle the chance of the cell that contains described dna molecular.This can realize by cell growth and the metabolism that changes target cell, and can be by secretion therapeutic product, to adjacent cells generation effect.The invention provides continuous activation or with the activatory selection of antibiotic Modulatory character.The present invention also provides the novel expression vector that is used for gene therapy part or transitivity mammary gland, ovary and carcinoma of prostate.

A kind of restriction and strengthen original strategy at tumor treatment gene expression on room and time also is provided, and its form is the expression vector that is designed for part or transitivity mammary gland, ovary and carcinoma of prostate.

In one embodiment of the invention, provide a kind of and continued method with reinforcing gene expression by activation heating or light inducible promoter.In one of this method changes,, but with the concentration of the antibiotic that acts on the fusion rotein that contains the tetracycline reactive component (tetracycline or derivatives thereof), come the lasting level of regulator gene expression with heat or photoactivation promoter.

In another embodiment of the present invention, provide a kind of structure to be used for the method for the carrier of gene therapy activation scheme.

In another embodiment of the present invention, provide and be used for reducing in heating not and the improved carrier expressed of irradiating cell background not.

In another embodiment of the present invention, provide the improved carrier of expressing in the normal irrelevant cell that reduces heating and shone.

In another embodiment of the present invention, provide a kind of expression vector that local and transitivity mammary gland and ovarian cancer are handled in gene therapy that is used for.

In another embodiment of the present invention, provide the expression vector that is used for gene therapy processing part and metastatic prostate cancer.

From the description of the preference of the present invention that hereinafter provides for open purpose, can clear and definite other aspects, features and advantages of the present invention.

The accompanying drawing summary

Said some example with reference to the accompanying drawings can make above-mentioned feature of the present invention, advantage and purpose and other content become obviously, but understood in detail can obtain the more detailed description of the present invention of above short summary.These accompanying drawings form the part of description.Yet what be noted that description of drawings is preference of the present invention, can not think the scope that has limited them.

Fig. 1 has shown the sketch map of plasmid pDATH-X (dominant negative, antisense, TET-ON controlled thermal shock protein plasmid)-p53, and it is made of following four boxes.(1) TET-ON is the fusions (Gossen M., etc., science, 268:1766-1769 (1995)) of the coded sequence in the amino acid/11-207 of tetracycline (tet) repressor protein and last 130 the aminoacid transcription activating territories of herpes simplex virus VP16 PROTEIN C-end.In box 1, the TET-ON sequence is placed under the control of HSP and tet operon binding site and pCMV.(2) HSP is the heat shock promoter, heat shock response element (260-30) (Voellymy by human heat shock 70 gene promoters, R., Deng, Proc.Natl.Acad.Sci.USA, 82:4949-4953 (1985)) and minimum CMV promoter, pCMV (Gossen M., Deng, science, 268:1766-1769 (1995)) linking to each other constitutes.In box 2, therapeutic gene X is placed under the control of tetp-pCMV promoter.(3) ptet is the tet operon, reverse repetition (the Gossen M and the Bujand H. of 19 base pairs (bp) that comprises the operon O2 of TNiO, Proc.Natl.Acad.Sci.USA, 89:5547-5551 (1992)), combine tet repressor protein and TET-ON on it.In box 3, antisense TET-ON is placed under the control of pCMV promoter.(4) antisense TET-ON is an antisense sequences, is made of the complementary series of preceding 80 bases of the TET-ON sequence that comprises ATG.In box 4, dominant negative TET-ON is placed under the control of pCMV promoter.This dominant negative TET-ON comprises the tet-repressor protein, but does not have the VP16 trans-activation domain, and it is under the control of pCMV promoter.There are not heat or light time, because the seepage of minimal promoter pCMV is expressed the background level that produces TET-ON.

Fig. 2 has described the pDATE carrier.Plasmid pDATE-X (dominant, antisense, the controlled EGR promoter expression of TET-ON plasmid) is made of following 4 boxes: 1) in box 1, the TET-ON sequence is under the control of EGRp, tetracycline operator binding site and pCMV; 2) in box 2, therapeutic gene X is under the control of tetp-pCMV promoter; 3) in box 3, antisense TET-ON is under the control of pCMV promoter; With 4) in box 4, dominant TET-ON is under the control of pCMV promoter." TET-ON " is the fusions of the coded sequence of the amino acid/11-207 of tet repressor protein and herpes simplex virus VP16 PROTEIN C-130 aminoacid transcription activating domains of end." EGRp " but be to contain the fragment-425 of EGR-1 promoter of 4 CArG functional domains copy to+65 radiation-induced promoteres that constitute." ptet " is the tet operon, contains the inverted repeat of 19bp of the operon O2 of TN10, and tet repressor protein and TET-ON combine with it, and this sequence is connected with minimum CMV promoter pCMV." antisense Tet-on " is the sequence that comprises that the complementary series of preceding 80 bases of the TET-ON sequence of ATG constitutes." dominant TET-ON " is made of the coded sequence of the tet repressor protein amino acid/11-207 under the control of pCMV promoter." M " is chicken matrix of lysosome binding site, works the position effect of separating each box.

Fig. 3 has described the structure of pRIBs-X (irradiation can be induced, mammary gland-specific promoter) expression vector.The pRIBS carrier is made of 4 boxes.Difference between box gene 1 and the above-mentioned carrier only is that it contains " Gal-DBD-mx ", this is the coding proteic N-end of yeast GAL4 (amino acid/11-a 147) DNA calmodulin binding domain CaM, be blended in alkaline helix-loop-helix-leucine zipper (bHLHLZ) territory (mx of Max, aminoacid 8-112), frame (ORF) is read with the integrated open of SV40 polyadenylic acid in the back.Box gene 2 comprises minimum CMV promoter (mCMVp), " antisense GAL-DBD-mx " (it is and the complementary antisense thing of Gal-DBD-mx sequence) and " IRES " (internal ribosome entry site) and " Gal-DBD " (with Gal-DBD-mx competition pGAL binding site).Box gene 3 comprises " VP16-TA-mc " (terminal preceding 11 the amino acid whose integrated opens of the N-of coding Gal4 (amino acid/11-147) are read frame), the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of proteic 130 the amino acid whose C-of herpes simplex virus VP16, the bHLHLZ functional domain of c-Myc (aminoacid 350-439) then is SV40 polyA.The fusion gene VP-16TA-mc that produces begins under the control of c-erbB-2 promoter (perbB2) from first ATG.Box gene 4 contains " GALp ", is made of 5 17 of Gal4 poly-DNA-binding sites.The TET-ON sequence is under the control of GALp-ptet promoter, and therapeutic gene X is connected by IRES with TET-ON; Box gene 5 contains antisense TET-ON, and this sequence is made up of the complementary series of preceding 80 bases of TET-ON sequence that comprise ATG, under the control of pCMV promoter.Box gene 6 contains a dominant TET-ON, and it comprises the coded sequence of the tet-repressor protein amino acid/11-207 under the control of pCMV promoter.

Fig. 4 has shown the structure of pRIPS-GFP (irradiation can be induced, prostate specific promoter) expression vector.PRIPS is made of 6 boxes.Box gene 1 and aforementioned bearer different only are to contain " Gal-DBD-mx ", this is that an integrated open is read frame, alkaline helix-loop-helix-leucine zipper (bHLHLZ) territory (mx of coding and Max, aminoacid 8-112) yeast GAL4 albumen N-end (amino acid/11-147) DNA of Rong Heing is in conjunction with the territory, and the back is with SV40 polyA.Box gene 2 contains minimum CMV promoter (mCMVp), " antisense Gal-DBD-mx " (it is and the complementary antisense construct of Gal-DBD-mx sequence), " IRES " (internal ribosome entry site) and " Gal-DBD " (with Gal-DBD-mx competition pGAL binding site).Box gene 3 comprises " VP16-TA-mc ", it is an open reading frame that merges, terminal preceding 11 aminoacid of the N-of coding Gal4 (amino acid/11-147), the back is with the nuclear localization signal of SV40 large T antigen, the trans-activation domain of 130 aminoacid C-ends of hsv protein VP16, the bHLHLZ territory of c-Myc (aminoacid 350-439), the back is with SV40 polyA.The fusion gene VP16-TA-mc that produces is from first ATG, under the control of prostate basic protein gene promoter " pProbasin ".Box gene 4 contains " GALp ", and it comprises 5 copies of the 17 poly-DNA binding sites of Gal4.The TET-ON sequence is under the control of GALp-tet promoter, and the therapeutic gene X is connected by IRES with TET-ON; Box gene 5 contains the antisense sequences of TET-ON, and this sequence is made of the complementary series of preceding 80 bases of the TET-ON sequence that comprises ATG, under the control of pCMV promoter.Box gene 6 contains dominant TET-ON, and the coded sequence of the tet repressor protein amino acid/11-207 under being controlled by the pCMV promoter constitutes.

Fig. 5 is the sketch map of pRIBS-GFP action model.

Fig. 6 has illustrated that the HSV promoter is weak.But it has summed up the result who tests the heat inducible system that contains the hsp70 promoter in expression treatment gene, p53 and TNF α.Fig. 6 A shows the plasmid structure of two gene p53 and TNF α.Fig. 6 B has described the p53 transcriptional activity.In order to analyze the inducibility of hsp promoter, with Post-2-CAT (containing CAT coded sequence) with consensus sequence p53 binding site with plasmid pHSP.3p53CD1 or to contrast pHSP.3 carrier cotransfection Proliferation of Human Ovarian Cell separately be SKOV3 (it has the p53 homozygous deletion).After the transfection 36 hours, heating or do not heat cell.Measure the CAT activity after 24 hours.In SKOV3 parental generation non-transfected cells (swimming lane 2, heating; Swimming lane 1, heating) in almost or can't see activity.Similarly, use the pHSP.3 carrier separately, heat or do not heat (swimming lane 3 and 4) and all can't see activity.Use the pHSP.3p53 plasmid, heating (swimming lane 6) back 24 hours visible high-caliber CAT activity.Yet,, also have basic p53 expression (about 25%) even do not heat (swimming lane 5).

Fig. 7 has described heating or photodynamic therapy (PDT) is induced TNF α.The coded sequence sub-clone of TNF α is gone into plasmid pHSP.3, and be transfected into the SKOV3 cell.Select with G418 and to isolate stable colony.45 ℃ are heated or are not heated cell.Handled back 6 hours, with the TNF alpha levels in the Genzyme TNF α ELISA kit measurement culture fluid.Show TNF α because of heating induction 4 times of raisings, PDT3 doubly improves, justacrine.Yet background is expressed more (27%).

Fig. 8 has shown that the p53 by the feedforward reaction expresses kinetics in the H358 lung cancer cell line, and wherein a, b, c, d and e represent the p53 level that the feedforward reaction reached after 10 hours.After the heat shock 6 hours, handle transfectional cell with the doxycycline of various dose.Different time points behind the adding doxycycline is with p53 antibody staining cell.For each point, catch the digital image of 50 immunostaining cells with the Nikon microscope.The protein content of expressing in each cell is directly proportional with staining power, is expressed as I=1/T (wherein T is measuring of transillumination/unit are).This figure shows the result with one of experiment of 0.01-0.1 μ g/ml doxycycline.

Fig. 9 has described pHIBS-X (hot derivable mammary gland-specific promoter) expression vector.PHIBS is made of 6 boxes.Box gene 1 and aforementioned bearer different only are that it contains " Gal-DBD-mx ", this is that an integrated open is read frame, alkaline helix-loop-helix-leucine zipper (bHLHLZ) territory (mx of coding and Max, aminoacid 8-112) yeast GAL4 albumen N-end (amino acid/11-147) DNA of Rong Heing is in conjunction with the territory, and the back is with SV40 polyA.The fusion gene GAL-DBD-mx that produces is heated and can induces the control of HSP promoter.Box gene 2 contains minimum CMV promoter (mCMVp), " antisense Gal-DBD-mx " (it is and the complementary antisense construct of Gal-DBD-mx sequence), " IRES " (internal ribosome entry site) and " Gal-DBD " (with Gal-DBD-mx competition pGAL binding site).Box gene 3 comprises " VP16-TA-mc ", it is that an integrated open is read frame, terminal preceding 11 aminoacid of the N-of coding Gal4 (amino acid/11-147), the back is with the nuclear localization signal of SV40 large T antigen, the trans-activation domain of 130 aminoacid C-ends of hsv protein VP16, the bHLHLZ territory of c-Myc (aminoacid 350-439), the back is with SV40 polyA.The fusion gene VP16-TA-mc that produces is from first ATG, under the control of c-erbB-2 promoter " perbB2 ".Box gene 4 contains " GALp ", comprises 5 copies of the 17 poly-DNA binding sites of Gal4.The TET-ON sequence is under the control of GALp-tet promoter, and the therapeutic gene X is connected by IRES with TET-ON; Box gene 5 contains antisense TET-ON, and this sequence is made of the complementary series of preceding 80 bases of the TET-ON sequence that comprises ATG, under the control of pCMV promoter.Box gene 6 contains dominant TET-ON, and the coded sequence of the tet repressor protein amino acid/11-207 under being controlled by the pCMV promoter constitutes.

Figure 10 has illustrated the structure of pHIPs-GFP (hot derivable prostate specific promoter) expression vector.The pHIPS carrier comprises 6 boxes.Box gene 1 and aforementioned bearer different only are that it contains " Gal-DBD-mx ", this is that an integrated open is read frame, alkaline helix-loop-helix-leucine zipper (bHLHLZ) territory (mx of coding and Max, aminoacid 8-112) yeast GAL4 albumen N-end (amino acid/11-147) DNA of Rong Heing is in conjunction with the territory, and the back is with SV40 polyA.The fusion gene GAL-DBD-mx that produces can induce under the control of HSP promoter in heat.Box gene 2 contains minimum CMV promoter (mCMVp), " antisense Gal-DBD-mx " (it is and the complementary antisense construct of Gal-DBD-mx sequence), " IRES " (internal ribosome entry site) and " Gal-DBD " (with Gal-DBD-mx competition pGAL binding site).Box gene 3 comprises " VP16-TA-mc ", it is that an integrated open is read frame, terminal preceding 11 aminoacid of the N-of coding Gal4 (amino acid/11-147), the back is with the nuclear localization signal of SV40 large T antigen, the trans-activation domain of 130 aminoacid C-ends of hsv protein VP16, follow by SV40 polyA in the bHLHLZ territory of c-Myc (aminoacid 350-439).The fusion gene VP16-TA-mc that produces is from first ATG, under the control of prostate basic protein gene promoter (pProbasin).Box gene 4 contains " GALp ", comprises 5 copies of the 17 poly-DNA binding sites of Gal4.The TET-ON sequence is under the control of GALp-tet promoter, and therapeutic gene X is connected by IRES with TET-ON; Box gene 5 contains antisense TET-ON, and this sequence is made of the complementary series of preceding 80 bases of the TET-ON sequence that comprises ATG, under the control of pCMV promoter.Box gene 6 contains dominant TET-ON, and the coded sequence of the tet repressor protein amino acid/11-207 under being controlled by the pCMV promoter constitutes.

Detailed Description Of The Invention

Term used herein " heating " refers to the heat energy that any method produces, and comprises microwave.

Term used herein " light " refers to the luminous energy of frequency in visible and invisible wide spectrum scope, comprises the ionizing irradiation that any method produces.This can comprise irradiation source, if can launch the particulate radionuclide of γ or β, or pass through linear accelerator.

According to the present invention, can use conventional molecular biology, microbiology and recombinant DNA technology in the art technology.These technology have been proved absolutely in the literature.For example see Maniatis, Fritsch ﹠amp; Sambrook, " molecular cloning: laboratory manual (1982); " dna clone: hands-on approach " volume I and II (D.N.Glover compiles, 1985); " oligonucleotide is synthetic " (M.J.Gait compiles, 1984); " nucleic acid hybridization " [B.D.Hames ﹠amp; S.J.Higgins compiles (1985)]; " transcribe and translate " [B.D.Hames ﹠amp; S.J.Higgins compiles (1984)]; " animal cell culture " [R.I.Freshney compiles (1986)]; " immobilized cell and enzyme " [IRL Press (1986)]; B.Perbal." practice guideline of molecular cloning " (1984).

The present invention is directed to a kind of to the localized area, as the novel gene therapy method of tumor.According to above-mentioned purpose, provide a kind of and be heated and/or photoactivation by containing, and the plasmid of the element that the existence and the concentration of antibiotic (tetracycline and derivant thereof) is reacted, and the mechanism of composing type activation and regulate gene expression.In regulate gene expression, with heat or light-initiated expression, but only at antibiotic (tetracycline and derivant thereof) constitutive expression gene when existing.Antibiotic concentration has been controlled the level and the persistent period of gene expression.

For gene expression is limited to tumor cell, two kinds of mechanism are provided, be used for the expression of suppressor gene normal cell by target in heat or light target.For not being exposed to heat or light, absorbed the normal cell of this plasmid accidentally, because the expression of this therapeutic gene that the background activity of promoter causes, be structured in the plasmid, can be hot or photoinduced, the antisense of the dependent transcriptional activator of antibiotic and the constitutive expression of dominant DNA sequence suppress.For having absorbed this plasmid, be exposed to the normal cell of heat or light then, there is another kind of mechanism to prevent the expression of this therapeutic gene.I.e. " double cross " system by use modifying, wherein antibiotic dependent transcription activator this in the expression of tissue-specificity activator and be exposed to heat or the control of light under.Therefore, only be exposed to heat or light at the same time, and have the expression of this therapeutic gene in the cell of expression tissue idiosyncratic transcription factor.

In one embodiment of the invention, provide a kind of recombinant vector pDATH-X (dominant, antisense, TET-ON controlled thermal shock promoter plasmid), be used to reduce the background level of expression.This carrier is made of following box: (a) fusions of the coded sequence in tetracycline repressor protein amino acid/11-207 and herpes simplex virus VP16 PROTEIN C-130 amino acid whose transcription activatings of end territory; (b) heat shock promoter, by the heat shock response element (260 to 30) of human heat shock 70 gene promoters, with minimum cytomegalovirus promoter, pCMV connects and composes; (c), constitute by the reverse repetition (sequence) of the O2 operon 19bp of TN10 with tet repressor protein and the bonded tet operon of TAKON; (d) antisense sequences is made of the complementary series of preceding 80 bases of the TAKON sequence that comprises ATG.

Provide in another embodiment of the present invention to be implemented under heat or the control of light inducible promoters, the method for continuous expression gene comprises step: the carrier that will contain described gene is introduced host organisms; And apply heat or luminous energy.In another embodiment of the present invention, described host living beings is the people.

In another embodiment of the present invention, a kind of recombinant vector pDATE-X is provided (dominant, antisense, the controlled EGR promoter expression of TET-ON plasmid), described carrier comprises following box: (a) in box 1, the TET-ON sequence is under the control of EGRp, tetracycline operator binding site and pCMV; (b) in box 2, therapeutic gene X is under the control of tetp-pCMV promoter; (c) in box 3, antisense TET-ON is under the control of pCMV promoter; (d) in box 4, dominant TET-ON is under the control of pCMV promoter.

An alternative embodiment of the invention provides recombinant vector pRIBs-X, (shining derivable mammary gland-specific promoter) expression vector, described carrier contains following box: (a) box 1 contains " Gal-DBD-mx ", it is an open reading frame that merges, coding combines the territory with yeast GAL4 albumen (Gal-DBD) N-end (amino acid/11-147) DNA that Max alkalescence helix-loop-helix-leucine zipper domain (aminoacid 8-112) merges, follow by SV40polyA-, but the fusion gene GAL-DBD-mx of generation is under the control of radiation-induced Egr-1 promoter; (b) box 2 contains minimum CMV promoter, " antisense Gal-DBD-mx " (with the complementary antisense construct of Gal-DBD-mx sequence), " IRES " (internal ribosome entry site) and " Gal-DBD " (with Gal-DBD-mx competition pGAL binding site); (c) box 3 is made up of " VP16-TA-mc ", it is that an integrated open is read frame, preceding 11 aminoacid (amino acid/11-147) of the terminal coding of N-Gal4, the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, alkaline helix-loop-helix-leucine zipper domain of c-Myc (aminoacid 350-439), the back fusion gene VP-16-TA-mc that produces with SV40 polyA-is from first ATG, under the control of c-erbB2 promoter " perB2 "; (d) box 4 contains " Galp ", 5 copies of the 17 poly-DNA binding sites of Gal4.The TET-ON sequence is under the control of GALp-ptet promoter, and therapeutic gene X is connected by IRES with TET-ON; (e) box 5 contains antisense TET-ON, and this sequence is made of the complementary series of preceding 80 bases of the TET-ON sequence that comprises ATG, under the control of pCMV promoter; (f) box 6 contains dominant TET-ON, is made of the coded sequence of amino acid/11-207.

The variant of front carrier also is provided, and wherein the perbB2 promoter is replaced by whey acidic protein promoter or stromelysin 3 promoteres.

An alternative embodiment of the invention provides a kind of method for the treatment of local and transitivity mammary gland and ovarian cancer, comprising: patient is used the pRIBs-X expression vector (or its variant) that contains cytotoxic gene.Representational cytotoxic gene is a tumor necrosis factor.

Also (irradiation can be induced at reorganization pRIBs-X in the present invention, the prostate specific promoter) expression vector, described carrier contains following box: (a) box 1 contains " Gal-DBD-mx ", it is an open reading frame that merges, coding combines the territory with yeast GAL4 albumen N-end (amino acid/11-147) DNA that Max alkalescence helix-loop-helix-leucine zipper domain (aminoacid 8-112) merges, the back is with SV40polyA-, but the fusion gene GAL-DBD-mx of generation is under the control of radiation-induced Egr-1 promoter; (b) box 2 contains minimum CMV promoter, " antisense Gal-DBD-mx " (with the complementary antisense construct of Gal-DBD-mx sequence), " IRES " (internal ribosome entry site) and " Gal-DBD " (with Gal-DBD-mx competition pGAL binding site); (c) box 3 is made up of " VP16-TA-mc ", it is that an integrated open is read frame, preceding 11 aminoacid (amino acid/11-147) of the terminal coding of N-Gal4, the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, alkaline helix-loop-helix-leucine zipper domain of c-Myc (aminoacid 350-439), the back fusion gene VP-16-TA-mc that obtains with SV40 polyA-is from first ATG, under the control of prostate basic protein gene promoter " pProbasin "; (d) box 4 contains Galp, 5 copies of the 17 poly-DNA binding sites of Gal4.The TET-ON sequence is under the control of GALp-ptet promoter, and therapeutic gene X is connected by internal ribosome entry site with TET-ON; (e) box 5 contains antisense TET-ON, and this sequence is made of the complementary series of preceding 80 bases of the TET-ON sequence that comprises ATG, under the control of pCMV promoter; (f) box 6 contains dominant TET-ON, is made of the coded sequence of amino acid/11-207.Also considered the variant of aforementioned bearer, replaced prostate basic protein promoter with the prostate specific antigen promoter.

An alternative embodiment of the invention provides a kind of method for the treatment of local and metastatic prostate cancer, comprising: patient is used the pRIPs-X (or its variant) that contains cytotoxic gene.Representational cytotoxic gene is a tumor necrosis factor.

In another embodiment of the present invention, a kind of recombinant expression carrier pHIBs-X (hot derivable mammary gland-specific promoter) is provided expression vector, described carrier contains following box: (a) box 1 contains Gal-DBD-mx, it is an open reading frame that merges, coding combines the territory with yeast GAL4 albumen N-end (amino acid/11-147) DNA that Max alkalescence helix-loop-helix-leucine zipper domain (aminoacid 8-112) merges, the back is with SV40polyA-, and the fusion gene GAL-DBD-mx of generation is under can the control of thermoinducible heat shock protein promoter; (b) box 2 contains minimum CMV promoter, antisense Gal-DBD-mx (with the complementary antisense construct of Gal-DBD-mx sequence), internal ribosome entry site and Gal-DBD (with Gal-DBD-mx competition pGAL binding site); (c) box 3 is made up of " VP16-TA-mc ", it is that an integrated open is read frame, preceding 11 aminoacid (amino acid/11-147) of the terminal coding of N-Gal4, the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, alkaline helix-loop-helix-leucine zipper domain of c-Myc (aminoacid 350-439), the back fusion gene VP16-TA-mc that produces with SV40 polyA is from first ATG, under the control of c-erbB2 gene promoter " perbB2 "; (d) box 4 contains GaLp, 5 copies of the 17 poly-DNA binding sites of Gal4.The TET-ON sequence is under the control of GALp-ptet promoter, and therapeutic gene X is connected by internal ribosome entry site with TET-ON; (e) box 5 contains antisense TET-ON, and this sequence is made of the complementary series of preceding 80 bases of the TET-ON sequence that comprises ATG, under the control of pCMV promoter; (f) box 6 contains dominant TET-ON, is made of the coded sequence of amino acid/11-207.Also considered the variant of aforementioned bearer, replaced the perbB2 promoter with whey acidic protein promoter or stromelysin 3 promoteres.

An alternative embodiment of the invention provides a kind of method for the treatment of local and transitivity mammary gland and ovarian cancer, comprising: patient is used the pHIBs-X expression vector (or its variant) that contains therapeutic genes.Representational therapeutic genes is a tumor necrosis factor.

An alternative embodiment of the invention provides a kind of recombinant expression carrier pHIPs-X (hot derivable prostate specific promoter) expression vector, described carrier contains following box: (a) box 1 contains Gal-DBD-mx, it is that an integrated open is read frame, coding combines the territory with yeast GAL4 albumen N-end (amino acid/11-147) DNA that Max alkalescence helix-loop-helix-leucine zipper domain (aminoacid 8-112) merges, the back is with SV40polyA-, and the fusion gene GAL-DBD-mx of generation is under the control of the derivable heat shock protein promoter of heat; (b) box 2 contains minimum CMV promoter, antisense Gal-DBD-mx (with the complementary antisense construct of Gal-DBD-mx sequence), internal ribosome entry site and Gal-DBD (with Gal-DBD-mx competition pGAL binding site); (c) box 3 is made up of " VP16-TA-mc ", it is that an integrated open is read frame, preceding 11 aminoacid (amino acid/11-147) of the terminal coding of N-Gal4, the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, alkaline helix-loop-helix-leucine zipper domain of c-Myc (aminoacid 350-439), the back fusion gene VP16-TA-mc that produces with SV40 polyA-is from first ATG, under the control of prostate basic protein gene promoter " pProbasin "; (d) box 4 contains GaLp, 5 copies of the 17 poly-DNA binding sites of Gal4.The TET-ON sequence is under the control of GALp-ptet promoter, and therapeutic gene X is connected by internal ribosome entry site with TET-ON; (e) box 5 contains antisense TET-ON, and this sequence is made of the complementary series of preceding 80 bases of the TET-ON sequence that comprises ATG, under the control of pCMV promoter; (f) box 6 contains dominant TET-ON, is made of the coded sequence of amino acid/11-207.Also considered the variant of aforementioned bearer, replaced prostate basic protein promoter with the prostate specific antigen promoter.

In another embodiment of the present invention, provide a kind of method for the treatment of local and metastatic prostate cancer, having comprised: patient is used the pHIPs-X expression vector (or its variant) that contains therapeutic genes.Representational therapeutic genes is a tumor necrosis factor.

Considered especially to prepare the pharmaceutical composition of the present invention that is used for gene therapy.Thus, said composition contains acceptable carrier on carrier of the present invention and the materia medica.Have the experiment that the personnel of general technology need not be too much in the cancer chemotherapy field and just can determine the suitable dose of dispenser and approach easily.For gene therapy, the interested gene that is included in one of plasmid of the present invention can pass to target cell by viral vector or liposome.

The those skilled in the art's of cancer biology field level has improved in recent years greatly.Those of ordinary skills can make up and utilize the plasmid of this novel method to carry out gene therapy according to the spirit of this description.

Provide following example and be for different embodiments of the invention are described, and do not mean and limit the present invention in any form.

Embodiment 1

PDATE carrier: the model of structure and effect

Fig. 2 is the sketch map of pDATE carrier.The pDATE-X plasmid works by the feedforward reaction, amplifies the expression of TET-ON and X.In the time need not shining,, the very weak background of EGRp will cause TET-ON mRNA synthetic because expressing.The translation of this mRNA of having followed expression decreased of antisense TET-ON RNA.In addition, leaking the TET-ON albumen of translating is non-activity when not having tetracycline.When tetracycline exists, (translation) TET-ON albumen that the leaked activity that become, but the dominant negative TET-ON albumen of constitutive expression (same-DNA binding site of competition ptet promoter) has prevented the feedforward reaction.

Insert two chicken matrix of lysosome attachment sites (MAR), with the effect of the position of Seperating box (McKnight, R.A. is etc., Mol.reprod ﹠amp; Dev.44:179-184 (1996)).Though when minimum CMV promoters driven antisense and dominant negative TET-ON expression, their may be optional, if with stronger promoter, when driving their expression as people β actin promoter, may need MAR.

When the cellular exposure of carrying pDATE-X in when irradiation, the first peak that TET-ON transcribes takes place, cause Synthetic 2-4 times to the TET-ON of background level, substantially exceeded dominant negative TET-ON.When tetracycline existed, this excessive TET-ON albumen combined with the tetp promoter then, was connected with the coded sequence of TET-ON and X with this promoter, and participated in the feedforward reaction.This reaction is controlled by the tetracycline level.Same, the X expression is enhanced and prolongs, and is removed until tetracycline, needs how long just begin once more the feedforward reaction with tetracycline when the proteic half-life of this time point TET-ON will determine no longer to be exposed to irradiation.

But this carrier utilization feedforward reaction realizes and has kept high-caliber inducible gene expression.This feedforward feature has overcome the instantaneity and the faint property of inducible promoters.When feedforward reaction is limited to a few hours, in heating with do not heat the TET-ON level that reaches in the cell and be very different.Therefore may add and remove tetracycline by other, strengthen TET-ON, regulate the difference of the TET-ON level that increases in irradiation and the non-irradiated cell.Yet,,, be difficult in vivo accomplish because the absorption of the interior tetracycline of the inhomogeneous and Different Individual body of tetracycline level is different with discharge in the tissue though can in cell culture, accurately control adding and remove tetracycline.Therefore, key is will to minimize by the seepage that TET-ON expresses with antisense and dominant negative box, does not significantly improve its level thereby feedforward is not reflected in the irradiating cell.

As needs, available much better than promoter is replaced the pCMV minimal promoter as people β actin promoter, drives the expression of antisense and dominant negative TET-ON, the effect of further meticulous this carrier of adjustment.In addition, can increase the copy number of antisense and dominant coded sequence.

In order to induce TET-ON to express in the body, used oxytetracycline, because half-life weak point in its body.In the people, after single agent is oral, has reached the oxytetracycline peak plasma concentration in the time of 2-4 hour and (for example seen Goodman; The therapeutic materia medica of Gilman ' s basis).Therefore can monitor TET-ON expression as the oxytetracycline concentration function.Oxytetracycline is very short action time, the only 9 hours half-life in the body (half-life of doxycycline is 18 hours relatively).At 24 hours ends, the oxytetracycline level was reduced to below 25% of input (about 10-30% never is absorbed, and is discharged from activity form).

Embodiment 2

PDATH carrier: the model of structure and effect

Fig. 1 is the sketch map of pDATH-X carrier.This carrier works in the identical mode of pDATE-X carrier, except having replaced the Egr-1 promoter with the HSP promoter, replaces light/ionizing irradiation with heat.

Embodiment 3

Amplify and the checking of the notion of continuous expression TET-On and p53 with the feedforward inducible promoters

Gene expression notion in order to verify that further hot derivable tetracycline feedforward is amplified has made up two plasmids.Wild type p53 cDNA sub-clone is gone into ptet-splice carrier (Gibco BRL) (this carrier places p53 under the control of tetp promoter (regulating and controlling sequence by the tetracyclin resistance operon of minimum hCMV promoter upstream constitutes)), made up plasmid " ptet-splice p53wt ".The CMV promoter of replacing among the ptet-on (Clontech) with HUMAN HEAT SHOCK PROTEINS Hhsp HSP70 promoter and the tetp promoter of 300bp has made up plasmid " HSP-tetp-TET-ON ".

In the RPMI+10% hyclone, cultivate H358, a kind of non--small cell lung cancer cell system with p53 homozygous deletion.With 50 micrograms " ptet-splice p53wt " and 10 micrograms " HSP-tetp-TET-ON ", use BRL cell polarizer at 1180 μ F and 240V, the cell of cotransfection 107 logarithmic growths in the 0.8mlRPMI+6mM glucose.The inoculation transfectional cell grew to 25% and is paved with in 36 hours, wherein half of 45 ℃ of heat shocks is 20 minutes then.After the heat shock 6 hours, handle cell with the doxycycline of various dose.Different time points after adding doxycycline with monoclonal p53 antibody DO-1 (Santa Cruz Biologicals), uses immunoperoxidase cell staining reagent box (Vector) and diaminobenzidine (DAB) to make the immunohistochemical staining cell.At each time point, catch the digital image of 50 immunostaining cells with the Nikon microscope.The protein content and the staining power of expressing in each cell are proportional, are expressed as I=1/T, and wherein T is measuring of projection light/unit are.Fig. 8 has shown the result with one of this experiment that the 0.01-0.1 microgram/the ml doxycycline carries out.

When heating back 6 hours (at this moment inductive TET-ON level should reach its peak value) adds 0.1 mcg/ml, reach after 10 hours the p53 that amplifies more than 12 times (curve a and b, Fig. 8).At this moment, as the substantial level of TET-ON showed, doxycycline had also begun the feedforward reaction in not heating cell.Yet, because this amplification is from lower level, the amplification level of 10 hours TET-ON only reach low-level (curve c and d, Fig. 8).

The level that the alternate scheme that adds with tetracycline in feedforward system and remove is regulated and control inductive p53 is possible.At this moment, think after removing, do not heat TET-ON in the cell (as Fig. 8 level [c]) and fall back background level [e], and the TET-ON level [a] in the cell that heated also should descend with similar ratio (equaling [c]-[e]).Yet, because this level ([a]-[c]-[e]) is more much higher than not heating in the cell [e], add tetracycline will be again from much higher level ([a]-[c]-[e]) begin to be heated feedforward reaction the cell.Same, low-level ([c]) that the level of background p53 will keep or reach in the time of 10 hours in Jia Re the cell not, or lower, and the p53 level that heats in the cell will continue rising.Therefore, be instantaneous though TNF α that HSP directly drives and p53 express, the expression that feedforward system drives continues, and continues, as long as tetracycline exists.Because tetracycline vivo degradation speed has determined tetracycline to add intravital scheme, it is important understanding the half-life of TET-ON in tumor cell.

In vivo, the pharmacokinetics of tetracycline in different tissues is uneven.The preferred concentration of tetracycline will cause TET-ON background higher in some tissue to be expressed in the particular organization.For example, in the people, removed the oxytetracycline of 10-35% by kidney, wherein sizable amount is discharged with activity form.Therefore, it is desirable at the very start the background expression is being minimized, to prevent that amplification is out of control in non-destination organization.But pDATE and pDATH inducible system adopt the antisense TET-ON of constitutive expression to suppress the background level of TET-ON translation, and with the TET-ON competition that dominant TET-ON comes and leaked expression, express to suppress background.Consistent with the background that suppresses, the time that adds tetracycline only is subjected to the desired level of therapeutic gene expression and the influence of persistent period, and is not subjected to suppress in normal not irradiating cell the influence of the needs of background expression.

Embodiment 4

The reduction of background expression

Use the 300bpHSP promoter, need not heat or the background expression of light be with about 25% of heat or light finding level.In order to reduce this background level ,-80 of HSP and minimum pCMV promoter is connected to+30.Because the background that pCMV is lower is expressed, this promoter is preferred.In addition, the higher amplification that it allows therapeutic gene to express is not subjected to the restriction of more weak HSP promoter, and this promoter can be used for causing the explosive reaction of heat or light.

In order further to overcome the problem that background is expressed, two boxes are introduced plasmid pDATH.The TAKON antisense sequences is placed under the control of pCMV promoter.The antisense sequences that this composing type produces can have adopted mRNA to combine with any TAKON that transcribes from background, and prevents that it is translated.In box #4, provide the another kind that background is transcribed to blockade, wherein will have the DNA binding site but do not have the dominant negative TAKON of transcription activating domain to place under the control of pCMV.This causes background to transcribe the generation that drives TAKON and dominant TAKON, competes the ptet binding site then.

Embodiment 5

The monitoring of p53 expression

In order to ensure the antisense TAKON and the dominant negative TAKON albumen that produce proper level, monitoring p53 expression is demarcated the copy number and the intensity that reduce the required promoter of background.The first, when tetracycline does not exist, separate the cell line of carrying pDATH.Monitor the ptet-EGFP of p53 level or cotransfection then, to determine that reducing background expresses the required antisense TAKON of pDATH and the copy number of dominant TAKON of mixing.

Embodiment 6

The expression vector pRIBs of treatment part and transitivity mammary gland and ovarian cancer

As mentioned above, in such promoter, as to shine derivable Egr-1 gene promoter control gene down be transient expression usually, and be low-level.This makes them not be suitable for treatment of cancer.In order to overcome these problems, designed expression vector pRIBs-X (shining derivable mammary gland-specific promoter).

With feedforward reaction and tetracycline dependency trans-activator, Tet-On places under the control of tetracycline promoter (tetp), afterwards with GAL-4 promoter (pGAL) thus optimized gene expression dose.Instantaneous transcribing at the pGAL place causes causing synthetic low-level Tet-On, and it combines with tetp in the presence of tetracycline then.Then, amplify by feedforward reaction Tet-On and himself express and the expression of connected therapeutic gene.The expression of therapeutic gene is subjected to the control (Fig. 3) of 6 box genes in the pRIBs carrier.In box 1, fusion gene GAL-DBD-mx (the HLH-LZ territory of max combines the territory with the DNA-of GAL-4 and merges) is subjected to the regulation and control of EGRp.The background of GAL-DBD-mx is expressed the inhibition of dominant GAL-DBD in the antisense GAL-DBD-mx that is subjected to constitutive expression and the box 2.In box 3, merge in the HLH-LZ territory of the transcription activating domain of hsv protein VP16 and c-Myc.The fusion gene VP16-TA-mc that produces expresses in the breast tumor cell of overexpression c-erbB-2 under the control of c-erbB-2 promoter.By replenishing VP16-TA-mc albumen, the GAL-DBD-mx fusion rotein combines with transcribing of pGAL promoter (box 4), and activates it and transcribe.

In irradiating cell not, the translation of background GAL-DBD-mx mRNA reduces, and dominant negative GAL-DBD (not having mx) competitiveness occupies the GALp in the box 4, the Tet-On expression of having blockaded.After the irradiation, instantaneously induce 3-4 GAL-DBD-mx doubly, and temporarily overcome the inhibition of box 2.GAL-DBD-mx replenishes VP-16-TA-mc (leucine zipper of VP16 trans-activation domain and myc is under the control of c-erbB-2 promoter) to GALp, and activates low-level Tet-On and transcribe, and begins the feedforward reaction.

For example in the therapeutic scheme that uses pRIBs-TNF α, can liposome complex or give tumor or normal cell as the recombinant virus systemic delivery.Need not shine and tetracycline, not express TNF α.The systemic administration oxytetracycline is followed X-ray and is shone known metastatic tumour position then.As a result, induced the TNF alpha expression with X-ray, and amplified by oxytetracycline or keep at tumor locus.Even not every tumor cell all can absorb pRIBs-TNF α, but be exposed under the TNF α of excretory very high concentration at the contiguous tumor cell of the cell of picked-up.The TNF alpha expression of breast tumor cell is only given in the design of pRIBs-TNF α, and is not contained in irradiated Normocellular expression in the X-ray light path.So, if any system toxicity is arranged, also only limit to the low-level TNF α that diffuses out from tumor cell.In addition or replace TNF α, can use another therapeutic gene of called after X with the pRIBS carrier.

Fig. 3 has shown the structure of pRIBs-GFP-1, and Fig. 5 has summed up action model.In irradiating cell not, box 2 has suppressed background GAL-DBD-mx in two ways expresses and function.The antisense thing of GAL-DBD-mx has suppressed the translation of background GAL-DBD-mx mRNA, and GAL-DBD albumen has played the effect of dominant mortifier by competing the pGAL promoter with GAL-DBD-mx.In irradiating cell, the instantaneous 3-4 GAL-DBD-mx doubly that induced expresses, and has overcome the inhibition of box 2.GAL-DBD-mx replenishes VP-16-TA-mc (fusion rotein of the leucine zipper of VP16 trans-activation domain and Myc is under the control of c-erbB-2 promoter) to GALp, and activates the transient expression of trans-activator TET-ON.In the presence of tetracycline, activate Tet-ON, it combines with the tetp promoter, and this promoter of trans-activation (Gossen, M., etc., science, 268:1766-1769 (1995)), in the feedforward reaction, amplify own level and GFP.When not having irradiation or tetracycline, the background of no TET-ON and GFP is expressed.

Embodiment 7

Stably express pRIBs-GFP cell line and different generation of planting body

The antisense that has 1 and 4 copy respectively and two kinds of pRIBs-GFP plasmids of dominant box gene have been made up, pRIBs-GFP-1 and pRIBs-GFP-4, and stable transfection to go into fibrosarcoma cell be HTB152 and breast tumor cell line SK-BR-3 and MDAMB231, with analyzed in vitro.With 5 * 10 ⁶The SCID mice is gone in individual cell xenotransplantation.Though all three-type-person's cell lines have formed PD tumor, only SK-BR-3 expresses high-caliber c-erbB-2.In fact, single-chain antibody in anti--erbB-2 born of the same parents of downward modulation cell surface erbB-2, only in SK-BR-3, but not apoptosis-induced in MDA-MB-231 (Chumakov A.M., etc., oncogene 8:3005-3011 (1993)).

Therefore pRIBs-GFP-1 and-4 plasmids are used as model, optimize in nude mice with the different condition of planting body of cytotoxic gene treatment transitivity breast tumor.Owing to only do not inducing the cytotoxic gene that is connected in EGRp in the irradiating cell, eliminated the toxicity of irradiating cell not.Yet, importantly prevent to express in the normal cell of cytotoxic gene in the X-ray light path.These different in c-erbB-2 expresses three kinds of cell lines show, express with tissue or tumor-specific promoters control VP16-TA-mc, this expression only can be limited in the non-irradiated breast tumor cell, and the normal cell through irradiation of the vital organ that is not arranged at metastatic cancer cell is expressed.

As shown in Figure 3, assembled the pRIBs-GFP plasmid.GAL-DBA-mx and VP16-TA-mc from the mammal two-hybrid system, modify and get (Fearon, E.R., etc., Proc.Natl.Acad.Sci.USA, 89:7958-7962 (1992)).Two kinds of plasmids have been tested, pRIBs-GFP-1 and pRIBs-GFP-4 (having antisense and the dominant GAL-DBD of 1 and 4 copy) by minimum CMV promoters driven.

With pRIBs-GFP and whole three cell lines of SVneo plasmid co-transfection.By in G418, selecting, separated the cell line of stably express pRIBs-GFP-1 and pRIBs-GFP-4.For the body inner analysis, with individual each cell line, stably express pRIBs-GFP plasmid 5 * 10 ⁶Cell is implanted SCID mice flank (4 every group), and makes it long to diameter 0.5cm.The protein extracting of the tumor of grinding in the EBC buffer, and with the quantitative protein content of RIA, has been analyzed without irradiation or there is not the external and different expression of planting in the body of the GFP of oxytetracycline.

Measured external GFP with western blot analysis and can induce level, and with 0-4Gy,, used the RIA quantitative assay after using 0-2 μ g/ml oxytetracycline then with VarianClinac X-ray generator irradiating cell.With the data show of HSPp, the feedforward reaction is very effective, and 0.01 μ g/ml enough induced p53 to express in 10 hours increases by 9 times.Shine after 6 hours intraperitoneal injection 0-15 μ g/g oxytetracycline.Inject (totally 24 hours) in the back 3 hours interval, take out tumor mass, measure TET-ON and GFP amount with respect to the actin total amount.In order to reach the GFP of higher or reduced levels, regulate oxytetracycline DM TET-ON level and repeat these experiments.By analyzing three hours plasma concentration in the interval, the speed of removing oxytetracycline is drained in monitoring.

Embodiment 8

With WAPp or ST3p targeted metastatic breast tumor

Selected the c-erbB-2 promoter to come this pRIB-X notion of preliminary identification, because human cancer overexpression c-erbB-2 is relevant with poor prognosis.Yet a kind of specific promoter can not solve the problem of the different breast tumor of treatment.Therefore, with GAL-DBD-mx expression whey acidic protein promoter, WAPp (McKnight, R.A., etc., Mo1.reprod ﹠amp; Dev., 44:179-184 (1996)) or stromelysin 3 promoter ST3p (Ahmad, A. is etc., cancer international magazine 73:290-296 (1997)) targeting also be important in the transitivity breast tumor.WAPp is targeted to galactophore epithelial cell with expression, and ST3p will express targeting in the substrate-metalloproteases that adjoins tumor-secreted stromal cell.

Replace the c-erbB-2 promoter with WAPp or ST3p, reconstruct pRIBs.The WAP of screening mammary gland and other tumor cell line and ST3 are high and low to express.Adopt WAP and/or ST3 to express the expression of different cell line test GFP.

Show, the WAP promoter is very single-minded (Tzeng YJ. to lactogenic mammal epithelial cell in the transgenic animal, Deng, oncogene 16 (16): 2103-2114 (1998)), and shown that stromelysin 3 promoter ST3p only express in adjoining the substrate fibroblast of cancerous cell.The evidence prompting, the generation of the stromal cell mesostroma-metalloproteases (comprising ST3) that relates in the neoplasm metastasis process is stimulated by cancerous cell.Therefore, the VP16-TA-mc targeting will cause the therapeutic gene product to express near the transitional cell tumor and discharge in stromal cell.Must be noted that by pRIBs-X being passed to c-erbB2 and obtained the treatment specificity that increases with the liposome that has wrapped up antibody.

Embodiment 9

The expression vector pRIPs of treatment part and metastatic prostate cancer

As mentioned above, usually only transient expression and level are very low to place the gene that shines under the derivable Egr-1 gene promoter control.This makes them not be suitable for treatment of cancer.In order to overcome these problems, designed expression vector pRIPs-X (shining derivable prostate specific promoter).

The pRIPs carrier contains 6 boxes.Box gene 1 is different with aforesaid carrier, only be that it contains " Gal-DBD-mx ", it is the OPF that merges, merge in coding and alkaline helix-loop-helix-leucine zipper (bHLHLZ) territory of Max, afterwards with the N-terminal DNA of the yeast GAL4 albumen (GAL-DBD) of SV40poly A in conjunction with territory (amino acid/11-147).Box gene 2 contains minimum CMV promoter (mCMVp), " antisense Gal-DBD-mx " (it is and the complementary antisense construct of Gal-DBD-mx sequence), " IRES " (it is an internal ribosome entry site) and " Gal-DBD " (it and Gal-DBD-mx competition pGAL binding site).Box gene 3 contains " VP16-Ta-mc ", it is the ORF that merges, preceding 11 aminoacid at N-terminal coding Gal4 (amino acid/11-147), the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, the bHLHLZ territory of c-Myc (aminoacid 350-439), the back is with SV40polyA.The fusion gene VP16-TA-mc that produces is from first ATG, under prostate basic protein gene promoter " pProbasin " control.Box gene 4 contains " GALp ", is made of 5 copies of the 17 poly-DNA binding sites of Gal4.The TET-ON sequence is under the control of GALp-ptet promoter, and therapeutic gene X is connected with TET-ON by an IRES; Box gene 5 contains antisense TET-ON, and it is the sequence that the complementary series by preceding 80 bases of the TET-ON sequence that comprises ATG constitutes, under the control of pCMV promoter.Box gene 6 contains dominant TET-ON, and the coded sequence of the tet repressor protein amino acid/11-207 under being controlled by the pCMV promoter constitutes.In the variant of other pRIPs-X, use PSA, the promoter region of prostate specific antigen or other prostate specific gene is replaced the prostate basic protein.

Embodiment 10

The expression vector pHIBs-X of treatment part and transitivity mammary gland and ovarian cancer

Designed expression vector pHIBs-X, it is made of 6 boxes.Box gene 1 and aforementioned bearer different only are that it contains " Gal-DBD-mx ", this is the OPF that merges, alkaline helix-loop-helix-leucine zipper (bHLHLZ) territory (mx of coding and Max, aminoacid 8-112) merge, the back with the N-terminal DNA of the yeast GAL4 albumen (GAL-DBD) of SV40poly A in conjunction with territory (amino acid/11-147).The gene GAL-DBD-mx that the produces derivable HSP promoter control of being heated.Box gene 2 contains minimum CMV promoter (mCMVp), " antisense Gal-DBD-mx " (it is and the complementary antisense construct of Gal-DBD-mx sequence), " IRES " (it is an internal ribosome entry site) and " Gal-DBD ", (it and Gal-DBD-mx competition pGAL binding site).Box gene 3 contains " VP16-Ta-mc ", it is the ORF that merges, preceding 11 aminoacid at N-terminal coding Gal4 (amino acid/11-147), the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, the bHLHLZ territory of c-Myc (aminoacid 350-439), the back is with SV40polyA.The fusion gene VP16-TA-mc that produces begins to be subjected to the control of c-erbB-2 promoter " perbB2 " from first ATG.Box gene 4 contains " GALp ", is made of 5 copies of the 17 poly-DNA binding sites of Gal4.The TET-ON sequence is under the control of GALp-ptet promoter, and therapeutic gene X is connected with TET-ON by an IRES; Box gene 5 contains antisense TET-ON, and it is the sequence that the complementary series by preceding 80 bases of the TET-ON sequence that comprises ATG constitutes, under the control of pCMV promoter.Box gene 6 contains dominant TET-ON, and the coded sequence of the tet repressor protein amino acid/11-207 under being controlled by the pCMV promoter constitutes.

The pHIBs-X expression vector is identical with the pRIBs-X plasmid, and in box gene 1, the Egr-1 promoter among the pRIBs-X is replaced by the HSP70 promoter.When tumor is exposed to when hot pHIBs-X specificity targeted local and transitivity mammary gland and ovarian tumor.

Embodiment 11

The expression vector pHIPs-X of treatment part and metastatic prostate cancer

Figure 10 has illustrated the structure of pHIPs-GFP (heat can be induced, the prostate specific promoter) expression vector.This carrier contains 6 boxes.Box gene 1 and aforementioned bearer different only are that it contains " Gal-DBD-mx ", it is the OPF that merges, alkaline helix-loop-helix-leucine zipper (bHLHLZ) territory (mx of coding and Max, aminoacid 8-112) merge, the back with the N-terminal DNA of the yeast GAL4 albumen (GAL-DBD) of SV40poly A in conjunction with territory (amino acid/11-147).The fusion gene GAL-DBD-mx that produces is subjected to and can thermoinducible HSP promoter controls.Box gene 2 contains minimum CMV promoter (mCMVp), " antisense Gal-DBD-mx " (it is and the complementary antisense construct of Gal-DBD-mx sequence), " IRES " (it is an internal ribosome entry site) and " Gal-DBD " (it and Gal-DBD-mx competition pGAL binding site).Box gene 3 contains " VP16-Ta-mc ", it is the ORF that merges, preceding 11 aminoacid at N-terminal coding Gal4 (amino acid/11-147), the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, the bHLHLZ territory of c-Myc (aminoacid 350-439), the back is with SV40polyA.The control that the fusion gene VP16-TA-mc that produces is begun from first ATG by prostate basic protein gene promoter (pProbasin).Box gene 4 contains " GALp ", is made of 5 copies of the 17 poly-DNA binding sites of Gal4.The TET-ON sequence is under the control of GALp-ptet promoter, and therapeutic gene X is connected with TET-ON by an IRES; Box gene 5 contains antisense TET-ON, and it is the sequence that the complementary series by preceding 80 bases of the TET-ON sequence that comprises ATG constitutes, under the control of pCMV promoter.Box gene 6 contains dominant TET-ON, and the coded sequence of the tet repressor protein amino acid/11-207 under being controlled by the pCMV promoter constitutes.

The pHIPs-X expression vector is identical with the pRIPs-X plasmid, and in box gene 1, the Egr-1 promoter among pRIBs-X and the pRIPs-X is replaced by the HSP70 promoter.When tumor is exposed to when hot pHIPs-X specificity targeted local and metastatic prostate tumor.

Any patent or the publication mentioned in this description are declarative to the technical staff who the present invention relates to the field.These patents and publication are incorporated herein for your guidance with same degree, as each independently publication be want special and the introducing that separates for your guidance.

Person skilled in the art will readily appreciate that the present invention is very suitable for implementing purpose, and obtain described result and interests, and mentioned in this article those.Embodiment described herein and method, program, processing, molecule and specific compound are representative with the preference, are exemplary, rather than will limit the scope of the invention.What those skilled in the art incited somebody to action changes and other purposes, all within the spirit of the present invention that claim limits.

Claims

1. recombinant vector pDATH-X, it is dominant, antisense, TET-ON controlled thermal shock protein promoter plasmid, it is characterized in that this carrier contains following box gene:

(a) fusions of the coded sequence of tetracycline repressor protein amino acid/11-207 and herpes simplex virus VP16 PROTEIN C-last 130 aminoacid transcriptional activation domains of end;

(b) a heat shock promoter, described promoter is connected and composed with minimum giant cell promoter pCMV by the heat shock response element (260 to 30) of human heat shock 70 gene promoters;

(c) a tet operon is made of the inverted repeat of 19 bases of the operon O2 of tet repressor protein and the bonded TN10 of TAKON; With

(d) antisense sequences is by comprising that complementary seriess ATG, preceding 80 bases of TAKON sequence constitute.

One kind under the control of heat or light inducible promoters, realize the method for gene continuous expression, it is characterized in that the step that this method comprises is:

The carrier that will contain described gene is introduced host organisms; With

Apply heat or luminous energy.

3. method as claimed in claim 2 is characterized in that described host organisms is the people.

4. recombinant vector pDATE-X, it is dominant negative, antisense, the controlled EGR promoter expression of TET-ON plasmid, it is characterized in that this carrier contains following box gene:

(a) box 1 is included in the TET-ON sequence under the EGRp control, tetracycline operator binding site and pCMV;

(b) box 2 is included in the therapeutic gene X under the control of tetp-pCMV promoter;

(c) box 3 is included in the antisense TET-ON under the control of pCMV promoter; With

(d) box 4 is included in the dominant negative TET-ON under the control of pCMV promoter.

5. recombinant vector pRIBs-X, it is derivable, the mammary gland-specific promoter expression vector of irradiation, it is characterized in that this carrier comprises following box gene:

(a) box 1 contains " Gal-DBD-mx ", it is that an integrated open is read frame, the alkaline helix-loop-helix of coding and Max aminoacid 8-112-leucine zipper domain merges, the back with the DNA of the proteic-terminal amino acid 1-147 of yeast GAL4 of SV40 polyA in conjunction with territory Gal-DBD, from the derivable Egr-1 promoter control of fusion gene GAL-DBD-mx exposure;

(b) box 2 contains minimum pCMV promoter, and " antisense Gal-DBD-mx ", it is and the complementary antisense construct of Gal-DBD-mx, internal ribosome entry site IRES and " Gal-DBD ", and it competes the pGAL site with Gal-DBD-mx;

(c) box 3 contains " VP16-TA-mc ", it is that an integrated open is read frame, preceding 11 aminoacid at N-terminal coding Gal4 amino acid/11-147, the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, alkaline helix-loop-helix-leucine zipper domain of c-Myc aminoacid 350-439, the back is with SV40 polyA, and the fusion gene VP16-TA-mc that obtains begins under the control of c-erbB2 promoter " perB2 " from first ATG;

(d) box 4 contains " GaLp ", 5 copies of the 17 poly-DNA binding sites of Gal4, and wherein the TET-ON sequence is connected with TET-ON by IRES with a therapeutic gene X under the control of GALp-ptet promoter;

(e) box 5 contains antisense TET-ON, and it is by under pCMV promoter control, comprises the sequence that the complementary series of preceding 80 bases of the TET-ON sequence of ATG constitutes; With

(f) box 6 contains the dominant negative TET-ON of the coded sequence formation of amino acid/11-207.

6. recombinant vector as claimed in claim 5 is characterized in that, has replaced the perbB2 promoter with the whey acidic protein promoter.

7. recombinant vector as claimed in claim 5 is characterized in that, has replaced the perbB2 promoter of box 3 with stromelysin 3 promoteres.

8. recombinant vector as claimed in claim 5 is characterized in that, described gene X is the gene of codes for tumor necrosin ﹠.

9. method for the treatment of local and transitivity breast carcinoma and ovarian cancer, it is characterized in that the method comprising the steps of: the patient to these treatments of needs uses the described expression vector of claim 5.

10. method for the treatment of local and transitivity breast carcinoma and ovarian cancer, it is characterized in that the method comprising the steps of: the patient to these treatments of needs uses the described expression vector of claim 6.

11. a method for the treatment of local and transitivity breast carcinoma and ovarian cancer, it is characterized in that the method comprising the steps of: the patient to these treatments of needs uses the described expression vector of claim 7.

12. a recombinant vector pRIPs-X, it is derivable, the prostate specific promoter expression vector of irradiation, it is characterized in that this carrier comprises following box gene:

(a) box 1 contains " Gal-DBD-mx ", it is that an integrated open is read frame, the alkaline helix-loop-helix of coding and Max aminoacid 8-112-leucine zipper domain merges, the back with the DNA of the proteic-terminal amino acid 1-147 of yeast GAL4 of SV40 polyA in conjunction with territory Gal-DBD, the derivable Egr-1 promoter control of the fusion gene GAL-DBD-mx exposure of generation;

(b) box 2 contains minimum pCMV promoter, antisense Gal-DBD-mx, and it is and the complementary antisense construct of Gal-DBD-mx, internal ribosome entry site IRES and GaI-DBD, it and Gal-DBD-mx competition pGAL site;

(c) box 3 contains " VP16-TA-mc ", it is that an integrated open is read frame, preceding 11 aminoacid at N-terminal coding Gal4 amino acid/11-147, the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, alkaline helix-loop-helix-leucine zipper domain of c-Myc aminoacid 350-439, the back is with SV40 polyA, and the fusion gene VP16-TA-mc of generation begins under the control of prostate basic protein promoter " pProbasin " from first ATG;

(f) box 6 contains the dominant TET-ON of the coded sequence formation of amino acid/11-207.

13. recombinant vector as claimed in claim 12 is characterized in that, has replaced described prostate basic protein promoter of box 3 with the prostate specific antigen promoter.

14. recombinant vector as claimed in claim 12 is characterized in that, described gene X is the gene of codes for tumor necrosin ﹠.

15. a method for the treatment of local and metastatic prostate cancer, it is characterized in that the method comprising the steps of: the patient to these treatments of needs uses the described expression vector of claim 12.

16. a method for the treatment of local and metastatic prostate cancer, it is characterized in that the method comprising the steps of: the patient to these treatments of needs uses the described expression vector of claim 13.

17. a recombinant expression carrier pHIBs-X, it is derivable, the mammary gland-specific promoter expression vector of heat, it is characterized in that this carrier comprises following box gene:

(a) box 1 contains Gal-DBD-mx, it is that an integrated open is read frame, the alkaline helix-loop-helix of coding and Max aminoacid 8-112-leucine zipper domain merges, the back with the DNA of the proteic-terminal amino acid 1-147 of yeast GAL4 of SV40 polyA in conjunction with territory Gal-DBD, the fusion gene GAL-DBD-mx of the generation derivable heat shock protein promoter control of being heated;

(b) box 2 contains minimum pCMV promoter, antisense Gal-DBD-mx, and it is and the complementary antisense construct of Gal-DBD-mx, internal ribosome entry site and Gal-DBD, it and Gal-DBD-mx competition pGAL binding site;

(c) box 3 contains " VP16-TA-mc ", it is that an integrated open is read frame, preceding 11 aminoacid at N-terminal coding Gal4 amino acid/11-147, the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, alkaline helix-loop-helix-leucine zipper domain of c-Myc aminoacid 350-439, the back is with SV40 polyA, and the fusion gene VP16-TA-mc of generation begins under the control of c-erbB2 promoter " perB2 " from first ATG;

(d) box 4 contains GALp, 5 copies of the 17 poly-DNA binding sites of Gal4, and wherein the TET-ON sequence is connected with TET-ON by internal ribosome entry site with a therapeutic gene X under the control of GALp-ptet promoter;

18. recombinant vector as claimed in claim 17 is characterized in that, has replaced the perbB2 promoter of box 3 with the whey acidic protein promoter.

19. recombinant vector as claimed in claim 17 is characterized in that, has replaced the perbB2 promoter of box 3 with stromelysin 3 promoteres.

20. recombinant vector as claimed in claim 17 is characterized in that, described therapeutic gene is a tumor necrosis factor.

21. a method for the treatment of local and transitivity mammary gland and ovarian cancer, it is characterized in that the method comprising the steps of: the patient to these treatments of needs uses the described expression vector of claim 17.

22. a method for the treatment of local and transitivity mammary gland and ovarian cancer, it is characterized in that the method comprising the steps of: the patient to these treatments of needs uses the described expression vector of claim 18.

23. a method for the treatment of local and transitivity mammary gland and ovarian cancer, it is characterized in that the method comprising the steps of: the patient to these treatments of needs uses the described expression vector of claim 19.

24. a recombinant expression carrier pHIPs-X, it is derivable, the prostate specific promoter expression vector of heat, it is characterized in that described carrier comprises following box gene:

(b) box 2 contains minimum pCMV promoter mCMVp, antisense Gal-DBD-mx, and it is and the complementary antisense construct of Gal-DBD-mx, internal ribosome entry site and Gal-DBD, it and Gal-DBD-mx competition pGAL binding site;

(c) box 3 contains " VP16-TA-mc ", it is that an integrated open is read frame, preceding 11 aminoacid at N-terminal coding Ga14, the back is with the nuclear localization signal of SV40 large T antigen, the terminal trans-activation domain of 130 amino acid whose C-of hsv protein VP16, alkaline helix-loop-helix-leucine zipper domain of c-Myc aminoacid 350-439, the back is with SV40 polyA, and the fusion gene VP16-TA-mc of generation begins under the control of prostate basic protein gene promoter " pProbasin " from first ATG;

25. recombinant vector as claimed in claim 24 is characterized in that, has replaced prostate basic protein promoter with the prostate specific antigen promoter.

26. recombinant vector as claimed in claim 24 is characterized in that, described therapeutic gene is a tumor necrosis factor.

27. a method for the treatment of local and metastatic prostate cancer, it is characterized in that the method comprising the steps of: the patient to these treatments of needs uses the described expression vector of claim 24.

28. a method for the treatment of local and metastatic prostate cancer, it is characterized in that the method comprising the steps of: the patient to these treatments of needs uses the described expression vector of claim 25.